Vertical and longitudinal gradients in HNA-LNA cell abundances and cytometric characteristics in the Mediterranean Sea

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1 Biogeosienes, 8, , doi:1.519/g Author(s) 211. CC Attriution 3. Liense. Biogeosienes Vertil nd longitudinl grdients in - ell undnes nd ytometri hrteristis in the Mediterrnen Se F. Vn Wmeke 1, P. Ctl 2,3, M. Pujo-Py 2,3, nd P. Leron 2,3 1 CNRS, Université de l Méditerrnée, Lortoire de Miroiologie, Géohimie et Eologie Mrines, UMR6117, Cse 91, Cmpus de Luminy, Mrseille edex 9, Frne 2 UPMC Univ Pris 6, Lortoire ARAGO Oservtoire Oénologique, Bnyuls/mer, Frne 3 CNRS, UMR7621, LOMIC, Oservtoire Oénologique, Bnyuls/mer, Frne Reeived: 29 Otoer 21 Pulished in Biogeosienes Disuss.: 1 Novemer 21 Revised: 18 June 211 Aepted: 2 June 211 Pulished: 13 July 211 Astrt. Heterotrophi terioplnkton undne nd prodution were investigted with depth (down to thypelgi lyers) nd with longitude (from.9 E to 32.7 E) long ruise trk ross the Mediterrnen Se in erly summer 28. Aundnes nd flow ytometri hrteristis (green fluoresene nd side stter signls) of high nulei id () nd low nulei id () teril ells were determined using flow ytometry. Contrry to wht is generlly oserved, the reltive importne of ells, s perent of totl ells, (%, rnge 3 69 %) ws inversely relted to teril prodution (rnge.15 ng C l 1 h 1 ) lthough the negtive reltion ws wek (log log regression r 2 =.19). The % s well s the men side stter of group inresed signifintly with depth in the meso nd thypelgi lyers. Vertil strtifition plyed n importnt role in influening the distriution nd hrteristis of teril ells espeilly with regrd to lyers loted ove, within or elow the deep hlorophyll mximum. Within given lyer, the reltionships etween the flow ytometri hrteristis nd environmentl vriles suh s hlorophyll-, nutrients or teril prodution hnged. Overll, the reltionships etween nd ells nd environmentl prmeters differed vertilly more thn longitudinlly. 1 Introdution Flow ytometry is powerful tool to study miroil ommunities. In smples from quti environments, terioplnkton ells tend to luster into t lest 2 or 3 distint frtions Correspondene to: F. Vn Wmeke (frne.vn-wmeke@univmed.fr) sed on differenes in the side stter signl (SSC, relted to the size, density nd morphology of the ells) nd in the reltive green fluoresene (relted to the nulei id ontent of the ells). These frtions re more generlly nmed (high nulei id) nd (low nulei id) ells (Leron et l., 21). Mny ppers hve reported undnes of nd ells over wide rnge of environmentl smples, from oligotrophi to eutrophi environments, sine the pioneering work of Li et l. (1995). However, ftors influening the reltive importne of to ells oth within nd mong quti systems re unler. Comprisons mong studies re hindered s different protools re used (differenes in instrument, smple fixtion nd storge, nulei id stin, et.). Thus, omprisons hve generlly een mde on popultion undne, nd few hve onsidered ytometri hrteristis. Within systems, vriety of ptterns hve een reported. Corzo et l. (25) exmined distriutions with depth through 1 m wter olumn in the Drke Pssge nd Gerlhe rits during the ustrl summer. SSC nd green fluoresene of ells were losely relted to hlorophyll- wheres those of ells were not. In ontrst, Sherr et l. (26) showed tht the rtios / for undne nd green fluoresene were remrkly stle long lrge inshore-offshore grdients in the northest Pifi Oen, onluding tht ells were not more responsive thn ells to hlorophyll vriility. When exmined over rod rnge of trophi onditions (teril prodution overing orders of mgnitude, from.1 to 11.6 µg C l 1 h 1 nd hlorophyll-.2 to 69 µg l 1 ), ytometri hrteristis hnged gretly in different eosystems nd long produtivity grdients (Bouvier et l., 27). The ptterns did not support the simple, dihotomous view of nd teri s tive nd intive ells, s Pulished y Copernius Pulitions on ehlf of the Europen Geosienes Union.

