Marie-Claude Jolly-Saad

Jean François Pastre

Sébastien Nomade

Séverine Fauquette

The Zanclean palaeofloras around the Mont-Dore strato-volcano: A window into upper Neogene vegetation and environments in the Massif Central (Puy de Dome, France)

Keywords:

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

Introduction

Tertiary volcanogenic deposits of the Puy de Dôme (Massif central, France) have been explored for a long time and are a place of interest to palaeobotanists because of the abundance and well-preserved micro-and macrofossil plants. The earliest written record of Mont-Dore region fossil flora dates back to the end of the 19 th century [START_REF] Boulay | Flore pliocène du Mont Dore (Puy-de-Dôme)[END_REF]. This work summarized all the discoveries that pointed vegetated Tertiary landscapes from the French Massif central (Varennes, Perrier, Niac, Pas de la Mougudo, Ceyssac, Saint-Vincent, Joursac, La Gratade and Pont de Chocol).

During the first half of the 20 th century, [START_REF] Grangeon | Découverte d'un nouveau gisement de plantes d'âge Villafranchien, dans les cinérites du lac Chambon (Puy-de-Dôme)[END_REF] reported some fossil plants in Chambon Lake outcrops and detailed the taxonomic studies; the flora which should belong to this locality contains a small assemblage of 9 species: Bambusa cambonensis N. Boul. (Arundinaria metake Sieb.), Alnus glutinosa Gaertn., Fagus silvatica L., Fagus pliocenica Sap., Populus tremula L., Ulmus effusa Willd., Acer pseudocampestre Ung., Acer Nicolai N. Boul. (affinity A. opulifolium Willd.), Carpinus betulus L., and one family, Poaceae. Later, this study was completed by [START_REF] Elhai | Flore pollinique des cinérites du lac Chambon (Puy-de-Dôme)[END_REF] with a microfossil analysis. La Gratade and Pont de Chocol were studied by [START_REF] Grangeon | Découverte d'un nouveau gisement de plantes fossiles dans les cinérites et argiles à Diatomées de la Gratade, Massif du Mont-Dore (Puy-de-Dôme)[END_REF]; most of the fossil flora samples come from La Gratade outcrop, with 23 taxa identified as Torreya nucifera Sieb. et Zucc., Potamogeton quinquenervis N. Boul., Salix sp., Populus tremula L., Populus alba L., Betula macrophylla Heer, Alnus glutinosa Gaertn., Carpinus orientalis Mill. (fruits and leave), Carpinus, Corylus avellana L., Fagus sylvatica Sap., Castanea vesca Gaertn., Quercus hispanica Rer., Quercus sp., Zelkova ungeri Kovats, (crenata and acuminata), Ulmus sp., Juglans regia L., Carya sp., Acer laetum C.A. Mey, Acer sp., and Tilia sp. Then a detailed microfloral study was produced by [START_REF] Elhai | Flore pollinique des argiles ligniteuses du pont de Chocol, vallée des Roches Tuilière et Sanadoire[END_REF] from the lignite clay from Pont de Chocol. The microflora of La Gratade has not been previously studied. Today, all these sites are widely scattered in the literature, most of them are not updated and not well constrained by radiometric dates. The more recent and detailed updates concern only the Miocene plant assemblage of Murat in the French Massif Central [START_REF] Roiron | La macroflore d'âge miocène supérieur des diatomites de Murat (Cantal, France). Implications paléoclimatiques[END_REF][START_REF] Legrand | Inventaire de la macroflore du Miocène supérieur de la diatomite de Murat (Cantal, Massif central, France). Inventory of the Upper Miocene macroflora from the diatomite of Murat (Cantal, Massif central, France)[END_REF][START_REF] Legrand | Complément à l'inventaire de la macroflore du Miocène Supérieur de la diatomite de Murat (Cantal, Massif central, France)[END_REF].

Recent harvests of macroflora were gathered by J.F. Pastre from 3 localities: Chambon Lake (LCh), La Gratade (LG) and Pont de Chocol (PTC), combined with new radiometric datations, bring us to complete and synthesize the results of former inventories. Despite the local character of such fossil records and the inequitable representation of ancient vegetation, they provide invaluable information about the history and distribution of the floristic elements and offer a great potential for identifying past environmental elements, making a contribution to a new picture of the palaeofloristic biodiversity in this area. The results of the palaeoenvironmental investigations are partly comparable but also complete earlier investigations with the description of 28 new taxa in Chambon Lake outcrop, as well as two species in La Gratade and Pont Chocol. The aims of the present study were: (i) to document, describe and determine the three new collections of plant fossils of Chambon Lake, Pont Chocol and La Gratade outcrops which were recently collected by one of us (JFP), (ii) to compare these fossil plant collections to the one recovered and published by [START_REF] Grangeon | Contribution à l'étude de la paléontologie végétale du massif du Coiron (Sud-Est du Massif Central Français)[END_REF]Rudel (1959, 1962) from the same area, (iii) to compare the Zanclean flora of these aeras to other selected Pliocene floras in the Massif central region and from Western Europa, and (iv) to infer the palaeoecology of the three area during the early Pliocene, using evidence from macrofossils and published data from dispersed pollen and spores.

Location and geological settings

The three fossiliferous outcrops studied in this article are all located in the French Massif central, in the periphery of the Mont-Dore strato-volcano (Fig. 1). They were deposited in lacustrine environments reworking contemporaneous pyroclastites and giving tuffites deposits.

The fossiliferous outcrop of the Chambon Lake (Altitude 866 m asl) is situated on the right bank of the Couze Chambon river downstreams from the Chambon Lake and to the West of the strombolian cone of the Tartaret (45.575° N, 2.929° E; Fig. 1). It was discovered in 1985 in trenches of ca. 1 m-depth dig for the electrification of a campground. The foliar imprints are contained in nodules of tuffites up to 30 cm in diameter. These nodules are higgledy-piggledy incorporated in a tufaceous silty matrix which also contains some blocks of bedded sandy tuffites with volcanic minerals (alkali feldspars, biotite...). These facies characterize a secondary outcrop due to a major landslide localized in the N-E part of the lake and giving the hills situated to the east and bordering the lake. This landslide originated in the Dent du Marais cirque situated to the North and bordered by the diatreme of basaltic breccia of the Saut de la Pucelle [START_REF] Besson | La Dent du Marais et la structure de Farges : éruptions de type maar-diatrème sur les marges orientales des Monts-Dore[END_REF]. If this landslide, which is not covered by the scoriae of the Tartaret, is probably Holocene in age, the material embedded in this landslide is much older. Alkali-feldspars found in the sandy tuffites of the outcrop and attributed to a contemporaneous pyroclastic activity are used hereafter to infer the age of the foliar imprints thanks to 40 Ar/ 39 Ar dating method. If this outcrop offers a particular context due to its nodules, it is probably contemporaneous of the cinerites of the lake Chambon, which outcrop sporadically on the northern shore of the lake and in the hills situated near the parking of the beach [START_REF] Besson | Les formations volcaniques du versant oriental du Massif du Mont-Dore (Massif central français), feuille Veyre-Monton 5-6[END_REF].

The fossiliferous outcrops of the Pont de Chocol and La Gratade are situated near the hamlet of La Gratade in the valley of the brook of Fontsalade, ca. 3 km to the South of the town of Rochefort-Montagne. The outcrop of Pont de Chocol (961 m asl) is located in the bed of the stream of Fontsalade, 50 m downstream the bridge on the path of the hamlet of Chez Chocol (45.656° N, 2.808° E; Fig. 2). It corresponds to bedded grey tuffites rich in foliar imprints. Other tuffites can be found in some places on the left bank slope where they are found associated with weathered trachytic pumices beds as well as small blocks of trachyte.

The outcrop of La Gratade (941 m asl) belongs to the same sedimentary unit. It is situated at about the half height of a small ravine tributary of the brook of Fontsalade, ca. 100 m to the NW of the mill of La Gratade (45.660° N, 2.806° E; Fig. 2). It is made of white to pale greybedded tuffites outcroping on ca. 2 m high that we also samples for 40 Ar/ 39 Ar dating purpose.

The granitic basement outcrops in the valley to the South and to the North of the study area. The position of the tuffites, which are several tens of metres thick, is anachronistic. It is very probable that they correspond to a lacustrine infilling of a maar crater opened in the basement. The first formation of the Mont-Dore sensu stricto which covers theses tuffites corresponds to the "Grande Nappe" (= ignimbrite) of rhyolitic fibrous pumices which can be seen along the road D80, north from La Gratade and which is dated at 2.77 ± 0.07 Ma [START_REF] Nomade | New 40 Ar/ 39 Ar constraints for the « Grande Nappe », the largest rhyolitic eruption from the Mont-Dore Massif (French Massif central)[END_REF].

Material and methods

Macroflora

Plant fragments were recovered by splitting clay containing the impression/compression fossils. Minimal preparation with fine needles was performed to fully expose the plant organs when needed. Cuticles are often present but were not studied here. The images were processed and measurements obtained using Nikon binocular loupe and Canon Elements. The taxonomic determinations established in this study are based exclusively on the gross morphological characters of the gathered specimens, whereas special attention is paid to the morphological details of lamina venation and margin outline of the leaves. The morphological terminology is based on the descriptions of [START_REF] Hickey | Classification of the architecture of dicotyledonous leaves[END_REF], [START_REF] Dilcher | Approaches to the identification of angiosperm leaf remains[END_REF][START_REF]Manual of leaf architecturemorphological description and categorization of dicotyledonous and net-veined monocotyledonous angiosperms[END_REF]. Two major treatments serve as a basis for the data presented here: besides several preliminary papers consulted [START_REF] Grangeon | Contribution à l'étude de la paléontologie végétale du massif du Coiron (Sud-Est du Massif Central Français)[END_REF][START_REF] Roiron | La macroflore d'âge miocène supérieur des diatomites de Murat (Cantal, France). Implications paléoclimatiques[END_REF][START_REF] Legrand | Inventaire de la macroflore du Miocène supérieur de la diatomite de Murat (Cantal, Massif central, France). Inventory of the Upper Miocene macroflora from the diatomite of Murat (Cantal, Massif central, France)[END_REF][START_REF] Legrand | Complément à l'inventaire de la macroflore du Miocène Supérieur de la diatomite de Murat (Cantal, Massif central, France)[END_REF], foliage are compared with and herbarium specimens and living material. The analyses are based on detailed comparisons of individual elements with the living analogues according to detailed morphological and anatomical studies. For palaeoenvironmental reconstruction, the Nearest Living Relative method [START_REF] Mosbrugger | The coexistence approach -A method for quantitative reconstructions of Tertiary terrestrial palaeoclimate data using plant fossils[END_REF] assumes that the fossil taxon had a climate tolerance and habitat similar to that of its nearest living relative.

All the studied new macrofossil specimens are housed at the LGP (CNRS, Meudon). Ancient collections were not revised in this work.

Microflora

The three samples were first treated with 37% hydrochloric acid (HCl) to dissolve the carbonate, and after washing, the residue was treated with 52-62% hydrofluoric acid to dissolve silica. The residue obtained by chemical dissolution of the mineral sample was further processed with KOH to extract the organic material from the mineral. The organic residue material was washed and centrifuged at 3600 rpm for 10 min. The residue from the casting ring was applied to a glass slide, dried, and after the addition of glycerine gelatine, it was covered with a cover slip. A transmitted light microscope (Leica) with ×400 and ×1000 magnifications (without oil immersion) was used for identification, and ×400 was used for the counting of pollen and spores. Pollen and spore identification was accomplished using Henri Elhaï reference database. Other published keys and pollen atlases were also used as well as atlases of recent pollen [START_REF] Reille | Pollen et spores d'Europe et d'Afrique du nord[END_REF][START_REF] Reille | Pollen et spores d'Europe et d'Afrique du nord[END_REF].

Ar/Ar sampling and analytical method

About 500 g of the pale grey-bedded tuffites (La Gratade hereafter) and of the sandy tuffites (Mdo hereafter) from La Gratade and Chambon Lake sites were sampled. After wet sieving, we kept the 600 µm to 1 mm fraction. From this fraction we picked 20 pristine Kfeldspars crystals for each sample. These crystals were irradiated 1:30 hours in the Β1 tube of the Osiris reactor (Irradiation 35; CEA Saclay Center, France). After irradiation, 9 and 10 crystals for Mdo and Gratade samples were individually loaded in a copper sample holder.

