David J Polette

Didier Batten

Néraudeau

France Polette
email: france.polette@univ-rennes1.fr

David J Batten
email: david.batten@manchester.ac.uk

Didier Néraudeau
email: didier.neraudeau@univ-rennes1.fr

Re-examination of the palynological content of the Lower Cretaceous deposits of Angeac, Charente, south-west France: age, palaeoenvironment and taxonomic determinations

Keywords: verrucate spores, Lygodiaceae, Schizaeaceae, megaspores, Wealden facies, Berriasian, Valanginian, Charentes

Further to the work published by Néraudeau et al. six years ago, palynological matter recovered from the lignitic bone bed of Angeac in Charente (south-west France) has been reexamined in order to provide more evidence of its age, the previous Hauterivian-Barremian interpretation being regarded as controversial. The samples come from four sedimentological units, An2-An5, which together yielded an assemblage of 34 species, taxonomically dominated by palaeoecologically significant lygodiaceous verrucate spores, including eight species attributable to the genus Concavissimisporites and three species of Trilobosporites. A few bisaccate pollen grains, including Vitreisporites pallidus, have been recovered, but the gymnospermous pollen spectrum is clearly dominated by the cheirolepidiaceous genus Classopollis, its abundance diminishing progressively upwards from units An4 to An2. The great abundance and diversity of verrucate forms, along with the presence of other spores

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aim of our paper is, therefore, to re-assess the composition of the continental palynological assemblage recovered from Angeac in the light of this recent development. We also discuss the taxonomy of Early Cretaceous verrucate spores.

Geological setting

The Angeac site (45° 37' 59.08" N-0° 5' 7.27" W) is situated 700 m north of the village of Angeac-Charente, between Angoulême and Cognac (Fig. 1A). The lignitic beds of "Wealden-facies" underlie Pleistocene alluvium laid down by the Charente River. Seven sedimentological units, An1-An7, were described by [START_REF] A N U S C R I P T A C C E P T E D Accepted Manuscript | The Hauterivian-Barremian lignitic bone bed of Angeac (Charente, south-west France): stratigraphical, palaeobiological and palaeogeographical implications[END_REF] from the top to the base respectively. An1 is composed of 0.10-0.20 m of fine white sand, with abundant fish micro-remains: it was mainly exposed during the first excavation campaigns of 2010 and 2011. An2 comprises 0.20-0.40 m of yellow and grey calcareous and marl deposits, with numerous dinosaur bones, other vertebrate remains, ostracods, charophytes, and pieces of wood (Fig. 1B). The stratification of this bed is difficult to determine because it has been affected by the trampling of dinosaurs (dinoturbation). An3 is a calcareous conglomerate 0.20-40 m thick, with abundant vertebrate remains, ostracods, charophytes, insect coprolites, pieces of wood and other plant debris [START_REF] A N U S C R I P T A C C E P T E D Accepted Manuscript Colin | Termite coprolites (Insecta: Isoptera) from the Cretaceous of western France: a palaeoecological insight[END_REF][START_REF] A N U S C R I P T A C C E P T E D Accepted Manuscript | The Hauterivian-Barremian lignitic bone bed of Angeac (Charente, south-west France): stratigraphical, palaeobiological and palaeogeographical implications[END_REF][START_REF] Benoit | A review of the Upper Jurassic-Lower Cretaceous charophytes from the northern Aquitaine Basin in south-west France[END_REF]. An4 is about 0.5-1.20 m thick. It is composed of blue-grey lignitic clay, oxidized at the top, and has yielded numerous plant and well-preserved vertebrate remains. The base of An5 is not visible. It is more than 0.7 m thick and composed of green clay that is devoid of macrofossils, and includes sub-angular limestone blocks that are likely to have been reworked into this deposit [START_REF] A N U S C R I P T A C C E P T E D Accepted Manuscript | The Hauterivian-Barremian lignitic bone bed of Angeac (Charente, south-west France): stratigraphical, palaeobiological and palaeogeographical implications[END_REF]. An6 and An7 consist of grey limestone and red clay respectively, but they were only seen in 2010, after the initial trench was extended laterally.

They did not yield any fossils.

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Material and methods

Material and laboratory processing methods

Six samples were processed by E. Masure in order to provide the palynological results presented in [START_REF] A N U S C R I P T A C C E P T E D Accepted Manuscript | The Hauterivian-Barremian lignitic bone bed of Angeac (Charente, south-west France): stratigraphical, palaeobiological and palaeogeographical implications[END_REF]. Duplicates of these preparations (LEM1-LEM6) were sent to one of us (DJB) in 2011 and these have now been (re)-examined for this paper. LEM1 comes from the An2 unit (Fig. 1B). LEM2 and LEM3 are from the An3 unit. LEM2 corresponds to the organic fraction in which the size of the particles ranges between 10 and 63 µm, whereas LEM3 includes particles ranging in size from 10 and 100 µm. LEM4 comes from the boundary between units An3 and An4. LEM5 is from the boundary between An4 and An5, and LEM6 comes from within the An5 unit. There was no palynological preparation from the An4 unit. However, a sample from this unit was processed in 2010 by DJB at Aberystwyth University, Wales (preparation QPR 3669: QPR is an Aberystwyth University, palynological preparation identifier), using a standard procedure involving immersion of the sample in 10% HCl followed by digestion in 58-62% HF and brief oxidation of the organic residue in fuming HNO 3 , washing after each stage until the residue was neutral (for further information, see [START_REF] Batten | Extraction techniques: small palynomorphs[END_REF].

Four samples were also processed in Aberystwyth for their mesofossil content: preparations MFP422 from the boundary between An3 and An4, and MFP415, MFP423 and MFP512 from unit An4. All yielded very small assemblages of megaspores. Their extraction involved initial soaking of 100 g of rock in warm water on a hot plate followed by immersion of the sample in a solution of Na 4 P 2 O 7 in order to disaggregate it as much as possible before sieving over a 70 µm mesh to reduce its bulk prior to standard palynological processing using HCl and HF. The megaspores were picked out of the aqueous residues under a

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stereomicroscope and housed dry in micropalaeontological slides. Some of the specimens recovered were mounted on scanning electron microscope (SEM) stubs to which carbon tabs had been fixed, coated with platinum-palladium, and examined under a Hitachi S47-100 SEM in the Institute of Biological, Environmental and Rural Sciences (IBERS), Aberystwyth University.

Specimen counts

For palynological preparations LEM1-LEM4, and QPR 3669, 250 specimens were counted from the slides under a light microscope. LEM5 yielded just 94 specimens, and only a few examples of Classopollis were recovered from LEM6.