2 185 F. Vn Wmeke et l.: Vertil nd longitudinl grdients in - hypothesized y erly ell sorting experiments (Servis et l., 1999; Leron et l., 21). The generl onlusion from the literture is tht teri re lrger nd more tive on ell sis thn teri. Overll, despite onsiderle mount of work, the ontriution of ells to totl teril prodution is still sujet of muh dete (Longneker et l., 26; Sherr et l., 26; Shrek nd Lts, 27; Morn et l., 21). The Mediterrnen Se is lrgely oligotrophi over muh of the yer, nd is hrterized y grdient of oligotrophy from the oligotrophi st to the ultr-oligotrophi Est. The BOUM (Biogeohemistry from the Oligotrophi to the Ultr-oligotrophi Mediterrnen) ruise rried out in erly summer 28, offered the opportunity to exmine teril undne nd prodution with depth over lrge longitudinl grdient. Indeed, to our knowledge, informtion ville in Mediterrnen Se out nd ytometri group distriutions nd properties is restrited to stern, ostl nd shelf res (Leron et l., 21; Joux et l., 25; Shrek nd Lts, 27). Very limited dt on nd distriutions within open se res re ville: the Dyfmed site in the N.W. Mediterrnen hs een studied on different time sles ut with no reports on vriility of ytometri properties like SSC or fluoresene (Mével et l., 28; Winter et l., 29). In this study we nlyzed lrge numer of smples to investigte flow ytometri hrteristis in reltion to teril prodution nd environmentl prmeters (temperture, totl hlorophyll-, phosphte, nitrte + nitrite). Smples were gthered from surfe to mesopelgi wters from 3 sttions ross the Mediterrnen Se. The im of this study ws to explore the ftors tht determine the vriility in undnes nd ytometri properties of nd ells in Mediterrnen Se wters, inluding the deep se nd the Estern Bsin. Flow ytometri hrteristis of the terioplnkton were exmined within lyers loted ove, within nd elow the deep hlorophyll mximum, s well s longitudinlly mong different regions. 2 Mteril nd methods 2.1 udy re nd smple olletion This work ws rried out during the BOUM ruise on the R/V Atlnte during June July 28. The ruise ws plnned s trnset of sttions enompssing lrge longitudinl grdient in the Mediterrnen Se (Fig. 1). Smples were olleted using rosette of 2 12 l Niskin ottles mounted on CTD rosette system. Sensors on the CTD provided profiles for temperture, slinity, nd fluoresene t eh sttion. The depth of the deep hlorophyll mximum (dm) ws determined from the fluoresene profile. Totl hlorophyll- (TChl-) ws nlyzed using HPLC (Rs et l., 28) nd the dt for vertil nd horizontl A Si B E C Fig. 1. Mp of the BOUM trnset. All sttions were smpled for teril undnes nd TChl- wheres only sttions indited with ode in red were smpled for teril prodution. Blues hrters nd lines seprtes the different regions ompred (see Mteril nd Method setion). Figures 1 nd 3 were drwn with Oen Dt View progrm (Shlitzer, 29). profiles re presented in Moutin et l. (211) nd Cromet et l. (211). Phosphte (PO 3 ) nd nitrte + nitrite (NO 3 + NO 2 ) were nlysed using utomted olorimetri tehnique s desried in Pujo-Py et l. (211). Detetion limits for the proedures were.1 µm for PO 3 nd.2 µm for NO 3 + NO 2. The numered sttions were oupied riefly nd smpled t different times of the dy. All these numered sttions were investigted for teril undne nd only one hlf of them for teril prodution (1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 2, 25). Sites designted y letters (A, B nd C) were gyre sttions, loted inside ntiyloni gyres nd eh ws smpled over dy period. At eh gyre sttion, up to depth profiles for teril undne nd prodution were otined, three t 9:.m., nd one t 2:.m. (lol time). The whole dt set ws used to desrie teril undne over 5 vertil profiles (93 smples) while reltionships with BP were exmined using 25 profiles (198 smples). 2.2 Flow ytometri nlysis of teri Aliquots of 1.8 ml were tken for heterotrophi terioplnkton (sensus strito referring to heterotrophi prokryotes), fixed with 2 % (w/v) formldehyde (PFA) solution, stored for t lest 3 min t room temperture, frozen in liquid nitrogen nd then stored t 8 C until smples ould e proessed on shore. Smples were left to thw t room temperture nd stined with SYBR Green I (Invitrogen Moleulr Proes) t.25 % (vol/vol) finl onentrtion for 15 min t room temperture in the drk. Counts were performed using the FsCliur flow ytometer (Beton Dikinson) equipped with n ir-ooled rgon lser (88 nm, 15 mw). The sheth fluid ws filtered (<.2 µm) sewter. ined teril ells, exited t 88 nm, were disriminted nd enumerted ording to their right-ngle light stter (SSC, relted to ell size) nd green fluoresene (53/3 nm). The speed Biogeosienes, 8, , 211

