Lambert Clément
email: clement.lambert@univ-brest.fr

Penaud Aurélie
email: aurelie.penaud@univ-brest.fr

Vidal Muriel

Klouch Khadidja

Gregoire Gwendoline

Ehrhold Axel

Eynaud Frédérique

Sabine Schmidt

Ragueneau Olivier

Siano Raffaele

Clément Lambert

Human-induced river runoff overlapping natural climate variability over the last 150 years: Palynological evidence (Bay of Brest, NW France)

Keywords: Pollen grains, Dinoflagellate cysts, Estuarine dynamics, River runoff, Climate variability

For the first time a very high resolution palynological study (mean resolution of 1 to 5 years) was carried out over the last 150 years in a French estuarine environment (Bay of Brest; NW France), allowing direct comparison between the evolution of landscapes, surface water, and human practices on Bay of Brest watersheds, through continental (especially pollen grains) and marine (phytoplanktonic microalgae: cysts of dinoflagellates or dinocysts) microfossils. Thanks to the small size of the watersheds and the close proximity of the depositional environment to the mainland, the Bay of Brest represents an ideal case study for palynological investigations. Palynological data were then compared to published palaeo-genetic analyses conducted on the same core and to various available instrumental data, allowing us to better characterize past environmental variability since the second half of the 19th century in Western Brittany. We provide evidence of some clues of recent eutrophication and/or pollution that affected phytoplankton communities and which appears linked with increased runoff (higher precipitations, higher percentages of riparian forest pollen, decline of salt marsh-type indicators, and higher values of the XRF Ti/Ca signal), mainly explained by the evolution of agricultural practices since 1945 superimposed on the warming climate trend. We assume that the significant relay observed between dinocyst taxa: Lingulodinium machaerophorum and Spiniferites bentorii around 1965 then followed by Spiniferites membranaceus after 1985, attests to a strong and recent eutrophication of Bay of Brest surface waters induced by high river runoff combined with abnormally elevated air temperatures, especially obvious in the data from 1990. The structure of the dinocyst community has thus been deeply altered, accompanied by an unprecedented increase of Alexandrium minutum toxic form at the same period, as confirmed by the genetic quantification. Despite this recent major anthropogenic forcing, the fossil pollen sequence also records natural climate variability. We highlight, for the first time, a possible connection between climate (AMO modes) and fossil pollen records (especially tree pollination rates) in coastal sediments using tree percentage fluctuations as an indirect

Introduction

During the past few decades, coastal environments have been increasingly studied worldwide due to natural fluctuations (e.g. climate variability and geomorphological changes) and anthropogenic pressures (e.g. eutrophication, soil artificialisation, CO 2 rejects) in order to better evaluate human forcing and future climate trends (e.g. [START_REF] A C C E P T E D M A N U S C R I P T 34 Ipcc | Climate Change 2014: Synthesis Report. Contribution of Working Groups I. II and III to the Fifth Assessment Report of the intergovernmental panel on Climate Change[END_REF]. Furthermore, the effects of soil fertilization on the composition and structure of microbial coastal communities is one of the major topics of present-day environmental sciences, taking into account that changes in pH, moisture, and nutrient rates are assumed to greatly affect biodiversity over many trophic levels (Guillaud and Bouriel, 2007;Diaz and Rosenberg, 2008).

North-western European coastal environments are interesting case studies because of their connection to the main Atlantic atmospheric (e.g. North Atlantic Oscillation, NAO; and East Atlantic pattern, EA) and oceanic (e.g. Atlantic Multidecadal Oscillation, AMO; and Atlantic Meridional Overturning Circulation, AMOC) patterns that govern regional and global climates over different timescales. In Brittany (NW France), weather regimes and North Atlantic Sea Surface Temperatures (SST), are the main mechanisms that drive precipitation patterns and related river discharges (Tréguer et al., 2014;[START_REF] Ruprich-Robert | Combined influences of seasonal East Atlantic Pattern and North Atlantic Oscillation to excite Atlantic multidecadal variability in a climate model[END_REF], as well as storm surges (van Vliet-Lanoë et al., 2014). As a major consequence, this region is today characterized by intense soil weathering, leading to intensive nutrient discharges in the coastal waters [START_REF] A C C E P T E D M A N U S C R I P T 36 Meybeck | Global coastal segmentation and its river catchment contributors: A new look at land-ocean linkage[END_REF]Tréguer and De La Rocha, 2013).

In this context, palynological studies can provide essential information about past environmental conditions through combined analyses of phytoplanktonic microalgae (especially dinoflagellate cysts or dinocysts) and pollen grains (Turon, 1984;[START_REF] Sanchez-Goñi | European climatic response to millennial-scale changes in the atmosphere-ocean system during the last glacial period[END_REF]Turon et al., 2003;Mudie et al., 2007;[START_REF] García-Moreiras | Modern pollen and non-pollen palynomorph assemblages of salt marsh and subtidal environments from the Ría de Vigo (NW Iberia)[END_REF]. However,

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M A N U S C R I P T 5 these studies, that are numerous for the Holocene, rarely cover the last century for which environmental changes can also be approached by instrumental data allowing comparison with palynological proxies.

Concerning pollen analysis, a well-known bias exists (i.e. over or under-representation)

regarding the continental surface covered by vegetation and fossil pollen percentages calculated from modern sediment archives (e.g. [START_REF] A C C E P T E D M A N U S C R I P T 32 Gaillard | Pollen/landscape calibrations based on modern pollen assemblages from surface-sediment samples and landscape mapping-a pilot study in South Sweden[END_REF][START_REF] Mazier | Two hundred years of land-use change in the South Swedish Uplands: comparison of historical map-based estimates with a pollen-based reconstruction using the landscape reconstruction algorithm[END_REF][START_REF] Ganne | Present-day palynomorph deposits in an estuarine context: the case of the Loire Estuary[END_REF], as recently discussed for Bay of Brest watersheds versus modern sediments retrieved within the bay [START_REF] Lambert | Modern palynological record in the Bay of Brest (NW France): signal calibration for palaeo-reconstructions[END_REF]. Furthermore, some studies highlight a large influence of climate-driven processes on the pollination rates of some trees (van Vliet et al., 2002;[START_REF] García-Mozo | Quercus pollen season dynamics in the Iberian Peninsula: response to meteorological parameters and possible consequences of climate change[END_REF]Besancenot and Thibaudon, 2012), as well as on the transport of pollen grains through temporal variability of precipitation and thus river runoffs.

These fluvial discharge variations in water volume and nutrient load, according to natural and/or anthropogenic factors, may also be responsible for the recrudescence of toxic algal blooms over the past decades (Klouch et al., 2016 a,b). Dinocyst analysis then represents an indirect approach to the reconstruction of the palaeobiodiversity of dinoflagellate communities and thus of one of the major components of primary productivity known to be very sensitive to environmental changes. Both signals (marine for dinocysts and continental for pollen grains) are often studied together in palaeoenvironmental reconstructions (Combourieu-Nebout et al. 1998;Turon et al., 2003;[START_REF] Sangiorgi | Reconstructing 150 years of eutrophication in the north-western Adriatic Sea (Italy) using dinoflagellate cysts, pollen and spores[END_REF], constituting a land-sea continuum involving a large number of natural (oceanic and atmospheric) and human forcings. The comparison between the palynological data carried out along the last century and the instrumental dataset collected in the same area will allow us to thus precisely identify the different factors driving pollen and dinocyst variations, and to

A C C E P T E D M A N U S C R I P T 6 better interpret these palynological tools for future Holocene palaeoecological reconstructions.

In this study, a core from the outlet of the Daoulas Estuary in the Bay of Brest has provided a very high-resolution palynological record from 1870 AD to present. The Daoulas Bay records today the most intense toxic bloom of dinoflagellates Alexandrium minutum within the Bay of Brest (Klouch et al. 2016a). Furthermore, the palaeobiodiversity of dinoflagellate communities has been recently reported on the same sampled Daoulas core with molecular data (Klouch et al., 2016b), suggesting a great increase in bloom frequency of this toxic species A. minutum over the past decades. We then expect to find clues of recent eutrophication (i.e. nutrient enrichment of the aquatic system; [START_REF] Andersen | Coastal eutrophication: recent developments in definitions and implications for monitoring strategies[END_REF] in the dinocyst assemblages study.

Main objectives of this study, connected to societal impacts for coastal ecosystems and North Atlantic climate knowledge, consist in: i) comparing palynological proxies and instrumental data available at a North Atlantic (SST, AMO modes), regional (atmospheric NAO/EA modes), and local scales (river flows, atmospheric temperatures),

ii) deciphering natural climate variability from recent huge human impacts on Bay of Brest watersheds by cross-correlating the whole set of data including pollen, dinocysts, but also all other non-pollen palynomorphs such as freshwater microalgae.