The sample holder was then put into a double vacuum Cleartran window. Each crystal was then fused using a Synrad CO2 laser at 15% of nominal power (ca. 25 W). The extracted gas was purified for 10 min by two hot GP 110 and two GP 10 getters (ZrAl). Argon isotopes ( 36 Ar, 37 Ar, 38 Ar, 39 Ar and 40 Ar) were analysed by mass spectrometry using a VG5400 equipped with an electron multiplier Balzers 217 SEV SEN coupled to an ion counter.

Neutron fluence J for each sample was calculated using co-irradiated Alder Creek Sanidine (ACs-2 hereafter) standard with an age of 1.193 Ma [START_REF] Nomade | Alder Creek Sanidine (ACs-2): A Quaternary 40 Ar/ 39 Ar standard[END_REF] and the total decay constant of Steiger and Jäger (1977). J-values computed from standard grains is 0.00067860 ± 0.00000339 and 0.00068750 ± 0.00000344 for Mdo and La Gratade, repectively. The 40 Ar/ 39 Ar age is a not a direct method and relies on the precise measurements of the 39 Ar produced in the nuclear reactor; for this, we used a natural K-feldspars standard (i.e., crystals extracted from rock of known age) that was irradiated under the same conditions than the sample of interest. The J-value was therefore the weighted mean and corresponding standard deviation based on the measurements of 3 to 6 standard crystals (in our case, Alder creek sanidine). This J flux factor was used to quantify the Neutron induced 39 Ar made from 39 K in the nuclear reactor that can varied as a fonction of the irradiation duration and position of the sample with respect to the irradiation schuttle and cannot be predicted from previous irradiation.

Mass discrimination was estimated by analysis of Air pipette throughout the analytical period, and was relative to a 40 Ar/ 36 Ar ratio of 298.56 [START_REF] Lee | A redetermination of the isotopic abundances of atmospheric Ar[END_REF]. Procedural blank measurements were computed after every two or three sample crystals, depending on the beam measured. For 10 min static blank, typical backgrounds are ca. 2.0-3.0×10 -17 and 5.0-6.0×10 -19 mol for 40 Ar and 36 Ar, respectively. The precision and accuracy of the mass discrimination correction was monitored by weekly measurements of air argon of various beam sizes.

Results

40 Ar/ 39 Ar datations

Full analytical details for individual crystal are given in Tables S1 andS2 (Appendix A). Results are presented as probability diagrams in Fig. 3. In this figure, individual crystal age uncertainties are given at 1σ-level and the weighted mean age uncertainties are quoted at 2σ-level, including J uncertainties. In both sample we found only one main population of crystal as shown by the unimodal probability diragrams (Fig. 3). Despite a slight excess scattered, meaningful weighted mean ages of 3.94 ± 0.04 Ma (MSWD = 1.9) and 4.46 ± 0.05 Ma (MSWD = 2.0) can be calculated for La Gratade and Mdo, respectively. It is worth noting that three crystals from Mdo displayed very low precision (Fig. 3) due to their low radiogenic content (i.e., 38 to 78%; see Appendix A for details). Removing these crystals from the weighted mean age does not change the weighted mean age reported above for this sample. These two ages are interpreted as the age of eruption of the volcanic material found in the two studied sites.

Floristic composition

Pollen analysis and palynofacies

All studied samples yielded well preserved sporomorphs suitable for detailed palynological analysis in Table 1, showing that the floras are in agreement with the 40 Ar/ 39 Ar datations. In detail, pollen grains of gymnosperms and angiosperms were very frequent in all samples. Fern and cryptogam spores were rare. In all, 437 pollen grains (of which 3% are herbaceous taxa) were identified at Chambon Lake, 414 pollen grains (of which 6% herbaceous) at Pont de Chocol, and 359 pollen grains (of which 11% herbaceous) at La Gratade, excluding the Fungus spores and the Algae. The number of terrestrial spermatophyte taxa found in each level varies from 30 to 40 species. The list of taxa is fairly similar in the four sites as well as between samples from each site (Table 1).

The vegetation indicates a forest physiognomy in the 3 different deposits, with Arboreal Pollen (AP) ranging from 88 to 97%. Poaceae are rare (0.7-1.7%); other herbaceous species are represented by few pollens of Cyperaceae, Apiaceae, Rosaceae, Nuphar, Caryophyllaceae, Amaranthaceae, Leguminosae, Papaveraceae, Helianthemum, Lamiaceae, and Ranunculaceae.

The maximum diversity is reached in Pont de Chocol deposits. Ericaceae frequency increase is noted in La Gratade and Pont de Chocol outcrops.

In the different palynological samples, Pinus diploxylon type is always dominant with pollen values between 25 and 34% while Cathaya is recorded with only 1-1.6% and Pinus haploxylon with values between 0.8 and 2.3%. Pinus are mainly accompanied by Picea (5.7-14.5%); higher frequencies are recorded in the Chambon Lake. Picea pollen percentages lower than 5% in extant vegetation are considered as an indication of regional presence [START_REF] Hicks | Present and past pollen records of Lapland forests[END_REF][START_REF] Huntley | An atlas of past and present pollen maps for Europe: 0-13000 years ago[END_REF]; two different Tsuga species recorded together between 3 and 7.2%, Abies 4.4-10% and Sciadopitys 6% in LCh, 1% in La Gratade, but the taxa is absent from Pont de Chocol.

Pollen grains of Cupressaceae (including "Taxodioid" pollen types) are represented with few pollens, respectively 0.7-2.3% and 0.4-4.6%, with lower frequencies recorded in La Gratade and Pont de Chocol aeras. Angiosperms are more diversified. Deciduous oaks were always present with low values of 3.6% and peaking at 6% in Pont de Chocol and 12% in La Gratade. Carya percentages are between 2.1 and 5%. Others arboreal taxa are Fagus with range frequencies of 1-5.8%, Pterocarya (1.7-2.4%), Juglans (1-3%), and Ulmus-Zelkova (1.6-2.6%). Nyssa is recorded with only one pollen in LCh and PTC. The absence of Engelhardia (Sapotaceae) is worth to be mentioned. Non pollen palynomorphs (NPP) were scarced; among them the freshwater Botryococcaceae algae Botryococcus braunii Kützing, 1849 is a common element in the different deposits, with 6-13.4% in Chambon Lake and Pont de Chocol and a lower representation in La Gratade (1.2 %). Several epiphyllous fungi are observed.

Several remarks can be expressed. The forests found in both successions are always relatively dense, with a thick forest canopy. Herbs which have entomophillous pollen dissemination are practically absent in the three outcrops (4-12%); the list of present-day taxa extinct in the region is the same or very similar, showing the presence of many trees living today in warm-temperate Asia, North America and Caucasia associated with still extant species. Very similar vegetations are shown at both sites with, however, sigthly differences in the frequencies of the different taxa. There is an indication for a pollen rain coming from several vegetational belts, as it results from the mixing of montane, mesophilous and hygrophilous elements. A homogenous forest formation characterizes the three outcrops.

Sciadopitys is not recorded in La Gratade deposit. Sporomorph assemblages show the apparent predominance of "arctotertiary" (including warm temperate and temperate) and cosmopolitan floristic elements. The palaeotropical and palaeotropical/warm temperate taxa were represented by a single pollen of Nyssa both in La Gratade and Pont de Chocol outcrops.

Concerning the presence of some arboreal taxa, few observations are useful to interpret the different records and to locate them in the chronological scale. First of all, we note the permanence of many mesothermic taxa such as Carya, Juglans, Pterocarya, Liquidambar, and Zelkova. Liquidambar disappeared from The Netherlands at the end of the Reuverian or at the beginning of the Praetiglian in Koningsbosch [START_REF] Zagwijn | Aspects of the Pliocene and early Pleistocene[END_REF] and in Normandie [START_REF] Clet-Pellerin | Le Plio-Pléistocène en Normandie : apports de la palynologie[END_REF]; it is absent in altitude but remains present in south Europa [START_REF] Bertini | Messinian to Zanclean vegetation and climate of Northern and Central Italy[END_REF][START_REF] Suc | Reconstruction of Mediterranean flora, vegetation and climate for the last 23 million years based on an extensive pollen dataset[END_REF], Engelhardtia disappeared at the end of the Miocene in The Netherlands [START_REF] Van Der Hammen | The floral record of the late Cenozoic of Europe[END_REF]. "Taxodioid" Cupressaceae disappeared in Netherlands at 2.5 Ma [START_REF] Zagwijn | The Pliocene-Pleistocene boundary in western and southern Europe[END_REF], during the Praetiglian in Normandy [START_REF] Clet-Pellerin | Le Plio-Pléistocène en Normandie : apports de la palynologie[END_REF], at 3 Ma in Languedoc [START_REF] Suc | Contribution à la connaissance du Pliocène et du Pléistocène inférieur des régions méditerranéennes d'Europe occidentale par l'analyse palynologique des dépôts du Languedoc-Roussillon (sud de la France et de la Catalogne[END_REF], and at 1.6 Ma in North Italy [START_REF] Bertini | Messinian to Zanclean vegetation and climate of Northern and Central Italy[END_REF][START_REF] Suc | Reconstruction of Mediterranean flora, vegetation and climate for the last 23 million years based on an extensive pollen dataset[END_REF].

The optical characterization of the micro-organic matter (palynofacies) in Table 1 highlights different environments: in Chambon Lake, two classes of organic matter are recognized. The first one shows a high percentage of particulate organic matter (POM = 85%) composed of diversified terrestrial plant fragments (mainly yellow-brown wood and cuticles, palynomorphs dominated by pollen contribute only to 10% of the POM total). It also contains opaque phytoclasts (up to 5% of the total of the organic matter). The second class is the amorphous organic matter with no recognizable elements (AOM = 15%).

La Gratade organic matter content is composed with POM (80%) dominated by sporomorphs and AOM which contributes for 20%. In Pont de Chocol, 3 classes are described: POM (50%), AOM (45%) and carbonized particules (COM = 5%).

Zooclasts and fungus spores are absent from the different palynofacies records.

The Recent collectings at three sites, Chambon Lake, La Gratade, Pont de Chocol in the Mont-Dore (Puy de Dôme, France) has resulted in the recovery of numerous plant specimens (Table 2;. The palaeobotanical dataset is mainly composed of foliages (impressions, sporadically carbonized compressions) and less frequently of fruits and seeds.

The plant fossil material of Chambon Lake is abundant and with an excellent quality of preservation. Most of the leaves are entire; they are preserved in a fine, regularly laminated sediment, while in other sites the deposited material is quite damaged and is characterized by numerous fragmented and small sized samples.

Most of the recorded angiosperm genera are present in the pollen analysis. In previous macroflora researches, only 9 taxa have been described from the Lake Chambon outcrops: Bambusa cambonensis, Alnus glutinosa Gaertn., Fagus silvatica L., Fagus pliocenica Sap., Populus tremula L., Ulmus effusa Willd., Acer pseudocampestre Ung., Acer Nicolai N.B. (aff.

A. opulifolium Willd.), Carpinus betulus L., and Poaceae [START_REF] Marty | Florule miocène et géologie des environs de Lugarde (Cantal)[END_REF][START_REF] Grangeon | Découverte d'un nouveau gisement de plantes d'âge Villafranchien, dans les cinérites du lac Chambon (Puy-de-Dôme)[END_REF]. With the new collection described in this paper the former floristic record is extended by 23 additional taxa. Angiosperms, which apparently prevail in macroflora, are documented either by numerous leaves and leaflets or less frequently by fruits and seeds or needles (Table 2). Fagaceae are by far the most diversified and the most abundant in the Chambon Lake assemblage. It comprises at least one more beech species: F. grandifolia type (Fig. 5(A,B The Chambon Lake macroflora assemblage, apart from the great number of specimens, is considered to be fairly diverse with a well preserved macroflora comprising 33 taxa identified at this stage (former study and this paper; Table 2) Herbaceous remains, excluding Poaceae, are totally absent from the macroremain assemblages of the three deposits. Ferns are recorded in Pont de Chocol with one sample (Pteridium aquilinum (L.) Kuhn). Buxaceae and gymnosperms are lacking in the small macrofloral collection from La Gratade and Pont de Chocol, but this absence does not mean that they were not present in the palaeoflora.