3 F. Vn Wmeke et l.: Vertil nd longitudinl grdients in green fluoresene 5 m, surfe 12 m, dm 2 m, elow dm side stter 3 m, deep Fig. 2. Exmples of four ytogrms otined fter SyBR green I stining. The dt omes from tion B (Ionin Se): surfe (5 m), t the dm (125 m), elow dm (2 m) nd in deep lyers (3 m). Time of nlysis ws set to 1 min for the 3 first lyers, nd 3 min for the 3 m lyer. Windows for nd ells re drwn nd were djusted from one smple to nother. : 1 µm eds. of the nlysis ws dependnt on the teril undne of the smple, typilly the volume nlyzed ws round 2 µl (low speed). The susequent ell onentrtion estimtion ws determined from the flow rte, whih ws lulted y weighing one tue of milliq wter efore nd fter 5 min run of the ytometer (this flow rte lirtion ws done fter eh five smple tues). Fluoresent eds (1.2 µm, Polysienes Europe) were systemtilly dded to eh smple s n internl stndrd nd used to normlize vlues of single ell vriles. Thus, the fluoresene nd side stter vlues of ells were stndrdized to those of the referene eds to ount for potentil differenes in mesurement onditions (ll smples were nlysed during 6 month-period y the sme nlyst). In plot of green fluoresene versus red fluoresene we were le to distinguish photosyntheti prokryotes whih were removed from nonphotosyntheti prokryotes. Bteri with high nulei id ontent ( ells) were disriminted from teri with low nulei id ontent ( ells). Eh sugroup ws delimited on the SSC versus green fluoresene plot y drwing gte, nd ell undne ws determined in eh sugroup (Fig. 2). Windows were djusted for eh individul smple. Totl undne is determined s + nd the perentge of ell undne (%) ws lulted s /( + ). 2.3 Bteril prodution Bteril prodution (BP sensus strito referring to heterotrophi prokryoti prodution ) ws determined y [ 3 H] leuine inorportion pplying the entrifugtion method (Smith nd Azm, 1992). Duplite 1.5 ml smples nd one trihloreti id (TCA) killed ontrol for lnk orretion were inuted with mixture of [,5 3 H]leuine (Perkin Elmer, speifi tivity 115 Ci mmol 1 ) nd nonrdiotive leuine t finl onentrtions of 16 nd 7 nm, respetively. Smples were inuted in the drk t the respetive in situ tempertures for 2 5 h depending on the expeted tivity. Preliminry heks showed tht the inorportion of leuine ws liner with time. Inutions were stopped y dding of TCA to finl onentrtion of 5 %. To filitte the preipittion of proteins, ovine serum lumin (BSA, Sigm, 1 mg L 1 finl onentrtion) ws dded prior to entrifugtion whih ws rried out t 16 g for 1 min. After disrding the superntnt, 1.5 ml of 5 % TCA ws dded nd the smples were vigorously shken using vortex nd then entrifuged gin. After disrding the superntnt, 1.5 ml of 8 % ethnol ws dded nd then the smples were shken nd entrifuged gin. The superntnt ws disrded, nd 1.5 ml Ultimgold MW dded in the entrifuge tue. The rdiotivity inorported into the pellet ws ounted using Pkrd LS 16 Liquid Sintilltion Counter. A ftor of 1.5 kg C mol leuine 1 ws used to onvert the inorportion of leuine to ron equivlents, ssuming no isotopi dilution. This ws heked on 3 osions with onentrtion kinetis (rnge of onentrtions 3 to 5 nm, isotopi dilution etween 1.1 nd 1.7). Ativities within meso- nd thy-pelgi lyers were lso investigted t sttions A, B nd C, etween 25 nd 91 m t C, 25 3 m t B nd m t A. These deeper smples, where lower tivity ws expeted, were treted y the filtrtion tehnique (Kirhmn, 1993). dded 1 nm finl onentrtion of [,5 3 H]leuine in 5 ml smples. Two duplites nd one formlin-killed lnk were inuted in the drk t in situ temperture for 15 2 h. Live smples were terminted y formlin ddition (1 % finl onentrtion) nd ll smples were filtered onto.2 µm Millipore GS/WP filters efore TCA extrtion (5 % finl) nd 8 % ethnol rinse diretly on the filter tower. Filters were dissolved in 1 ml of ethyl ette efore ddition of Ultim Gold MV sintilltion oktil. Errors ssoited with the vriility etween replite mesurements (hlf the differene etween the two replites) verged 6 % nd 8 % for BP vlues determined using the entrifuge (surfe smples) nd filtrtion (deep smples) methods, respetively. 2. tistil nlysis nd groups of dt For omprison of eh prmeter etween different regions or vertil lyers, sttistil nlysis ws done y post ho Fisher PLSD test fter n ANOVA test. Aundne vriles Biogeosienes, 8, , 211