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Bay of Brest environmental settings

Geomorphological and geographical contexts

The Bay of Brest is located in north-western Brittany (NW France, W Europe; Fig. 1a [START_REF] Ballèvre | Palaeozoic history of the Armorican Massif: models for the tectonic evolution of the suture zones[END_REF]. The study area has subsided since the Eocene and continues to slightly subside (0.02 to 0.04 mm/yr.) (Ziegler, 1992;Bonnet et al., 2000;[START_REF] Goslin | L'évolution du niveau marin relatif le long des côtes du Finistère (Bretagne, France) de 8000 BP à l'actuel : entre dynamiques régionales et réponses locales[END_REF], a value which can be considered as negligible at the timescale of the Holocene (40 mm/10,000 yrs.;

Goslin, 2014). The river system has been established since the Tertiary (Hallegouet et al., 1994).

During the Quaternary, the Bay of Brest was submitted to alternations of glacial lowstands, river valleys being subsequently deepened by erosive processes, and interglacial highstands characterized by sediment infilling; the last transgressive episode thus corresponding to the Holocene [START_REF] Lambert | Modern palynological record in the Bay of Brest (NW France): signal calibration for palaeo-reconstructions[END_REF]. Bathymetry highlights submarine channels in the Bay of Brest that attest to palaeo-fluvial systems (Fig. 1a; [START_REF] Gregoire | Modern morpho-sedimentological patterns in a tide-dominated estuary system: The Bay of Brest (west Britanny, France)[END_REF]. Palaeo-channels of the two main current rivers, the Aulne and Elorn, are about 30 and 15 m deep respectively, and converge in the west at a trough of about 50 m deep (Troadec et al., 1997). Nevertheless, at present, the depth of the Bay of Brest does not exceed 10 m deep for approximately 60% of its surface [START_REF] Monbet | Bilan des connaissances océanographiques en rade de Brest[END_REF].

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Today, the Bay of Brest is characterized by a macrotidal influence with a maximum tidal amplitude of 8m (Troadec et al., 1997). Granulometry of modern sediments reflects specific hydrodynamic conditions mainly related to tidal currents [START_REF] Gregoire | Modern morpho-sedimentological patterns in a tide-dominated estuary system: The Bay of Brest (west Britanny, France)[END_REF]. In its westernmost part, the Bay of Brest is connected to the Atlantic Ocean through the "Goulet", a strait of about 1.8 km large and 50 m deep. Marine waters flow into the Bay of Brest at each new tidal cycle with a current speed reaching 1 m/s, mainly oriented S-E. In the stream direction, sedimentation is mainly composed of sands and gravels [START_REF] Gregoire | Modern morpho-sedimentological patterns in a tide-dominated estuary system: The Bay of Brest (west Britanny, France)[END_REF]. On either side of this axis, the current speed is strongly reduced and grain-size is characterized by fine to very fine sediments (silts and clays) in small bays (Troadec et al., 1997;[START_REF] Gregoire | Modern morpho-sedimentological patterns in a tide-dominated estuary system: The Bay of Brest (west Britanny, France)[END_REF]. The lack of interbedded sands and high energetic sedimentary structures in these bays argues for in situ deposits, an assumption reinforced by 210 Pb profiles carried out on modern sediments [START_REF] Gregoire | Modern morpho-sedimentological patterns in a tide-dominated estuary system: The Bay of Brest (west Britanny, France)[END_REF][START_REF] Lambert | Modern palynological record in the Bay of Brest (NW France): signal calibration for palaeo-reconstructions[END_REF]Klouch et al., 2016b). In its easternmost part, the Bay of Brest receives its main freshwater supplies from the Aulne, the Elorn, and from the smaller Daoulas River (Fig. 1a). Bay of Brest watersheds (Fig. 2a) are characterized by 2,000 km of waterways and most of their runoff flows into the Bay of Brest through the Aulne River (114 km long; 1,224 km² of watershed and 20.40 m 3 /s. of annual debit; Troadec et al., 1997). The Aulne and Elorn River's outflows contribute to up to 85% of the total river discharges in the Bay of Brest (Delmas and Treguer, 1983). The salinity of the Bay of Brest is 34.5% and shows virtually no evolutionary trend over the last 20 years (Tréguer et al., 2014).

Environmental settings of Bay of Brest watersheds

The department of Finistere is one of the leading agricultural regions of France, with a cultivated and/or grazed surface of about 60% (Quéré et al., 2008). At the scale of Bay of In the 19 th century, the rural landscape was essentially composed of cultivated parcels with relatively low vegetation and few trees. From the second half of the century, between 1850 and 1950, the network of hedgerow meadows grew considerably and saw the breaking up of agricultural land into many small cultivated plots delimited by slopes and hedges composed of bushes and shrubs. This new landscape mosaic dramatically wins over the heathlands area.

Since the second half of the 20 th century, the department of Finistere has increasingly recorded intensive agriculture practices and a mechanization of farming techniques; the department being placed today among the most mechanized in France (Flatrès, 1963). Recent studies showed that agriculture and particularly animal husbandries (intensive livestock of pig, poultry, and cattle farms, since the end of the Second World War ; Flatrès, 1963) are the main contributors (between 60 and 80%) to phosphorus flows (phosphates) and high nitrate concentrations (often largely exceeding the national standard of 50 mg/l) through soil leaching into Brittany rivers [START_REF] Lemercier | La pollution par les matières phosphorées en Bretagne : sources, transfert et moyens de lutte[END_REF]Huet et al., 2005;[START_REF] Aurousseau | Evolution de la qualité des eaux en nitrate en Bretagne, synthèse régionale au 30 Septembre 2007[END_REF].

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Despite the decreasing number of farms over the 20 th century, total cultivated areas increased until the 1990s. More specifically, in the 1960-70s, the department held the French record of the use of fertilizers, especially of nitrogen and phosphoric acids (Flatrès, 1963)

Climatological context

General overview

The Bay of Brest and its watersheds are subjected to an oceanic climate characterized by mild temperatures (slight seasonal variations on the coastline and small daily temperature differences, with an average of 15°C in summer and around 7.5 °C in winter (Fig. 1c), and high regular rainfalls (with an average of 200 rainy days/yr. in Western Brittany and 800 to

A C C E P T E D M A N U S C R I P T 11
1,000 mm/yr. measured in the Bay of Brest;Troadec et al., 1997 (Fig. 1c). Wind conditions, usually during autumn and winter, are mainly blowing from the southwest (Fig. 1d). Minor northeast and northwest wind components can also appear in the spring and summer (Troadec et al., 1997) Tréguer et al., 2014;[START_REF] Ruprich-Robert | Combined influences of seasonal East Atlantic Pattern and North Atlantic Oscillation to excite Atlantic multidecadal variability in a climate model[END_REF]. It is however unclear how these mechanisms interact with each other, one influencing the other in various and complex feedbacks (Tréguer et al., 2014;[START_REF] Ruprich-Robert | Combined influences of seasonal East Atlantic Pattern and North Atlantic Oscillation to excite Atlantic multidecadal variability in a climate model[END_REF]. In Brittany, the abovementioned weather regimes, combined with Atlantic SST, mainly explain precipitation patterns and in turn river discharges (Tréguer et al., 2014), as well as storm surges (van Vliet-Lanoë et al., 2014).

North Atlantic climatic oscillations

Through atmospheric connections and air/ocean exchanges, SST variations in the North Atlantic Ocean (i.e. varying AMO modes with cycles of around 70 years) seem to control a large part of the multidecadal climate variability over the continents bordering the North Atlantic area [START_REF] Knight | Climate impacts of the Atlantic multidecadal oscillation[END_REF][START_REF] Mccarthy | Ocean impact on decadal Atlantic climate variability revealed by sea-level observations[END_REF][START_REF] Ruprich-Robert | Combined influences of seasonal East Atlantic Pattern and North Atlantic Oscillation to excite Atlantic multidecadal variability in a climate model[END_REF].

Furthermore, the North Atlantic circulation (AMOC strength) appears to be a main precursor for SST variations (Msadek and Frankignoul, 2009) and then also for AMO oscillations.

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12 Stronger AMOC will result in stronger heat transport from the tropics to northern latitudes of the North Atlantic Ocean (i.e. higher North Atlantic SST), thus implying positive modes of the AMO (McCarthy et al., 2015;[START_REF] Ruprich-Robert | Combined influences of seasonal East Atlantic Pattern and North Atlantic Oscillation to excite Atlantic multidecadal variability in a climate model[END_REF].

Also, NAO modes must be considered on smaller time scales (i.e. pseudo-cycles of around 20 years; Hurrell, 1995). The NAO is the major natural atmospheric variability in the North Atlantic region, defined as differences of winter atmospheric pressures at sea level between the Iceland Low and the Azores High. Negative NAO modes are characterized by weakened mid-latitude westerlies as well as colder and drier winters above Brittany with less storminess, the opposite occurring during positive NAO modes (Cayan, 1992;Hurrell, 1995). The EA pattern is the second prominent mode of low-frequency atmospheric variability over the North Atlantic region, whatever the month considered, with a subtropical relationship. The EA is structurally analogous to the NAO mechanism and consists of a north-south dipole of anomaly centres spanning the North Atlantic Ocean from east to west [START_REF] Barnston | Classification, seasonality and persistence of low-frequency atmospheric circulation patterns[END_REF][START_REF] Ruprich-Robert | Combined influences of seasonal East Atlantic Pattern and North Atlantic Oscillation to excite Atlantic multidecadal variability in a climate model[END_REF]. Both phenomena (NAO and EA) may not be directly correlated and the dominant forcing acting on the study area is not yet understood.

