Longi udinal a iabili y o diazo oph
abundances in he sub opical
No h A lan ic Ocean
MAR BENAVIDES1,2*, PIA H. MOISANDER3, MEAGHAN C. DALEY3, ANTONIO BODE4AND JAVIER ARI
´STEGUI2
1
AIX MARSEILLE UNIVERSITE
´,CNRS/INSU,UNIVERSITE
´DE TOULON,IRD,MEDITERRANEAN INSTITUTE OF OCEANOGRAPHY (MIO)UM 110,98848 NOUME
´A,
NEW CALEDONIA,
2
INSTITUTO DE OCEANOGRAFI
´AYCAMBIO GLOBAL,UNIVERSIDAD DE LAS PALMAS DE GRAN CANARIA,35017 LAS PALMAS DE GRAN CANARIA,
SPAIN,
3
DEPARTMENT OF BIOLOGY,UNIVERSITY OF MASSACHUSETTS DARTMOUTH,NORTH DARTMOUTH,MA 02747,USA AND
4
CENTRO OCEANOGRA
´FICO DE A
CORUN
˜A,INSTITUTO ESPAN
˜OL DE OCEANOGRAFI
´A,APDO 130,15080 ACORUN
˜A,SPAIN
*CORRESPONDING AUTHOR: ma .bena[email p o ec ed]
Recei ed Sep embe 17, 2015; accep ed Decembe 11, 2015
Co esponding edi o : John Dolan
Diazo ophy- ela ed s udies in he No h A lan ic ha e la gely ocused on i s wes e n opical a ea, lea ing he sub-
opics and he eas unde sampled. We s udied he longi udinal dis ibu ion o T ichodesmium, UCYN-A, UCYN-B, he
pu a i e Gammap o eobac e ium g-24774A11 and Richelia (He 1) along 24.58N, using quan i a i e polyme ase chain
eac ion on di e en size ac ions (10, 10–3 and 3–0.2 mm) and addi ional ilamen coun s o T ichodesmium.
T ichodesmium was he mos abundan phylo ype, ollowed by UCYN-A, g-24774A11 and He 1, wi h maximum abun-
dances o 8.8 !10
5
, 2.0 !10
5
, 3.3 !10
3
and 3.4 !10
2
ni H copies L
21
, espec i ely, whe eas UCYN-B was mos ly
unde ec ed. A clea shi in he diazo oph communi y was obse ed a !308W, coinciding wi h he ansi ion
be ween he No h A lan ic Sub opical Gy e bounda y and inne co e. This ansi ion zone di ided he ansec in o
an eas e n hal domina ed by UCYN-A and wes e n hal domina ed by T ichodesmium and g-24774A11. g-24774A11
was only de ec ed in he 10–3 mm ac ion, sugges ing hei associa ion wi h la ge mic obes o agg ega es. Ou
esul s indica e ha ypical size ac iona ion by 10 mm is no op imal o econciling diazo oph phylo ypes o N
2
ix-
a ion a es and ha non-cyanobac e ial diazo ophs may con ibu e impo an ly o bulk diazo ophic ac i i y in he
wes e n sub opical No h A lan ic.
KEYWORDS: ni H; qPCR; N
2
ixa ion; upwelling; No h A lan ic Sub opical Gy e
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INTRODUCTION
The biological ixa ion o a mosphe ic dini ogen (N
2
)
p o ides signi ican inpu s o eac i e ni ogen o aqua ic
ecosys ems and hence con ibu es o phy oplank on
p ima y p oduc ion (Ka l e al., 2002). This p ocess is pe -
o med by specialized p oka yo es called diazo ophs,
dis ibu ed among b oad phylogene ic g oups om
cyanobac e ia and o he bac e ia o a chaea (Zeh e al.,
2003). The occu ence o N
2
ixa ion in he ocean was i s
desc ibed in he No h A lan ic basin and a ibu ed o he
ilamen ous cyanobac e ium T ichodesmium (Dugdale e al.,
1961). In he ollowing decades, i has become appa en
ha he diazo ophic communi y is also composed o
dia om-diazo oph symbioses (DDAs), unicellula cyano-
bac e ia (UCYN, di ided in o h ee g oups UCYN-A,
-B, -C) and non-cyanobac e ial diazo ophs (including
bo h bac e ia and a chaea) (Zeh e al., 2003). N
2
ixed
by UCYN and DDAs a imes locally exceeds ha o
T ichodesmium, highligh ing he impo ance o hese diazo-
ophs in N
2
ixa ion s udies (e.g. Ra en e al., 2015).
The di e en diazo ophic phylo ypes a e he e oge-
neously dis ibu ed in he No h A lan ic Ocean be-
cause o geog aphical di e ences in he en i onmen al
ac o s ha con ol hem (Bena ides and Voss, 2015).
T ichodesmium is known o needing ni a e-poo , wa m,
s a i ied wa e s and is abundan in he opical wes e n
A lan ic whe e hese condi ions pe sis (Capone e al.,
2005). UCYN-A a e small pho ohe e o ophic cyanobac-
e ia (,1mm) no capable o ca ying ou he
oxygen-e ol ing s eps o pho osyn hesis o ixing ino -
ganic ca bon (Zeh e al., 2008), and hey a e commonly
ound in symbiosis wi h a euka yo ic alga (Thompson
e al., 2012). UCYN-A seem o be able o ix N
2
a ela-
i ely low empe a u es and high ino ganic ni ogen con-
cen a ions (e.g. ,228C and .1.5 mM)(Tu k e al.,
2011;Ra en e al., 2015). UCYN-B a e es ic ed o
wa m opical wa e s (e.g. Moisande e al., 2010;Tu k
e al., 2011), whe eas UCYN-C a e o en associa ed wi h
coas al a eas (Langlois e al., 2008;Tu k e al., 2011).