Analysis of verrucate spores

Owing to their morphological resemblance to the products of the extant fern Lygodium, Cretaceous verrucate spores are considered to belong to the Schizaeales [START_REF] Bolchovitina | Fossil and contemporaneous spores of the Family Schizaeaceae[END_REF]Ivanova in Samoilovitch and Mtchedlishvili, 1961;[START_REF] Fensome | Taxonomy and biostratigraphy of schizaealean spores from the Jurassic-Cretaceous boundary beds of the Aklavik Range, District of Mackenzie[END_REF] see Discussion, Section 5). From the 1950s to the late 1980s, tens of species of eight genera (Concavissimisporites, Converrucosisporites, Impardecispora, Maculatisporites, Rubinella, Trilobosporites, Tuberositriletes and Varirugosisporites) were erected to accommodate upper Mesozoic verrucate spores, principally on the basis of the outline of their amb, and the size and arrangement of the verrucae on a specimen. This led to considerable overlap between generic and specific diagnoses [START_REF] Potonié | Die Gattungen der paläozoischen Sporae dispersae und ihre Stratigraphie[END_REF][START_REF] Delcourt | Les spores et grains de pollen du Wealdien du Hainaut[END_REF][START_REF] Döring | Trilete Sporen aus dem Oberen Jura und dem Wealden Norddeutschlands[END_REF]Venkatachala et al., 1969;Dörhöfer, 1977), as previously underlined and discussed by [START_REF] Batten | Systematics of pre-Quaternary palynomorphs[END_REF].

The history of classification of these spores is well summarized by Fensome (1987, pp. 19, 20), who broadened the diagnosis of Concavissimisporites, and considered

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Tuberositriletes and Impardecispora to be junior synonyms. Tuberositriletes was erected by [START_REF] Döring | Trilete Sporen aus dem Oberen Jura und dem Wealden Norddeutschlands[END_REF] to accommodate Mesozoic forms similar to Converrucosisporites Potonié et Kremp, 1954, because he considered the Carboniferous holotype of the type species of the latter to be an atypical developmental stage. Venkatachala et al. (1969) designated Impardecispora apiverrucata [START_REF] Couper | British Mesozoic microspores and pollen grains: a systematic and stratigraphic study[END_REF] as the type of Impardecispora. This species had previously been accommodated in Trilobosporites, a genus that is distinguishable from Concavissimisporites by the fact that it possesses one or several valvae at each apex, and/or displays a more or less complete network of muri in the apical region [START_REF] Fensome | Taxonomy and biostratigraphy of schizaealean spores from the Jurassic-Cretaceous boundary beds of the Aklavik Range, District of Mackenzie[END_REF]. [START_REF] Srivastava | Microspores from the Fredericksburg Group (Albian) of the southern United States[END_REF] considered Maculatisporites Döring, 1964 to be a junior synonym of , but Fensome (1987, p. 19) noted that the morphological features of the type species, M. undulatus [START_REF] Döring | Trilete Sporen aus dem Oberen Jura und dem Wealden Norddeutschlands[END_REF] do not clearly demonstrate this. The genera Rubinella and Varirugosisporites were erected by [START_REF] Maljavkina | Determination key of Jurassic and Cretaceous spores and pollen[END_REF], emended by Potonié, 1960) and [START_REF] Döring | Die sporenpalaontologische Gliederung des Wealden im Westmecklenburg (Struktur Werle)[END_REF] respectively to accommodate Jurassic verrucate forms, but have rarely been used by other authors. The diagnoses of both are very broad and can easily be accommodated within [START_REF] Fensome | Taxonomy and biostratigraphy of schizaealean spores from the Jurassic-Cretaceous boundary beds of the Aklavik Range, District of Mackenzie[END_REF]

Concavissimisporites

emended diagnosis of Concavissimisporites.

A few years later, Waksmundzka (1992) adopted an even more reductive approach and placed Converrucosisporites, Concavissimisporites, Converrucitriletes, Gemmatriletes, Tuberositriletes, Maculatisporites, Rubinella and Varirugosisporites in synonymy with Verrucosisporites. She considered the basis for separation of these genera was insufficient, this being mainly the general outline of the spores. However, Verrucosisporites was established by [START_REF] Ibrahim | Sporenformen des Aegirhorizonts des Ruhr-Reviers[END_REF], and emended later by [START_REF] Potonié | Die Gattungen der paläozoischen Sporae dispersae und ihre Stratigraphie[END_REF] and [START_REF] Krutzsch | Mikropaläontologische (sporenpaläontologische) Untersuchungen in der Braunkohle des Geiseltales[END_REF], among others, to accommodate Palaeozoic spores covered with warts or small verrucae, which more closely resemble those produced by the Osmundaceae than the Lygodiaceae. Also, placing in synonymy too many taxa can defeat the purpose of achieving a sensible, stratigraphically and biologically relevant taxonomy. Indeed, Waksmundzka (1992,

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p. 47) noted that the spores belonging to her emended version of Verrucosisporites are imprecisely related to the Pteridophyta. Hence, we do not accept her solution to the problem of too many genera for verrucate miospores. We prefer to base our identifications on Fensome's major revision (1987) which, despite his synthesis and practical approach, has never been discussed, most of the genera noted above still being encountered in the literature (e.g., [START_REF] Burden | Illustrated key to genera of Lower Cretaceous terrestrial palynomorphs (excluding megaspores) of western Canada[END_REF][START_REF] Song | Fossil Spores and Pollen of China[END_REF][START_REF] Dejax | Palynology of the dinosaur-bearing Wealden facies in the natural pit of Bernissart (Belgium)[END_REF]. This not only renders comparisons between assemblages challenging, but also diminishes the stratigraphic significance of the species, because too many names are available for the same morphotype and closely similar forms.

As a result, it was decided to carry out a Principal Components Analysis (PCA) of the verrucate spores in the Angeac palynomorph assemblages, one of the purposes being to determine whether Concavissimisporites can truly be regarded as encompassing a morphological group that is well separated from Trilobosporites (see Section 4.2.). Indeed, since numerous verrucate spores were recovered from the samples corresponding to sedimentological units An2-4, we decided to identify them a priori following Fensome's emended diagnoses as species of either Concavissimisporites or Trilobosporites. The PCA was then carried out using 120 specimens from the three units (50, 20, and 50 for An2, An3 and An4, respectively). The purposes of this statistical analysis were to see whether it is possible to (1) obtain several morphological groups that would cluster according to the diagnoses of already established species, (2) determine those groups that can be divided into two or more genera, and (3) ascertain whether there are any significant morphological differences between the specimens recovered from the different lithological units.