However, it is known that positive (/negative) modes of NAO and East Atlantic patterns are associated with above (/below)-average precipitations over north-western Europe (Tréguer et al., 2014).

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13

Material and methods

Study core: sedimentological characteristics and age model

Small bays of the Bay of Brest are characterized by low hydrodynamics and mean current sedimentation rates of around 0.15 cm/yr. [START_REF] Gregoire | Modern morpho-sedimentological patterns in a tide-dominated estuary system: The Bay of Brest (west Britanny, France)[END_REF]. In this study, core I_09 was collected in the Daoulas estuary (48°20'46.6''N; 4°17'41.208''W; 3 m depth; 58 cm long), using a manual PVC corer of 60 cm in length, during the SERABEQ I cruise (2014, Albert-Lucas vessel, IFREMER). Core I_09, mainly characterized by clayey sediments (Fig. 2), was sampled immediately after coring in order to minimize oxidation for genetic analysis (detection of ancient DNA in sediments; Klouch et al., 2016b). The final age model (cf. Klouch et al., 2016b for the detailed methodology) was based on 210 Pb xs (T 1/2 = 22.3 years)

and 137 Cs depth profiles [START_REF] Schmidt | Present-day sedimentation rates on the southern and southeastern Australian continental margins[END_REF]. The core ranges from 1870 (+/-6.5) AD to 2013 (+/-1.3) AD, with a mean sediment accumulation rate of about 0.4 cm/yr. (Klouch et al., 2016b). Sampling, each centimeter from 0 to 17 cm, then every 2 centimeters downwards, provides a study resolution of 1 to 5 years.

Palynological study

Laboratory procedure

For palynological treatments, we sampled an aliquot (3-4 cc) of sediment among 40 residues kept refrigerated after DNA analyses (Klouch et al., 2016b). Samples were treated at the EPOC laboratory (University of Bordeaux, Pessac, France) following a standard protocol described by [START_REF] Rochon | Distribution of recent dinoflagellate cysts in surface sediments from the North Atlantic Ocean and adjacent seas in relation to sea-surface parameters[END_REF] that includes cold 10-25-50% HCl and cold 45-70% HF to remove, respectively, carbonate and siliceous fractions (http://www.epoc.u-

A C C E P T E D M A N U S C R I P T 14
bordeaux.fr/index.php?lang=fr&page=eq_paleo_pollens). Then, after sieving at 10µm, all final residues were mounted between slides and coverslips with glycerin. Palynomorph concentrations were calculated with the marker grain method consisting of adding one tablet containing 20,848 Lycopodium spores before palynological treatments; these exotic spores being then counted in parallel with studied palynomorphs [START_REF] Stockmarr | Tablets with spores used in absolute pollen analysis[END_REF][START_REF] Rochon | Distribution of recent dinoflagellate cysts in surface sediments from the North Atlantic Ocean and adjacent seas in relation to sea-surface parameters[END_REF][START_REF] Mertens | Determining the absolute abundance of dinoflagellate cysts in recent marine sediments: The Lycopodium marker-grain method put to the test[END_REF].

Palynological identification

For each analyzed sample, a minimum of 300 pollen grains and 150 dinocysts have been systematically counted, using a Leica DM 2500 at magnifications of X630 and X1000, in order to provide robust assemblages from a statistical point of view (Fatela and Taborda, 2002). Taxa identification follows Beug (1961), Faegri and Iversen (1989), [START_REF] Moore | Pollen analysis[END_REF], [START_REF] A C C E P T E D M A N U S C R I P T 38 Reille | Pollen et spores d'Europe et d'Afrique du Nord : supplément 1[END_REF], for pollen grains and [START_REF] Rochon | Distribution of recent dinoflagellate cysts in surface sediments from the North Atlantic Ocean and adjacent seas in relation to sea-surface parameters[END_REF] for dinocysts.

Pollen and dinocyst percentages were calculated independently for each taxonomical group, on a main sum excluding non-identified taxa. Other Non-Pollen Palynomorphs (NPP) were only expressed in absolute concentrations (number of palynomorphs/cm 3 of dry sediments).

The "continental" versus "marine" (C/M) ratio, that includes selected continental and freshwater (Halodinium, Pediastrum, Concentricystes, amoebae) versus selected marine (foraminiferal linings, Radiosperma corbiferum) NPP microrests, has been used in this study to discuss continental versus marine influences. Finally, we calculated the number of taxa per sample (S) using the "Past version 1.75b" software (Hammer et al., 2001), allowing us to discuss the pollen and dinocyst diversity through time.

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15

Foraminiferal analysis

In this study, a minimum of 300 benthic foraminiferal counts were performed on the fraction >150 µm (Leica M60 binocular, X60 magnification) so as to express in percentages the number of specimens exhibiting morphological anomalies (atrophied, hypertrophied or overlapped chambers). Indeed, shell deformations are known to represent excellent tracers of water pollution [START_REF] Alve | Benthic foraminiferal responses to estuarine pollution: a review[END_REF]Polovodova and Schönfeld, 2008;[START_REF] Martins | The response of benthic foraminifera to pollution and environmental stress in Ria de Aveiro (N Portugal) /La respuesta de los foraminíferos bentónicos a la contaminación y el estrés ambiental en la Ría de Aveiro (N de Portugal)[END_REF].

A C C E P T E D M A N U S C R I P T 16

Palynological results from I_09 Daoulas core

Palynomorph diversity and concentrations

Considering pollen assemblages, 51 different taxa have been identified (Appendix 1) with an average diversity of 26 taxa per slide (Fig. 3). In parallel, 18 different dinocyst taxa have been identified (Appendix 1) with an average diversity of 7 taxa per slide. Finally, 19 other NPP have been determined and include different types of spores, freshwater and marine microalgae, amoebae, and copepod eggs (Fig. 3; Appendix 1).

From the base of the core to 35 cm, palynomorph concentrations are rather low (i.e. 4,263 spores/cm 3 ; 7,285 cysts/cm 3 ; 31,344 pollen/cm 3 ), always below the mean value of each dataset (Fig. 3). Then, at 35 cm, significant increases in pollen, dinocyst, and spore concentrations are noticeable with values 3, 5, and 4 times higher, respectively. Also, this limit underlines a re-increase (after the slight occurrence noted between 53 and 47 cm) of freshwater microalgae (especially Concentricystes and Halodinium) and other continental markers (cf. C/M ratio; Fig. 3), while H. problematica (estuarine algae) and amoebae concentrations are characterized by a drastic drop. This leads us to consider this limit as a major transition identified as limit A in Fig. 3. From limit A, all palynomorph concentrations are characterized by strong amplitudes between high values (i.e. between 10,000 and 20,000 spores/cm 3 ; 30,000 and 50,000 cysts/cm 3 ; 80,000 and 100,000 pollen/cm 3 ) and lower ones, typical of those recorded before 35 cm. However, a second major transition can be identified at 17 cm, referred to as limit B in Fig. 3. This limit evidences the highest freshwater fossil concentrations, up to 2,000 algae/cm 3 , and thus the highest C/M ratio recorded throughout the core. Amoebae concentrations sharply increase after 13 cm (up to 1,000 amoebae/cm 3 ) while R. corbiferum concentrations strongly drop, reaching below average values for the entire core

A C C E P T E D M A N U S C R I P T 17 (i.
e. 111 algae/cm 3 ). The same trend is observed for foraminiferal linings. Regarding the diversity of pollen (between 18 and 33 different taxa) and dinocyst (between 2 and 12 different taxa) assemblages (Fig. 3), an increase of pollen (-dinocyst) diversity is especially observed from limit B onwards (-limit A).

Pollen assemblages

Major taxa (those exceeding a threshold of 2% among the whole assemblage) are depicted in [START_REF] Lambert | Modern palynological record in the Bay of Brest (NW France): signal calibration for palaeo-reconstructions[END_REF] in the Daoulas Bay (identified with red dots in Fig. 4). Also, for Alnus, Chenopodiaceae, and Poaceae, top values from the I_09 core are obviously similar to mean modern values reconstructed in the area [START_REF] Lambert | Modern palynological record in the Bay of Brest (NW France): signal calibration for palaeo-reconstructions[END_REF].

Throughout the sequence, Alnus displays an obvious increasing trend, from around 5% until 27 cm to systematically above 10% after 27 cm, with peaks reaching 20-25% especially after limit B. A similar trend is observed for Betula at 27 cm (1.5 to 3%). This leads us to consider a third major transition in between the two limits described above, and referred to as limit C in Fig. 4. It is also worth noting the slight increasing percentages of Fraxinus, Salix, and Hedera after limit B (17cm), a period also characterized by the persistence of the highest

A C C E P T E D M A N U S C R I P T 18
percentages of Alnus ever recorded in the core (i.e. systematically above 12%). Finally, Quercus does not present a linear homogeneous trend but rather exhibits several fluctuations of an amplitude of 10-15% around a mean value throughout the record. Among herbaceous taxa, Poaceae are clearly anti-correlated with trees (i.e. especially Quercus) and similarly marked by several fluctuations, ranging between 10 and 20%. Lower-average values are noted after limit B, while Chenopodiaceae and Plantago maritima are characterized by decreasing trends, especially obvious after limit A. Percentages of other herbaceous taxa are rather uniform all along the core even if characterized by weak amplitudes.