DDAs need a sou ce o silica o he syn hesis o he us -
ule o he dia om hos ; hus silica in he i e plumes o
he Amazon and Congo i e s may p omo e hem (Fos e
e al., 2007;2009;Sub amaniam e al., 2008). The dis i-
bu ion o non-cyanobac e ial diazo ophs in he No h
A lan ic seems o be ex ensi e, spanning a wide la i udin-
al ange (Luo e al., 2012), al hough hei ecology and
en i onmen al cons ain s a e unknown (Fa nelid e al.,
2011), pa ly because o he di icul ies in disce ning
cyanobac e ial om non-cyanobac e ial N
2
ixa ion a es
in
15
N
2
ace incuba ions (Bena ides and Voss, 2015).
Diazo ophic bac e ia om he open ocean clus e wi h a
wide ange o phylogene ic g oups, including Fi micu es,
Alpha-, Be a-, Gamma- and Del ap o eobac e ia (Zeh
e al., 2003). The Gammap o eobac e ia-a ilia ed phylo-
ype g-24774A11 was eco e ed om he Sou h China
Sea (Moisande e al., 2008), and he same diazo oph
phylo ype (also called Gamma A o UMB) has been
eco e ed a high equencies om se e al en i onmen s
such as he A lan ic, Paci ic and Indian Oceans and he
Medi e anean Sea (Bi d e al., 2005;Chu ch e al., 2005a;
Tu k e al., 2011;Moisande e al., 2014;Shiozaki e al.,
2014;Langlois e al., 2015). A leas h ee di e en pub-
lished quan i a i e polyme ase chain eac ion (qPCR)
p ime se s a ge his phylo ype clus e ha has some
mic odi e si y (Moisande e al., 2014), and hese p ime s
ha e been e med Gammap o eobac e ia (Chu ch e al.,
2005a), Gamma-A (Langlois e al., 2008) and g-24774A11
(Moisande e al., 2008). Each o hese h ee p ime se s
a ge s a sligh ly di e en a ian in he clus e (Moisande
e al., 2014).
The dis ibu ion and abundance o all o hese diazo-
ophic phylo ypes ha e been p e iously epo ed in he
No h A lan ic by qPCR assays o he ni H gene (which
encodes o he ni ogenase i on p o ein componen o
he ni ogenase enzyme complex) (e.g. Langlois e al.,
2008;Fos e e al., 2009;Goebel e al., 2010). The e is a
geog aphic bias in he dis ibu ion o hese s udies, wi h
mo e samples aken in he wes e n han in he eas e n
No h A lan ic Ocean (Mon oya e al., 2007). Mo eo e ,
mo e sampling has been conduc ed in he opics com-
pa ed wi h highe la i udes, lea ing he sub opical and
empe a e la i udinal zones wi h sca ce da a (Bena ides
and Voss, 2015). Mos ni H da a a ailable in he No h
A lan ic a e es ic ed o he la i udinal band loca ed
be ween he Equa o and !158N, and he e is a wide
gap in obse a ions in he a eas comp ised by 20–408N
and 40–808W, as can be obse ed in Fig. 3 o Luo e al.
(2012). This gap impedes a comp ehensi e assessmen o
he spa ial dis ibu ion o di e en diazo oph species in
he No h A lan ic.
Wi h he aim o con ibu ing o he ew published
s udies on he longi udinal a iabili y o diazo ophy in
he No h A lan ic (Vos s e al.,2004;Mon oya e al.,2007),
we quan i ied he abundance o he main diazo ophic phy-
lo ypes along 24.58N om10 o808Wandexamined hei
geog aphical dis ibu ions in pa allel wi h di e en en i on-
men al condi ions and phylo ype-speci ic cons ain s.
METHOD
Hyd og aphy, nu ien s and chlo ophyll
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R/V Sa mien o de Gamboa,sailingwes wa ds om he
No hwes A ican coas o he No h Ame ican coas along
24.58N(Fig.1). Tempe a u e, salini y and chlo ophyll-
luo escence da a we e eco ded wi h a SeaBi d 911 plus
CTD equipped wi h a Sea-Tech luo ome e . The CTD
was moun ed on a Gene al Oceanics ose e ame
equipped wi h 24 Niskin bo les (12 L olume).
Samples o nu ien analysis we e aken wi h he 12 L
Niskin bo les, s o ed in 15 mL polyp opylene ubes and
immedia ely ozen a 2208C un il analysis asho e. The
concen a ions o ni a e plus ni i e, phospha e and
silica e we e de e mined wi h a Technicon segmen ed- low
au oanalyse . S anda d me hods we e modi ied o ob ain a
de ec ion limi o 2 nmol L
21
(Raimbaul e al., 1990).
De ails on he concen a ions and dis ibu ions o nu ien s
can be ound elsewhe e (Bena ides e al., 2013b;
Mompea
´ne al., 2013) and will be used he e only wi h he
pu pose o in e p e ing he dis ibu ion o diazo ophs.
The mixed laye dep h (MLD) was es ima ed om an
inc ease in wa e column densi y (
s
) o 0.125 kg m
23
wi h espec o su ace alues (Bena ides e al., 2013a).
Chlo ophyll a(Chl a;mgm
23
) da a we e ob ained om
he Na ional Ae onau ics and Space Adminis a ion
(NASA) Godda d Ea h Sciences Da a and In o ma ion
Se ices Cen e Gio anni (NASA GES DISC) online
da abase and a e aged o he mon hs o Feb ua y and
Ma ch 2011.
DNA collec ion, ex ac ion and qPCR assays
Samples o DNA we e aken a 15 s a ions along he
ansec (Table I). Su ace seawa e samples (!5 m) we e
collec ed using a 30 L Niskin bo le. Ten li es we e ans-
e ed o acid-washed da kened ca boys wi h a ba bed
auce . Acid-washed acuum ubing was connec ed o he
auce , and h ee sepa a e il e holde s (Wha man) we e
connec ed in-line con aining 47 mm diame e whi e 10, 3
and 0.2 mm polyca bona e il e s (GE-Osmonics Po e ics).