4 1856 F. Vn Wmeke et l.: Vertil nd longitudinl grdients in - nd ytometri hrteristis s well s BP nd environmentl prmeters (temperture, hlorophyll, nutrients) were log 1 trnsformed in order to hieve normlity nd homogeneity of vrines efore mking orreltions or ANOVA. Respeting reommendtions of Sokl nd Rohlf (1981), dt sets inluding vlues etween zero nd one were multiplied y 1 (green fluoresene) or 1 (SSC) efore log trnsformtion to void negtive hrteristis in the logrithms. PO 3 nd NO 3 + NO 2 dt sets lso inluded mny zeros nd their trnsformtion ws done pplying the formul LOG(dt*1 + 1). Model II regression ws used to exmine reltionships etween 2 prmeters when oth (used in X nd Y xis) were sujeted to mesurement vriility. Model I ws used to exmine the reltionship etween ny Y environmentl dependnt vrile nd n X independent vrile s temperture or depth. Note tht the oeffiient of determintion is the sme whtever the model used. Reltionships were exmined vertilly following different groups of smples: surfe smples loted etween the surfe nd the deep hlorophyll mximum (dm) lyer, dm smples t the dm depth, elow dm orresponds to smples tken etween the dm nd 25 m, deep orresponds to smples tken elow 25 m. Indeed, the dm lyer represents n idel eologil oundry in our study whih enled dt to e ompred from sttions ross the Mediterrnen Se, where the trnspreny of wter, depths of nutrilines nd dm vried gretly (Pujo Py et l., 211; Cromet et l., 211). Longitudinlly, we ompred groups defined s follows: (i) inluding North stern Mediterrnen Se sttions (25 to 27) whih were loser to the ône river with higher hlorophyll iomss nd teril undnes nd hlorophyll mximum etween 3 nd 5 m; (ii) inluding sttions 19 to 2 nd A loted in open stern Mediterrnen with deeper dm depths (etween 69 nd 96 m); (iii) with sttions 1 to 17 (within the Siily strit) nd sttion 18 whih is in the stern sin ut its intermediry lyers re influened y the flow of susurfe Estern wters (Pujo- Py et l., 211); nd (iv) E inluding sttions 1 to 13 nd sttions B nd C loted in open, Estern Mediterrnen (Fig. 1). 3 Results 3.1 Distriution nd vriility of iologil nd physio-hemil prmeters The sttions were loted etween 3.2 N,.93 E (lose to ône river mouth) nd 33.7 N, 32.7 E (South of Cyprus, depth (m) teril prodution (ng C l 1 h 1 ) A B C dm depth Fig. 3. Contour plot of teril prodution long the BOUM trnset, from the most Northwestern sttion (left) to the most Estern sttion (right) nd for the 25 m lyer. Depth of the deep hlorophyll mximum (dm) is lso indited (red dots). The r t 7 in the Siily strit shows the ottom depth (116 m). on the se mount Erthostène, Fig. 1). All sttions were situted within the ontinentl slope or open Se (exept t the Siily rit: 1, 382 m nd 17, 116 m nd lose to the one river mouth: 27, 15 m), with ottom depths down to 3321 m (st 12). The diel vriility of se surfe temperture per sttion ws round 1 C. Surfe temperture (5 m) rehed mximum of 27.5 C (st 12 in the Levntine Se) with lowest tempertures t 27 (17.2 C, lose to the ône river mouth) nd deresing with depth down to 13.3 C in the Estern Bsin nd 12.8 C in the stern Bsin (Tle 1). There ws lssil st Est grdient of the deepening of the depth of the dm (Fig. 3), whih vried from 5 m t the western sttion 27 to 15 m t estern sttion B. Mximum TChl- onentrtion ws found t sttion 25, with 1.7 µg TChl- l 1 t 5 m depth. Bsed on integrted TChl- (onentrtions mesured down to 25 m), the most oligotrophi sttion ws sttion 8, with 16.1 mg TChl- m 2 wheres the rihest ws sttion 25 (55.3 mg TChl- m 2 ). Surfe lyers were low in PO 3 nd in NO 3 + NO 2 throughout the trnset (see Fig. 3 in Pujo-Py et l., 211), lthough t some sttions (st C in Levntine Bsin, 19, 2, 21, 22, nd 27 in the stern Bsin), PO 3 vlues were slightly ove the detetion limit. PO 3 deresed signifintly in the surfe lyers from the region st ( :.12 ±.13 µm) to region Est ( E :.8 ±.1 µm, p =.2) s well s in elow dm lyers ( :.16 ±.1 µm, E :.±. µm, p <.1); nd in the deeper lyers ( :.35 ±.3 µm, E :.15 ±.5 µm, p <.1). There ws no sttistil differene in the men PO 3 onentrtion in the dm lyer mong regions. The mens of NO 3 + NO 2 did not hnge signifintly mong the different sins for surfe nd dm lyers, ut signifintly deresed in elow dm lyers from (.8 ± 2.7 µm) to E (2.± 1. µm) regions. Signifint hnges in the mens etween Estern nd stern Bsins within the elow dm lyers were minly Biogeosienes, 8, , 211

5 F. Vn Wmeke et l.: Vertil nd longitudinl grdients in Tle 1. Rnges of slinity, temperture, totl hlorophyll- (TChl-), phosphte (PO 3 ), nitrte + nitrite (NO 3 + NO 2 ), totl teril undne (totl ), nd teril prodution (BP) for the dt set used for omprison of undne nd ytometri hrteristis of nd ells. Mens, oeffiient of vrition nd numer of dt for eh group of wter olumn prtition: lyers ove the deep hlorophyll mximum (surfe), lyers t the deep hlorophyll mximum ( dm ), lyers elow the dm ut ove or equl to 25 m ( elow dm ), nd lyers elow 25 m deep. dl: elow detetion limits. Slinity PSU temperture C TChl- µg l 1 PO 3 µm NO 3 + NO 2 µm totl 15 ml 1 BP ng C l 1 h 1 N min dl dl mx surfe (1.8 %, n = 192) (18 %, n = 192) (7 %, n = 111) (155 %, n = 18) (392 %, n = 18) (33 %, n = 192) (6 %, n = 11) dm (1.6 %, n = 5) (7 %, n = 5) (67 %, n = 3) (112 %, n = 38) (13 %, n = 38) (3 %, n = 5) (61 %, n = 2) elow dm (1.2 %, n = 21) (8 %, n = 21) (137 %; n = 15) (98 %, n = 175) (79 %, n = 175) (9 %, n = 21) (96 %, n = 5) deep (. %, n = 55) (5 %, n = 55) (35 %, n = 55) (28 %, n = 55) (11 %, n = 55) (123 %, n = 1) due to the deepening of nutrilines in the Est, nd for deep lyers were due to the different origin of deep wters msses in Estern nd stern Bsins (see Pujo-Py et l., 211). 