Ten variables were taken into account (Fig. 2): S: size of specimen (= maximum diameter).

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SA: smallest value of the reflex, acute or obtuse angle formed by the two tangents of the starting and arrival points of the arc or curve formed by the outline of the interradial region of a spore.

LA: largest value of the reflex, acute or obtuse angle formed by the two tangents of the starting and arrival points of the arc or curve formed by the outline of the interradial region of a spore.

MiD/MaD: ratio between the minimum and maximum distances from the centre to the edge of the spore (excluding the verrucae).

mDV: mean diameter of verrucae on one spore; 32 verrucae were counted on each specimen, when possible. Most of the means have been calculated based on approximately 20 values. mHV: mean height of verrucae on one spore.

STDV: standard deviation of the diameter of verrucae on one spore.

STHV: standard deviation of the height of verrucae on one spore.

ET: exine thickness. PC: per cent cover of the verrucae on the proximal face of a spore. 0.15: 0-30%; 0.45: 31-60%; 0.75: 61-94%; 0.95: 95-100%.

All of the values have been divided by the size of the specimen, and scaled. They were measured on clear images of the specimens under a light microscope. The angles were measured using the free java application OnScreenProtractor. The various lengths and diameters were measured using the software Inkscape. The PCA was carried out using the software Past.

Results

Palynological assemblages

Altogether 34 taxa (excluding those placed in comparison apart from Concavissimisporites sp. cf. C. crassatus) have been identified at genus or species level in the seven samples examined (Fig. 3). The most common of these are the cheirolepidiaceous pollen grain Classopollis (Fig. 4C,D (Fig. 4A). This is the only assemblage in which a specimen of Cicatricosisporites hallei Delcourt et Sprumont was found (Fig. 4M). The sample taken from the boundary between An3 and An4 yielded 16 species, five of which are exclusive to this sample: Callialasporites dampieri (Balme) Dev (Fig. 4B), Densoisporites microrugulatus Brenner (Fig. 4K), cf.

Foraminisporis wonthaggiensis (Cookson et Dettmann) Dettmann (Fig. 4L), Microreticulatisporites sp. cf. M. diatretus Norris (Fig. 4I), and Podocarpidites ellipticus Cookson (Fig. 4F). The assemblage from the lignitic clays of An4 is the most diverse, having yielded 21 species, three of which were recorded only from this level: Retitriletes sp. (Fig. 4O), Cerebropollenites mesozoicus Couper, and Vitreisporites pallidus (Reissinger) Nilsson (Fig. 4E). This sample yielded proportionally the greatest abundance of Classopollis (Fig. 7), but also, and in common with An2, the most diverse verrucate spore assemblage (nine species). The An4/An5 sample yielded a moderately diverse, albeit small assemblage of 15 species, including six verrucate taxa. Overall, the relative abundance of Classopollis tends to decrease upwards in the succession from An4/An5 to An2, whereas the abundance of the verrucate and smooth walled spores increases greatly in this direction. Also, significant in this context is the fact that specimens identified as Trilobosporites are more abundant in the palynomorph assemblage recovered from An2 than in the older deposits, and that overall, verrucate spores in the An2 assemblage are generally larger and more sparsely sculptured than those recovered from the other units (see PCA plots, Figs 8,9).

Megaspores

The four samples processed for their mesofossil content yielded only a few megaspores and fragments thereof, most of which are referable to the genus Erlansonisporites (Figs. 10, 11A, B), and faecal pellets, probably attributable to the activities of termites (Colin et Erlansonisporites sp. sensu Batten, 1975, pl. 13, fig. 6, a selaginellalean megaspore from the upper Berriasian-Valanginian Ashdown Formation in Sussex, south-east England (e.g., Batten, 2011): this is consistent with the suggested revised age determination of the Angeac deposits herein. Numerous microspores, all exhibiting the same morphology, are lodged within the muri of its reticulate sculpture, especially on the proximal surface close to the triradiate flange. In the dispersed state, these are probably attributable to the small spore genus Patellasporites, or perhaps Uvaesporites.

The specimens of Minerisporites sp. are atypical for this genus in that, although zonate, they have a virtually smooth surface and a triradiate flange that is of uneven elevation, indeed it can be partly spinose (Fig. 11C). The outer edge of the zona is also uneven to weakly spinose, features that might equally suggest a representative of Henrisporites, albeit again atypical. More specimens are required before this form can be satisfactorily identified.

Systematic palaeontology of the verrucate spores

The PCA2 plot (Fig. 9) supports [START_REF] Fensome | Taxonomy and biostratigraphy of schizaealean spores from the Jurassic-Cretaceous boundary beds of the Aklavik Range, District of Mackenzie[END_REF] taxonomic approach to the identification of verrucate spores, the specimens from Angeac only being attributable to two for Concavissimisporites apiverrucatus, specimens assigned to a particular species are clustered together, although the boundaries between the clusters are not always clearly defined. It was thought that the semi-quantitative variable corresponding to the per cent cover of verrucae (PC) might be biasing the results of the PCA in assuming too much importance compared to the other variables. Thus, another PCA was run without the per cent cover variable, but the results were very similar, indicating that this did not significantly affect the plot.

Genus Concavissimisporites [START_REF] Delcourt | Les spores et grains de pollen du Wealdien du Hainaut[END_REF], emend. Fensome, 1987. Type species: Concavissimisporites verrucosus [START_REF] Delcourt | Les spores et grains de pollen du Wealdien du Hainaut[END_REF]emend. Delcourt et al., 1963. Concavissimisporites apiverrucatus [START_REF] Couper | British Mesozoic microspores and pollen grains: a systematic and stratigraphic study[END_REF] Döring, 1965 (Fig. 5A, B) Material. Angeac units An2, An3/4, An4, An4/5: 11 specimens, 57 (72) 105 µm in diameter.

Remarks. Concavissimisporites apiverrucatus is differentiated from the other species of this genus in that the sculpture of the apices is slightly to distinctly coarser than over the poles and in interradial regions. In the case of the Angeac specimens, the polar and interradial regions are almost smooth (Fig. 5B), granulate (Fig. 5A), or sculptured with verrucae. Specimens attributed to this species are the only ones that do not form a united cluster on PCA2 (Fig. 9),

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principally because the main diagnostic characters mentioned above have not been considered as a variable, these being very difficult to calculate.