Dinocyst assemblages

Dinocyst assemblages (Fig. 4) are largely dominated by Spiniferites bentorii (mean value of 32% for the whole core), Spiniferites belerius (mean value of 25%), and Lingulodinium machaerophorum (mean value of 21% assemblages, with a particular prevalence of the latter species (over 80%) in estuarine domains and inland waterways [START_REF] Morzadec-Kerfourn | La signification écologique des dinoflagellés et leur intérêt pour l'étude des variations du niveau marin[END_REF][START_REF] Morzadec-Kerfourn | Les kystes de Dinoflagellés[END_REF].

L. machaerophorum is characterized by above-average values (20 to 40%) until limit A. From this limit, and especially limit C, L. machaerophorum percentages significantly drop, reaching values below the average. It is worth noting that L. machaerophorum is a species that has

A C C E P T E D M A N U S C R I P T 19
commonly been interpreted as a proxy for stratified waters in various contexts (estuaries and fjords: [START_REF] Morzadec-Kerfourn | La signification écologique des dinoflagellés et leur intérêt pour l'étude des variations du niveau marin[END_REF]Dale et al. 1999;Dale, 2009;Ganne et al., 2016, and even in marine environments associated with typical climate intervals of higher fluvial discharges: Zaragosi et al., 2001;Penaud et al., 2009). On the contrary, a generally increasing trend of S.

bentorii percentages is noted in our study, with clear fluctuations anti-correlated with S.

belerius ones. Among other characteristic features, S. membranaceus is continuously present with above-average values from limit B (17 cm). Today, S. bentorii is commonly associated to warm waters (Zonneveld et al., 2013) and anthropogenic-induced nutrient-enriched areas (Pospelova et al. 2002). It is also worth noting that the decreasing profile of L.

machaerophorum is extremely close to the one depicted by Chenopodiaceae percentages in the pollen record (Fig. 4). Interestingly, Chenopodiaceae are typical herbaceous plants from tidal-influenced environments. This halophile vegetation commonly grows on salt marshes together with Poaceae and Asteraceae. Before limit A, the association of Poaceae, Chenopodiaceae, and L. machaerophorum is a clear signal of the dominance of salt marsh environments. After limit A, and especially after limit B, the salt marsh signature is less pronounced in favour of a palynological signal mainly characterized by the Alnus -S. membranaceus association.

A C C E P T E D M A N U S C R I P T 20

Discussion

Climate forcing on tree pollination rates

Mean tree pollen percentages(i.e. AP, 58%; Figs. 4 and5) recorded in core I_09 are similar to the tree signature currently recorded in Bay of Brest modern sediments (57%; [START_REF] Lambert | Modern palynological record in the Bay of Brest (NW France): signal calibration for palaeo-reconstructions[END_REF]. However, since 1870, these values fluctuate throughout I_09 core with amplitude of 10-20%, mainly explained by Quercus percentages (in addition to Alnus at the top of the core). More precisely, a significant increase (+/-15%) is observed between 1890 and 1930 (cf. raw data in Fig. 5), as well as from 1975 upwards, the Poaceae signature being then anticorrelated as expected (particularly noteworthy for cereals). These variations can be partially explained by the agricultural practices (see section 2.1 this paper) with increasing hedgerows up to 1950 (mosaic landscape) and their drop during the 1970's (grouping of agricultural surfaces). However, this is not consistent with the increase of pollen trees percentages from 1985 to now and raises question regarding Quercus (Alnus in a lesser extend) variations.

Therefore, natural modes of North Atlantic climate changes may be involved.

In our study, the observed fluctuating arboreal pollen percentages appears closely related to the oceanic AMO index (Enfield et al., 2001) as well as to the atmospheric seasonality parameter (i.e. mean difference between summer and winter temperature) (Fig. 5).

The multidecadal variability characteristic of the AMO reflects changes in North Atlantic SST [START_REF] Kerr | A North Atlantic climate pacemaker for the centuries[END_REF] have contributed to the boost of tree pollination rates, and may then be responsible for the significant increases observed in arboreal percentages.

Major runoff increase over the last 60 years

In parallel with the natural climate variability described above, higher percentages of Alnus, together with increasing freshwater palynomorph, pollen, and dinocyst concentrations, as well as the decreasing trend of R. corbiferum (marine algae), are observed from about 1945 (limit A; Fig. 6), and even more obviously since 1965 (limit C; Fig. 6). This trend is correlated with the XRF Ti/Ca signal, commonly interpreted as a detrital terrigenous advection signal through runoff and soil leaching (e.g. [START_REF] Bahr | Late glacial to Holocene climate and sedimentation history in the NW Black Sea[END_REF][START_REF] Mollier-Vogel | Rainfall response to orbital and millennial forcing in northern Peru over the last 18 ka[END_REF][START_REF] Steinke | Mid-to late-Holocene Australian-Indonesian summer monsoon variability[END_REF]. A recent study carried out on Bay of Brest modern sediments leads us to consider Alnus as a fluvial discharge indicator, based on its present-day distribution, which is correlated to river mouths [START_REF] Lambert | Modern palynological record in the Bay of Brest (NW France): signal calibration for palaeo-reconstructions[END_REF]. Its fluvial discharge significance is thus confirmed, especially since this taxon has not been found to increase on the corresponding watersheds over the last decades (National Botanical Conservatory of Brest, personal

A C C E P T E D M A N U S C R I P T 22 communication).
In parallel with the Alnus trend, the combined increase of other trees typical from the riparian forest (especially Salix, Betula and Fraxinus; Fig. 6) suggests an increase in fluvial discharge and/or in precipitation over the past 60 years.

In parallel with these observations, the meteorological station of Rennes evidences a clear increase of precipitation in Brittany, well correlated with the XRF Ti/Ca ratio, probably arguing for higher runoffs on Bay of Brest watersheds and then increased Aulne River flows (Brittany DREAL; Fig. 6). For example, when looking more into details, lower Ti/Ca values recorded at around 1972-1976, 1982-1987, and 2003-2008, are well correlated with lower values of river flows and precipitations, giving strength to our Ti/Ca signal as an indirect proxy for runoff changes through time. It is also worth noting that higher precipitations, river flows, and riparian forest percentages are recorded after 1985, synchronously with the highest atmospheric temperatures (mean annual temperatures of about 12.5°C since 1985) recorded at the meteorological station of Rennes (dataset available since 1930; Fig. 7). Interestingly, it has been demonstrated that the recent rise in global air temperatures can promote a significant increase in runoff worldwide, caused by an acceleration of the hydrological cycle [START_REF] Labat | Evidence for global runoff increase related to climate warming[END_REF]. One may advance with good reason that what is valid worldwide might not be true locally. However, the EA pattern is known to affect the rainfall budget in Western Europe [START_REF] Ruprich-Robert | Combined influences of seasonal East Atlantic Pattern and North Atlantic Oscillation to excite Atlantic multidecadal variability in a climate model[END_REF] and is also well correlated to the XRF Ti/Ca signal (Fig. 6).

From these sedimentological data (XRF Ti/Ca signal) and palynological evidences (riparian forest, freshwater fossils, palynomorph concentrations), combined with recent instrumental data (EA pattern, as well as air temperatures, precipitations, and river flows across Brittany), three major periods (i.e. pluri-decadal trends) of increased runoff and therefore fluvial inputs can be highlighted, first after the Second World War (limit A; Fig. 6), then after 1965 (limit C;

A C C E P T E D M A N U S C R I P T

23 Fig. 6), and finally after 1985 (limit B; Fig. 6). However, the warming context and the increasing rainfall pattern previously described are not the only parameters to take into account. Superimposed on these climate trends, other mechanisms may be involved from 1945 onwards. Indeed, higher river flows have been accentuated by increasing artificial agricultural surfaces in Bay of Brest watersheds (Fig. 7). After the Second World War, the implementation of a new agricultural policy led to the reunification of agricultural holdings (i.e. gathering of cultivated plots). The destruction of hedgerows then modified the erodability of soils and significantly contributed to runoff increases in Bay of Brest watersheds due to the absence of embankments (Flatrès, 1976;[START_REF] Merot | Influence du réseau de haies des paysages bocagers sur le cheminement de l'eau de surface[END_REF]. This limit is also clearly noticeable on the XRF Ti/Ca signature that obviously increases around 1945. From this period onwards, Chenopodiaceae and Plantago maritima percentages noticeably drop as a consequence of the local loss of the salt marsh signature in favor of a fluvial-type signal dominated by the riparian forest (Fig. 6).