The end o he line was connec ed o a wa e je aspi a o
acuum pump (Eyela). The ac ions e ained we e hus
.10, ,10– .3 (he ea e abb e ia ed as 10–3 mm) and
,3–.0.2 mm (he ea e abb e ia ed as 3–0.2 mm),
espec i ely. A e il a ion, he il e s we e ans e ed o
s e ile sc ewcap c yo ials and s o ed a 2808C un il ana-
lysis.
DNA was ex ac ed using he DNeasy Plan Mini Ki
(Qiagen), as modi ied by Moisande e al. (2008). The
abundance o diazo ophs was de e mined using TaqMan
qPCR assays and p e iously published p ime –p obe se s
o T ichodesmium,UCYN-A(Chu ch e al.,2005a), UCYN-B
(Moisande e al., 2010), he Gammap o eobac e ium
g-24774A11 (Moisande e al., 2008) and He 1 (Richelia-
Rhizosolenia DDAs) (Chu ch e al., 2005b). T ichodesmium
ichomes usually measu e .80 mm and we e he e o e
only analysed on he .10 mm ac ion. He 1 on he
.10 and 10–3 mm ac ions, UCYN-A and UCYN-B
we e analysed in he 10–3 and 3–0.2 mm ac ions, and
he g-24774A11 we e analysed in all h ee size ac ions.
The qPCR was un in 20 mL eac ions ha consis ed
o 10 mL ABI TaqMan Gene Exp ession Mas e Mix,
6.4 mL nuclease- ee wa e , 0.5 and 0.25 mM inal con-
cen a ions o p ime s and p obe, espec i ely, and
1.6 mL DNA empla e. All samples we e un in duplica e.
Ten- old dilu ions o linea ized plasmids con aining he
ele an ni H a ge s we e used as s anda ds and we e
included wi h each 96-well pla e un. The eac ions we e
un on a S epOnePlus Real-Time PCR sys em (Li e
Technologies). S anda d cu es we e made by plo ing
linea eg ession o he h eshold cycle (C
) and log gene
copies pe eac ion using duplica e s anda ds anging
om 10
8
o 10
0
gene copies. Ampli ica ion e iciencies
we e .90% o all eac ions. Duplica e no empla e
con ol wells we e included in all pla es un and did no
show ampli ica ion in any uns. Inhibi ion es s we e
ca ied ou o all samples by combining he sample and
1.6 mL o 10
5
s anda d as a empla e. The e iciencies o
inhibi ion es s anged om 97.3 o 101.73%, and hus
we conside ha ou samples we e no inhibi ed. The
limi o de ec ion (LOD) and de ec ed bu no quan i i-
able (DNQ) limi s used we e one and eigh gene copies
pe eac ion, espec i ely. Samples ha we e below LOD
we e designa ed a alue o 0 in he da a se , whe eas gene
copies highe han LOD bu less han DNQ we e desig-
na ed a conse a i e alue o 1 ni H gene copy pe li e.
Fig. 1. Map o s a ions whe e CTD p o iles we e pe o med (“CTD s a ions”, labelled wi h an as e isk) and s a ions whe e samples o
ni H analyses we e aken (“ni H s a ions”, labelled wi h an open symbol and s a ion numbe ), supe imposed on chlo ophyll aconcen a ion da a (Chl
a; mg m
23
).
M. BENAVIDES ET AL.
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T ichodesmium
ilamen coun s
Samples we e collec ed by owing a 40 mm mesh size
plank on ne om 200 m o he su ace a 43 s a ions
(Mompea
´ne al., 2013), om which 6 s a ions (24, 45, 85,
91, 97 and 104; Fig. 1) we e coinciden wi h hose whe e
DNA was sampled. The olume o seawa e il e ed a
each s a ion was 14 m
3
.T ichodesmium ilamen coun s we e
made on aliquo s o he sample p ese ed in glu a alde-
hyde (25% inal concen a ion) using a FlowCam sys em
(Fluid Imaging Technologies), ollowing he ecommenda-
ions by A
´l a ez e al.(2014). Samples we e un in he
au oimage mode using a 4!lens (40!o e all magni ica-
ion) and a low cell o 300 mm dep h. P io o analysis,
he samples we e sc eened wi h a 100 mm nylon mesh o
p e en clogging o he FlowCam cell. Resul s a e epo ed
as numbe o ichomes pe li e o seawa e .
N
2
ixa ion a es
The ac iona ed (.10 and ,10 mm) N
2
ixa ion was
assayed wi h he dissol ed
15
N
2
me hod acco ding o
Moh e al. (2010), as desc ibed in Bena ides e al. (2013b).
RESULTS
Hyd og aphy, nu ien s and Chl
a
da a
Tempe a u e and salini y we e low nea he coas al up-
welling o No hwes A ica (!198C and 36.8, espec -
i ely; Table I). F om eas o wes , empe a u e inc eased
om ca. 20 o ca. 258C a 628W and hen dec eased
sligh ly ( o !238C) un il he wes e nmos s a ion.
Fluo escence alues we e highes o he No hwes
A ican coas (Table I) and dec eased wes wa ds mi o -
ing he dis ibu ion o Chl a(Fig. 1). The su ace concen-
a ions o ni a e plus ni i e, phospha e and silica e we e
maximal o he No hwes A ican coas (12.61, 0.73
and 19.34 mM, espec i ely; Table I) and also dec eased
wes wa ds along he ansec . Fu he de ails on hyd o-
g aphic a iables measu ed along he ansec can be
ound elsewhe e (Bena ides e al., 2013b;Mompea
´ne al.,
2013). The MLD was !80 m in he eas e n end o he
ansec , and i deepened in he cen al pa o he No h
A lan ic Sub opical Gy e (NASG), shoaling owa ds he
wes e n end o he ansec wi h alues !60 m (Table I).