Occurrence. This species occurs widely in rocks ranging in age from latest Jurassic to Albian (e.g., [START_REF] Fensome | Taxonomy and biostratigraphy of schizaealean spores from the Jurassic-Cretaceous boundary beds of the Aklavik Range, District of Mackenzie[END_REF][START_REF] Burden | Illustrated key to genera of Lower Cretaceous terrestrial palynomorphs (excluding megaspores) of western Canada[END_REF]. In France, it has been recorded, as Trilobosporites apiverrucatus Couper, from "Wealden" (Delcourt andSprumont, 1959), lower Valanginian (Vakhrameev andKotova, 1980), upper Barremian-lower Aptian [START_REF] Herngreen | Palynology of a Wealden section (Lower Cretaceous) in the "Carrière de Longueville", the Boulonnais (France)[END_REF]) and upper Albian [START_REF] Bardet | L'ichthyosaure de Bédeille" (Ariège, France): examen palynologique de la gangue et mise au point stratigraphique[END_REF] deposits.

Concavissimisporites sp. cf. C. crassatus [START_REF] Delcourt | Les spores et grains de pollen du Wealdien du Hainaut[END_REF][START_REF] Delcourt | Revision of some Lower Cretaceous microspores from Belgium[END_REF] (Fig. 5L,N)

Material. Angeac unit An2: three specimens 61 (63) 67 µm in diameter.

Remarks. Specimens Q35.4 and B28.4 (Fig. 5L,N) fit the description given by Delcourt et al. (1963, p. 285), bearing in mind the changes made by Fensome to the generic diagnosis (1987, p. 19). No formal size range is given by Delcourt et al., but their illustrated specimen is 78 µm in diameter. Delcourt and Sprumont (1955, p. 26) stated that the "radius" of their form crassatus is 40-55 µm. As a result, we only compare our specimens to this species because they are smaller.

On the PCA2 (Fig. 9), the squares pertaining to C. sp. cf. crassatus are situated at the edge of the cluster that corresponds to C. montuosus. It could be argued that these specimens are also identifiable as C. sp. cf. C. montuosus, the limiting character being the small size of the verrucae.

Occurrence. Concavissimisporites crassatus was originally described from Wealden-type strata in Belgium [START_REF] Delcourt | Les spores et grains de pollen du Wealdien du Hainaut[END_REF][START_REF] Delcourt | Revision of some Lower Cretaceous microspores from Belgium[END_REF]. It has not been encountered previously in France, but has been found in Albian strata of the Bay of Biscay [START_REF] Batten | Miospores and other acid-resistant microfossils from the Aptian/Albian of holes 400A and 402A, DSDP-IPOD LEG 48, Bay of Biscay[END_REF], Berriasian-mid-Valanginian strata in the Netherlands [START_REF] Burger | Palynology of uppermost Jurassic and lowermost Cretaceous strata in the eastern Netherlands[END_REF], upper Berriasian, and upper Barremian-lower Aptian rocks in Germany (Dörhöfer, 1977;[START_REF] Lister | Palynostratigraphy of the Barremian-Aptian succession in the Wiechendorf 1/86 and Hoheneggelsen KB 9 Boreholes in the Lower Saxony Basin, North Germany[END_REF], and within Cenomanian deposits of Spain (Solé de [START_REF] Solé De Porta | Palinología de dos secciones del Cenomaniense de los alrededores de Oviedo (España)[END_REF]. It was also recorded by [START_REF] Fensome | Taxonomy and biostratigraphy of schizaealean spores from the Jurassic-Cretaceous boundary beds of the Aklavik Range, District of Mackenzie[END_REF] from Upper Jurassic to lower Valanginian strata in western Canada.

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Concavissimisporites exquisitus [START_REF] Singh | Lower Cretaceous microfloras of the Peace River area, northwestern Alberta[END_REF]) Fensome, 1987 (Fig. 5C,D)

Material. Angeac units An2, An3, An3/4, An4, An4/5: 19 specimens, 47 (59) 75 µm in diameter.

Remarks. This species is defined by its triangular to convex outline, coupled with bulbous verrucae 6-10 µm wide. In his original diagnosis, Singh (1971, p. 116) stated that the verrucae are spaced 1-4 µm apart. The verrucae on the specimens from Angeac are more closely spaced overall, being separated by no more than 1 µm. The size-range given by Singh is 68 (71) 84 µm, but this is based on only four specimens, and the only specimen of this species recovered by Fensome is 59 µm in diameter (1987, pl. 4, fig. 11). Some relatively small, rounded specimens, such as R40.2 (Fig. 5E), are referred to Concavissimisporites sp. cf. C. exquisitus because they show proportionally smaller verrucae. They are more similar to Verrucosisporites major (Couper) Burden et Hills, a form that occurs widely in Jurassic and Cretaceous strata.

The cluster corresponding to this species on the PCA2 plot is very close to that representing Concavissimisporites ferniensis (Fig. 9), which is, however, distinguished from C. exquisitus in having significantly lower verrucae separated by narrow canals that form a more or less distinct negative reticulum.

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Occurrence. This species was originally recovered from Albian strata in Alberta, Canada [START_REF] Singh | Lower Cretaceous microfloras of the Peace River area, northwestern Alberta[END_REF], but [START_REF] Fensome | Taxonomy and biostratigraphy of schizaealean spores from the Jurassic-Cretaceous boundary beds of the Aklavik Range, District of Mackenzie[END_REF] found it in upper Jurassic deposits. It has not been encountered previously in France, but [START_REF] Mohr | New palynological information on the age and environment of Late Jurassic and Early Cretaceous vertebrate localities of the Iberian Peninsula (eastern Spain and Portugal) Berliner Geowissenschaftliche Abhandlungen[END_REF] mentioned its presence in upper Tithonian-lower Berriasian strata in Portugal. (Pocock, 1970[START_REF] Fensome | Taxonomy and biostratigraphy of schizaealean spores from the Jurassic-Cretaceous boundary beds of the Aklavik Range, District of Mackenzie[END_REF] (Fig. 5F-H) Material. Angeac units An2, An3, An3/4, An4: seven specimens 45 (56) 78 µm in diameter.

Concavissimisporites ferniensis

Remarks. For differences between Concavissimisporites ferniensis and C. exquisitus, see remarks above under the latter species. In common with C. exquisitus, the specimens assigned to C. ferniensis are very similar to Verrucosisporites major, which however differs in usually being smaller and in lacking a negative reticulum (see [START_REF] Couper | British Mesozoic microspores and pollen grains: a systematic and stratigraphic study[END_REF].