Major trophic disturbances since 1990

After 1965 (limit C), and especially 1985 (limit B), an atypical palynological signature is observed with several indicators (Fig. 7). Pollen assemblages have already been discussed as characterized by the highest riparian forest signature from 1965, with high percentages observed around 1970,1995,2005, and at the top of I_09 core (Fig. 5). Regarding dinocyst assemblages (Fig. 4), a non-ambiguous relationship can be highlighted between L. machaerophorum and S. bentorii across limit C (1965) and then between S. bentorii and S. [START_REF] Morzadec-Kerfourn | Les kystes de dinoflagellés dans les sédiments récents le long des côtes Bretonnes[END_REF][START_REF] Ganne | Present-day palynomorph deposits in an estuarine context: the case of the Loire Estuary[END_REF], L. machaerophorum is known to be very sensitive to nitrate and phosphate inputs, while these inputs can also benefit other species, such as S. bentorii Zonneveld et al., 2013). A sharp increase of L. machaerophorum was observed in Norwegian fjords in response to the increased watershed eutrophication (Dale et al., 1999;Dale, 2009),

(
while Pospelova et al. (2002) showed that, in some American anthropized estuaries, L.

machaerophorum rather negatively responds to anthropogenic nutrient enrichment, in favor of other species such as S. bentorii. No consensus thus exists on a fossil dinocyst assemblagetype that would reflect nutrient enrichment in disturbed estuarine domains (Pospelova et al., 2002). There is no particular species that can be used to characterize high nutrient loading in NW Atlantic estuaries. Indeed, the different responses according to the studied estuaries can be explained by the different physico-chemical configurations (e.g. salinity, pH) (Price et al. record (Fig. 7). We indeed observe a significant increase in amoebae concentrations (i.e.

continental markers; [START_REF] Mitchell | Testate amoebae analysis in ecological and palaeoecological studies of wetlands: past, present and future[END_REF][START_REF] Lambert | Modern palynological record in the Bay of Brest (NW France): signal calibration for palaeo-reconstructions[END_REF]. In addition, benthic foraminifera are known to strongly react to anthropogenic pollution. Shell deformations may indicate an immediate reaction to a human-induced environmental stress [START_REF] Alve | Benthic foraminiferal responses to estuarine pollution: a review[END_REF]Polovodova and Schönfeld, 2008;[START_REF] Martins | The response of benthic foraminifera to pollution and environmental stress in Ria de Aveiro (N Portugal) /La respuesta de los foraminíferos bentónicos a la contaminación y el estrés ambiental en la Ría de Aveiro (N de Portugal)[END_REF]. Here, despite the low proportion and diversity of benthic foraminifera in core I_09, an increasing trend is observed with up to 6% of deformed shells, particularly since 1990. Finally, the increase in toxic algal blooms has previously been detected and quantified in the same core (Klouch et al., 2016b;cf. Figs. 2 and 7), especially from 1990, by the presence of fossilized DNA (qPCR method) of a particular dinoflagellate taxon, Alexandrium minutum, known to produce virulent toxins poisoning shellfish [START_REF] Aylagas | Evaluation of marine phytoplankton toxicity by application of marine invertebrate bioassays[END_REF].

Interestingly, the main factor controlling phytoplankton community developments in the Bay of Brest can be directly related to river-induced fertilization (Quéguiner and Tréguer, 1984). Nitrate inputs in the Bay of Brest are well correlated with river flows (Delmas and Treguer, 1983) as evidenced in Figs 6 and 7. We then suggest that the increases in fluvial discharges since the late 1980s may have brought huge amounts of fertilizers that promoted an enhancement of the dinocyst diversity and total dinocyst concentrations (Fig. 3), probably promoting dinoflagellate productivity as also recorded by Harland et al. (2004) in Sweden.

This may be also responsible for the growth of toxic dinoflagellate species (A. minutum; Klouch et al., 2016b) in Bay of Brest surface waters. Consequently, in addition to the obvious increased runoff for half a century, an increase in nitrogen fluxes, and probably of eutrophication since 1985-1990 (i.e. nutrient enrichment of the aquatic system; [START_REF] Andersen | Coastal eutrophication: recent developments in definitions and implications for monitoring strategies[END_REF], in parallel with an increase in atmospheric temperatures has certainly contributed to the drastic alteration of plankton communities, as evidenced by the dinocyst relay and deformed benthic foraminiferal shells. It is also interesting to note that the Daoulas Bay, where the maximum occurrences of toxic algal blooms are observed today within the Bay of Brest (Klouch et al., 2016a), also corresponds to the site where maximal percentages of agriculture-type pollen markers are observed in Bay of Brest modern sediments [START_REF] Lambert | Modern palynological record in the Bay of Brest (NW France): signal calibration for palaeo-reconstructions[END_REF], confirming the link between increased runoff on agricultural soils and the disturbance of the phytoplankton community. This may be related to the small size of the watershed and the isolation of the Daoulas estuary from main tidal currents.

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A C C E P T E D M A N U S C R I P T 27

Conclusion

The high resolution palynological study conducted over the past 150 years in the Bay of Brest

  ,b) and is a shallow semi-enclosed basin of 180 km² surrounded by a 230 km long coastline. Its bedrock consists of Proterozoic igneous rocks in the north and Brioverian (Neoproterozoic, possibly Early Cambrian) to Palaeozoic (Ordovician, Devonian and locally Carboniferous) sediments in the south and east. Present-day low reliefs (few hills reach 330m high) are inherited from the peneplanation of the high Hercynian chain (Chauris and Plusquellec, 1980;

Fig. 4 .

 4 Fig. 4. Among trees, deciduous Quercus (mean value of 20% for the whole core), Corylus

  membranaceus across limit B (1985). In 1965, no particular climate shifts may explain this connection. In addition, it has been demonstrated a close relationship between A C C E P T E D M A N U S C R I P T 24 Chenopodiaceae and L. machaerophorum trends (Figs. 4 and 6), arguing for a common origin of their signature. Both palynological taxa may thus indicate salt marsh-like environments, and their decline argues for a change in fluvial dynamics probably also responsible for a trophic change in the dinoflagellate community. From 1965 onwards, it is notable that each recent peak of Alnus is associated with an even more pronounced drop of L. machaerophorum percentages, confirming the relationships between both fluvial and salt marsh signals, respectively. Despite its ecological coastal and estuarine preferences (Morzadec-Kerfourn,

  2017). Our results appear similar to those obtained byPospelova et al. (2002), who concluded that L. machaerophorum does not respond positively to human inputs in shallow estuaries, having fewer consequences on water mass stratification than in deep Norwegian fjords(Dale et al., 1999; Dale, 2009). Moreover, from 1985 onwards, other non-pollen palynomorph, micropaleontological, and genetic signals argue for atypical environmental conditions when compared with the whole A C C E P T E D M A N U S C R I P T 25

  Nitrate fluxes, respectively recorded in the Elorn and Mignonne rivers since 1970 and 2000 (source: SAGE Elorn), allow us to compare the evolution of agricultural practices (i.e. fertilizer uses) on Bay of Brest watersheds with our fossil data (Fig.7). This indirect eutrophication indicator sharply increases from 1970 to 1990, then reaching a plateau of abnormally high values approaching "standard" thresholds at that time. Various remediation policies were then successfully applied to lower nitrate fluxes in Bay of Brest watersheds from 1990 to present. Nitrogen fertilizers are indeed widely used in Brittany and particularly in the Daoulas watershed, which is characterized by the largest nitrogen introductions recorded in the region (above 35 kg/ha; Agreste -Draaf Bretagne). Nitrate concentrations in Brittany rivers then often exceed the national standard of 50 mg/l[START_REF] Joanny | Qualité du milieu marin littoral-Editions lFREMER[END_REF], and largely dominate at the North Atlantic scale(Howarth et al., 1996), mainly coming fromA C C E P T E DM A N U S C R I P T 26 agriculture (98% : use of fertilizers, extension of intensive farming;[START_REF] Aurousseau | Evolution de la qualité des eaux en nitrate en Bretagne, synthèse régionale au 30 Septembre 2007[END_REF].

(

  NW France) has highlighted the relationship between natural climate variability, human pressures, and fossil palynomorphs preserved in sediments (especially pollen grains and dinoflagellate cysts). Varying SST in the North Atlantic Ocean, by driving atmospheric temperatures across Europe, may have influenced tree pollination rates deduced from arboreal pollen percentage fluctuations. Superimposed on natural climate modes, increased human influence on watersheds is perceptible since 1945. Changes in agricultural practices, particularly the regrouping of cultivated areas and the reduction of hedgerows, led to a runoff increase, recorded through our palynological (increase of riparian forest as well as reduction of Chenopodiaceae and L. machaerophorum percentages) and sedimentological (XRF Ti/ Ca increase) markers. Also, changes in the structure of the dinocyst community (major trophic relay between L. machaerophorum and S. bentorii around 1965 followed then by S. membranaceus around 1985), and the intensification of toxic algal blooms (A. minutum) detected by genetic analyses, appear directly correlated to the increase of fertilizer use for agriculture over the past decades. Such changes caused strong environmental and health impacts. Here, for the very first time, a palynological study conducted in parallel with a genetic study provides clear driving mechanisms regarding the major causes responsible for recent phytoplankton (dinoflagellates and their cysts) changes affecting European coastal ecosystems. It demonstrates the strong potential of such micropaleontological approaches which allow drawing a comprehensive view of modern human induced environmental alterations in a retrospective frame.