3.2 Totl teril undne nd prodution Totl teril undnes rnged from.15 to ml 1 (Tle 1). BP rnged from.1 ng C l 1 h 1 (65 m, sttion C) to 3.9 ng C l 1 h 1 (Siily rit, 5 m, sttion 15), nd 1 ng C l 1 h 1 t the sttion smpled for BP losest to the ône river mouth (25 m, sttion 25, Fig. 3). The reltionship etween teril iomss (BB, lulted ssuming 12 fgc per ell) nd BP ws high (r 2 =.7) when the dt were pooled ll together (dt not shown). r 2 ws lower ut remined signifint for different lyers (r 2 =.2,.5 nd.7 for surfe, dm nd elow dm lyers, respetively). The slopes otined for the whole dt set (.57) s well s for the seprte wter-olumn lyers (..55) suggest ottom-up ontrol of teril iomss following Billen et l. (199) nd Duklow (1992). TChl- ws wekly linked to BP (29 % of the vriility explined, reltion not shown). Notly in different lyers, there ws no reltionship etween TChl- nd BP for surfe lyers, nd wek reltionships for the dm nd elow dm lyers (r 2 =.52 nd.53). There ws not ny reltionship etween BP nd onentrtion of PO 3 (log trnsformed dt, p >.5) regrdless of lyer. 3.3 nd teril undne All smples hd two nd ell frtions disernle y fluoresene versus SSC ytogrms. ell undnes rnged from to ml 1, nd those of ells from to ml 1. Both ox-plot distriutions of nd ells showed high vriility within the surfe nd dm lyers (Fig., ). Neither nor ell undnes were sttistilly different in the surfe when ompred to the dm lyers (ANOVA, threshold hosen t p =.1), ut signifintly deresed elow the dm nd in the deep lyers (ANOVA, p <.1). The derese in nd ell undnes with depth (log-log regressions undne-depth) were signifint for dm, elow dm nd deep lyers (r 2 rnged.33.66, for ll regressions p <.1). Surprisingly, the perentge of ell undne (% = /( + )) inresed signifintly with depth in the meso- nd thypelgi zones (log-log regression in the deep lyers, r 2 =.59, n = 55, Fig. 5), wheres for the other lyers the orreltion ws insignifint. Longitudinl vriility ws exmined in the different vertil lyers. Within the surfe lyer st- Est grdient ws visile for oth nd undnes, with signifintly higher vlues in the region nd lower vlues in E ompred to region (Fig. 6, ). However, these differenes in undnes mong different regions were onomitnt for oth groups s longitudinlly, % did not hnge (see for exmple % distriution in surfe lyers Fig. 7). BP ws responsile for 65 % nd 7 % of the vriility in nd ell undne, respetively, ut the positive slope of the log-log regression ws higher for the ells (.62 versus.53, with model II, figure not shown). Consequently, % deresed slightly with inresing BP (the log-log regression ws rely signifint, r 2 =.19, Fig. 8). However, the log-log reltion etween % nd BP ws insignifint for surfe, elow dm nd deep lyers, ut BP explined (with positive slope) 21 % of the vriility of % in the dm lyers (Tle 2). Biogeosienes, 8, , 211

6 1858 F. Vn Wmeke et l.: Vertil nd longitudinl grdients in - Tle 2. Reltionships etween undne of ells, undne of ells nd % versus TChl-, versus BP, versus solule retive phosphorus (PO 3 ) nd versus nitrte + nitrite (NO 3 + NO 2 ) onentrtions. Dt were trnsformed efore fitting with liner regressions (see methods). n: numer of dt, r 2 : determintion oeffiient, nd: not determined, ns: not signifint (Signifint threshold set t p =.5). For lrity, sign of slopes for wek orreltions (r 2 <.1) were not indited. The numer of dt used in the regressions for eh type of environmentl vrile nd for eh lyer re indited in Tle 1. reltion with Thl- reltion with BP reltion with PO 3 reltion with NO 3 + NO 2 Sign of slope r 2 sign of slope r 2 sign of slope r 2 sign of slope r 2 ll dt undne surfe ns ns dm ns ns elow dm deep nd ns ns ns ll dt undne surfe ns ns dm ns ns elow dm deep nd ns ns ns % ll dt surfe ns ns ns ns dm ns ns elow dm.2 ns ns ns deep nd ns ns ns TChl- ppered to explin lrge vriility in ell undnes. Both nd ell undnes showed signifint, positive slope following TChl- onentrtions (slopes model II:.31, r 2 =.59;.3, r 2 =.6, respetively). The orreltions of undnes with Thl- were lso signifint within the 3 hlorophyll lyers when onsidered independently ( surfe, dm nd ove dm ). TChl- explined slightly the vriility of % only t the dm lyer (r 2 =.12, Tle 2). Aundnes of nd were lso inversely relted to onentrtions of phosphte nd nitrte + nitrite (r 2 rnged.1 to.9, Tle 2). Some of the % vriility ws explined y vriility of PO 3 (r 2 =.21) nd NO 3 + NO 2 (r2 =.2). Suh reltionships were minly due to the o-vrition with depth s this reltionship wekened nd/or disppered when lyers were onsidered seprtely (Tle 2). Finlly, plotting the dt ll together ( surfe, dm, elow dm nd deep ) the log-log reltionship etween %, ell undnes nd ell undnes with temperture ws wekly signifint (r 2 =.19,.25 nd.19, respetively). However, there ws no signifint reltionship etween %, ell undnes or ell undnes with temperture in the surfe lyers where the temperture rnge ws the highest. 