Occurrence. This species has only been recovered from Jurassic sedimentary rocks in western Canada [START_REF] Fensome | Taxonomy and biostratigraphy of schizaealean spores from the Jurassic-Cretaceous boundary beds of the Aklavik Range, District of Mackenzie[END_REF], but very similar forms, identified as Leptolepidites major Couper, have been found in various French Jurassic, Albian and Cenomanian strata [START_REF] Fauconnier | Les dinoflagellés de l'Albien et du Cénomanien inférieur du Bassin de Paris. Répartition stratigraphique et relations avec la nature du dépôt[END_REF][START_REF] Fechner | Die Cenoman-Ablagerungen Nördlich von Simeyrols (Dept. Dordogne, SW-Frankreich): Sedimentologie, Palaontologie, Palynologie[END_REF]Bignot et al., 1994).

Concavissimisporites montuosus [START_REF] Döring | Trilete Sporen aus dem Oberen Jura und dem Wealden Norddeutschlands[END_REF][START_REF] Fensome | Taxonomy and biostratigraphy of schizaealean spores from the Jurassic-Cretaceous boundary beds of the Aklavik Range, District of Mackenzie[END_REF] (Fig. 5J,K,M) Material. Angeac units An2, An3, An3/4, An4, An4/5: 26 specimens 52 (72) 105 µm in diameter.

Remarks. This species is characterized by being sculptured with typically well-spaced verrucae 2-5 µm in diameter. It is the most common verrucate spore in the Angeac succession. A few specimens have been referred to Concavissimisporites sp. cf. C. montuosus (e.g., N26.1, not illustrated), because they have more closely spaced and smaller verrucae.

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Occurrence. The stratigraphic range of Concavissimisporites montuosus extends from upper Oxfordian to Albian. It is a distinctive element in many Volgian-Valanginian palynofloras [START_REF] Fensome | Taxonomy and biostratigraphy of schizaealean spores from the Jurassic-Cretaceous boundary beds of the Aklavik Range, District of Mackenzie[END_REF]. It has not been found in France previously, but has been recovered, as Converrucosisporites montuosus Döring, from lowest Cretaceous strata in north-western Germany [START_REF] Dörhöfer | Discrimination and correlation of highest Jurassic and lowest Cretaceous terrestrial palynofloras in North-West Europe[END_REF]. According to these authors, an abundance of this form is characteristic of their upper Berriasian palynofloral suite "Hils 1". Dörhöfer, 1977 (Fig. 5I) Material. Angeac units An2, An3: two specimens 59 and 66 µm in diameter.

Concavissimisporites robustus

Remarks. Concavissimisporites robustus differs from the other species encountered in having a more or less uniformly thick exine coupled with poorly delineated verrucae that are either sparsely or densely distributed. The two specimens recovered from Angeac are weakly sculptured. The PCA2 plot shows that they cluster closest to the specimens identified as C. uralensis, mainly because of the scarcity of the verrucae on the proximal face.

Occurrence. This species was originally described from the upper Berriasian palynofloral suites "Hils 1" and "Hils 2" in north-western Germany (Dörhöfer, 1977). It has also been recorded from uppermost Jurassic strata in western Canada [START_REF] Fensome | Taxonomy and biostratigraphy of schizaealean spores from the Jurassic-Cretaceous boundary beds of the Aklavik Range, District of Mackenzie[END_REF] and middle Berriasian deposits in eastern Maryland, USA [START_REF] Doyle | Palynological evidence for Berriasian age of basal Potomac Group sediments, Crisfield Well, eastern Maryland[END_REF]. [START_REF] Bolchovitina | Fossil and contemporaneous spores of the Family Schizaeaceae[END_REF]) Fensome, 1987 (Fig. 6D) Material. Angeac units An2, An4: three specimens 56 (68) 90 µm in diameter.

Concavissimisporites uralensis

Remarks. This species can appear very similar to the specimens identified here as Concavissimisporites apiverrucatus: they differ in being more sparsely sculptured. They also

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differ from specimens of C. robustus in having a thinner exine. It could be argued that they are intermediate between C. apiverrucatus and C. robustus. However, the PCA clustered all three specimens together, quite far away from the other clusters, which is why we decided to regard them as belonging to a distinct species (see Section 6.2 for the biological relevance of this decision).

Occurrence. Concavissimisporites uralensis was originally described from Barremian-Aptian strata in western Kazakhstan [START_REF] Bolchovitina | Fossil and contemporaneous spores of the Family Schizaeaceae[END_REF]. It has also been recorded from Ryazanian (lowest Berriasian) strata in western Canada [START_REF] Fensome | Taxonomy and biostratigraphy of schizaealean spores from the Jurassic-Cretaceous boundary beds of the Aklavik Range, District of Mackenzie[END_REF]. [START_REF] Delcourt | Les spores et grains de pollen du Wealdien du Hainaut[END_REF]emend. Delcourt et al., 1963 (Fig. 6A,B)

Concavissimisporites verrucosus

Material. Angeac units An3, An4, An4/5: six specimens 73 (81) 100 µm in diameter.

Remarks. This species differs from the other verrucate spores from Angeac by its large size, and in having relatively small, closely spaced verrucae. The specimen D20.4 (Fig. 6C) is considered to be an intermediate form between C. verrucosus and C. montuosus, because it is sculptured with relatively large, closely spaced verrucae.

Occurrence. The stratigraphic range of C. verrucosus extends from Middle Jurassic to Lower Cretaceous. In western Europe, it occurs principally within lowest Cretaceous and "Wealden"-type strata [START_REF] Burger | Palynology of uppermost Jurassic and lowermost Cretaceous strata in the eastern Netherlands[END_REF][START_REF] Bolchovitina | Distribution of Schizaeaceous spores in Jurassic and Lower Cretaceous deposits of Eurasia[END_REF][START_REF] Dörhöfer | Discrimination and correlation of highest Jurassic and lowest Cretaceous terrestrial palynofloras in North-West Europe[END_REF]. Genus Trilobosporites Pant, 1954ex Potonié, 1956emend. Fensome, 1987 Type species: Trilobosporites hannonicus [START_REF] Delcourt | Les spores et grains de pollen du Wealdien du Hainaut[END_REF] Potonié, 1956

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Trilobosporites aequiverrucosus Dörhöfer, 1977 (Fig. 6I) Material. Angeac unit An4: one specimen, 100 µm in diameter.

Remarks. This species differs from Trilobosporites canadensis in having proportionally smaller unfused, closely spaced verrucae in polar and interradial regions. As stated by Dörhöfer (1977, pp. 53, 54), this species is considered to include many specimens previously attributed to Trilobosporites bernissartensis (Delcout et Sprumont) Potonié, for which the type and size of the sculptural elements were not specified. The re-illustrations of the holotype and paratypes in Delcourt et al. (1963, pl. 43, figs. 11-14) are considered inadequate, because several morphotypes are displayed. As a result, Dörhöfer considered that a precise interpretation of T. bernissartensis is not possible.