  Turon, J.-L., 1984. Le palynoplancton dans l'environnement actuel de l'Atlantique nord-oriental : évolution climatique et hydrologique depuis le dernier maximum glaciaire. Université de Bordeaux I. Turon, J.-L., Lézine, A.-M., Denèfle, M., 2003. Land-sea correlations for the last glaciation inferred from a pollen and dinocyst record from the Portuguese margin. Quat. Res. 59, 88-96. van Vliet, A.J., Overeem, A., De Groot, R.S., Jacobs, A.F., Spieksma, F., 2002. The influence of temperature and climate change on the timing of pollen release in the Netherlands. Int. J. Climatol. 22, 1757-1767. van Vliet-Lanoë, B., Goslin, J., Hallégouët, B., Hénaff, A., Delacourt, C., Fernane, A., Franzetti, M., Le Cornec, E., Le Roy, P., Penaud, A., 2014. Middle-to Late-Holocene storminess in Brittany (NW France): Part I-morphological impact and stratigraphical record. The Holocene 24, 4, 434-453. Zaragosi, S., Eynaud, F., Pujol, C., Auffret, G.A., Turon, J.-L., Garlan, T., 2001. Initiation of the European deglaciation as recorded in the northwestern Bay of Biscay slope environments (Meriadzek Terrace and Trevelyan Escarpment): a multi-proxy approach. Earth Planet. Sci. Lett. 188, 493-507.

Figure 1 :

 1 Figure 1: Locations of : a) the Bay of Brest in north-western France, as well as of : b) I_09

Figure 2 :

 2 Figure 2: I_09 core photograph and X-Ray radiography, with the sampling procedure carried

Figure 3 :Figure 4 :

 34 Figure 3: Depth chart representing dinocysts, pollen grains, spores and major non-pollen

Figure 5 :

 5 Figure 5: Comparison between selected pollen percentages (cereals, Quercus, sum of Alnus,

Figure 6 :

 6 Figure 6: Comparison between palynological data from I_09 core, data from literature, and

  ; warmer SSTs being recorded across the entire North Atlantic basin during AMO

	1994) with milder climate conditions recorded over Western Europe during AMO +
	(McCarthy et al., 2015), or tree-ring data (Delworth and Mann, 2000; Gray et al., 2004).
	Furthermore, at the studied timescale, fluctuations observed in core I_09 cannot be strictly
	attributed to the growth or decline of pollen-producing young plants (National Botanical
	Conservatory of Brest, personal communication). Since weather conditions are known to
	greatly influence current tree pollination rates (van Vliet et al., 2002; Garcia-Mozo et al., 2006; Besancenot and Thibaudon, 2012), we therefore suggest that an increase of North M A N U S C R I P T Atlantic SST (AMO +), by favoring milder springs and summers in Western Europe, may
	A C C E P T E D
	positive modes (AMO +), and conversely during AMO negative modes. Basin wide Atlantic
	SST variations therefore play an important role in multidecadal climate changes observed in
	the northern hemisphere as recorded through averaged air temperatures (Schlesinger et al.,
	21

  This study was supported by the French CNRS and is a contribution to the 2015-2016 INSU Finally, we would like to thank Katalin Kovacs for the English editing of the manuscript, as well as Michel Aïdonidis (Head of the Weather Station at Brest) and the National Botanical Conservatory of Brest for constructive exchanges. Frankignoul, C., 2009. Atlantic multidecadal oceanic variability and its influence on the atmosphere in a climate model. Clim. Dyn. 33, 45-62. Mudie, P.J., Marret, F., Aksu, A.E., Hiscott, R.N., Gillespie, H., 2007. Palynological evidence for climatic change, anthropogenic activity and outflow of Black Sea water during the late Pleistocene and Studies on the Phytoplankton in the Bay of Brest (Western Europe). Goberville, E.,Barrier, N., L'Helguen, S., Morin, P., Bozec, Y., Rimmelin-Maury, P., Czamanski, M., Grossteffan, E., Cariou, T., others, 2014. Large and local-scale influences on physical and chemical characteristics of coastal waters of Western Europe during winter. J. Mar. Le Goff, R., others, 1997. Etat des lieux et des milieux de la rade de Brest et de son bassin versant. Phase Préliminaire Contrat Baie Rade Brest Communauté Urbaine Brest.

	project EC2CO-LEFE: « CAMOMI : Convergences / Approches croisées des signaux MOléculaires et MIcropaléontologiques pour décrypter les forçages anthropiques et climatiques en milieu côtier (Rade de Brest) ». This work was supported by the «Laboratoire Besancenot, J.-P., Thibaudon, M., 2012. Changement climatique et pollinisation. Rev. Mal. Respir. 29, 1238-1253. Beug, H.-J., 1961. Leitfaden der pollenbestimmung. Fisch. Stuttg. 1, 63. Bonnet, S., Guillocheau, F., Brun, J.-P., Van Den Driessche, J., 2000. Large-scale relief development related to Quaternary tectonic uplift of a Proterozoic-Paleozoic basement: The Armorican deVernal , A., Henry, M., Bilodeau, G., 1999. Techniques de préparation et d'analyse en micropaléontologie. Cahiers du GEOTOP 3, 41. Delmas, R., Treguer, P., 1983. Evolution saisonnière des nutriments dans un écosystème eutrophe d'Europe occidentale (la rade de Brest). Interactions marines et terrestres. Oceanol. Acta 6, 345-356. Tréguer, P., Syst. 139, 79-90. Msadek, R., Holocene: Centennial-to decadal-scale records from the Black and Marmara Seas. Quat. Int. 167, Tréguer, P.J., De La Rocha, C.L., 2013. The world ocean silica cycle. Annu. Rev. Mar. Sci. 5, 477-501.
	d'Excellence» LabexMER (ANR-10-LABX-19) and co-funded by a grant from the French government under the program « Investissements d'Avenir ». We thank the UMR CNRS 5805 EPOC (Talence) for palynological laboratory procedures (Muriel Georget) and radionuclide measurements, the laboratory IFREMER-Marine Geosciences (Plouzané) for having set out the means for recovering the sediment core (SERABEQ cruises; G. Gregoire PhD thesis, 2016), and the laboratory IFREMER-DYNECO (Plouzané) for having provided samples necessary for the study. A C C E P T E D M A N U S C R I P T Massif, NW France. Journal of Quaternary Research 105, B8, 19273-19288. Cayan, D.R., 1992. Latent and sensible heat flux anomalies over the northern oceans: The connection to monthly atmospheric circulation. J. Clim. 5, 354-369. Chauris, L., Plusquellec, Y., (coord.) 1980. Carte Geol. France (1/50 000), feuille Brest (274). Ed. B.R.G.M. Orléans deglaciation in the central Mediterranean Sea: palaeovegetation and palaeohydrological evolution. QSR, 17(4-5), 303-317. Dale, B., 2009. Eutrophication signals in the sedimentary record of dinoflagellate cysts in coastal waters. J. Sea Res. 61, 103-113. A C C E P T E D Mar. Micropaleontol. 45, 169-174. Flatrès, P., 1963. La deuxième" Révolution agricole" en Finistère. Études Rural. 5-55. Flatrès, P., 1976. Rapport de synthèse-Géographie. Bocages Hist. Ecol. Econ. 21-30. Flatrès, P., 1979. Paysages ruraux européens, in : Standing European Conference, Rennes, Université A C C E P T E D Massachusetts (USA). Sci. Total Environ. 298, 81-102. Price, A.M., Coffin, M.R., Pospelova, V., Latimer, J.S., Chmura, G.L., 2017. Effect of nutrient pollution on dinoflagellate cyst assemblages across estuaries of the NW Atlantic. Marine Pollution Bulletin, 121(1-2), 339-351. Quéguiner, B., Tréguer, P., 1984. Seasonal Variations in Composition, Biomass and Production in Relation to Hydrological and A C C E P T E D Collins, M., Botzet, M., Carril, A.F.Combourieu-Nebout, N., Paterne, M., Turon, J.L., Siani, G., 1998. A high-resolution record of the last M A N U S C R I P T Delworth, T.L., Mann, M.E., 2000. Observed and simulated multidecadal variability in the Northern Hemisphere. Clim. Dyn. 16, 661-676. Diaz, R.J., Rosenberg, R., 2008. Spreading dead zones and consequences for marine ecosystems. science 321, 926-929. M A N U S C R I P T 73-90. Penaud, A., Eynaud, F., Turon, J.L., Zaragosi, S., Malaizé, B., Toucanne, S., Bourillet, J.-F., 2009. What forced the collapse of European ice sheets during the last two glacial periods (150kaB. P. and 18ka cal BP)? Palynological evidence. Palaeogeogr. Palaeoclimatol. Palaeoecol. 281, 66-78. Polovodova, I., Schönfeld, J., 2008. Foraminiferal test abnormalities in the western Baltic Sea. J. Foraminifer. Res. 38, 318-336. M A N U S C R I P T Troadec, P.,
	Dale, B., Thorsen, T.A., Fjellsa, A., 1999. Dinoflagellate cysts as indicators of cultural eutrophication in de Haute Bretagne. Chemical Features (1981-1982). Bot. Mar. 27, 449-460.
	the Oslofjord, Norway. Estuar. Coast. Shelf Sci. 48, 371-382.
	Quéré, E., Magnanon, S., Ragot, R., Gager, L., Hardy, F., 2008. Atlas floristique de Bretagne : la flore
	du Finistère. Ed. Siloë, 693 pp.
	30 31 37 39