N
2
ixa ion a es ( om Bena ides e al., 2013b) we e in a
simila ange in he .10 mm ac ion (0.009–
0.385 nmol N L
21
h
21
) and in he ,10 mm ac ion
(0.011–0.335 nmol N L
21
h
21
), al hough he o me
we e gene ally highe han he la e along he ansec .
N
2
ixa ion a es we e highes o he No hwes A ican
coas and main ained alues !0.3 nmol N L
21
h
21
Table I: Measu ed alues o co e pa ame e s ( empe a u e, salini y and luo escence) and ino ganic nu ien concen a ions (ni a e plus ni i e
-NO
3"þNO2"-;silica e - SiO2";and phospha e - PO43"-;Bena ides e al., 2013b;Mompea
´ne al., 2013) a s a ions whe e
diazo oph phylo ypes we e su eyed
S a ion
Da e (dd/mm
in 2011)
La i ude
(8N)
Longi ude
(8W)
Su ace
empe a u e (8C)
Su ace
salini y
Su ace luo escence
( ela i e uni s)
MLD
(m)
NO
3
2
þNO
2
2
(mM)
PO
4
32
(mM)
SiO
2
(mM)
.10 mmN
2
ixa ion a es
(nmol N L
21
h
21
)
a
,10 mmN
2
ixa ion a es
(nmol N L
21
h
21
)
a
1 28/01 27.78 13.34 19.59 36.77 1.61 75.50 12.47 0.73 19.34 0.385 0.335
13 29/01 27.02 15.70 20.70 36.96 1.20 87.41 0.05 0.58 0.66 0.210 0.167
24 01/02 25.63 20.00 21.73 37.06 1.19 123.16 0.07 0.65 0.58 0.191 0.143
36 05/02 24.50 26.54 21.57 37.24 1.05 129.12 0.11 0.20 0.12 0.261 0.237
41 07/02 24.50 29.59 22.44 37.42 0.93 123.16 0.01 0.58 0.86 0.302 0.181
45 09/02 24.50 32.04 22.81 37.45 0.92 99.33 0.01 0.65 0.79 0.271 0.151
58 13/02 24.50 38.49 23.07 37.53 0.85 117.21 0.13 0.39 0.74 0.178 0.018
77 18/02 24.50 47.27 23.41 37.39 0.86 93.37 0.05 0.42 0.20 0.243 0.116
85 21/02 24.50 50.96 23.99 37.24 0.76 81.46 n/a n/a n/a 0.093 0.023
91 23/02 24.50 54.03 24.26 36.99 0.79 57.62 n/a n/a n/a 0.104 0.041
97 25/02 24.50 57.72 23.99 36.73 0.80 39.74 n/a n/a n/a 0.108 0.051
104 27/02 24.50 62.01 24.92 36.28 0.83 63.58 n/a n/a n/a 0.142 0.065
110 01/03 24.50 65.68 23.82 36.73 0.76 57.62 n/a n/a n/a 0.168 0.088
129 07/03 26.20 73.82 23.81 36.78 1.07 51.66 0.19 0.16 0.12 0.024 0.033
139 09/03 26.20 76.15 22.69 36.77 0.89 75.50 0.12 0.17 n/a 0.089 0.061
n/a indica es da a no a ailable.
a
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wes wa ds un il !328W and hen dec eased s eadily
owa ds he wes e n end o he ansec (Table I).
Longi udinal dis ibu ion o diazo ophic
phylo ypes
The di e en diazo ophic phylo ypes showed a iabili y
in hei longi udinal dis ibu ion, wi h a shi in hei pa -
e ns occu ing a !308W (Fig. 2). T ichodesmium (only
assayed in he .10 mm ac ion) had he highes ni H
copy L
21
numbe s de ec ed o e all in his s udy, showing
low abundances eas o !308W, bu inc easing om
!10
4
ni H copies L
21
o a maximum o 8.8 !10
5
ni H
copies L
21
owa ds he wes e n end o he ansec
(Fig. 2a). T ichodesmium ichomes (as coun ed by he
FlowCam om samples eco e ed wi h a plank on ne ,
see Me hods) we e absen be ween s a ion 1 and !158W,
peaked a !288W wi h !3!10
5
ichomes L
21
and
hen dec eased s eadily un il 7–9 !10
3
ichomes L
-1
a
he wes e nmos s a ions o he ansec (Fig. 2a).
The pu a i e Gammap o eobac e ium g-24774A11
was de ec ed in he .10 and 10–3 mm ac ions, wi h
maximum abundances in he o me up o 3.3 !10
3
ni H copies L
21
, al hough hei longi udinal dis ibu ion
and abundance we e e y simila in bo h size ac ions
(Fig. 2a and b). In he .10 mm ac ion, g-24774A11
showed a s able longi udinal pa e n a 10
2
ni H copies
L
21
, whe eas in he 10–3 mm ac ion, hei dis ibu ion
was simila o ha o T ichodesmium, wi h non-de ec able
abundances a longi udes eas o !308W and an inc eas-
ing pa e n wes o his poin , s abilizing a 10
2
ni H
copies L
21
un il 808W (Fig. 2b). g-24774A11 we e no
de ec ed in he 3–0.2 mm ac ion (Fig. 2c). He 1 we e
de ec ed a low abundances (6.2 !10
1
–3.4 !10
2
ni H
copies L
21
; Fig. 2a and b) a s a ions loca ed eas o
!308W, wi h he excep ion o s a ion 104 a 628W, whe e
hey we e de ec ed a 3.8 !10
1
ni H copies L
21
(Fig. 2a).
The abundance o UCYN-A dec eased longi udinally
om eas o wes (Fig. 2b and c). They we e de ec ed
om he eas e nmos s a ion un il !508W in he
10–3 mm ac ion (Fig. 2b), bu only un il !308W and
a highe abundances (!10
4
ni H copies L
21
) in he
3–0.2 mm ac ion (Fig. 2c). UCYN-B we e only de ec ed
in one sample (s a ion 91, 548W) a a low abundance
(!10
2
ni H copies L
21
).