Occurrence. The species was originally described from upper Berriasian-lower Valanginian beds in western Germany (Dörhöfer, 1977). As Trilobosporites bernissartensis, it has been found in Wealden-type strata in Belgium [START_REF] Delcourt | Les spores et grains de pollen du Wealdien du Hainaut[END_REF][START_REF] Delcourt | Revision of some Lower Cretaceous microspores from Belgium[END_REF], and in Purbeck and Wealden beds in southern England and stratigraphically equivalent deposits in western France [START_REF] Couper | British Mesozoic microspores and pollen grains: a systematic and stratigraphic study[END_REF][START_REF] Lantz | Etude des spores et pollens d'un échantillon purbeckien de l'île d'Oléron[END_REF][START_REF] Deák | Microfossiles organiques" du Wealdien et du Cénomanien dans un sondage de Charente-Maritime[END_REF]. It has also been reported from lowest Aptian strata in south-eastern France (de [START_REF] De Reneville | Palynologie du stratotype du Barrémien[END_REF], and within upper Barremian-lower Aptian strata in northern France [START_REF] Herngreen | Palynology of a Wealden section (Lower Cretaceous) in the "Carrière de Longueville", the Boulonnais (France)[END_REF].

However, the specimen illustrated by Herngreen (1971, pl. 3, fig. 1)

is in fact attributable to

Trilobosporites hannonicus (Delcourt et Sprumont) Potonié, so we do not accept this record of occurrence.

Trilobosporites aornatus Döring, 1965 (Fig. 6E, F) Material. Angeac units An2, An3, An4: three specimens 68 (78) 96 µm in diameter.

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Remarks. The morphology of the specimens recorded as Trilobosporites aornatus have a maculate sculpture and a large verruca or rounded valva in equatorial radial regions. K28.3 (Fig. 6G) is referred to Trilobosporites sp. cf. T. aornatus, because of the presence of a few verrucae close to each of the angles, in common with the specimen illustrated by Norris as Trilobosporites obsitus Norris (1969, pl. 106, fig. 7) (see Section 5.2. for the biological relevance of this species).

Occurrence. [START_REF] Döring | Die sporenpalaontologische Gliederung des Wealden im Westmecklenburg (Struktur Werle)[END_REF] described this species from Upper Jurassic-lowermost Cretaceous strata in eastern Germany. In southern England, it is typical of the upper Berriasian-lower Valanginian palynofloral suite C of [START_REF] Norris | Miospores from the Purbeck Beds and marine Upper Jurassic of southern England[END_REF], and as discussed by [START_REF] Dörhöfer | Discrimination and correlation of highest Jurassic and lowest Cretaceous terrestrial palynofloras in North-West Europe[END_REF]. In North America, it ranges into the lower Barremian [START_REF] Burden | Illustrated key to genera of Lower Cretaceous terrestrial palynomorphs (excluding megaspores) of western Canada[END_REF].

Trilobosporites canadensis Pocock, 1962 (Fig. 6H) Material. Angeac units An2, An3, An3/4, An4, An4/5: 11 specimens 69 (88) 105 µm in diameter.

Remarks. Trilobosporites canadensis is characterized by its coarse interradial and polar verrucae, which sometimes merge to form elongate elements, along with one or two large, pronounced, strongly projecting verrucae or valvae in equatorial radial regions. This is the most common form of Trilobosporites at Angeac, especially in the An2 unit.

Occurrence. This species was originally described from "Neocomian" deposits in western Canada [START_REF] Pocock | Microfloral analysis and age determination of strata at the Jurassic-Cretaceous boundary in the Western Canadian plains[END_REF]. In Western Europe, it has been reported from upper Berriasian-lower Valanginian beds in western Germany (Dörhöfer, 1977), probable Berriasian Purbeck beds in southern England (as T. cf. canadensis; [START_REF] Hunt | Miospores from the Portland Stone Formation and the lower part of the Purbeck Formation (Upper Jurassic/Lower Cretaceous) from Dorset, England[END_REF], Tithonian-Berriasian deposits in Portugal [START_REF] Mohr | New palynological information on the age and environment of Late Jurassic and Early Cretaceous vertebrate localities of the Iberian Peninsula (eastern Spain and Portugal) Berliner Geowissenschaftliche Abhandlungen[END_REF] and within upper Barremian-lower Aptian strata in northern France [START_REF] Herngreen | Palynology of a Wealden section (Lower Cretaceous) in the "Carrière de Longueville", the Boulonnais (France)[END_REF]. However, the morphology of the specimens illustrated in Herngreen's paper (pl. 3, figs. 2-4) is closer to that of Concavissimisporites montuosus than to T. canadensis.

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Discussion

Age determination and comparisons

We discuss the previous identification in Néraudeau et al. ( 2012) of the dinoflagellate cyst Odontochitina imparilis (Duxbury) Jain et Khowaja-Ateequzzaman in the Angeac assemblage. In his description of Muderongia imparilis, Duxbury (1980, p. 128) indicated the presence of three well-developed horns as a diagnostic feature, with the lateral horn being greatly reduced. A lateral horn seems to be present on the Angeac specimen, but it is folded behind the periblast. There are two antapical horns, one shorter than the other. The longer of the two is very corroded. A vestigial apical horn is present. The original description also states that the endoblast and periblast are joined together in the epicystal region, which does not seem to be the case of the Angeac specimen, the endoblast being proportionally much smaller than the periblast.

The species has usually been found within Hauterivian-Barremian deposits [START_REF] Duxbury | Barremian phytoplankton from Speeton, East Yorkshire[END_REF][START_REF] Jain | Reappraisal of the genus Muderongia Cookson & Eisenack[END_REF][START_REF] Leereveld | Hauterivian-Barremian (Lower Cretaceous) dinoflagellate cyst stratigraphy of the western Mediterranean[END_REF], hence the previous attribution of a Hauterivian-Barremian age to the Angeac deposits. However, the species has also been recorded from upper Valanginian strata (Burger, 1996). Although the poor preservation of the Angeac specimen renders the identification questionable, the short range of the species may well be related to the general scarcity of this form and linked to specific environmental conditions (Masure, pers. comm. 2017), so a find of O. imparilis in older deposits is not impossible.

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The Angeac specimen is also similar to Muderongia simplex Alberti, which has one apical, two lateral, and two antapical horns. The holotype of this species is 151 µm long, which is almost the same as the Angeac specimen at 152 µm. However, they differ in that the latter possesses a proportionally small endoblast, and does not display a second lateral horn, although it is possible that has been destroyed or is obscured by folding. The stratigraphic range of Muderongia simplex extends from upper Tithonian to upper Valanginian [START_REF] Riding | A taxonomic study of the dinoflagellate cyst Muderongia simplex Alberti 1961 and related species[END_REF].