, Drange, H., Jouzeau, A., Latif, M.,

Masina, S., Otteraa, O.H., Pohlmann, H., Sorteberg, A., others, 2006

. Interannual to decadal climate predictability in the North Atlantic: a multimodel-ensemble study. J. Clim. 19, 1195-1203. Enfield, D.B., Mestas-Nuñez, A.M., Trimble, P.J., 2001. The Atlantic multidecadal oscillation and its relation to rainfall and river flows in the continental US. Geophys. Res. Lett. 28, 2077-2080. Faegri, K., Iversen, J., 1989. Textbook of pollen analysis (4th edn by Faegri, K., Kaland, PE & Krzywinski, K.). Wiley, New York. Fatela, F., Taborda, R., 2002. Confidence limits of species proportions in microfossil assemblages. Pospelova, V., Chmura, G.L., Boothman, W.S., Latimer, J.S., 2002. Dinoflagellate cyst records and human disturbance in two neighboring estuaries, New Bedford Harbor and Apponagansett Bay,

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M A N U S C R I P T 33 Gregoire, G., Le Roy, P., Ehrhold, A., Jouet, G., Garlan, T. 2017. Control factors of Holocene sedimentary infilling in a semi-closed tidal estuarine-like system: the bay of Brest (France).

Marine Geology, 385, 84-100. Guillaud, J.F., Bouriel, L., 2007

Appendix caption

Appendix 1: Exhaustive table of palynological results (individual counts).

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A


Ilaria Ciofini

Maria Gabriella Chiariello

Federico Coppola

Fulvio Perrella

Marika Savarese

Umberto Raucci
email: umberto.raucci@unina.it

Nadia Rega
email: nadia.rega@unina.it

An electron density based analysis to establish the electronic adiabaticity of proton coupled electron transfer reactions

Keywords: Density based indexes, Proton Coupled Electron Transfer, Electronic Adiabaticity Degree, Non Adiabatic Coupling, Density Functional Theory

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Proton Coupled Electron Transfer (PCET) reactions are at the origin of many fundamental biological [1][2][3][4][5][6][7][8][9][10] (i.e. photosynthesis, enzymatic reactions) and electrochemical [11][12][13] processes, thanks to their versatile nature. The transfer of electrons and protons represents the leitmotif of PCET. [14][15][16][17][18][19] The general PCET definition covers several different mechanisms and kinetics, controlled by the coupling between the transferred electrons and protons. In the last few years, many efforts were devoted to provide an universal classification of the complex family of PCET reactions. 15,[18][19][20][21][22][23][24][25][26][27] This is essential for supporting the interpretation of experimental data and also to drive the design of efficient charge transfer molecular machines suitable for catalysis and artificial photosynthesis. [28][29][30][START_REF] Llansola-Portoles | From Molecules to Materials[END_REF][START_REF] Gust | [END_REF][33][START_REF] Berardi | [END_REF] The classification of the PCET reactions proposed by Hammes Shiffer relies on the electronic, vibrational and vibronic adiabaticity degree between the transferred electron and proton. [20][21][22][23] Concerted PCET reactions, where no stable intermediates are involved, are basically distinguished as Hydrogen Atom Transfer (HAT) or Electron Proton Transfer (EPT), depending on the electronic adiabaticity degree of the process. 23 In a HAT process the reaction evolves only in the ground electronic state and it is electronically adiabatic. In this case the vertical gap between the ground and the first excited state is larger than the thermal energy. Otherwise, if the ground and the first excited state are strongly coupled together the reaction is electronically non-adiabatic. EPT reactions fall into this category. An adiabaticity parameter, defined as the ratio of the proton tunneling time and the electronic transition time, was introduced and widely adopted to distinguish between HAT and EPT mechanisms. 20,21,35,36 Moreover, considering that the electronic adiabaticity is strictly related to the charge redistribution along the proton transfer coordinate, several diagnostic analyses were also performed. 36,37 For example, the adiabaticity degree of PCET processes was evaluated analyzing the dipole moment variation along the reaction coordinate. 36,37 Another less intuitive and more computational demanding analysis, based on the evaluation of the non-adiabatic coupling (NAC) vectors between the ground and the excited electronic state, was also proposed. 36,37 The first order non-adiabatic coupling ma-trix elements between the | Ψ m > and | Ψ n > electronic states can be written as follows: [38][39][40]

τ ξ mn = <Ψm| ∂ ∂ξ Vne|Ψn> En-Em
where ξ represents a Cartesian nuclear coordinate, Vne is the electronnucleus attraction operator, while E m and E n are the energy eigenvalues of | Ψ m > and

| Ψ n >, respectively.
The computation of NAC vectors is crucial to characterize regions of the Potential Energy Surface (PES) where two or more electronic states become degenerate (i.e. conical intersection points), and to follow the excited state non-adiabatic dynamics of the system. [40][41][42][43][START_REF] Domcke | Conical Intersections: Theory, Computation and Experiment[END_REF][START_REF] Handy | [END_REF][46][47][START_REF] Tully | Modern Methods for Multidimensional Dynamics Computations in Chemistry[END_REF][START_REF] Petrone | [END_REF][50][51][52][53][54][55] In the case of PCET reactions, large magnitude of the NAC vectors and a drastic change in the dipole moment along the reaction coordinate are observed for non-adiabatic reactions, whereas adiabatic processes show small magnitude of NAC and a slight variation of the dipole moment. 36,37 In this context, the classification of PCET processes in terms of electronic adiabaticity would greatly benefit from a suitable tool able to condensate both the information of non-adiabatic coupling and dipole moment variation. These properties are intrinsically related to the ground and the excited state electron density distributions. Thus, we propose here an electron density based analysis to discriminate among concerted PCET reactions belonging to different adiabaticity regimes.

In detail, the D CT electron density based index 56,57 was employed as key instrument to quantify the ground-excited state electronic reorganization occuring during the PCET process.

This index simply represents the distance between the barycenters of charge distributions associated respectively to the increase and the decrease of the electron density upon a vertical excitation. 57 Recently, it was successfully applied to analyze excited state reactivity (i.e. excited state proton transfer reactions) in combination with both static and dynamical approaches. [58][59][60][61][62][63][64][65][66] The information on the charge rearrangement after the vertical excitation is innate in the D CT index, which is related to the ground-excited state dipole moments variation by construction. 57 Moreover, it allows to quantify the electron density redistribution between two electronic states, containing information indirectly related to the non-adiabatic coupling.

PES regions characterized by a strong electronic coupling are expected to show an high similarity between the ground and the excited state electron densities. In these points the 4 ground-first excited state electronic reorganization is small, corresponding to low values of D CT . The opposite behavior is expected in electronically uncoupled regions of the PES.

Relying only on the evaluation of the ground and the excited state electron densities, the D CT index is cheaply computed compared to the time-demanding NAC computation. Most importantly, it can be directly evaluated and used in combination to any quantum chemical method, such as DFT or post-Hartree Fock based. 67 In this paper, this density based index was applied to discriminate the different adiabaticity degree in the PCET reaction of a prototype system, the 3-Methylphenoxyl/phenol one, hereafter named mMphenoxyl/phenol. For a related system, i.e. the phenoxyl/phenol dimer, a recent study of Harshan and co-workers showed that, depending on the geometrical arrangement of the two aromatic rings, the PCET reaction takes place with a HAT or an EPT mechanism. 35 For this system the existence of two transition states corresponding to an open and a stacked geometry was proved, as well as the fact that the PCET reaction is electronically non-adiabatic for the open conformation, while it can be classified as electronic adiabatic reaction in the case of the stacked geometry. 35 We simulated the reaction pathway for both the open and the stacked configurations of the mMphenoxyl/phenol dimer by the integration of the intrinsic reaction coordinate at density functional theory (DFT) level. [START_REF] Parr | Density-Functional Theory of Atoms and Molecules[END_REF] The ground and first excited state PESs and their electron densities were also computed at DFT and TD-DFT 69,70 level of theory.

The D CT analysis along the reaction coordinate, suggested that this index provides a fairy picture of the degree of adiabaticity for the examined cases. A very nice agreement with the conclusions reported by Harshan and co-workers was also found.

Methods

The proton exchange reaction of the mMphenoxyl/phenol system was investigated at DFT and TD-DFT levels of theory. [START_REF] Parr | Density-Functional Theory of Atoms and Molecules[END_REF][START_REF] Stratmann | [END_REF][70] More in details, the M06-2X 71 /6-311+G(d,p) level was employed to locate the ground state transition state (TS) for both the open and the stacked conformations. The TS nature was confirmed by a subsequent frequency calculations (see Figure S1 for the displacement vectors along the normal mode with immaginary frequency).

A completely planar TS could not be optimized for the open arrangement, therefore the coplanarity between the phenyl rings was enforced constraining the CC OM -C O C dihedral angle to be planar (C OM and C O are the carbon atoms bound to O M and O, respectively, Figure 1).