Vegetation reconstruction

There is no apparent correspondance between the macro-and microfossil records. (Elhaï andRudel, 1961, 1965;[START_REF] Elhai | Découverte d'un nouveau gisement de plantes fossiles au pont de Cladel, près de Rochefort-Montagne (Puy-de-Dôme)[END_REF] this study) is difficult to interpret, as suggested by [START_REF] Favre | A contribution to deciphering the meaning of AP/NAP with respect to vegetation cover[END_REF] and not well aligned with the presence of some heliophilious taxa (Leguminosae) which are recorded in the different outcrops.

Four main vegetation types well known in the Neogene are identified. The aquatic vegetation type is absent from the macroflora; diversified aquatic plants (Potamogeton, Sparganium, Nuphar) and abundant Botryoccocus records point to the presence of a waterbody with stagnant or slowly moving water (Guy-Ohlson, 1992) according to palynological analysis. They are only recorded with high frequencies in the two deposits of Chambon Lake and Pont de Chocol. Only few taxa represent the riparian azonal vegetation with Alnus sp., Alnus glutinosa, Alnus stenophylla, Betula macrophylla, Betula subpubescens, Betula sp., Salix sp., Fraxinus sp., and Acer laetum. Pollen and macroremains are encountered in all deposits. The occurrence of Sciadopitys pollen may indicate a swampy vegetation as found in the Neogene of Bresse where for the first time in Western Europe macroremains support a different ecology than extant species [START_REF] Philippe | A palm-tree and Sciadopitys swamp-forest from the Neogene of Bresse (Eastern France)[END_REF]. Possibly, Sciadopytis may underline a moist broad similar to that occupied by the extant species S. verticillata, an endemic of Southern Japanese mountains under leaved forest, temperate maritime climate with high rainfall and moisture. Sciadopitys lives there under more severe thermal conditions, i.e., it withstands three to six months of cold weather (mean temperature less than 15°C) [START_REF] Willard | Palynological record from the North Atlantic Region at 3 Ma: a vegetational response to a period of global warmth[END_REF].

Most of the elements dominating the assemblage (deciduous Fagaceae, Carpinus, etc.) belong to the broad-leaved forests outside swampy and flooded habitats and covered large parts of the studied aeras. Diversified Fagus species dominated the flora in the area of Chambon with a clear predominance of Fagus sylvatica while the deciduous oaks and especially Quercus roburoides and Quercus hispanica were predominating locally in the forest of PTC and LG. Some, more likely mesophytic woody elements are better represented, e.g., Zelkova, Acer spp., deciduous Juglandaceae, namely Carya, Pterocarya and Juglans may have entered both mesophytic and non-flooded areas together with Ulmus, Fraxinus and Salicaceae. Some shrubs (cf. Buxaceae) are relatively rare in the fossil macrofloras. In areas with poor substratum, the pine trees were probably forming pure stands, although the high amount of pollen in the assemblage allows us to suppose that this element was spreading also on sites with higher soil fertility forming a mixed oak-beech-pine forest and belonged to mesophytic representatives of mixed coniferous and broad-leaved deciduous forests.

The last vegetation type constitutes the highland vegetation with the development of conifer forests with Picea, Abies and Tsuga.

Termophilous elements (megathermic and mega-mesothermic elements sensu [START_REF] Fauquette | Latitudinal climatic gradients in the Western European and Mediterranean regions from the Mid-Miocene (c. 15 Ma) to the Mid-Pliocene (c. 3.5 Ma) as quantified from pollen data[END_REF]) such as some Hamamelidaceae, Lauraceae, Sapotaceae, Symplocos and others, which may indicate the Mixed Mesophytic Forest of East Asia, are largely absent. As it is revealed by previous and present studies, the assemblage is floristically characterized by the apparent predominance of 'arctotertiaty' elements [START_REF] Mai | Palaeofloristic changes in Europe and the confirmation of the Arctotertiary -Palaeotropical geofloral concept[END_REF]Ferguson and Knoblock, 1998) whereas thermophilic elements are quite rare. The simultaneous occurrence of these units and elements attests to a relatively humid, warm-temperate climate. Modern analogues of the studied outcrops might be the rich mesophytic deciduous forest of the Eastern part of North America, Northern China and Caucasus.

Discussion

Pliocene plant assemblages in the French Massif central occur at localities variously dated and in various stage of exploration. Based on published fossil floras for most of them, it appears that most micro-and macroflora assemblages may be less stratigraphically controlled than assumed in several former studies, as demonstrated by [START_REF] Bellon | Datations absolues des laves d'Auvergne par la méthode K-Ar[END_REF] and [START_REF] Gibert | Données géochronologiques K-Ar sur quelques gisements à plantes du massif Volcanique néogène du Cantal (Massif central France)[END_REF] with the first radiometric controls for several Miocene outcrops in Cantal (Massif central, France).

Based on palynological data, the discussed floristic assemblages of the Chambon Lake, La Gratade and Pont de Chocol areas emphazise the presence of temperate deciduous elements mixed with abundant Pinaceae as typical for the Neogene of Europe. A "Villafranchian" age (3.58-1.7 Ma) was estimated for these three assemblages by comparison with the detailed climatic reconstructions established in The Netherlands by [START_REF] Zagwijn | Aspects of the Pliocene and early Pleistocene[END_REF] for the last 5 myr based on pollen analysis, which stimulated comparable palynological researches. The new 40 Ar/ 39 Ar dates obtained for Chambon Lake (4.46 ± 0.05 Ma) and La Gratade (3.94 ± 0.04 Ma) places the deposits in the Zanclean (= Brunssumian; 5.32-3.58 Ma).

The Zanclean is first subdivided into three climatic periods: warm (Brunssumian A from 5.33 to ca. 4.75 Ma, cooler (Brunssumian B from ca. 4.75 to ca. 4.00 Ma), and the warmest phase (Brunssumian C from ca. 4.00 to ca. 3.60 Ma) [START_REF] Suc | Plio-Pleistocene correlations between the northwestern Mediterranean region and northwestern Europe according to recent biostratigraphic and palaeoclimatic data[END_REF][START_REF] Zagwijn | Palynostratigraphie du Plio-Pléistocène d'Europe et de Méditerranée nord-occidentales : correlations chronostratigraphiques, histoire de la végétation et du climat[END_REF][START_REF] Suc | Zanclean (∼Brunssumian) to early Piacenzian (∼ early-middle Reuverian) climate from 4° to 54° north latitude (West Africa, West Europe and West Mediterranean areas)[END_REF]. Recently, new ages extrapolated from the climatostratigraphic interpretation of the DSDP site 380 pollen record were proposed for the Brunssumian A (5.45-4.08 Ma), the Brunssumian B (5.08-3.94 Ma), and the Brunssumian C (3.94-3.37 Ma) by [START_REF] Popescu | Pliocene and Lower Pleistocene vegetation and climate changes at the European scale: Long pollen records and climatostratigraphy[END_REF].

Pinus has an important place in the palynological records, whereas Abies, Picea, Sciadopitys and Tsuga are also important components of the vegetation. The deciduous component of the forest is diversified with Acer, Betula, Carpinus, Corylus, Fagus, Carya, Pterocarya, Juglans, Quercus, Tilia, Ulmus, and Zelkova, with high abundance of Sequoiatype (concurrent with abundant Taxodium-type) and regular occurrence of Symplocos and Sapotaceae [START_REF] Zagwijn | Aspects of the Pliocene and early Pleistocene[END_REF][START_REF] Roche | Geo-Phytostratigraphie et paléoenvironnements du Néogène de l'Entre-Sambre-et-Meuse et du Condoz (Belgique). Evolution paléoclimatique du subtropical humide au tempéré froid[END_REF]. These taxa are sometimes associated with several morphotypes (Tricolporopollenites edmundi, T. librarensis, T. megaexactus and T. microhenrici) which cannot be considered as valid taxa [START_REF] Suc | Reconstruction of Mediterranean flora, vegetation and climate for the last 23 million years based on an extensive pollen dataset[END_REF].

By comparing our data with micro-and macrofloras recognized in nearby regions with similar datations, it appears that the floristic composition of the lower Pliocene is not uniform. Some analogies might also be drawn with the record from the same altitudinal range in the upper Miocene lacustrine diatomite deposits from Murat (Massif central, Cantal) dated by the K/Ar method to 5.34 ± 0.3 Ma [START_REF] Roiron | La macroflore d'âge miocène supérieur des diatomites de Murat (Cantal, France). Implications paléoclimatiques[END_REF], corrected in Kovar-Eder et al., 2006) . integerrimum, A. sanctae-crucis, A. campestre, A. opulifolium, A. platanoides, A. tricuspidatum, H. helix, Ilex sp. (aff. Ilex cornuta) The microfossil analysis [START_REF] Durand | Le dépôt de la diatomite de Sainte-Reine (Cantal) débute au Pliocène supérieur et permet de déceler les traces du refroidissement prétiglien[END_REF] gives the following results: Pinus (59%), Picea (31%), Abies (1%), Cupressaceae, Tsuga, Carya, Pterocarya (total 3.5%), Sciadopitys, Sequoia, Taxodium, Nyssa (total 3%), Betula, Alnus, Quercus, Ulmus, Fagus (total 2%), and herbaceous plants (1%). The thermophilic elements such as "Taxodioid" Cupressaceae are scarce while mesophilic taxa are abundant as Alnus, Carpinus, Acer, Zelkova, deciduous Quercus, Carya, etc. Lauraceae are recorded by Legrand (2011) The Massif central results do not fit well with the general evolution of leaf and microfloras described from different Pliocene deposits of lowland from Western Europe. These different deposits include only a few common elements. In Northwestern Europe localities, high Sequoia content are recorded at Susteren [START_REF] Zagwijn | Aspects of the Pliocene and early Pleistocene[END_REF] and La Londe [START_REF] Clet-Pellerin | Le Plio-Pléistocène en Normandie : apports de la palynologie[END_REF]) but are absent from the Mont-Dore and Cantal records.

Pliocene of the so-called "Haguenau terrace" and lowlands of the Rhine River in Northern Alsace, France, has yielded numerous fossil plant remains [START_REF] Geissert | Le pliocene et le quaternaire au nord de Strasbourg. Note preliminaire sur la decouvert de nouveaux végétaux pliocènes a Auenheim (Bas-Rhin)[END_REF][START_REF] Geissert | Caractéristiques paléobotaniques du Pliocène et du Quaternaire en Basse-Alsace[END_REF]Geissert and Ménillet, 1979;[START_REF] Geissert | Die « Saugbaggerflora », eine Frucht-und Samenflora aus dem Grenzbereich Miozan-Pliozan von Sessenheim im Elsass (Frankreich)[END_REF]) dated from the early to late Pliocene without independent dating. Most of the elements dominating the Auenheim assemblage show high similarities with the Mont-Dore outcrops and decribed as Fagus, deciduous Quercus, Carpinus, Salicaceae, and Ulmaceae, belonging to the deciduous mesophytic to moist riparian broad-leaved forests outside swampy and flooded habitats. Only a few woody elements are swamp plants, Taxodium, Alnus, Nyssa, and Fraxinus. Mesophytic woody elements are better represented, e.g., Zelkova, Acer spp., Buxus, Carpinus, and Rosaceae. Carya and Pterocarya may have entered both mesophytic and riparian non-flooded areas together with Ulmus, Zelkova, Fraxinus, Gleditsia, and Salicaceae. Also all recorded gymnosperms except Taxodium, such as Ginkgo, Pinaceae, and Torreya belong to mesophytic representatives of mixed coniferous and broad-leaved deciduous forests in the Northern Hemisphere.

Thermophilous elements, such as evergreen Fagaceae, Lauraceae and others, which would indicate the Mixed Mesophytic Forest of East Asia, are largely absent. Exceptions are Cathaya and Craigia, two relict living genera, which deviate from their ancestors in their more thermophilous character [START_REF] Kvacek | The Pliocene leaf flora of Auenheim, Northern Alsace (France)[END_REF]Teodoris et al. 2009).

Conifers are represented by some more boreal elements, such as Picea and Abies, typical of the mixed coniferous and broad-leaved deciduous forests. This is a characteristic feature of most late Pliocene floras of Europe. Rare subtropical/evergreen elements occur; typical plants of the European Pliocene (Sassafras, Torreya) are also present, which may correspond to the altitudinal level of the Auenheim site.