Co ela ions wi h en i onmen al a iables
and eas -wes di e ences
The signi ican Spea man co ela ions (P,0.00042
a e applying a Bon e oni co ec ion o mul iple com-
pa isons; McDonald, 2014) be ween he abundance o
diazo ophic phylo ypes wi h en i onmen al pa ame e s
( empe a u e, salini y, oxygen, luo escence and nu ien s)
a e shown in Table II. Some signi ican co ela ions
we e ound be ween di e en diazo ophs. UCYN-A
om he 3–0.2 mm ac ion we e nega i ely co ela ed
wi h g-24774A11 om he 10–3 mm ac ion and wi h
T ichodesmium ni H copies (analysed only in he .10 mm
ac ion). In e es ingly, T ichodesmium ni H copies we e no
signi ican ly co ela ed wi h hei co esponding ich-
ome coun s, al hough isually some co espondence was
appa en in he wo me hods (Fig. 2a). The la e we e
ins ead co ela ed posi i ely wi h g-24774A11 (in bo h
he .10 and 10–3 mm ac ions).
Some diazo ophs also showed signi ican co ela ions
wi h en i onmen al pa ame e s. A nega i e co ela ion
was obse ed be ween empe a u e and he ni H gene
copies o UCYN-A om he 3–0.2 mm ac ion. Posi i e
co ela ions we e also obse ed be ween salini y and
T ichodesmium ichome coun s, and hence he co ela ion
o he la e was nega i e wi h empe a u e ( empe a u e
and salini y we e nega i ely co ela ed). UCYN-A e-
co e ed om he 3–0.2 mm ac ion we e posi i ely
co ela ed wi h oxygen and luo escence (no e ha luo -
escence and oxygen we e co ela ed). No signi ican
co ela ions we e ound wi h ino ganic nu ien s.
I we di ide he ansec in o wo hal es (wes and eas
o 27.158W o s a ion 37) and pool he obse a ions
Fig. 2. Abundance o he diazo ophic phylo ypes (ni H copies L
21
)
T ichodesmium,g-24774A11, He 1 and UCYN-A e ained on (a) he
.10 mm, (b)3mm and (c) 0.2 mm ac ions. The abundance o
T ichodesmium ichomes (log
10
ichomes L
21
) is supe imposed on (a)
and scale shown on he igh y-axis. 163 !183 mm
2
(200 !200 DPI).
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wi hin each o he hal es, s eep g adien s in ni H abun-
dances a e demons a ed: he abundance o UCYN-A in
he 3–0.2 mm ac ion, g-24774A11 in he 10–3 mm
ac ion and T ichodesmium ni H and ilamen coun s we e
signi ican ly di e en be ween he eas e n and wes e n
hal es (Wilcoxon es P¼0.05, 0.02, 0.006 and 0.009,
espec i ely).
DISCUSSION
Longi udinal a iabili y o diazo oph
phylo ypes
Ou esul s sugges ha he sub opical A lan ic Ocean
has a clea eas -wes di ide a !308W, whe e he p od-
uc i i y o su ace wa e s dec eases as depic ed by Chl a
concen a ions in Fig. 1, he deep chlo ophyll maximum
deepens and he p esence o measu able phospha e con-
cen a ions wi hin he eupho ic zone disappea s (Table I;
see also Fig. 2 in Mompea
´ne al., 2013). This is a physical
sepa a ion caused by he ansi ion om he upwelling-
in luenced wa e s o he No hwes A ican coas o he
NASG, which a ec s he s uc u e and ac i i y o plank-
onic communi ies (e.g. He na
´ndez-Leo
´ne al., 1999).
Ou da a show ha his physical ba ie also a ec s
diazo ophs, in a phylo ype-speci ic manne .
T ichodesmium was he mos abundan diazo oph phy-
lo ype de ec ed by qPCR assays along he ansec , ol-
lowed by UCYN-A and g-24774A11, in ag eemen wi h
p e ious s udies in he No h A lan ic (e.g. Langlois e al.,
2008;Ra en e al., 2015). T ichodesmium was ound a low
abundances eas o !308W es ima ed by ei he qPCR o
ilamen coun s (Fig. 2a), ag eeing wi h p e ious s udies
(He na
´ndez-Leo
´ne al., 1999;Fe na
´ndez e al., 2012), and
inc eased owa ds he wes in pa allel wi h inc easing
empe a u e and a shoaling MLD (Table I), consis en
wi h i s equi emen s o high ligh and oligo ophic con-
di ions (Ca pen e and Roennebe g, 1995). T ichodesmium
is known o mos equen ly h i ing in wa m, s a i ied
and ni a e-poo wa e s (Capone e al., 2005), whe eas in
he colde and nu ien - iche wa e s o he eas e n sub-
opical and opical No h A lan ic, i is almos absen o
p esen a e y low abundances and mainly ound as ee
ichomes ins ead o colonies (Fe na
´ndez e al., 2010;
Goebel e al., 2010;Bena ides e al., 2011).