3. Ptterns in ytometri prmeters ndrdized SSC of nd ells rnged from.93 to.265 nd from.78 to.185, respetively. ndrdized green fluoresene of nd ells rnged from.18 to. nd from.5 to.97, respetively. The oeffiient of vrition of SSC ws slightly higher for ells (21 %) thn for ells (17 %). This ws the sme for green fluoresene (15 % versus 12 %). Green fluoresene of ells deresed slightly with depth (log log regressions, r 2 =.2, n = 93, Fig. e nd Fig. 5 e,f). In mrked ontrst, the green fluoresene of the ells inresed signifintly with depth (r 2 =.51, dt Fig. f nd Fig. 5e, f) nd with nutrients (r 2 =.5 for phosphte, r 2 =.56 for nitrte + nitrite, reltion not shown). The ANOVA nlysis of SSC nd green fluoresene sed on the hlorophyll -defined depth lyers suggests tht this disrimintion riterion ws prtiulrly pproprite in explining the vriility in green fluoresene of ells (n = 89, F = 356), nd to lesser degree of ells (F = 32, Fig. e, f). This disrimintion riterion ws more or less le to explin vriility in SSC of the two groups (F = 9 for ells, F = 76 for ells, Fig., d). Biogeosienes, 8, , 211

7 F. Vn Wmeke et l.: Vertil nd longitudinl grdients in Side ter undnes (x 1 5 ml -1 ) surfe A C dm elow dm deep surfe dm elow dm deep B D depth (m) depth (m) % SSC d green fluoresene e f Green fluoresene E surfe dm elow dm deep F surfe dm elow dm deep Fig. 5. Vertil distriutions of (, ): perentge of ells; (, d): side stter signl (SSC); (e, f): green fluoresene signl. NO 3 + NO 2 or PO3 inside the different hlorophyll lyers). Within surfe lyer, regionl effets on SSC ws inexistent for (Fig. 6) or sre for (Fig. 6d). Green fluoresene of oth ytometri groups slightly deresed towrd Est ( nd E regions for, E region for, Fig. 6e, f). Fig.. Box plots showing distriution of (A, B): undnes, (C, D): side stter nd (E, F): green fluoresene of sugroups nd long different wter olumn lyers: ove the deep hlorophyll mximum ( surfe ), t the deep hlorophyll mximum ( dm ), elow the dm ut ove or equl to 25 m ( elow dm ), nd elow 25 m ( deep ). Groups onneted y the sme letter (in itlis nd in red) re not signifintly different t the.1 proility level. Lower to the upper vlues re indited, respetively, 1 %, 25 %, 5 % (medin), 75 nd 9 % perentiles. While green fluoresene hrteristis were very distint etween the nd ells regrdless of depth, SSC differenes were slight within surfe lyers ut surprisingly higher in meso nd thy pelgi lyers (Fig. 5d nd Fig. d). SSC deresed with depth in surfe lyers (log log regressions, model I, p <.1, r 2 =.37 for ells p <.1, r 2 =.6 for ells, Fig. 5). In ontrst, SSC inresed in deep lyers (p <.1, r 2 =.66 for ells, p <.1, r 2 =.27 for ells. This ws more pronouned for ells. Indeed, SSC of this group inresed 6 times more rpidly with depth thn the SSC of the ells (Fig. 5d). The ytometri properties (SSC, fluoresene) of nd ells showed low or inexistent reltionships with nutrient onentrtions when the depth effet ws removed (i.e. when looking for reltionships with Disussion.1 Links with resoures: hlorophyll nd nutrient vilility In given terioplnkton popultion, ells hve een onsidered s the very tive nd dynmi ytometrillydefined group with high ell-speifi tivity rtes (Servis et l., 1999; Leron et l., 21; Longneker et l., 25, 26). Therefore, the perentge of ells reltive to totl undne (%) is generlly relted to vriles trking the produtivity index of the eosystem, suh s hlorophyll stoks or teril prodution (Corzo et l., 25; Morn et l., 27). In our study, the perentge of deresed slightly, ut signifintly, with inresing BP. It showed lso very wek negtive orreltion with TChl. This is in ontrst with wht hs een reported for lrge sle produtivity grdients for whih the orreltion etween % nd hlorophyll is generlly positive (Bouvier et l., 27 with Chl- grdient from.1 to 69 µg l 1, Sherr et l., 26 with Chl- grdient from.2 to 2 µg l 1 ). For grdients muh loser to our ontext, the reported orreltions re muh more vrile, sometimes positive (Morn nd Clvo-Diz, 29), negtive (Nishimur et l., 25) or even showing no signifint orreltion (Corzo et l., 25). Biogeosienes, 8, , 211