Between North and South a thermic gradient is significant. The Northern Mediterranean area is characterized by dominance of arboreal pollen, suggesting a dense forest cover [START_REF] Suc | Zanclean (∼Brunssumian) to early Piacenzian (∼ early-middle Reuverian) climate from 4° to 54° north latitude (West Africa, West Europe and West Mediterranean areas)[END_REF] with the presence of mega-mesothermic elements (Cupressus, Cedrela, Laurus, Persea) from records in South localities of Cessenon (Zanclean) situated near Murviel (Hérault, France) [START_REF] Roiron | La macroflore d'âge miocène supérieur des diatomites de Murat (Cantal, France). Implications paléoclimatiques[END_REF], corrected in Kovar-Eder et al., 2006) and the early Pliocene plant assemblage of Pichegu (Gard, France) [START_REF] Roiron | La macroflore d'âge miocène supérieur des diatomites de Murat (Cantal, France). Implications paléoclimatiques[END_REF]Ambert, 1990, corrected in Kovar-Eder et al., 2006). The plant assemblage of Cessenon includes: Abies sp., Pinus sp., Glyptostrobus europaeus, cf. Sequoia, Cupressus cf. sempervirens, Laurus primigenia, P. leucophylla, Quercus coccifera, Quercus ilex, Q. hispanica (aff. sessiliflora), Q. kubinyii, Alnus ducalis, Populus tremula, Salix sp., Prunus sp., Cedrela sp., Nerium oleander, and Fraxinus cf. oxycarpa. The leaf assemblage contains only a few subtropical taxa such as Glyptostrobus or Cedrela, absent from the Mont-Dore flora, some riparian elements such as Populus, Salix and Platanus, and plants with Mediterranean nearest relatives such as Cupressus, Laurus nobilis, Q. ilex, Q. coccifera, Nerium, etc. This assemblage is compared with the Californian vegetation, the similar extant model where subtropical humid taxa live mixed with more xerophilic plants ( [START_REF] Kovar-Eder | Late Miocene to Early Pliocene vegetation of southern Europe (7-4 Ma) as reflected in the megafossil plant record[END_REF]. The microflora of Cessenon is more diversified and includes conifers such as Cathaya and Cedrus, mesophilic deciduous broad-leaved taxa such as Liquidambar, Parrotia, Carya, and Mediterranean elements such as Pistacia, Olea and Phillyrea [START_REF] Suc | Contribution à la connaissance du Pliocène et du Pléistocène inférieur des régions méditerranéennes d'Europe occidentale par l'analyse palynologique des dépôts du Languedoc-Roussillon (sud de la France et de la Catalogne[END_REF]. Hence at the beginning of the Pliocene, the climatic conditions in Languedoc show a decrease in summer rainfall [START_REF] Roiron | La macroflore d'âge miocène supérieur des diatomites de Murat (Cantal, France). Implications paléoclimatiques[END_REF].

The early Pliocene plant assemblage of Pichegu (Gard, France) includes (Roiron, 1991, corrected): Ginkgo adiantoides, Abies cf. spectabilis, Tsuga cf. caroliniana, Chamaecyparis polymorphum, D. ungeri, L. nobilis, L. primigenia, O. heeri, cf. Persea gratissima, P. leucophylla, L. europaea, Ulmus pyramidalis, Zelkova zelkovifolia (aff. Z. crenata and aff. Z. acuminata), Carya minor, Pterocarya denticulata, Fagus gussonii, F. pliocenica, Quercus faginea, Q. hispanica (aff. Q. sessiliflora), Q. ilex, Q. kubinyii, Q. cf. castaneifolia, Alnus ducalis, Carpinus betulus, C. orientalis, C. suborientalis, C. cf. tschonoskii, Populus alba, P. balsamoides, P. nigra, P. tremula, Salix sp., Acer tricuspidatum, A. sp., and Bambusa sp. The assemblage is rich in mesophilic taxa and riparian elements. Some xerophilic taxa such as Carpinus orientalis, Q. ilex and L. nobilis are present. In addition, the record contains megamesothermic taxa such as subtropical Lauraceae and Celastraceae. There are no taxodioids in the macrofossil record but the pollen diagram shows a decrease in Cupressaceae (including Taxodiaceae) from the base to the upper part of the section [START_REF] Suc | Contribution à la connaissance du Pliocène et du Pléistocène inférieur des régions méditerranéennes d'Europe occidentale par l'analyse palynologique des dépôts du Languedoc-Roussillon (sud de la France et de la Catalogne[END_REF]. The disappearance of taxodioids in Languedoc at that time is certainly caused by the Mediterranean-type climate with dry summers [START_REF] Suc | Origin and evolution of the Mediterranean vegetation and climate in Europe[END_REF][START_REF] Girard | Fossil mega-and micro-flora from Bernasso (Early Pleistocene, southern France): A multimethod comparative approach for paleoclimatic reconstruction[END_REF].

Conclusions

The plant material recovered from Chambon Lake, La Gratade and Pont Chocol (Puyde-Dôme) and newly dated to 4.46 ± 0.05 Ma and 3.94 ± 0.04 Ma by 40 K. LCh-13c: epiderm incertae sedis (petal?). Scale bars: 1 cm (A-G), 500 µm (I-K).

  )), associated with abundant leaves of F. sylvatica L. (Figs. 4(A), 5(I)) and two other different oak species (Q. Roburoides Gaud. (Fig. 5(C-E)) and Q. hispanica Rér. (Figs. 4(F), 5(F), 6(I, J)). Betulaceae are abundantly represented by diversified Alnus species including A. glutinosa Gaertn. (Fig. 4(J)), A. stenophylla Sap. et Mar. (Fig. 4(G-I)), Alnus sp., as well as Betula subpubescens Goepp., B. macrophylla (Goerpp) Heer (Fig. 4(B-E)), Betula alnoides Buch. Ham.ex D. Don (Fig. 8(D)), Betula sp. (Fig. 4(H)), Carpinus betulus L. (Fig. 9(G)) and Carpinus orientalis Sap. (Fig. 9(I)). Salicaceae (Salix sp.) are not common in the different deposits. Leaves of Ulmus braunii type (Fig. 8(A)), Zelkova ungeri aff. Z. acuminata Planch. (Fig. 7(C)) and Zelkova ungeri aff. Z. crenata Spach. (Figs. 5(A), 7(A, B, D)), Carya minor Sap. et Mar. (Figs. 6(G), 8(C)), and Pterocarya sp. (Fig. 8 (F, H, I)) are well documented. In addition, a few interesting, though very poorly represented accessory elements have been collected that correspond to the ancient morpho-species and well preserved fossil leaves of Acer laetum Unger (Fig. 6(A)), Acer integerrimum (Viv.) Mass. (Fig. 6(B)), Acer sanctaecrucis Stur (Roiron, 1991) (Fig. 6 (C, D)) as well as an unidentifiable Acer sp., Buxus pliocenica Saporta et Marion (two leaf specimens), and Cornus distans type (Fig. 7(G, H)). Leguminosae are represented by the morphotype element Leguminophyllum sp. 1 (Fig. 8(E)) and a set of unidentified taxa including Dicotyphyllum sp. 1 (Fig. 9(J)), D. sp. 2 (Fig. 9(E)), D. sp. 3 (Fig. 8(B)), D. sp. 4 (Fig. 7(I)), and D. sp. 5 (Fig. 6(H-K)). Few fruits or seeds of Carpinus suborientalis Sap. (syn. C. tschonoski Maxim, 1881) (Fig. 10(I)), Carpinus betulus L. (Fig. 10(D)), Betula sp. (Fig. 10(A-C)), Alnus sp. (Fig. 10(F)), Pinus sp. (Fig. 10(H)), Picea sp. (Fig. 10(G)) and Tsuga (Fig. 10(E)), as well as one needle of Abies ramesi Sap. (Fig. 9(K)) and one of Tsuga sp. (Fig. 7(J)) are recorded in the macroflora.

  , including at least 10 gymnosperms, the presence of some of them being demonstrated by both reproductive and few vegetative organs. With a few winged seeds and numerous pollen, Abies ramesii Sap., ?Tsuga or Picea sp. are poorly represented in the macroflora but are abundant in the pollen flora. Pinus L. and Sciadopitys (Thunb.) Siebold et Zucc. are only documented by well distinguishable pollen that are particularly numerous in all fossiliferous layers of the site. Comparatively, La Gratade and Pont de Chocol, both located between the Roche Tuilière and Sanadoire in the South and Rochefort-Montagne in the North, are more monotoneous. The great majority of the 23 plant taxa formerly described from La Gratade site show great similarities with the Chambon macroflora. Taxa identified by Grangeon and Rudel (1962) are Potamogeton quinquenervis N. Boul., Salix sp., Populus tremula L., Populus alba L., Betula macrophylla Heer, Alnus glutinosa Gaertn., Carpinus orientalis Mill. (fruits and leave), Carpinus paucinervia Andreansky, Corylus avellana L., Fagus sylvatica Sap., Castanea vesca Gaertn., Quercus hispanica Rer., Quercus sp. (groupe Q. drymeja), Zelkova ungeri Kovats (crenata and acuminata), Ulmus sp., Juglans regia L., Carya sp., Acer laetum C.A. Mey, Acer sp., Tilia sp., and Torreya nucifera Sieb. et Zucc. In the present paper only 10 taxa were identified (Table2); Quercus leaves are abundant but predominantly incomplete, the species was probably the local dominant species in the area with Alnus sp., A. stenophylla, Betula subpubescens, Fagus sylvatica, Fagus grandifolia, and Zelkova type Z. crenata. Among these species only Fagus grandifolia was not recorded previously.Pont de Chocol new record is a smaller and very similar assemblage with 12 taxa: Pteridium aquilinum (L.) Kuhn, Alnus glutinosa Gaertn., Alnus kefersteinii Ung., Alnus viridis?, Quercus hispanica, Quercus sp., Q. roburoides Ber., Ulmus type U. braunii, Zelkova type Z. acuminata Planch, and Dicotiphyllum sp. 3 which probably belongs to the Leguminosae.

  Macroflora indicates preferentially local vegetation while microflora records a wider environment and provides quantitative data. The combined analyses constitute a comprehensive part of environment and allow for a more accurate differentiated reconstruction of the vegetation types and environments. According to zonal and azonal vegetation of the different sites was quite diverse but not totally uniform. First, in the microand macro-analyses arboreal elements prevail and few plants are indicative of open landscapes; apart from taxa with a wide ecological range (Ulmus and Zelkova), the presence of few dispersed Leguminosae leaflet (found only in LCH and PTC) and reduced humid heliophilous forest with Sciadopitys, Nyssa and "Taxodioid" Cupressaceae might indicate the presence of more open areas. The minor contribution of herbaceous plants in the differrent palynological pollen records

  , Tilia tomentosa, Dombeyopsis lobata, Berberis sp. (cf. B. regeliana), Ceratophyllum demersum, Crataegus sp. (cf. C. douglasii), Prunus acuminata, Rosa sp. (cf. R. californica), cf. Photinia, and Bambusa sp.

  and Hamamelidaceae are lacking. This vegetation may indicate a climate change at the end of the Miocene with a continuous decline in Chambon Lake palaeoflora. The lack of Sassafras, Cedrela and Phellodendron differenciates the Chambon Lake from Murat and may indicate a new significant climate change between 5.34 and 4.4 Ma or an overall cooler aspect due to higher elevation and may relate to the cooler phase of the Zanclean B (= Brunssumian B) in Chambon Lake. In La Gratade and Pont de Chocol, pollen records prove otherwise that microthermic elements are regressing while the mesothermic members are increasing, as seen in the warmest phase of the Zanclean C.

  Ar/ 39 Ar are among the taxonomically richest lower Pliocene floras in the French Massif central. The plant assemblage of micro-and macrofloras added complementary data of both autochthonous and allochthonous origin. It comprises at least 76 different taxa (pollen flora and macroflora), a number considerably increased with the recent collect of macrofossil material. It consists mainly of broad-leaved deciduous angiosperms (Fagus, Quercus, Pterocarya, Carya, Ulmus, Zelkova, Betula, and Acer) and conifers (Pinus, Tsuga, Abies, Picea, and Cupressaceae including members of the former Taxodiaceae family). According to comparisons with the current data, the Zanclean palaeoflora indicates the occurrence of extensive riparian woods throughout the wetlands, together with deciduous mesophytic and mid altitude forests at the slopes of the surrounding hills, developed in a relatively warm temperate and humid climate. These new plant findings offer a unique opportunity to enrich our still fragmentary knowledge of lower Pliocene palaeovegetation and to gain a more accurate picture of the plant succession and palaeoenvironmental history of the French Massif central region.

Fig. 1 .