In his s udy, we de e mined he abundance o
T ichodesmium in wo ways: by quan i ica ion o ni H gene
copies in seawa e samples collec ed om he 5 m dep h
wi h Niskin bo les and il e ed on o 10 mm il e s
(.10 mm ac ion) and by e ically sampling he wa e
column h ough he op 200 m using a 40 mm mesh ne ,
ollowed by coun ing ichomes wi h a FlowCam (see
Me hods). Al hough he o e all eas -wes end was
Table II: Signi ican (P,0.00042) Spea man co ela ions (posi i e o nega i e) be ween diazo oph abundances, en i onmen al a iables and
nu ien concen a ions
UCYN-A
3mm
UCYN-A
0.2 mm
g-24774A11
10 mm
g-24774A11
3mm
T ichodesmium
ni H
T ichodesmium
coun s
He 1
10 mm
He 1
3mm Tempe a u e Salini y Oxygen Fluo escence Ni a e þni i e Phospha e Silica e
UCYN-A 3 mm
UCYN-A 0.2 mm22 2 þþ
g-24774A11
10 mm
þþ
g-24774A11
3mm
þ
T ichodesmium
ni H
T ichodesmium
coun s
þ
He 1 10 mm
He 1 3 mm
Tempe a u e 22 2
Salini y þ
Oxygen
Fluo escence
Ni a eþni i e
Phospha e þ
Silica e
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simila , T ichodesmium ni H copy numbe s did no
co ela e signi ican ly wi h T ichodesmium ichome coun s
(Table II and Fig. 2a). When di iding T ichodesmium ni H
copy numbe s by ichome coun s a s a ions whe e bo h
ypes o da a we e a ailable, each ichome would ha e
con ained be ween 0.7 and 4.5 ni H copies (wi h an ex-
cep ion o 19 ni H copies pe ilamen a s a ion 97; da a
no shown). This is one o wo o de s o magni ude ewe
han would be expec ed, gi en ha a 100 mm ilamen
has app oxima ely 70 cells (Ty ell e al., 2003). The
numbe o cells pe ichome is a iable (usually up o
100 cells, bu inding b oken ilamen s wi h ewe cells is
common; Bena ides e al., 2011). Pa allel compa ison o
he wo coun ing me hods was only possible o six s a-
ions, and he di e ences be ween hese me hods may
ha e a ec ed he esul s conside ably. Plank on ne s a e
gene ally p e e ed o e Niskin bo les o sampling
T ichodesmium when i s abundance is low (Chang, 2000),
gi en he eno mous di e ence in he olume o seawa e
il e ed in each case ( ens o li es om Niskin bo les
e sus hund eds o housands o li es concen a ed when
using ne ows). The di e ences obse ed be ween he
wo coun ing me hods a e also likely associa ed wi h he
sampling dep hs co e ed (5 m e sus in eg a ed o e
200 m), as well as he e ical mig a ion o T ichodesmium
colonies (Villa eal and Ca pen e , 2003), and/o hei
pa chy dis ibu ion in he wa e column (Ca pen e e al.,
2004;Da is and McGillicuddy, 2006).
UCYN-A de ec ed in he 10–3 mm ac ion we e
mo e abundan eas o !308W and only de ec ed in he
3–0.2 mm ac ion om samples collec ed be ween
!308W and he No hwes A ican coas (Fig. 2b and c).
UCYN-A we e he second mos abundan phylo ype in
ou s udy, highligh ing hei po en ial ole in bulk N
2
ix-
a ion ac i i y. UCYN-A usually appea in symbiosis wi h
a p ymnesiophy e om which i ob ains o ganic ca bon
(Thompson e al., 2012), al hough i has also been ound
in associa ion wi h la ge plank on cells such as dia oms
and dino lagella es (Thompson e al., 2014), and a imes
as ee-li ing cells, likely due o hei loose associa ion
wi h he su ace o hei hos (Thompson e al., 2012;
K upke e al., 2014). These di e en associa ion possibil-
i ies, as well as de achmen du ing sample handling,
would explain he p esence o UCYN-A in bo h he
10–3 and he 3–0.2 mm ac ions in ou s udy (Fig. 2b
and c), which is consis en wi h p e ious epo s (Agawin
e al., 2014). Uniden i ied diazo ophic cells in he
UCYN-A size ange we e also obse ed in agg ega es
using whole-cell hyb idiza ion echniques (Bena ides
e al., 2013a), which could con ibu e o hei gene copies
being ound in he 10–3 mm size ac ion in his s udy.
The associa ion o UCYN-A wi h phy oplank on cells
also likely explains hei appa en p e e ence o colde
and nu ien - ich wa e s compa ed wi h o he diazo-
ophs (Moisande e al., 2010). In he No h A lan ic,
UCYN-A ha e been ound in wa e s wi h empe a u es
anging om !128C o lowe o 238C(Langlois e al.,
2008;Rees e al., 2009;Ben zon-Tilia e al., 2014;K upke
e al., 2014;Sca o o e al., 2015). UCYN-A ha e also
been epo ed as he dominan diazo oph in he up-
welling wa e s o he No hwes coas o A ica wi h
empe a u es as low as 178C and high nu ien con-
cen a ions (Agawin e al., 2014), as well as o e he
Equa o ial upwelling wa e s whe e simila condi ions
a e ound (Fos e e al., 2009). Al hough ac i e UCYN-A
(as ni H ansc ip s) we e no ound in hese cold and
nu ien - ich wa e s (e.g. Fos e e al., 2009), he highe N
2
ixa ion ac i i y o he No hwes coas o A ica
(Bena ides e al., 2013b) and in he Equa o ial upwelling
whe e hey domina e (Sub amaniam e al., 2013) sugges s
ha hey con ibu e impo an ly o ixed ni ogen inpu s
in hese a eas. In ou s udy, UCYN-A we e de ec ed a
.10
4
ni H gene copies L
21
in wa e s wi h empe a u es
anging be ween !19 and 218C, wi h e y high nu i-
en concen a ions (e.g. ni a e plus ni i e .12 mM;
Table I). Howe e , al hough hese diazo ophs ha e
been de ec ed in highe la i ude coas al wa e s (o he
No heas Ame ican coas ; Mulholland e al., 2012),
hei ubiqui ous p esence in oceanic wa e s a la i udes
.30–408N was sugges ed o be unlikely (K upke e al.,
2014). The clea longi udinal seg ega ion in UCYN-A
abundances may also be d i en by i on-s ess alle ia ion
ia Saha an dus inpu s, which a e highe in his longi-
udinal ange o he ansec (Bena ides e al., 2013b)
and we e epo ed o limi N
2
ixa ion by UCYN-A
(K upke e al., 2015).