8 186 F. Vn Wmeke et l.: Vertil nd longitudinl grdients in - undnes (x 1 5 ml -1 ) Side ter Green fluoresene A C E, E E, E B D F, E E E Fig. 6. Box plots showing distriution of (A, B): undnes, (C, D): side stter nd (E, F): green fluoresene of sugroups nd in the surfe lyers ording regionl distriution: (sttions 27, 26, 25), (sttions 2, 23, A, 22, 21, 2, 19), (tions 18, 17, 16, 15, 1) nd E (sttions 13, 12, B, 1, 2, 3,, 5, 6, 7, 8, 9, 1, C). Groups onneted y the sme letter (in itlis nd in red) re not signifintly different t the.1 proility level % , A These very ontrsting results suggest tht the ulk tivity is not only ontrolled y ells sine some tive ells my e spred differentilly ross distint groups of terioplnkton or tht other environmentl vriles re etter preditors of the reltive importne of ells. In the strtified Mediterrnen Se, the dynmis of heterotrophi teril ommunities in surfe lyers is linked to the vilility of inorgni resoures, mostly P lone, sometimes N + P nd in few osions, N lone (Sl et l., 22; Vn Wmeke et l., 22). Consequently, we lso exmined the reltionships etween inorgni nutrient onentrtions nd the %. The % ws slightly more strongly orrelted with PO 3 or NO 3 + NO 2 onentrtions (r 2 =.21 nd.23, respetively) thn with TChl- (r 2 =.7). As the vertil vriility of nutrient onentr, E B surfe dm elow dm d deep Fig. 7. Box plots showing distriutions of % in the different regions for the surfe lyers (A) nd distriution of % in the different lyers (ll sttions inluded, (B)). Within ommon lyer, groups onneted y the sme letter (in itlis nd in red) re not signifintly different t the.1 proility level. tion is importnt, the longitudinl effet ould only e studied y ompring different lyers. However, lthough men nutrient onentrtions showed few sttistil differenes etween some regions (minly etween E nd ), we ould not find ny distint ptterns of % longitudinlly mong the different regions ompred. This ide is reinfored y the ft tht the % ws not relted to PO 3 or NO 3 + NO 2 onentrtion when onsidering lyers independently. This suggests tht nd distriutions re muh more ffeted y the vertil strtifition thn y the longitudinl distriution. During the BOUM ruise, the effets of inorgni nutrient dditions ws exmined in miroosm experiments in the three gyre sttions using surfe lyer wters (Tnk et l., 211). In the surfe smples (8 m depth), BP ws stimulted y N (sttion B) or N + P (sttion C, Tnk et l., 211). Nishimur et l. (25) hypothesized tht P limittion exerts more severe onstrints on the growth of teril groups with higher nulei id ontent. However t sttion B, N ddition resulted in 2 fold inrese of ell speifi tivity of ll ytometri groups, inluding, with low SSC nd with high SSC (Tlrmin et l., 211). Thus, it ppered tht ll ytometri groups reted on the sme wy to nutrient dditions, suggesting tht oth nd ells were nutrient limited. This ould explin why the % in surfe lyers did not hnge in the more oligotrophi onditions in the Est (Fig. 7). Both temperture nd nutrient limittion ould ply role in the distriution of ytometri groups. Indeed, referring to modelling study y Hll et l. (28), wrm-dpted speies ould hve lower miniml P ell quots thn old-dpted speies. This would onfer n dvntge to ells hving low nulei id ontent (s nulei ids re n importnt Biogeosienes, 8, , 211

9 F. Vn Wmeke et l.: Vertil nd longitudinl grdients in % 1 1 surf dm elow dm deep > 25m BP ng C l -1 h -1 Fig. 8. Reltionships etween teril prodution nd the perentge of ells. intrellulr soure of P) in wrm, resoure-limited environment. But the temperture role ould e disounted s the temperture rnge in the surfe lyers during our study ws gret (round 11 C: 26 C down to 15 C) nd ross this rnge the % ws not relted to the temperture. The ytometri properties of nd ells were ffeted differently elow the surfe lyers. Size stter of ells dereses, nd green fluoresene of ells inreses (wheres tht of deresed). Interestingly, Tlrmin et l. (211) showed, through 3 H leuine lelling oupled to ell sorting, tht the ontriution of ells to ulk leuine inorportion rtes ws higher in surfe wters wheres tht of ells ws higher t the viinity of the dm t the three sites A, B nd C. This is in ordne with the positive nd signifint reltionship etween BP nd % t the dm (Tle 2). Possile explntions for these trends re swith from nutrient to ron limittion whih is generlly oserved within the dm in strtified onditions (Sl et l., 22; Vn Wmeke et l., 22). This is ssoited with lrge hnges in txonomi omposition of heterotrophi prokryotes (Ghiglione et l., 28; Vn Wmeke et l., 29). Apprently the group enefits more of suh hnges thn the. This suggests, possily, group s etter driver of prodution when