The reaction path was, then, followed by integrating the intrinsic reaction coordinate (IRC) at the M06-2X/6-311+G(d,p) level, employing the Hessian-based Predictor Corrector integrator. [72][START_REF] Dykstra | Theory and Applications of Computational Chemistry: the first forty years[END_REF][START_REF] Hratchian | [END_REF] On the IRC structures single point calculations at CAM-B3LYP 75 /6-311+G(d,p)

and TD-CAM-B3LYP/6-311+G(d,p) level of theory were performed to define the ground and first excited energy profiles. The M06-2X functional was employed for the geometry optimizations because it includes dispersion effects, whereas excited state properties were evaluated using the CAM-B3LYP functional that provides a robust and reliable description of the charge transfer excitation. 76 Cartesian non adiabatic coupling vectors between the ground and first excited state were analitically computed according to the Refs. 38,40 as implemented in the Gaussian package. [START_REF] Frisch | Gaussian16 Revision A.03[END_REF] The D CT index was computed using relaxed densities.

All calculations were performed using Gaussian 16 suite programs. 3

Results and Discussion

The structural and energetic features of the PCET reactions are discussed in the next section, while the electron density based analysis is disclosed in section 3.2. The energy differences between the ground and first excited state surfaces are reported in Table S2 for reactants, transition states and products. In the case of the open geometry, the two surfaces are very close in energy near the transition state region (the vertical spacing at the TS is about 5 kcal/mol). On the other hand, they are well separated in the stacked conformation, with an energy difference of about 27 kcal/mol at the TS. These results obtained at CAM-B3LYP 75 and TD-CAM-B3LYP levels of theory on the mMphenoxylphenol system are in very nice agreement with both the CASSCF and CDFT-CI calculations reported by Harshan and co-workers on the phenoxyl-phenol couple. 35 Moreover, the CAM-B3LYP energy profiles reproduce the different regimes of adiabaticity recognized for the two geometries. Indeed, an electronically non-adiabatic process occurs in the case of the open conformation, while an electronically adiabatic reaction is observed for the stacked one.

Structural and energetic features

Electron density based analysis

The different electronical adiabaticity degree of the open and stacked conformations is now analyzed and described by means of the D CT electron density based index. Beside the absolute values of D CT , which are dependent on the distance between the two aromatic rings, is very interesting to analyze its derivatives with respect to the reaction coordinate.

The computed D CT values and its derivatives along the IRC path are reported for the two structural arrangements (open and stacked) in Figure 3a and 3b, respectively. The variation of the electronic density between the ground and first excited states is shown for reactants, transition states and products in the lower panel of Figure 3.

As it can be inferred from Figure 3a,D CT is minimum at the TS and its derivative is, in turn, zero in this point. This applies for the open and the stacked conformation as well.

Moreover, the open arrangement shows larger derivatives in proximity of the transition state region when compared to the stacked one (Figure 3b). As matter of fact, in the former case a very large electron density redistribution between the ground and first excited states occurs in the reactant and product zones, as it can be observed in the lower panel of Figure 3. This electronic rearrangement remains constant in these regions while it is very low at the transition state, where the two hydroxyl groups are mainly involved. More closely, a depletion of the electron density is observed on the carbon atoms, while it increases on both the phenolic oxygen (Figure 3). The ground-first excited state electronical reorganization can be quantified by the D CT index, which assumes constant values of about 3.6 Å in both the reactant and product zones, while a value of 0.14 Å is computed at the TS (see Table S2). As a result, D CT derivatives with respect to the reaction coordinate has its maximum variation in the TS region, where the non-adiabatic coupling is larger, while it is constant in the other zones of the PES.

On the other hand, a different behavior for the stacked dimer can be inferred from Figure 3a. Starting from a value of about 0.7 Å in the reactant region, D CT gradually increases, reaching a value of about 1 Å before the transition state, where it softly decreases at 0.45 Å. From here, this index starts to rise up to a value of about 1 Å to slowly decrease in the product zone, where it reaches again the value of 0.7 Å.

In turn the D CT derivative slightly grows up when moving from the reactant, while a steeper variation is observed at the transition state. From the density difference plot associated to the ground and first excited state along the reaction coordinate (see Figure S2 in SI)

it is clear that the ground-first excited state electronic reorganization increases when going from the reactant to the transition state region. More in detail, the electronic reorganization mainly affects the phenoxyl ring on the reactant structure (see lower panel of Figure 3 and Otherwise, when the reaction is electronically adiabatic, as in the case of the stacked conformation, the electronic coupling is lower at the transition state, and in turn the D CT derivatives is also smaller in this region.

Finally, the adiabaticity degree of the considered PCET reactions was examined analyzing the first-order non-adiabatic coupling vectors between the ground and the first excited states, and the trend of the root mean square (RMS) of NAC vectors and D CT along the reaction coordinate was compared.

The root mean square of the cartesian NAC vectors is reported in Figure 4a, while the derivatives of the NAC RMS respect to the reaction coordinate are shown in Figure 4b.

The dimensions of the NAC vectors are [length] -1 while the D CT index is simply a distance.

For this reason, we compared the root mean square of the NAC vectors with the inverse of D CT , 1/D CT , in Figure 4.

The inspection of Figure 4 shows a very different behavior for the two conformations, in agreement with the CASSCF results reported by Harshan and co-workers. 35 By comparing the variations of the NAC RMS and 1/D CT along the reaction path is clear that they strictly follow the same trend. Moreover, their derivatives along the reaction coordinate are also in very nice agreement (Figure 4b). These outcomes support and encourage This rather cheap and smart analysis provides a very promising diagnostic tool to get new insights on complex PCET mechanisms for real systems at low computational cost.

77

 77

Figure 1 :

 1 Figure 1: Schematic view and labelling of open and stacked conformations considered for reactants of the mMphenoxyl/phenol PCET reaction.

  Before describing in detail the electron density based analysis, we briefly illustrate structural and energetic features of the mMphenoxyl/phenol system, in both the open and stacked conformations. The main structural parameters of reactants, transition states and products are provided in TableS1of the Supplementary Information (SI). The open geometry shows a planar arrangement of the two aromatic rings, with an O-H-O M angle of 180 • at the TS structure, as illustrated in Figure1. On the other hand, the stacked TS is characterized by a O-H-O M angle of 166 • . The ground and first excited PESs obtained by integrating the intrinsic reaction coordinate (IRC) in the ground state are reported in Figure2for the open and stacked geometries. In both cases the proton exchange reaction proceeds with a barrier: about 13 kcal/mol is the barrier computed for the open form, while a lower value (about 8 kcal/mol) is found for the stacked conformation. Reactants and products are almost isoenergetic, although the proton is bound to the phenolic ring in a slightly more stable way.

Figure 2 :

 2 Figure 2: Ground (green line) and first excited (blue line) energy profiles (kcal/mol) along the IRC for the open (left panel) and stacked (right panel) conformations.

Figure 3 :

 3 Figure 3: Upper panel: D CT ( Å) and its derivatives with respect to the reaction coordinate along the IRC path are reported in panels (a) and (b) respectively. Black and red lines refer respectively to the open and the stacked conformations. Lower panel: difference density plots (positive and negative variation of the density is represented in green and blue, respectively) between the ground and first excited state computed for reactans, transitions states and products of the open and stacked geometries.

Figure

  Figure S2). Moving along the reaction coordinate also the other ring gets involved in the electron density redistribution. Inspection of the molecular orbitals involved in the excitation reveals a charge transfer transition from one aromatic ring to the other when moving along the IRC path. This leads, in turn, to the observed D CT growth and supports also the hypothesis of a HAT mechanism with a small amount of EPT character proposed for the stacked geometry by Harshan and coworkers.35 A specular behavior is observed when going from the transition state to the product, while at the TS the electronic rearrangement is minimum and the D CT falls down. On the other hand, in the stacked geometry the two aromatic rings are both involved in the electronic reorganization, due to their face to face arrangement.These results suggest that the D CT index and its variation along the reaction path are able to capture the different electronic adiabaticity degree shown by concerted PCET reactions.

  When an electronically non-adiabatic process occurs (open geometry), ground and excited state are strongly coupled, and the root mean square of the NAC vectors assumes large values in the transition state region. On the other hand, the coupling drastically decreases for the electronically adiabatic proton exchange reaction of the stacked arrangement, and low values of the NAC RMS are observed. Both the conformations show weakly coupled electronic states in the reactant and product zone.

Figure 4 : 4 Conclusions

 44 Figure 4: Panel (a) shows the root mean square of the cartesian non-adiabatic coupling vectors (bohr -1 ) between the ground and first excited electronic states. The derivatives of the NAC root mean square with respect to the reaction coordinate along the IRC path is reported in panel (b). The inverse of D CT , 1/D CT ( Å-1 ), and its derivatives with respect to the reaction coordinate along the IRC path are reported as inset in panel (a) and (b) respectively. Black and red lines refer respectively to the open and the stacked arrangements.

Acknowledgments U.R., M.G.C., F.C., F.P. and N.R. gratefully acknowledge funds from Regione Campania (L.R. 5, BRC3179) and Gaussian, Inc. (Wallingford, CT). I.C. acknowledges funding from the H2020 European Research Council (ERC CoG STRIGES n 648558).