 1 Fig. 1. Geographical and geological map of the Chambon Lake area (Massif Central, France), modified from Besson et al. (1977). 1: Scree; 2: Recent alluvium; 3: Landslide; 4: Basaltic fall of the Tartaret; 5: Strombolian scoriae of the Tartaret; 6: Basaltic breccia of the Saut de la Pucelle; 7: Cinerites of the Lake Chambon; 8: Nuées ardentes deposits; 9: Trachyte; 10: Soshonite lava-flow; 11: Basaltic lava-flows; 12: Oligocene clayey sands; 13: Cristalline basement; 14: 90 m-high cliff; 15: Summit depression; 16: Fossiliferous outcrop.

Fig. 2 .

 2 Fig. 2. Geographical and geological map of La Gratade and Pont de Chocol area (Massif Central, France), modified from Brousse and Tempier (1981) BRGM geological map 1/50,000 Bourg-Lastic (infoterre.brgm.fr). 1: Granitic basement (La Bourboule granite); 2: Tuffites from la Gratade; 3: "Grande Nappe" rhyolitic ignimbrite; 4: Volcano-sedimentary deposits; 5: Soshonite with pyroxene, amphibole and plagioclase phenocrystals; 6: Soshonite with pyroxene, biotite, plagioclase and sanidine phenocrystals; 7: Potassic trachybasalt (aphyric or with rare olivine, pyroxene and plagioclase phenocrystals); 8: Basalt with pyroxene, amphibole and plagioclase phenocrystals; 9: Tephrite with pyroxene, amphibole, plagioclase and haüyne phenocrystals; 10: Ankaramite with olivine and pyroxene phenocrystals; 11: Quaternary alluvium; 12: Fault; 13: Brook; 14: Habitations; 15: Fossiliferous outcrops: A, Pont-de-Chocol; B, La Gratade.

Fig. 3 .

 3 Fig.3.40 Ar/ 39 Ar relative probability distributions for the Chambon Lake and La Gratade outcrops.

Fig. 4 .

 4 Fig. 4. A. LCh-1a: Fagus sylvatica L. B, C. LCh-2a: Betula macrophylla Goerpp.; LCh-2b: Quercus hispanica D, E. LCh-2a: margin and veination details. F. Quercus hispanica Rér. G-I. LCh-2d: Alnus cf. stenophylla L.; H: LCh-31a: Betula sp. J. LCh-21: Alnus glutinosa L. Scale bars: 1 cm.

Fig. 7 .

 7 Fig. 7. A, B. LG-9b: Zelkova ungeri aff. crenata Spach. C. LCh-72: Zelkova ungeri aff. Zelkova acuminata (petiolate leaf fold). D. LCh-49: Zekova ungeri aff. Z. crenata Spach. E. LCh-39b: Zelkova ungeri aff. crenata Spach. F. LCh-39d: Zekova ungeri aff. Z. crenata Spach. G, H. LCh-37a: Cornus type distans. I. LCh-51: Dicotyphyllum sp. 4. J. LCh-30: Tsuga sp. needle. Scale bars: 1 cm (A-I), 2 mm (J).

Fig. 8 .

 8 Fig. 8. A. LCh-18: Ulmus braunii Heer. B. LCh-11: Dicotyphyllum sp. 3. C. LCh-48: Carya minor Sap. et Mar. D. LCh-19: Betula alnoides Buch.-Ham. ex D. Don. E. LCh-5: Leguminophyllum sp.1 cf. Cassia sp. F. LCh-42a: Pterocarya sp. G. PTC-22: Cuticular cells with laterocytic stomatal type. H, I. LCh-39d: Pterocarya sp. Scale bars: 1 cm (A-F, H), 25 µm (G).

Fig. 9 .

 9 Fig. 9. A-C. PTC-34A: Leguminophyllum sp. 2; A: General view; B, C: venation details. D. LCh-22: Leguminophyllum sp. 4. E. LCh-25a: Dicotyphyllum sp. 2. F, G. LCh-51b: Carpinus betulus L. H. Monocotyledon epiderm (?Bambusa sp.). I. LCh-20C: Carpinus suborientalis Sap. J. PTC-34b, Dicotyphyllum sp. 1. K. LCh-23: Abies ramesi Sap. Scale bars: 5 mm.

Fig. 10 .

 10 Fig. 10. A-C. LCh-2: Betula sp., seeds. D. LCh-30d: Carpinus betulus, akene. E. LCh-46: Tsuga sp., seed cone. F. LCh-30c: Alnus sp., strobili. G. LCh-2d: Picea sp. H. LCh-13d: Pinus sp. I. LCh-13a: Carpinus suborientalis Sap. J. LCh-13b: Black circular fungi on leaf.

Fig

  . Murat macroflora includes G. europaeus, Sequoia abietina, Abies ramesi, Pinus sp., Picea sp., A. ), Celtis australis, Populus tremula, Carya minor, Juglans regia, Phellodendron sp. (cf. P. amurense), Cedrela sp., A

ducalis, A. cecropiifolia, Alnus viridis, Alnus sp. (cf. A. kefersteinii), Betula sp., Carpinus suborientalis, Carpinus betulus, C. orientalis, Q. hispanica, Q. sp. (cf. Q. macranthera), Q. kubinyi, Z. zelkovifolia (aff. Z. crenata and Z. acuminata), Ulmus campestris, U. sp. (cf. U. fulva

Table and Figure captionsTable 1 .

 1 Palynological results of LCh, LG and PTC outcrops (Puy-de-Dôme, France).

Table 2 .

 2 Summary of the macrofloristic composition of the studied localities of Chambon lake, La Gratade, and Pont de Chocol, including the NLR (Near Living Relatives;

	Mosbrugger and Utescher, 1997) and frequency groups: *, single (1 specimen); **, 2-5
	specimens; ***, 6-15 specimens; ****, predominant (>16 specimens). Locality: LCh,
	Chambon Lake; LG, La Gratade; PTC, Pont de Chocol. Botanical organs studied: L, leave; S,
	seed; F, fruit.

Acknowledgements

We thankfully acknowledge Dr. Monique and Jean Dupéron (CR2P, MNHN and Paris 6 University) who spent a great deal of their time to help us with the identification of macroflora remains. We are grateful to Jean-Pierre Suc and Vincent Girard who contributed to the improvement of the manuscript.


Aurore Desmons

Elizabeth Thioulouse

Jean-Yves Hautem

Anne Saintier

Bruno Baudin

Antonin Lamazière

Claude Netter

Fathi Moussa
email: fathi.moussa@u-psud.fr

Direct liquid chromatography tandem mass spectrometry analysis of amino acids in human plasma

Keywords: Amino acid profiling, human plasma, LC-MS/MS, mixed-mode stationary phases

Here we describe a new HPLC-MS/MS method using a mixed mode stationary phase and a binary gradient of elution for the rapid separation and quantification of AAs in human plasma without derivatization or ion pairing reagent addition. The sample preparation procedure consists in a single dilution step after protein precipitation with sulfosalicylic acid. The proposed method allows for the unambiguous identification and analysis of 52 AAs and related compounds including the separation of isomers and isobars in an 18 min chromatographic run including the conditioning and the equilibration times. AAs were detected by selective reaction monitoring. Internal calibration was used for the quantification of 37 AAs, including 25 using the corresponding isotopically labeled internal standards. External calibration (no internal standard) was used for five additional analytes. Qualitative detection was achieved for the remaining compounds.

Validation studies evaluated accuracy, linearity, within-and between-run precision, lower limits of detection and quantification for 37 amino acids present in commonly used quality control samples. For within-run precision CVs averaged 3.8 % (n = 30) for all compounds. For between-run precision, CVs averaged 8.6 % for all compounds (n = 20). Correlation with the common standard ion-exchange chromatography with post-column derivatization method was also performed for 32 plasma samples. While the proposed method is at least 50 times more sensitive, the data showed good correlation with slopes equal or higher than 0.9 and correlation coefficients mostly higher than 0.90. The method was successfully applied for analysis of plasma samples for detection of inherited disorders of amino acid metabolism.

Introduction

The quantitative analysis of amino acids (AAs) in biological media including plasma, urines, and cerebrospinal fluid (CSF) is one of the earliest chromatographic methods commonly used in clinical laboratories [START_REF] Moore | Photometric ninhydrin method for use in the chromatography of amino acids[END_REF][START_REF] Moore | Automatic recording apparatus for use in the chromatography of amino acids[END_REF]. This analysis is mainly indicated for the diagnosis and the follow-up of inherited metabolic diseases involving amino acids also called congenital disorders of amino acid metabolism [START_REF] Saudubray | Inborn Metabolic Diseases: Diagnosis and Treatment[END_REF]. Amino acids patterns are also markers of hepatic, renal, and intestinal functions [START_REF] Saudubray | Inborn Metabolic Diseases: Diagnosis and Treatment[END_REF]. Besides, their analysis in plasma is routinely indicated for nutritional assessment in patients under haemodialysis [START_REF] Young | Anthropometry and plasma valine, amino acids, and proteins in the nutritional assessment of hemodialysis patients[END_REF][START_REF] Bossola | Anorexia and plasma levels of free tryptophan, branched chain amino acids, and ghrelin in hemodialysis patients[END_REF] or after bariatric surgery [START_REF] Nicoletti | Protein and amino acid status before and after bariatric surgery: a 12-month follow-up study[END_REF].

For more than sixty years after its development ion exchange chromatography (IEC) with post-column ninhydrin derivatization coupled to UV-Visible detection had been the conventional standard for amino acids assay [START_REF] Moore | Photometric ninhydrin method for use in the chromatography of amino acids[END_REF][START_REF] Moore | Automatic recording apparatus for use in the chromatography of amino acids[END_REF][START_REF] Parvy | Ionexchange chromatography and clinical criteria in the screening of the aminoacidopathies[END_REF]. Although robust, this method suffers three main drawbacks: 1-it is very time consuming (2 to 3 hours), 2it is not always specific due to drug interferences notably in urines, and 3-it requires a dedicated analyser. In addition, some AAs, such as homocitrulline, pipecolic acid, sulfocysteine, argininosuccinic acid, and saccharopine require specific separation methods [START_REF] Saudubray | Inborn Metabolic Diseases: Diagnosis and Treatment[END_REF].

Recent developments in reversed phase liquid chromatography (RPLC) and hyphenated techniques allowed the implementation of several RPLC methods mainly coupled to tandem mass spectrometry (LC-MS/MS) [START_REF] Willis | Automated pre-column derivatization of amino acids with ophthalaldehyde by a reagent sandwiching technique[END_REF][START_REF] Dietzen | Rapid comprehensive amino acid analysis by liquid chromatography/tandem mass spectrometry: comparison to cation exchange with post-column ninhydrin detection[END_REF][START_REF] Filee | Evaluation of physiological amino acids profiling by tandem mass spectrometry[END_REF][START_REF] Piraud | ESI-MS/MS analysis of underivatised amino acids: a new tool for the diagnosis of inherited disorders of amino acid metabolism. Fragmentation study of 79 molecules of biological interest in positive and negative ionisation mode[END_REF][START_REF] Piraud | A new reversed-phase liquid chromatographic/tandem mass spectrometric method for analysis of underivatised amino acids: evaluation for the diagnosis and the management of inherited disorders of amino acid metabolism[END_REF][START_REF] Harder | Quantification of 22 plasma amino acids combining derivatization and ion-pair LC-MS/MS[END_REF][START_REF]Matrix Effects in Mass Spectrometry Combined with Separation Methods -Comparison HPLC, GC and Discussion on Methods to Control these Effects[END_REF]. Gas chromatography coupled to MS has been also proposed for AAs analysis since 2012 [START_REF] Dettmer | Amino acid analysis in physiological samples by GC-MS with propyl chloroformate derivatization and iTRAQ-LC-MS/MS[END_REF]. Although more specific than IEC, all these methods require; however, either pre-column derivatization of the AAs present in the sample [START_REF] Parvy | Ionexchange chromatography and clinical criteria in the screening of the aminoacidopathies[END_REF][START_REF] Willis | Automated pre-column derivatization of amino acids with ophthalaldehyde by a reagent sandwiching technique[END_REF][START_REF] Dietzen | Rapid comprehensive amino acid analysis by liquid chromatography/tandem mass spectrometry: comparison to cation exchange with post-column ninhydrin detection[END_REF][START_REF] Dettmer | Amino acid analysis in physiological samples by GC-MS with propyl chloroformate derivatization and iTRAQ-LC-MS/MS[END_REF] or the use of ion-pairing agents (IPAs) [START_REF] Filee | Evaluation of physiological amino acids profiling by tandem mass spectrometry[END_REF][START_REF] Piraud | ESI-MS/MS analysis of underivatised amino acids: a new tool for the diagnosis of inherited disorders of amino acid metabolism. Fragmentation study of 79 molecules of biological interest in positive and negative ionisation mode[END_REF] or a combination of both [START_REF] Piraud | A new reversed-phase liquid chromatographic/tandem mass spectrometric method for analysis of underivatised amino acids: evaluation for the diagnosis and the management of inherited disorders of amino acid metabolism[END_REF], namely in order to enhance the retention and the separation of these highly polar compounds. Pre-column derivatization is costly and time-consuming and it requires controlled conditions to achieve robustness, reliability, and reproducibility. On the other hand, IPAs alter the robustness of the separation in term of reproducibility [START_REF] Liu | Transient Ion-Pair Separations for Electrospray Mass Spectrometry[END_REF][START_REF]Ion Pair Chromatograpy: A Critical Prespective[END_REF] and their continuous injection into the mass spectrometer irremediably leads to the contamination of the ion source.