Non-cyanobac e ial diazo ophs a e cosmopoli an,
and hei ac i i y may be d i en by ac o s o he han
hose con olling cyanobac e ial diazo ophs (Fa nelid
e al., 2011). In e es ingly, du ing ou s udy, g-24774A11
ni H copies we e de ec ed a abundances compa able o
hose o UCYN-A (Fig. 2). The g-24774A11 ha e been
shown o consis en ly exp ess hei ni H gene in he open
ocean (Moisande e al., 2014), sugges ing ac i e N
2
ix-
a ion. Thus, i hei pe cell N
2
ixa ion ac i i ies a e as
high as wi h UCYN-A, wi h equal cell numbe s hey
migh ha e a compa able con ibu ion o in si u N
2
ixa ion a es. The g-24774A11 we e ound exclusi ely in
he .10 and 10–3 mm size ac ions, implying he
a achmen o hese diazo ophs o la ge mic obes, pa -
icula e ma e ial o a sel -p oduc ion o o ganic agg e-
ga es. The associa ion o non-cyanobac e ial diazo ophs
wi h pa icles as a means o ob aining o ganic nu ien s
has been p e iously hypo hesized, and gammap o eo-
bac e ial ep esen a i es ha e been ound o ming agg e-
ga es in oxygena ed cul u es (Ben zon-Tilia e al., 2015).
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A pa icle-a ached mode o li e may p o ide se e al
ad an ages o plank onic bac e ial diazo ophs, such as
oxygen-deple ed mic ozones and he a ailabili y o
o ganic ma e (Riemann e al., 2010). Howe e , he lon-
gi udinal dis ibu ion o Chl aalong he ansec (Fig. 1)
sugges s ha o ganic pa icles we e mo e abundan eas
o !308W han on he wes e n hal o he ansec ,
which is opposi e o he dis ibu ion o g-24774A11 in
he .10 and 10–3 mm ac ions (Fig. 2a and b). This
makes he sel -p oduc ion o o ganic agg ega es he mos
plausible explana ion o hei longi udinal dis ibu ion
in ou s udy, which is consis en wi h a p e ious s udy
(Ben zon-Tilia e al., 2015). Ne e heless, he associa ion
o diazo ophic Gammap o eobac e ia wi h pelagic pa -
icles has no been s udied di ec ly, and we canno
assume ha he coloniza ion o pa icles by hese
mic obes is equal in space and ime; i is possible ha i
changes wi h pa icle size, chemical composi ion, among
o he cha ac e is ics. The il a ion me hod we used may
ha e con ibu ed o he obse a ion o high abundances
o g-24774A11 in he .10 and 10–3 mm size ac ions.
In ou pas s udies in which a pe is al ic pump sys em
was used, his phylo ype was no de ec ed in he
.10 mm size ac ion (Moisande e al., unpublished
da a). I is possible ha he wa e je acuum aspi a o
il a ion me hod used he e is mo e gen le han he pe i-
s al ic pump sys em and gene a es less bac e ial de ach-
men om pa icles o agg ega es. The high abundance
o g-24774A11 de ec ed oge he wi h p e ious s udies
(Moisande e al., 2014;Langlois e al., 2015) unde sco es
he cu en need o quan i y he con ibu ion o non-
cyanobac e ial diazo ophs o global N
2
ixa ion a es, as
well as o elucida e hei ecology and physiological
cons ain s. A p esen , i is ou inabili y o disce n cyano-
bac e ial om non-cyanobac e ial N
2
ixa ion ac i i y
ha keeps us om u he es ima ing he global impo -
ance o he la e (Bena ides and Voss, 2015), al hough
he measu able diazo ophic ac i i y in apho ic wa e s
(e.g. Bonne e al., 2013), as well as he enhancemen o
N
2
ixa ion a es upon he addi ion o pho osys em
II-blocking agen s o s op he ac i i y o au o ophic dia-
zo ophs (Raha e al., 2015), sugges s ha hei ac i i y
may be signi ican .
Simila o UCYN-A, He 1 diazo ophs (Richelia)
depend on he ecological cons ain s o hei hos
(Rhizosolenia dia oms). O e he ansec su eyed in his
s udy, He 1 we e ound a low abundances eas o
!308W in he NASG bounda y a ea (Fig. 2), coinciding
wi h he highes phospha e and silica e concen a ions o
he ansec because o he p oximi y o he No hwes
A ican coas al upwelling (Table I). He 1 a e usually
es ic ed o a i e plume o coas al uno -a ec ed a eas
such as he Amazon and he Congo Ri e plumes (Fos e
e al., 2007,2009) and he No h Ame ican coas a
highe la i udes (e.g. .308N; Ra en e al., 2015). Thei
maximum abundances measu ed du ing ou s udy
(!10
2
ni H copies L
21
; Fig. 2) a e below hose in he
Amazon and Congo Ri e plumes (up o 10
5
and 10
3
ni H copies L
21
, espec i ely; Fos e e al., 2007,2009),
and o he s udies pe o med in open-ocean wa e s o he
No h A lan ic ha e gene ally also ound low abundances
(,10
1
ni H copies L
21
;Luo e al., 2012; also see Fig. 3 in
Bena ides and Voss, 2015). O e all, he low abundances
and he low equency o de ec ion among samples o e
he ansec sugges ha hei con ibu ion o bulk N
2
ix-
a ion in his a ea is minimal. I also needs o be no ed
ha al hough he ni H gene copies a e de ec ed in each
cell o he e ocys ous diazo ophs, only some o he cells
a e he e ocys s and hus N
2
- ixing. I only he numbe o
N
2
- ixing cells is conside ed, he numbe s epo ed he e
should be di ided by he es ima ed ege a i e cell: he e o-
cys equency, which o Richelia has been epo ed o be
app oxima ely 5 (Fos e e al., 2009). Taking in o accoun ,
his con e sion would educe he con ibu ion o his g oup
o N
2
ixa ion u he .