Dating Lower Cretaceous deposits using miospores can be challenging, the forms encountered often being long ranging. For instance, Aequitriradites verrucosus and Pilosisporites trichopapillosus are present through all of the Lower Cretaceous in western Europe. In France, A. verrucosus has only been reported recently within Cenomanian deposits [START_REF] A N U S C R I P T A C C E P T E D Accepted Manuscript | Palaeobotanical investigation of a Cenomanian clay lens in Hucheloup Quarry, Maine-et-Loire, NW France: taxonomic, stratigraphic and palaeoenvironmental implications[END_REF]. [START_REF] Batten | Miospores and other acid-resistant microfossils from the Aptian/Albian of holes 400A and 402A, DSDP-IPOD LEG 48, Bay of Biscay[END_REF] recorded it from Aptian-Albian strata in the Bay of Biscay. It occurs rarely elsewhere in Europe but has nonetheless been documented from Aptian and Wealden deposits in England (Ruffell and Batten, 1994 and Batten unpublished, respectively) and in Wealden-type facies in Germany [START_REF] Döring | Trilete Sporen aus dem Oberen Jura und dem Wealden Norddeutschlands[END_REF].

Pilosisporites trichopapillosus ranges from uppermost Jurassic [START_REF] Couper | Jurassic and Lower Cretaceous palynology of the Netherlands and adjacent areas. Verhandelingen van het Geologisch-Mijnbouwkundig Genootschap voor Nederland en Kolonien[END_REF] to Upper Cretaceous [START_REF] Robaszynski | The Campanian-Maastrichtian boundary in the chalky facies close to the type-Maastrichtian area[END_REF], but is more frequently recovered from Lower Cretaceous deposits [START_REF] Bolchovitina | Distribution of Schizaeaceous spores in Jurassic and Lower Cretaceous deposits of Eurasia[END_REF]Dörhöfer, 1977;[START_REF] Batten | Miospores and other acid-resistant microfossils from the Aptian/Albian of holes 400A and 402A, DSDP-IPOD LEG 48, Bay of Biscay[END_REF][START_REF] Doubinger | Une microflore du Barrémien dans la Province de Valencia, Espagne[END_REF][START_REF] Batten | Aspects of palynomorph distribution, floral provinces and climate during the Cretaceous[END_REF]. In France, it has only been documented from Tithonian strata [START_REF] Couper | Jurassic and Lower Cretaceous palynology of the Netherlands and adjacent areas. Verhandelingen van het Geologisch-Mijnbouwkundig Genootschap voor Nederland en Kolonien[END_REF].

Nevertheless, dating can be more precise if the composition of entire assemblages is taken into account. A few attempts at stratigraphic correlations using Lower Cretaceous miospore assemblages were made during the late 1960s and early 1970s. A method introduced by Hughes and Moody-Stuart (1969) consisted of using several types of Cicatricosisporites to correlate between different beds in a Wealden borehole and an outcrop

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in south-east England. Their abandonment of Linnaean nomenclature in favour of biorecords [START_REF] Hughes | A method of stratigraphic correlation using Early Cretaceous miospores[END_REF][START_REF] Hughes | The need for agreed standards of recording in palaeopalynology and palaeobotany[END_REF] meant that their method did not gain widespread support among palynologists [START_REF] Batten | Systematics of pre-Quaternary palynomorphs[END_REF]. Regardless, we could not apply a similar approach to the Angeac assemblages using species of Cicatricosisporites because only a few specimens of this taxon were recovered.

Another method involved the use of palynological assemblage-types for correlating Wealden deposits in southern England [START_REF] Batten | Use of palynologic assemblage-types in Wealden correlation[END_REF]. Seventeen assemblages were described, taking into account preservation state, diversity of miospores, average size of trilete spores, abundance of brown wood, and the abundance of certain taxa. The total Angeac assemblage (i.e. the overall composition of the assemblages recovered from An2-5) is very similar to Batten's Assemblage Type 15 (AT 15). This was characterized by the common presence of Concavissimisporites, coupled with a "Trilete spore content dominated by average-sized or large forms; miospore diversity average or large; general state of preservation of miospores good or fair; brown wood and cuticle P, C or F [present, common, or frequent]; Pilosisporites P or C [present or common], Cicatricosisporites C or V [common or very common]…..Trilobosporites occurs in more than 70% of the assemblages referable to this AT" (Batten, 1973, pp. 13, 14). The only difference from this description is the scarcity of Cicatricosisporites. Following the same study, and using current stratigraphic terminology (e.g., Batten, 2011), this assemblage-type has mostly been recovered from the Ashdown Formation, including the Fairlight Clays facies, and the Grinstead Clay Formation, all of which are part of the Hastings Group, which was deposited between the mid Berriasian and the late Valanginian.

The Angeac assemblage overall is also very similar to those recovered from the Bückeberg Formation, especially the upper Berriasian palynofloral suites "Hils 1" and "Hils 2", which are characterized by abundant Concavissimisporites montuosus, large

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Trilobosporites, and the relative scarcity of Cicatricosisporites (Dörhöfer, 1977;[START_REF] Dörhöfer | Discrimination and correlation of highest Jurassic and lowest Cretaceous terrestrial palynofloras in North-West Europe[END_REF]. In France, Berriasian palynological assemblages have been recovered from Jura (eastern France) and potentially Oléron island (Fig. 1). The Angeac assemblage is somewhat similar to that recovered by [START_REF] Médus | Palynologie des niveaux émersifs du Berriasien du Jura. Comparaison avec des spectres de Sardaigne et des Pyrénées[END_REF] from upper Berriasian strata of southern Jura in that the latter is characterized by an abundance of Densoisporites microrugulatus and Trilobosporites. It was compared to palynofloral suite "Hils 4" (Médus and Mojon, 1991, p. 57), but only a few illustrations were provided to support the identifications, and D. microrugulatus is not abundant in the Angeac assemblage. The uppermost Berriasian assemblage from Jura recovered earlier by Taugourdeau- [START_REF] Taugourdeau-Lantz | Un aperçu de l'environnement végétal pendant l'épisode régressif du Berriasien terminal dans le Jura méridional (France)[END_REF] is less similar, with few verrucate spores and several dinoflagellate cyst species in association. The palynoflora recovered from the "Purbeck beds" of Oléron island [START_REF] Lantz | Etude des spores et pollens d'un échantillon purbeckien de l'île d'Oléron[END_REF] includes similar forms of Trilobosporites, but the types of Concavissimisporites are less diverse, and the specimens illustrated seem comparatively small. The Angeac assemblage is, however, less clearly correlated with younger palynofloras, such as that recovered from the Wealden-facies, dinosaur-bearing pit of Bernissart (Belgium, [START_REF] Dejax | Palynology of the dinosaur-bearing Wealden facies in the natural pit of Bernissart (Belgium)[END_REF], considered to be Hauterivian in age. The samples from this pit yielded specimens of Trilobosporites that are similar to the species T. hannonicus (Delcourt et Sprumont) Potonié, which has not been recorded from Angeac, more species of Cicatricosisporites, and angiospermid pollen grains. Correlations with the assemblage recovered from Saint-Romain-de-Benet, regarded as Valanginian-Hauterivian in age, are quite difficult to make because of a lack of precision in the description of the assemblage as indicated by such statements as …. "on n'observe pratiquement pas de pollens d'Angiospermes" in Deák and Combaz (1967, p. 72), meaning that they "almost did not observe any angiosperm pollen grains". Their stratigraphic range chart (p. 71) is rather inaccurate because most of the species they note as restricted to the Valanginian-Hauterivian

