Longitudinal variability of diazotroph abundances in the subtropical North Atlantic Ocean

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 a ailable online a www.plank .ox o djou nals.o g #The Au ho 2016. Published by Ox o d Uni e si y P ess. All igh s ese ed. Fo pe missions, please email: jou nals.pe [email protected] Jou nal o Plank on Resea ch plank .ox o djou nals.o g J. Plank on Res. (2016) 0(0): 1–11. doi:10.1093/plank / b 121 JPR Ad ance Access published Janua y 18, 2016 a ULPGC - Uni e sidad de Las Palmas de G an Cana ia on Janua y 19, 2016h p://plank .ox o djou nals.o g/Downloaded om 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 a The leg 8 o he Malaspina ci cumna iga ion c uise ook place om 27 Janua y o 15 Ma ch 2011 onboa d he JOURNAL OF PLANKTON RESEARCH j VOLUME 0 j NUMBER 0 j PAGES 1–11 j 2016 2 a ULPGC - Uni e sidad de Las Palmas de G an Cana ia on Janua y 19, 2016h p://plank .ox o djou nals.o g/Downloaded om 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. j DIAZOTROPHS IN THE SUBTROPICAL NORTH ATLANTIC OCEAN 3 a ULPGC - Uni e sidad de Las Palmas de G an Cana ia on Janua y 19, 2016h p://plank .ox o djou nals.o g/Downloaded om 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 F om Bena ides e al. (2013b). JOURNAL OF PLANKTON RESEARCH j VOLUME 0 j NUMBER 0 j PAGES 1–11 j 2016 4 a ULPGC - Uni e sidad de Las Palmas de G an Cana ia on Janua y 19, 2016h p://plank .ox o djou nals.o g/Downloaded om 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). M. BENAVIDES ET AL. j DIAZOTROPHS IN THE SUBTROPICAL NORTH ATLANTIC OCEAN 5 a ULPGC - Uni e sidad de Las Palmas de G an Cana ia on Janua y 19, 2016h p://plank .ox o djou nals.o g/Downloaded om 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 JOURNAL OF PLANKTON RESEARCH j VOLUME 0 j NUMBER 0 j PAGES 1–11 j 2016 6 a ULPGC - Uni e sidad de Las Palmas de G an Cana ia on Janua y 19, 2016h p://plank .ox o djou nals.o g/Downloaded om 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). M. BENAVIDES ET AL. j DIAZOTROPHS IN THE SUBTROPICAL NORTH ATLANTIC OCEAN 7 a ULPGC - Uni e sidad de Las Palmas de G an Cana ia on Janua y 19, 2016h p://plank .ox o djou nals.o g/Downloaded om 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). JOURNAL OF PLANKTON RESEARCH j VOLUME 0 j NUMBER 0 j PAGES 1–11 j 2016 8 a ULPGC - Uni e sidad de Las Palmas de G an Cana ia on Janua y 19, 2016h p://plank .ox o djou nals.o g/Downloaded om 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. REFERENCES Agawin, N., Bena ides, M., Busque s, A., Fe iol, P., S al, L. J. and A ı ´s egui, J. (2014) Dominance o unicellula cyanobac e ia in he diazo ophic communi y in he A lan ic Ocean. Limnol. Oceanog .,59, 623–637. A ´l a ez, E., Moyano, M., Lo ´pez-U u ia, A., Noguei a, E. and Scha ek, R. (2014) Rou ine de e mina ion o plank on communi y composi ion and size s uc u e: a compa ison be ween FlowCAM and ligh mic oscopy. J. Plank on Res.,36, 170–184. Bena ides, M., Agawin, N., A ı ´s egui, J., Fe iol, P. and S al, L. J. (2011) Ni ogen ixa ion by T ichodesmium and small diazo ophs in he sub- opical no heas A lan ic. Aqua . Mic ob. Ecol.,65, 43–53. Bena ides, M., A ı ´s egui, J., Agawin, N. S. R., Lo ´pez Cancio, J. and He na ´ndez-Leo ´n, S. (2013a) Enhancemen o ni ogen ixa ion a es by unicellula diazo ophs s. T ichodesmium a e a dus deposi ion e en in he Cana y Islands. Limnol. Oceanog .,58, 267–275. Bena ides, M., B onk, D. A., Agawin, N. S. R., Pe ´ ez-He na ´ndez, M. D., He na ´ndez-Gue a, A. and A ı ´s egui, J. (2013b) Longi udinal a iabili y o size- ac iona ed N 2 ixa ion and DON elease a es along 24.58N in he sub opical No h A lan ic. J. Geophys. Res. Oceans, 118, 3406–3415. Bena ides, M. and Voss, M. (2015) Fi e decades o N 2 ixa ion esea ch in he No h A lan ic Ocean. F on . Ma . Sci.,2, 1–40. Ben zon-Tilia, M., Se e in, I., Hansen, L. H. and Riemann, L. (2015) Genomics and ecophysiology o he e o ophic ni ogen- ixing bac- e ia isola ed om es ua ine su ace wa e . mBio,6, e00929-–15. Ben zon-Tilia, M., T a ing, S. J., Man ikci, M., Knudsen-Lee beck, H., Hansen, J. O. R. L., Ma kage , S. and Riemann, L. (2014) Signi ican N 2 ixa ion by he e o ophs, pho ohe e o ophs and he e ocys ous cyanobac e ia in wo empe a e es ua ies. ISME J.,9, 273–285. M. BENAVIDES ET AL. j DIAZOTROPHS IN THE SUBTROPICAL NORTH ATLANTIC OCEAN 9 a ULPGC - Uni e sidad de Las Palmas de G an Cana ia on Janua y 19, 2016h p://plank .ox o djou nals.o g/Downloaded om