More recent developments in stationary phases, notably the advent of mixedmode sorbents [START_REF] Stevenson | Retention mechanism divergence of a mixed mode stationary phase for high performance liquid chromatography[END_REF][START_REF] Lui | Comparison of reversed-phase/cation-exchange/anionexchange trimodal stationary phases and their use in active pharmaceutical ingredient and counterion determinations[END_REF] may allow the separation of polar compounds without using derivatization or adding IPAs. These silica-based stationary phases are specifically designed for simultaneous separation of pharmaceutical drug substances and counter-ions, as well as mixtures of acidic, basic, and neutral compounds. Providing the adjustment of selectivity by changing mobile phase ionic strength, electrolyte type, pH, and organic solvent, such stationary phases may allow for simultaneous separation of basic, neutral, and acidic analytes, notably the retention of ionic and ionizable analytes such as AAs without using ion-pairing reagents. Hence, we focused on a new mixed-mode stationary phase in order to develop and validate a HPLC-MS/MS method of quantification of AAs in biological samples without derivatization or IPAs addition. The sample preparation procedure consists of a single dilution step after protein precipitation.

Material and methods

Calibration standards and control samples

Calibration standards were prepared by mixing Amino Acids Mixture Standard Solutions, Type B (γ-aminobutyric acid, anserine, arginine, carnosine, ethanolamine, hydroxyl-lysine, histidine, 1-methylhistidine, 3-methylhistidine, lysine, ornithine) and Type ANII (alanine, α-alanine, α-aminoadipic acid, α-aminobutyric acid, α-aminoisobutyric acid, aspartic acid, citrulline, cysteine, cystathionine, glutamic acid, glycine, hydroxyl-proline, isoleucine, leucine, methionine, phenylalanine, phosphorethanolamine, phosphoserine, proline, sarcosine, serine, taurine, threonine, tyrosine, valine) both from Wako (Pure Chemical Industries, Osaka, Japan) along with pH 2.8, 0.1 M ammonium formate individual solutions prepared from weighed standards (all from Sigma-Aldrich) of L-alloisoleucine, 5-aminolevulinc acid, L-argininosuccinic acid, L-asparagine, L-glutamine, L-homoctrulline L-pipecolic acid, L-saccharopine, S-sulfo-L-cysteine, and L-tryptophan. An individual solution of L-homocystine was prepared separately in 0.1 M HCl. The resulting stock solution was diluted with distilled water to final concentrations of 500 and/or 250, 100, 50 and 10 µM for all AAS except for sarcosine (1250, 625, 250, 125, and 25 µM), glutamine (1000, 500, 250, 125, 50, and 25 µM), and L-homocystine (250, 125, 50, and 25 µM). Hence, a panel of 52 amino acids and related compounds were analyzed (Figure 1 and Supplementary Tables 1 and2).

As internal standards we used a Metabolomics Amino Acid Mix Standard purchased from Cambridge Isotopes Laboratories (Tewksbury, Massachusetts, USA) as well as Taurine 13 C2 15 N, citrulline 13 CD4, tryptophan 13 C11 15 N2, asparagine 13 C4D3 15 N2, pipecolic acid 13 C6 15 N, glutamine 13 C5D5 15 N, gamma aminobutyric acid 13 C4 all supplied by Eurisotope (Saint-Aubin, France). Ornithine D6 was from Sigma-Aldrich (St Louis, Missouri, USA). In the end, an internal standard (IS) solution containing twenty-five internal standards was prepared for sample dilution and was used for quantitation. The IS solution was obtained by diluting the Metabolomics Amino Acid Mix Standard and pure solutions of the other amino acid isotopes with the eluent A (pH 2.8, 0.12 M ammonium formate and acetonitrile (98.2/1.2, v/v)). The concentrations of the IS in the initial mixes vary from 1.25 mM (for Cystine, for instance) to 2.5 mM (for arginine, alanine, aspartic acid, histidine, etc.). Thus, after dilution (1/200, v/v) in the eluent A, the final concentrations of the AA isotopes in the IS solution vary from 6.25 µM to 12.5 µM according to the considered IS. Internal quality controls, level 1 (L1) and level 2 (L2) batch numbers: 2017.0061 and 2017.0062 respectively, used for method validation were obtained from MCA Laboratory (Winterswijk, The Netherlands).

Plasma samples

This study using only plasma sample residues was performed according to French Public health regulations (Code de la Sante ́Publique -Article L1121-3, amended by Law n°2011-2012, December 29, 2011 -Article 5). Blood samples used for this study were received in the routine workload of the laboratory for amino acids assay as part of the follow-up or diagnosis of patients. No other biological investigation was performed than amino acids analysis.

Sample preparation

One hundred microliters of each calibrator, quality control, or plasma sample were mixed in a 1.5 mL Eppendorf tube with 100 μL of a 3 % sulfosalicylic acid solution. The samples were vortex mixed for 30 seconds, refrigerated at 4 °C for 30 minutes, and centrifuged at 12,000 RCF for 5 minutes. Fifty microliters of supernatant were added to 450 μL of internal standard solution and vortex mixed for 30 seconds.

Five μL of the resulting mixture were injected into the LC-MS/MS system.

LC-ESI-MS/MS

Liquid chromatography

Samples were analyzed on a Thermo Scientific™ UltiMate™ 3000 RS system equipped with an HPG pump, a column oven and a thermostated autosampler. The separation was performed on a 150 × 2.1 mm, 3 μm Thermo Scientific™ Acclaim™ Trinity P1 column, protected with a 10 x 2.1mm guard cartridge filled with the same stationary phase at 30 °C. The eluent A is composed of a mixture of pH 2.8, 0.12 M ammonium formate and acetonitrile (98.8/1.2, v/v) and the eluent B is composed of a mixture of pH 6.5, 0.10 M ammonium formate and acetonitrile (80/20, v/v). Ammonium formate and acetonitrile were purchased from Sigma-Aldrich (St Louis, Missouri, USA).

MS/MS analysis

Compounds were detected on a Thermo Scientific™ TSQ Endura™ triple quadrupole mass spectrometer equipped with a Thermo Scientific™ Easy-Max NG ion source with a heated electrospray ionization probe. Sheath gas was set at 45 arbitrary units, auxiliary gas at 15 arbitrary units, and spray voltage at 3500 V for positive ionization and at 2700 V for negative ionization. Vaporizer temperature was set to 370 °C and transfer tube temperature to 270 °C, while source fragmentation was applied at 15 V. Data was acquired in Selected Reaction Monitoring (SRM) mode using a resolution of 0.7 full width at half maximum (FWHM) for both quadrupoles with a 400 ms cycle time.

Ion exchange chromatography

For the analysis of AAs in plasma by the IEC conventional method we used the dedicated analyzer "AminoTac JLC-500 V JEOL (Tokyo, Japan)" following a previously described procedure [START_REF] Parvy | Ionexchange chromatography and clinical criteria in the screening of the aminoacidopathies[END_REF].

Method validation

The validation of the method was performed according to French Committee of accreditation (Cofrac) guidelines [based on ISO 3534-1 (1993) and ISO 5725-1 (1998) guidelines] for bioanalytical methods.

The specificity and the selectivity of the method are based on both MS/MS detection and chromatographic resolution. The accuracy of the proposed method is based on the closeness of agreement between the quantity values obtained by measurement with the estimated values (as provided by the Erndim EQA Program) of the analytes contained in the quality controls level 1 (L1) and level 2 (L2) used in this study, which are assumed to be calculated consensus values of the AAs.

The limit of quantification (LOQ) for each analyte was determined as the lowest value in the calibration curve giving an average % bias between nominal and back-calculated concentration within ± 20% and a % CV below 20% on 10 replicate injections of calibrators.

The linearity of the method was checked at least with six levels in the concentration ranges of LOQ to 125, 500, or 5000 µM according to the targeted AA.

Six independent determinations were performed at each concentration. Internal calibration was used for 37 compounds, 25 using the corresponding isotopically labeled internal standards. External calibration (no internal standard) was used for five additional analytes. Qualitative detection was achieved for the remaining compounds.

Within-and between-run precisions were performed for the 27 compounds that are present in the Internal quality controls, level 1 (L1) and level 2 (L2) plasma control material. For within-run precision, the controls were prepared and analyzed 30 times in the same day. For between-run precision, they were prepared 20 times in 15 different days.

Data was acquired and processed using Thermo Scientific™ TraceFinder™ (4.1 software).

Results and discussion

LC-MS/MS method development

Tandem mass spectrometry

In order to develop an MS/MS method, we used the product-ions can (PIS) mode by selecting the [M+H] + parent ion and scanning in a range from m/z = 30 to M+1 for each AA. This procedure was performed for collision energy values ranging from 5 to 40 eV.

Table 1 summarizes the MS/MS transitions of the studied AAs. As expected, there are several isomers and isobars exhibiting the same precursor ion. Hence their separation must be achieved, notably when their fragmentations produce the same product ions.

Selecting the separation conditions

Having in common a molecular structure consisting of a side chain R bearing an amino (-NH2) and a carboxylic (-COOH) functional groups, the problem with the LC-MS/MS analysis of AAs lies in the large diversity of their R type specific to each amino acid [START_REF] Saudubray | Inborn Metabolic Diseases: Diagnosis and Treatment[END_REF]. AAs differ according to R type, locations of functional groups, isoelectric point, and polarity. Therefore, their chromatographic separation with an MS-compatible mobile phase requires an appropriate stationary phase with several possibilities of interactions such as a mixed-mode stationary phase.

The first step of method development was column selection according to two selection criteria: resolution of isobaric compounds with an acceptable symmetrical profile for all chromatographic peaks. Preliminary investigations showed that except for the acclaim Trinity P1 stationary phase [START_REF] Lui | Comparison of reversed-phase/cation-exchange/anionexchange trimodal stationary phases and their use in active pharmaceutical ingredient and counterion determinations[END_REF], several bimodal columns (RP-WAX, RP-WCX) and trimodal columns failed to comply with both criteria. Hence, we focused on the Acclaim Trinity P1 stationary phase [START_REF] Lui | Comparison of reversed-phase/cation-exchange/anionexchange trimodal stationary phases and their use in active pharmaceutical ingredient and counterion determinations[END_REF], which exhibits multiple retention mechanisms including anion-exchange, cation-exchange, and reversedphase. This stationary phase consists of porous silica particles coated with charged nano-polymer beads including an inner-pore area modified with an organic layer that provides both reversed-phase and anion-exchange properties and an outer-pore surface with cation-exchange functionality. Among other similar stationary phases, we chose the Acclaim Trinity P1 because of its strong ion-exchange interactions together with its low RP retention [START_REF] Lui | Comparison of reversed-phase/cation-exchange/anionexchange trimodal stationary phases and their use in active pharmaceutical ingredient and counterion determinations[END_REF]. Indeed, the main objective was to obtain a sufficient retention for the more polar AAs while shortening the retention of less polar ones, in order to obtain a good separation in the shortest possible run-time.

In order to separate AAs on the chosen stationary phase we used the simplest MS-compatible mobile phase consisting of a mixture of ammonium formate buffer and acetonitrile. Then we focused on mobile phase composition including pH, and buffer concentration, as well as organic solvent type and concentration, temperature, and flow rate.