Rela ionship be ween he diazo ophic
communi y and N
2
ixa ion a es along
24.58N
The longi udinal a iabili y o N
2
ixa ion ac i i y in he
No h A lan ic (encompassing bo h i s eas e n and
wes e n basins) has only been add essed in a ew s udies
(Voss e al., 2004;Mon oya e al., 2007;Bena ides e al.,
2013b;Ra en e al., 2015). The size- ac iona ed (.10
and ,10 mm) N
2
ixa ion a es co esponding o wa e
samples in es iga ed in his s udy ha e been published
p e iously (Bena ides e al., 2013b) and a e also shown in
Table I.N
2
ixa ion a es in bo h ac ions dec eased
om eas o wes om !0.4 o 0.01 nmol N L
21
h
21
.
This pa e n is opposi e o wha would be expec ed om
aT ichodesmium-domina ed communi y; indeed, highe
a es owa ds he wes e n A lan ic ha e been obse ed
p e iously in s udies ocusing on hese cyanobac e ia (e.g.
Capone e al., 2005), bu o he bulk wa e N
2
ixa ion
s udies ound an opposi e longi udinal pa e n (e.g. Voss
e al., 2004). Bena ides e al. (2013b) di ided he ansec
in o eas e n and wes e n hal es a abou !458W, coin-
ciding wi h a change in sea su ace heigh alues. Despi e
his di ision ha esul ed in signi ican ly di e en N
2
ixa ion a es be ween he eas e n and wes e n hal es o
he ansec , he isual inspec ion o N
2
ixa ion a es
(Table I) shows ha he s eepes change in N
2
ixa ion ac-
i i y occu s a !328W, ag eeing wi h he shi obse ed
in he composi ion o he diazo ophic communi y in his
s udy (Fig. 2).
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P io epo s sugges ha in opical la i udes, UCYN-A
domina e he N
2
ixa ion ac i i y in he eas e n No h
A lan ic basin, whe eas T ichodesmium domina e in he
wes e n basin (Mon oya e al., 2007;Bena ides and Voss,
2015). This s udy sugges s ha a simila end in ni H phy-
lo ype dis ibu ions o UCYN-A and T ichodesmium is
ound also in sub opical la i udes. The dis ibu ion o ni H
copies in he di e en size ac ions measu ed sugges s ha
in sub opical la i udes Gammap o eobac e ia may also
con ibu e impo an ly o .3mmN
2
ixa ion in he
wes e n No h A lan ic basin. The p esence o small dia-
zo ophs such as Gammap o eobac e ia in he .10 and
10–3 mm size ac ions sugges s ha addi ional size ac-
iona ion o samples incuba ed wi h
15
N
2
wi h 3 mm il e s
could p o ide u he in o ma ion abou g oups con ibu -
ing o he N
2
ixa ion a es.
O e ou ansec , ,10 mmN
2
ixa ion a es con ibu-
ed on a e age 40% o bulk N
2
ixa ion (Bena ides e al.,
2013b), sugges ing an impo an ole o he diazo ophs
cap u ed on he 10–3 and 3–0.2 mm ac ions and
quan i ied in his s udy. Mo eo e , ,10 mmN
2
ixa ion
a es we e signi ican ly highe in he eas e n hal o he
ansec when compa ed wi h he wes e n hal , suppo ed
by he high abundance o UCYN-A de ec ed (Fig. 2). In
summa y, N
2
ixa ion a es may be d i en p ima ily by
UCYN-A in he eas e n pa o he ansec and by a
combina ion o T ichodesmium and g-24774A11 in he
wes e n pa .
CONCLUSIONS
This s udy co e ed wo a eas ep esen a i e o he condi-
ions a he bounda y and cen e o he NASG and p o-
ides a desc ip ion o he diazo ophic communi y in an
unde sampled a ea o he No h A lan ic Ocean. Ou
esul s indica e ha he ansi ion be ween he bounda y
and cen al NASG c ea es a sepa a ion in he composi ion
o he diazo ophic communi y, wi h UCYN-A domina -
ing he eas e n hal o he A lan ic and T ichodesmium
and g-24774A11 Gammap o eobac e ia domina ing he
wes e n pa .
The de ec ion o g-24774A11 in he .10 and
10–3 mm ac ions and no in he 3–0.2 mm ac ion
indica es ha hese diazo ophs occu in associa ion wi h
ei he la ge mic obes, exis ing ma ine pa icles, o sel -
p oduced mucilage ma ices; o ou knowledge, his has
no been p e iously epo ed. The il a ion me hod we
used may be mo e gen le han a pe is al ic pump sys em,
ha ing less bac e ial de achmen om pa icles. These
esul s also sugges ha size- ac iona ion s udies a e no
op imal o iden i ying he di e en diazo oph phylo-
ypes esponsible o he measu ed N
2
ixa ion a es and
suppo pas obse a ions ha non-cyanobac e ial diazo-
ophs a e p esen a conside able abundances in open-
ocean wa e s, hus dese ing u he s udy.
ACKNOWLEDGEMENTS
We hank he c ew and echnicians o he R/V Sa mien o
de Gamboa and he chie scien is o acili a ing he collec-
ion o samples o his s udy. We a e g a e ul o J. Lo enzo
and J. Va ela o sampling and coun ing T ichodesmium
ilamen s.
FUNDING
This wo k was suppo ed by p ojec s Consolide -Malaspina
(CSD2008-00077), CAIBEX (CTM2007-66408- CO2-02)
and HOTMIX (CTM2011-30010-CO2-01) o J.A. M.B.
was suppo ed by a pos doc o al ellowship om he People
P og amme (Ma ie Skłodowska-Cu ie Ac ions) o he
Eu opean Union’s Se en h F amewo k P og amme (FP7/
2007-2013) unde REA g an ag eemen numbe 625185.
P. H . M . w a s s u p p o e d b y N S F O C E 1 1 3 0 4 9 5 a n d u n d s
om he Uni e si y o Massachuse s Da mou h.
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