C is the resoure limiting BP..2 Deep lyers The reltionships etween the ytometri prmeters of nd ells were exmined throughout the wter olumn. One striking feture ws the inrese in the perentge of with depth in the meso nd thypelgi zones (r 2 =.59, n = 55, p <.1). Sine oth undnes of nd ells deresed with depth, this inrese in % ws due to the 2 times lrger derese in ell undnes with depth thn tht of ells. This inrese of % is ordne with other reports. In the Atlnti, the % hs een shown to inrese from 6 to 7 % t some sttions etween 6 75 m nd 1 m depth (Gsol et l., 29). In the deep North Atlnti (down to 38 m), Reinthler et l. (26) lso reported n inrese in the % with depth ut the dt were not shown. Vritions in nd groups with depth were shown to our following de-strtifition of the wter olumn (Winter et l., 29). Another interesting nd originl feture from our study ws the systemti inrese in SSC nd green fluoresene vlues of ells with depth in the meso nd thypelgi zones (Fig. 5). SSC of ells lso inresed with depth ut 6 times less thn tht of ells. SSC vlues of oth nd ells were not orrelted to BP in the deep lyers, nor to nutrient (PO 3, NO 3 + NO 2 ) onentrtions. Sine SSC vritions re sometimes relted to iovolume (Bouvier et l., 21; Felip et l., 27) the SSC inrese ould e interpreted s n inrese in ell iovolume ut this should e onsidered with ution. L Ferl et l. (2) investigted the iovolume nd lipopolyshride ontent of terioplnkton down to m in the Ionin Se nd the men ell volume vried in similr rnge in oth the euphoti nd photi zone. It is importnt to onsider tht SSC is omplex vrile not only nd diretly relted to ell iovolume ut lso depends on the ell struture nd the hemil omposition of the outer memrnes. The resons underlying the inrese in the % nd SSC of oth nd in deep lyers re not lerly known. They my e relted (i) to memrne properties hnges due to hydrostti pressure, (ii) to swith from Euteri to Arhe dominne nd, (iii) to differentil spets of predtion nd/or virl lysis on nd ells (Wilson et l., 21; Corzo et l., 25; Tmurini et l., 29; Winter et l., 29). 5 Conlusions To the est of our knowledge, this is the first investigtion of the ytometri properties of nd ells over lrge depth sles nd longitudinl grdients in the oligotrophi Mediterrnen Se. Although the proportions of the nd ells did not vry lot, their ytometri hrteristis showed distint properties nd reltionships with other environmentl vriles in distint hlorophyll- defined depth lyers, nd this might e relted to the swith from nutrient to ron limittion. % nd SSC of ells re distintly higher in the thypelgi lyers, suggesting tht the tivity in the two groups nd/or environmentl ontrols on nd ells differ vertilly. In ontrst, ytometri properties nd % vried little with longitude Biogeosienes, 8, , 211

10 1862 F. Vn Wmeke et l.: Vertil nd longitudinl grdients in - ross the lrge trnset smpled. Further investigtions will e needed to understnd these trends mong these ytometri groups. There is more nd more evidene suggesting tht different groups re not only relted to different speifi tivities ut proly lso phyologenetilly, perhps with some speifi groups deteted s or ells independent of their physiologil stte. The omintion of tools suh s ell sorting with tivity mesurements, susequent loning/sequening (Guilleult et l., 21), nd investigtion of individul ell C/N/P quots (Twining et l., 28) will e essentil to etter understnd the eologil mening of the uiquitous presene of nd ells in quti environments. Aknowledgements. This reserh ws founded y the Frenh INSU-CNRS nd y the SESAME projet (Southern Europen Ses: Assessing nd Modelling Eosystem Chnges), EC Contrt No GOCE-3699, funded y the Europen Commission s Sixth Frmework Progrmme. The uthors would like to thnk Thierry Moutin for ledership of the projet nd hief sientist of the BOUM ruise, ell Psrr nd Joséphine Rs for totl hlorophyll smpling nd nlysis, Clude Courties nd Christin Tmurini for disussions, John Doln for improvement of the English nd nonymous reviewers for their helpful omments in the first version of this ms. Edited y: C. Jenthon The pulition of this rtile is finned y CNRS-INSU. Referenes Billen, G., Servis, P., nd Bequevort, S.: Dynmis of terioplnkton in oligotrophi nd eutrophi quti environments: ottom-up or top-down ontrol? Hydroiologi, 27, 37 2, 199. Bouvier, T., Troussellier, M., Anzil, A., Courties, C., nd Servis, P.: Using light stter signl to estimte iovolume y flow ytometry, Cytometry,, , 21. Bouvier, T., Del Giorgio, P., nd Gsol, J.: A omprtive study of the ytometri hrteristis of high nd low nulei id terioplnkton ells from different quti eosystems, Environ. Miroiol., 9, , 27. 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