In order to separate all targeted AAs a gradient elution was necessary, of course because of their large differences in isoelectric points (Ip) and polarities.

Hence we chose the simplest procedure consisting of a binary system including an eluent A composed of a mixture of pH 2.8, 0.12 M ammonium formate and acetonitrile (98.2/1.2, v/v) and an eluent B consisting of a mixture of pH 6.5, 0.1 M ammonium formate and acetonitrile (80/20, v/v).

Preliminary investigations showed that for such a stationary phase, starting with an acidic pH for eluent A where AAs are mostly protonated is mandatory.

Considering the Ip values of most AAs, at pH approaching the mean Ip most polar AAs are not retained, while increasing the pH to obtain the anionic forms of AAs is incompatible with the stationary phase stability. The main objective was to achieve the retention of the more polar AAs while separating isomeric and isobaric ones, notably leucine, isoleucine, alloisoleucine, and hydroxyproline; alanine and sarcosine; and lysine and glutamine. Thus, we first studied the effects of eluent A pH between 2.6 and 3.0 as well as the ammonium formate concentration between 0.05 and 0.15 M. Eluent B composition was then developed to achieve the rapid elution of less polar and basic AAs.

The optimization of the gradient time, the purging period, the conditioning and the equilibration times were then performed in order to still reach optimum robustness and a run-time as short as possible. Given the specificity of the used selected reaction monitoring (SRM) detection mode, our main objective was to obtain maximum separation efficiency, in order to obtain a sufficient sensitivity, regardless of the resulting resolution except for isomers and isobars. The order of eluting compounds is not only determined by their isoelectric points or molecular weights (Table 1). A further examination of the elution order of some representative AAs (Supplementary Table 1) clearly shows that the side chain plays an unneglectable role. The most polar AAs (hydroxyproline, asparagine, glutamine, serine, threonine) except tyrosine and cysteine elute first followed by three neutral AAs (glycine, alanine, and proline) thus anticipating the elution of acidic AAs (aspartic, glutamic, and alpha-amino-adipic acids). The elution of the remaining neutral AAs (α-amino butyric acid, valine, methionine, leucine, and isoleucine) and citrulline occurs between 2.2 and 3.2 min, while γ-aminobutyric acid elutes at 4.5 min followed by the polar AAs tyrosine and cysteine at 4.5 and 4.9 min and then the neutral AAs phenylalanine and tryptophan at 5.3 and 9.7, respectively, while basic AAs elute between 10.9 and 13.5 min. These results clearly show that the outer-pore cation-exchange mechanism is not the only one involved in the separation. A reversed-phase mechanism is also involved as reflected by the observed retention times of some polar (citrulline, tyrosine, and cysteine) and some neutral AAs (γ-amino butyric acid, phenylalanine, and tryptophan). Also, the need of increasing the concentration of acetonitrile in addition to increasing pH in eluent B in order to accelerate the elution of tryptophan and basic amino acids confirms the existence of the reversed-phase retention mechanism. These results agree with those obtained previously with mixed-mode stationary phases for other compounds [START_REF] Stevenson | Retention mechanism divergence of a mixed mode stationary phase for high performance liquid chromatography[END_REF][START_REF] Lui | Comparison of reversed-phase/cation-exchange/anionexchange trimodal stationary phases and their use in active pharmaceutical ingredient and counterion determinations[END_REF].

Except for critical pairs of isomeric and isobaric AAs, most compounds elute as peaks that were overlapping but unambiguously identifiable by SRM. The best resolution with optimal robustness of critical AAs isomeric pairs sarcosine and alanine, and leucine, isoleucine, allo-isoleucine that are closely eluting is obtained at a pH range between 2.6 and 2.9 and a concentration of ammonium formate between 0.05 and 0.15 M. Decreasing pH below 2.7 improves the resolution but at the expense of the stability and column-life. Increasing pH above 2.9 resulted is a decrease of resolution leading to the co-elution of the critical isomer pairs. Similarly, the drop in the concentration of formate buffer below 0.1 M results in the decrease of the resolution accompanied by the alteration of peaks symmetry and a poor reproducibility of retention times. This phenomenon is obviously related to the need for a good buffering effect in order to obtain an appropriate compatibility between eluent A and the solvent of injection. Also, adding a low percentage of at least 1% of a polar solvent such as acetonitrile or methanol in eluent A is necessary in order to obtain a good peaks symmetry. This low percentage of organic solvent probably serves to solvate the hydrophobic sites of the stationary phase resulting in a better accessibility (less overlapping) of these sites of interaction. Methanol may also be used for this purpose but using acetonitrile resulted in a significant reduction of the run-time together with a better sensitivity notably due to the improvement of the peak shapes and the efficiency of the separation.

Figure 3 shows representative XIC chromatograms showing the separation of the main circulating AA isomers and other usual isobaric compounds present in plasma samples. The separation of leucine and isoleucine isomers as well as hydroxyproline and 5-aminolevulinic acid all with the precursor ion [M+H] + at m/z 132 (Fig. 3A) is satisfactory achieved (Rs ≥ 1.0 for leucine isomers). This is the same for alanine, sarcosine, and 3-aminopropanoic acid all exhibiting a parent ion [M+H] + at m/z 90 (Fig. 3B), as well as for aminobutyric acid isomers with the precursor ion m/z 104 (Fig. 3C).

Proposed method

Considered together these separation developments led us to adopt the following chromatographic conditions for the separation of the targeted AAs: separations are performed on a 150 × 2.1 mm, 3 μm Acclaim™ Trinity P1, protected with a 10 x 2.1 mm guard cartridge filled with the same stationary phase. Eluent A is composed of a mixture of pH 2.8, 0.12 M ammonium formate and acetonitrile (98.8/1.2, v/v) and the eluent B is composed of a mixture of pH 6.5, 0.10 M ammonium formate and acetonitrile (80/20, v/v).

The elution of the targeted AAs is performed in 18 min at 30 °C under the following conditions: 100 % A over 5 min, then eluent B is increased to 100 % from 5 to 7 min and held constant for 4 min. From 11 to 12 min eluent A is increased to 100 % and hold constant during 6 min. The flow rate is set at 0.3 mL/min from 0 to 12 min. Then the flow rate is increased to 0.45 mL/min over 0.5 min and held constant for 2 min. Finally, the flow rate is decreased to 0.3 mL/min over 0.5 min and held constant from 15 to 18 min.

Figure 2B shows a representative LC-MS/MS chromatogram of a plasma sample obtained with the proposed method. All the targeted AAs are separated in less than 11 min. The proposed stationary phase is able to retain the highly polar AAs as well as acidic AAs even with a starting mobile phase consisting of pH 2.8 ammonium formate buffer while achieving the separation of leucine and isoleucine isomers (Fig. 3A).

It is worthy to note that the 5-minute isocratic hold of the eluent A widely contributes to the robustness of the method in terms of retention time reproducibility and peak shapes. It can also enable easy method transfer between instruments with differing gradient dwell volume.

Optimization of sample preparation

The simplest and most appropriate sample preparation conditions were determined by focusing on the nature of the protein precipitating agent, the volume ratio of the precipitating agent, and the dilution level of the resulting supernatant in eluent A in order to minimize matrix interferences while avoiding: 1-altering the robustness of the method notably in terms of retention times reproducibility and separation efficiency, which are dependent on solvent of injection/eluent A compatibility, and 2-the excessive loss of sensitivity due to sample dilution.

To sum up, the sample preparation procedure consists of a single dilution (1/10, v/v) of the sample with the IS solution, after protein precipitation with 100 µL of a 3 % sulfosalicylic acid solution (1/1, v/v).

The precipitation of the sample can be achieved with 10 µL of a 30 % sulfosalicylic acid solution (9/1, v/v) resulting in lower dilution of the initial sample, but this may reduce the precision of the method. In the same way, it is mandatory to dilute the supernatant in the IS solution at least ten times, in order to avoid the robustness alteration of the method as well as an excessive matrix effect.

We also used other classical precipitating agents such as methanol but using sulfosalicylic acid resulted in better robustness of the separation together with minimum matrix effect.

LC-MS/MS identification of AAs in plasma samples

As shown from the XIC chromatograms (Fig. 2 and3), retention times, as compared to those of pure standards (Supplementary Fig. 1), and MS/MS transitions allow the unambiguous identification of all targeted AAs. For instance, the MS/MS transitions m/z 132 > 69 and m/z 132 > 43 allow the attribution of the corresponding peaks to isoleucine and leucine, respectively. Also, it is possible to confirm any MS transition from the ESI-MS spectra of the targeted AA by a secondary transition. This applies for the other targeted AAs.

Method validation

Specificity

The specificity of the method is obviously based on the MS/MS detection mode (3.2. section). In addition, it is well established that the SRM detection method ensures the highest specificity possible for the analysis of AAs and/or any other targeted compound in complex media such as biological ones, providing that the separation of isomers and isobaric compounds is achieved.

Accuracy, precision, and linearity

In order to accurately quantify the targeted AAs in plasma Internal Standard calibration was used for 37 compounds, 25 using the corresponding isotopically labeled internal standards. External calibration (no internal standard) was used for five additional AAs. Qualitative detection was achieved for the remaining compounds. Details of calibration approach, linearity range, and LOQ for each AA are reported in Table 2 (And supplementary Fig. 2).

The obtained results show good linearity over the considered concentration ranges, which allow the determination of most analytes in plasma samples with a single calibration curve for each analyte. The limits of detection (LOD) and quantification (LOQ) of the method were estimated for a signal-to-noise ratio of 3/1 and 10/1, respectively. Representative chromatograms at the LOQ are presented in Supplementary Figure 2. Considering the dilution of the sample in the mobile phase and the injected volume, these results show that the proposed method is at least 50 times more sensitive than the commonly used IEC methods.

For accuracy, within-run, and between-run precision study we used the 27 compounds present in the L1 and L2 control samples (Tables 3 and4, and Product certificate amino acids in the Supplementary material). Calibration was performed in water given that most of the studied compounds are endogenous. The obtained results show good accuracy for the two quality controls in plasma for the 27 studied compounds with recoveries mostly higher than 90 % and lower than 106 % except for a few AAs with low concentration levels close to the LOQ (Table 2), thus confirming the possibility to use this quantitation approach for plasma samples.

The use of internal standards corresponding to most of the compounds of biochemical interest corrects for the possible matrix effects that can be observed in the biological medium as compared to the calibrators resulting in a good precision for most compounds (Tables 3 and4).

For the within-run precision, CVs are lower than 7.6 % for both levels except for aspartic acid in L1 control. This is obviously due to the low level of aspartic acid in L1 (12 μmol/L), which is very close to the LOQ (10 μM; Table 2). For the between-run precision, all CVs of L2 control are lower than 11 % while for L1 some CVs can vary from 10.5 to 16.7 %. However, the between-run precision is overall acceptable because of the low concentration levels in the L1 control. This is true even for some AAs without dedicated internal standard such as for cystathionine.

Robustness and stability

The robustness of the method was evaluated in terms of variation of pH (2.7, 2.8, and 2.9 for the Eluent A, and 6.4, 6.5, and 6.6 for the eluent B), salt concentration (0.10, 0.12, and 0.15 M for both eluents A and B), and acetonitrile concentration (1.0, 1.2, and 1.4 % for eluent A, and 18, 20, and 22 % for eluent B) as well as for temperature (29, 30, and 31°C), flow rate (0.28, 0.30, and 0,032 mL/min), isocratic hold (4, 5, and 6 min) and gradient time (1, 2, and 3 min). The acceptance criteria were the reproducibility of retention times (CVs < 10 %, n = 6) along with good shapes and acceptable peak asymmetry (≤ 1.2), and more importantly, a sufficient resolution for leucine isomers (Rs ≥ 1.0) as well as for sarcosine and alanine (Rs ≥ 1.2) (Fig. 3).

The stability of AAs in plasma samples after protein precipitation with sulfosalicylic acid is well known [2, 11, 12, 14 -16]. In the case of cysteine, considering the acidic conditions for the protein precipitation, only cystine is present since cysteine is completely oxidized to cystine under these conditions. Cystine is indeed the oxidized dimer form of cysteine (Fig. 1); two moles of cysteine are used for the generation of one mole of cystine.

Correlation studies

Finally, in order to complete the evaluation of the proposed LC-MS/MS method we compared the results obtained for 32 plasma samples with the proposed method with those obtained with the IEC conventional standard for amino acids assay [START_REF] Willis | Automated pre-column derivatization of amino acids with ophthalaldehyde by a reagent sandwiching technique[END_REF].

Table 5 summarizes the obtained results. Apart from a few AAs where the concentration means were very low, mostly lower than 50 µM, such as for aminobutyric acid, tryptophan, cysteine, asparagine, and hydroxyproline (Supplementary Fig. 3), the correlations between the two methods are rather satisfactory with slopes equal or higher than 0.9 and correlation coefficients mostly higher than 0.90. We were not able to study the correlation for aspartic acid because the concentration levels in the 32 studied plasma samples were below the LOD of the IEC used method.

Although the number of plasma samples analysed for correlation purposes is not statistically significant to establish the AAs reference values with the LC-MS/MS method, it is worthy to note that the results obtained by the proposed method (dependent variable) are generally lower by nearly 10 % than those obtained by the IEC method (independent variable). This is probably due to the technological difference between the two methods as well as to the calibration procedures even though we used the same standard solution.

Analysis of two known cases of inherited metabolic disorders.

The proposed method was applied to the diagnosis of a known case of phenylketonuria [20] detected in neonatal newborn screening in a 20 days female.

Classical phenylketonuria is an inherited metabolism disorder characterized by an increase of phenylalanine level in plasma due to a phenylalanine hydroxylase deficiency. The analysis of the amino acids profile by the proposed method allowed to confirm a large increase of phenylalanine concentration in plasma (2270 µM, Supplementary Table 4 and Supplementary Fig. 4A).

The proposed method was also applied to the analysis of the amino acids profile of a suspected case of lysinuric protein intolerance [21], an inherited metabolism disorder due to a defect of renal transporter of dibasic cationic amino acids, in a 21-year-old man. The obtained plasma amino acids profile highlighted an increase of glutamine, and a slight decrease of lysine and cysteine, which lead us to analyse by the same method the amino acids profile in a urine sample of this patient.

The obtained data showed an increased urinary excretion of 3 basic amino acids: lysine, ornithine, and arginine (Supplementary Table 4 and supplementary Fig. 4B andC).

Conclusion

A robust HPLC-MS/MS method for the rapid determination of AAs in plasma without derivatization or IPAs addition was validated. The workflow used in this study comprises a simple offline protein precipitation followed by dilution of the sample supernatant with mobile phase containing the internal standards. The proposed method allows the separation of important isomers and the analysis of 52 compounds in an 18 min chromatographic run.

Accuracy and precision studies confirm the possibility of using aqueous calibrators to quantify at least 32 compounds in plasma. Besides, as long as internal standardization is used, this method is capable of quantifying any amino acid or related compound present in plasma over a wide range of concentrations, ranging from a few tens to a thousand μM.

Since the proposed method is necessarily more specific than IEC, it is now necessary to revisit the AAs reference values with the LC-MS/MS method. This work is in progress in our laboratory. Also, additional studies are in progress to adapt the proposed method for other biological matrices. * For phospho-ethanolamine the confirmation fragment 79 was acquired in negative mode with a parent ion corresponding to [M-H] -adduct with m/z=140

Figure 2 A

 2 Figure 2 A shows the representative extracted ion current (XIC) chromatogram

Figure captions Figure 1 .Figure 2 .Figure 3 .

 123 Figure captions Figure 1. Chemical structures of the analyzed compounds

Table 1 .

 1 M/MS transitions. Polarity positive, except for phospho-ethanolamine. 593 Retention time refers to the center of the retention window. NCP: No calibration 594

	595	performed.
		Compound	Retention time	Precursor	Product ions	Collision	Internal Standard
			(min)	ion (m/z)	(m/z)	energy (V)
		Alanine	2.0	90.1	44.4; 90.2	10; 5	Alanine 13 C3 15 N
		β-alanine	3.7	90.1	30.7; 72.2	10	Tyrosine 13 C9 15 N1
		α-aminoadipic acid	2.2	162.1	98.2; 144.1	16;	Alanine 13 C3 15 N
		α-aminobutyric acid	2.2	104.1	41.5; 58.3	20 ; 10	γ-aminobutyric acid 13 C4
		β-aminoisobutyric acid	3.9	104.1	57.0	20	γ-aminobutyric acid 13 C4
		γ-aminobutyric acid	4.5	104.1	69.2; 87.1	15;	γ-aminobutyric acid 13 C4
		5-aminolevulinic acid	4.5	132.1	68.2; 114.1	18;	γ-aminobutyric acid 13 C4
		Anserine	11.8	241.0	109.0	20	Lysine 13 C6 15 N2
		Arginine	13.5	175.1	70.2; 116.1	22;	Arginine 13 C6 15 N4
		Argininosuccinic acid	9.5	291.1	70.2; 116.1	31;	Tryptophan 13 C11 15 N2
		Asparagine	1.9	133.2	74.2; 87.1	16;	Asparagine 13 C4 D3 15 N2
		Aspartic acid	2.1	134.1	74.2; 88.1	15;	Aspartic acid 13 C4 15 N1
		Carnosine	12.1	227.1	110.1; 210.0	22;	Arginine 13 C6 15 N4
		Citrulline	2.3	176.2	70.3; 159.0	22;	Citrulline 13 C D4
		Cystathionine	8.2	223.1	88.1; 134.1	26;	Tryptophan 13 C11 15 N2
		Cysteine	4.9	122.1	59.2; 76.2	21;	NCP
		Cysteine-homocysteine disulfide	7	255,0	134.0	20	NCP
		Cystine	6	241.1	74.2; 152	27;	Cystine 13 C6 15 N2
		Ethanolamine	3.8	62.3	44.4; 62.3	10; 5	Tyrosine 13 C9 15 N1
		Glutamic acid	2.1	148.1	84.1; 130.1	15;	Glutamic acid 13 C5 15 N1
		Glutamine	1.9	147.1	84.1; 130.0	16;	Glutamine 13 C5 D5 15 N2
		Glutathione reduced	7.0	308.3	162; 179	16;	NCP
		Glycine	2.0	76.3	30.5; 76.3	10; 5	Glycine 13 C2 15 N1
		Histidine	10.9	156.1	83.2; 110.1	23;	Histidine 13 C6 15 N3
		Homocitrulline	3.2	190.0	127.1; 173.0	16;	Isoleucine 13 C6 15 N1
		Homocysteine	3.5	136.1	56.3; 90.1	18;	NCP
		Homocystine	9.6	269.1	88.2; 136.0	30;	Tryptophan 13 C11 15 N2
		Hydroxylysine	10.3	163.0	82.0; 128.0	20;	Ornithine D6
		Hydroxyproline	1.8	132.0	68.2; 86.2	18;	Proline 13 C5 15 N1
		Kynurenine	9.4	209.1	146.0; 192.0	18;	Histidine 13 C6 15 N3
		allo-isoleucine	2.9	132.1	69.2	16	Isoleucine 13 C6 15 N1
		Isoleucine	2.9	132.1	69.2	16	Isoleucine 13 C6 15 N1
		Leucine	3.2	132.1	43.4	24	Leucine 13 C6 15 N1
		Leucine, isoleucine and allo-isoleucine	3.0	132.1	86.2	10	Isoleucine 13 C6 15 N1; Leucine 13 C6 15 N1
		Lysine	10.9	147.1	84.2; 130.1	15;	Lysine 13 C6 15 N2
		Methionine	2.7	150.0	104.1; 133.0	10	Methionine 13 C5 15 N1

Table 3 .

 3 Accuracy and within-run precision (n = 30)

			Control Level 1			Control Level 2
	Analyte	conc. (µM) Theoretical	conc. (µM) Measured	(%) Recovery	conc. (µM) CV% Theoretical	conc. (µM) Measured	(%) Recovery	CV%
	Alanine	318.0	309.0	97.2	4.2	925.0	843.4	91.2	4.0
	2-aminobutyric acid	31.2	26.6	85.3	6.8	94.7	81.2	85.7	7.6
	Arginine	16.6	16.7	99.4	2.9	519.0	490.3	94.5	3.7
	Asparagine	107.7	92.7	86.1	3.8	222.0	181.0	81.5	3.4
	Aspartic Acid	12.0	14.3	119.2	10.5	99.6	89.6	90	4.7
	Citrulline	4.6	5.1	110.9	6.3	415.0	372.1	89.7	3.3
	Cystathionine	10.0	7.9	79	4.1	29.6	24.9	84.1	3.7
	Cystine	33.0	27.6	83.6	2.8	69.4	52.4	75.5	3.0
	Glutamic Acid	107.0	110.8	103.6	3.6	223.0	211.2	94.7	3.7
	Glutamine	575.0	577.8	100.5	2.6	1165.0	1100.3	94.4	3.2
	Glycine	516.0	523.6	101.5	4.2	1021.0	946.5	92.7	5.1
	Histidine	203.0	189.1	93.2	2.0	398.0	343.0	86.2	3.4
	Hydroxyproline	48.0	50.4	105	9.6	98.0	126.3	128.9	7.7
	Isoleucine	52.1	49.1	94.2	4.2	398.0	393.1	98.8	5.1
	Leucine	26.8	25.7	95.9	2.2	890.0	857.7	96.4	4.8
	Lysine	271.0	263.3	97.2	2.8	534.0	482.4	90.3	3.6
	Methionine	79.9	77.7	97.2	2.6	241.0	222.8	92.4	3.5
	Ornithine	159.0	155.2	97.6	2.5	639.0	569.9	89.2	3.0
	Phenylalanine	341.0	331.8	97.3	2.0	681.0	626.9	92.1	3.3
	Pipecolic Acid	44.6	47.1	105.6	2.1	92.6	90.3	97.5	3.4
	Proline	301.0	294.7	97.9	3.3	602.0	556.0	92.4	2.9
	Serine	154.0	150.9	98	3.7	463.0	411.7	88.9	5.0
	Taurine	213.0	216.8	101.8	3.7	417.0	361.6	86.7	3.2
	Threonine	205.0	193.4	94.3	2.7	408.0	396.4	97.2	4.0
	Tryptophan	116.0	117.4	101.2	2.7	292.0	291.1	99.7	3.3
	Tyrosine	234.0	229.0	97.9	2.2	927.0	877.4	94.6	3.2
	Valine	416.0	397.2	95.5	2.8	823.0	761.3	92.5	4.2

Table 4 .

 4

			Control Level 1			Control Level 2
	Analyte	Theoretical	Measured	Recovery	CV% Theoretical	Measured	Recovery	CV%
		conc. (µM)	conc. (µM)	(%)		conc. (µM)	conc.(µM)	(%)
	Alanine	318.0	318.6	100.2	5.9	925.0	938.9	101.5	7.0
	2-aminobutyric acid	31.2	28.1	90.1	17.1	94.7	90.9	95.9	8.3
	Arginine	16.6	16.7	100.6	10.5	519.0	501.3	96.6	6.8
	Asparagine	107.7	99.5	92.3	14.5	222	228.7	103	6.9
	Aspartic Acid	12.0	12.0	100.1	14.8	99.6	96.3	96.7	9.6
	Citrulline	4.6	4.9	105.5	10.5	415.0	395.3	95.3	5.4
	Cystathionine	10.0	8.3	83.0	15.2	29.6	45.5	153.7	6.9

Table 5 .

 5 Correlation study. LC-MS/MS vs IEC for 32 plasma samples

		Slope	Intercept	r 2
	Alanine	1.10	-16	0.95
	Aminobutyric acid	0.80	1.8	0.87
	Arginine	0.90	1.9	0.99
	Citrulline	0.90	0.9	0.98
	Cystine	0.80	3.0	0.83
	Glutamic acid	1.00	0.1	0.96
	Glutamine	1.10	-32.0	0.89
	Asparagine	0.67	7.3	0.73
	Glycine	1.00	1.8	0.98
	Histidine	0.90	-1.1	0.97
	Hydroxyproline	0.60	3.3	0.87
	Isoleucine	0.90	3.0	0.95
	Leucine	1.00	-12.1	0.97
	Lysine	1.00	0.5	0.94
	Méthionine	0.90	-5.0	0.96
	Ornithine	1.00	-4.9	0.97
	Phénylalanine	1.00	-3.1	0.99
	Proline	0.90	4.4	0.97
	Sérine	1.10	-17.7	0.86
	Taurine	1.10	-0.7	0.92
	Thréonine	1.00	2.3	0.94
	Tryptophan	0.80	6.4	0.93
	Tyrosine	1.00	-2.1	0.98
	Valine	0.90	2.6	0.99