Biological relevance of the verrucate spores

The family Schizaeaceae originally included five extant genera: Schizaea Sm., Actinostachys Wall., Mohria Sw., Anemia Sw., and Lygodium Sw., which are widely distributed in predominantly tropical or southern warm-temperate parts of the world (Tryon and Lugardon, 1991). However, [START_REF] Smith | A classification for extant ferns[END_REF] considered that these genera in fact belong to three families of the order Schizaeales: Schizaeaceae (for Schizaea and Actinostachys), Anemiaceae (for Anemia, including Mohria), and Lygodiaceae (for

Lygodium).

Unequivocal members of this order first appeared in the Early or Middle Jurassic, but they did not become widespread until the Early Cretaceous, as indicated by occurrences of their spores in numerous deposits throughout Eurasia [START_REF] Bolchovitina | Distribution of Schizaeaceous spores in Jurassic and Lower Cretaceous deposits of Eurasia[END_REF]Van Konijnenburg-Van Cittert, 2002). The Schizaeaceae produce monolete, bean-shaped spores, with diverse ornamentation. The Anemiaceae have trilete spores with coarse, compact ridges.

These have abundant Lower Cretaceous analogues, such as Cicatricosisporites, and specimens found in situ within fertile plant organs associated with the ferns Ruffordia, Pelletieria, and Schizaeopsis (e.g., [START_REF] Hughes | Descriptions of schizaeaceous spores taken from Early Cretaceous macrofossils[END_REF]. Lygodium spores are

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trilete, and can be tuberculate, verrucate or reticulate (Tryon and Lugardon, 1991). Thus, Early Cretaceous verrucate spores have often been compared to the genus Lygodium (e.g., in [START_REF] Delcourt | Les spores et grains de pollen du Wealdien du Hainaut[END_REF][START_REF] Bolchovitina | Fossil and contemporaneous spores of the Family Schizaeaceae[END_REF]Ivanova in Samoilovitch and Mtchedlishvili, 1961;[START_REF] Fensome | Taxonomy and biostratigraphy of schizaealean spores from the Jurassic-Cretaceous boundary beds of the Aklavik Range, District of Mackenzie[END_REF].

Mesozoic spores comparable to Lygodium have only been found in situ in Jurassic rocks in northern England (Van Konijnenburg-Van Cittert, 1981), but they have a reticulate sculpture. They have been encountered in sorophores from Turonian deposits in New Jersey, USA [START_REF] Gandolfo | Sorophores of Lygodium Sw. (Schizaeaceae) from the Late Cretaceous of New Jersey[END_REF], and compared to the extant species Lygodium palmatum (Bernh.) Sw. on account of their trilete, psilate spores, among other characters. Dispersed spores are much more abundant. [START_REF] Bolchovitina | Fossil and contemporaneous spores of the Family Schizaeaceae[END_REF] produced a comprehensive monograph on fossil and extant spores of the former family Schizaeaceae (now Schizaeales), in which she listed 62 species of extant Lygodium [START_REF] Smith | A classification for extant ferns[END_REF] considered that the Lygodiaceae comprise only about 25 species), remarked on and illustrated 27 of these, and listed, commented, described or re-described, and illustrated 46 species of dispersed fossil spores referable to Lygodium.

The spores of the extant species are usually described according to their shape, thickness of exine, colour and sculpture. The last of these may vary within a species, such as in L. flexuosum (L.) Sw. and L. japonicum (Thunb.) Sw., which produce smooth to slightly tuberculate spores. However, when a species also displays a coarser sculpture, several authors (e.g., [START_REF] Couper | British Mesozoic microspores and pollen grains: a systematic and stratigraphic study[END_REF][START_REF] Dettmann | Spore morphology of Anemia, Mohria, and Ceratopteris (Filicales)[END_REF] have considered associated smooth spores to be immature forms. Spore polymorphism has been recognized in a few anemiaceous species [START_REF] Dettmann | Spore morphology of Anemia, Mohria, and Ceratopteris (Filicales)[END_REF], but it would seem that in general mature spores of extant species of Lygodium can be well separated on the basis of their sculpture [START_REF] Bolchovitina | Fossil and contemporaneous spores of the Family Schizaeaceae[END_REF]Tryon and Lugardon, 1991; see also SEM micrographs of Lygodium spores in the Cornell University website).

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Hence, most of the morphological clusters of the verrucate spore species displayed by PCA2 (Fig. 9) could well also have biological significance. Specimens assigned to Concavissimisporites uralensis, which are defined by their weak sculpture, could in fact be immature forms of other species of Concavissimisporites. The same applies to Trilobosporites aornatus, which could comprise immature specimens of T. aequiverrucosus or T. canadensis.

Intermediate forms such as specimen K28.3 (Fig. 6G) would support this suggestion. The most abundant verrucate spore in the Angeac palynomorph assemblages, Concavissimisporites montuosus, is similar to the spores of the extant species Lygodium circinatum (Burm. f.) Sw., in that both display quite well spaced, massive verrucae. Specimens assignable to the genus Trilobosporites are somewhat similar to the spores of Lygodium merrillii Copel., which display coarse distal muri that appear as a thickening of the exine in apical regions on the proximal face. However, no spores of extant species of Lygodium seem to develop valvae, which is all the more reason to maintain Trilobosporites as a separate genus for fossil spores with this character. Some species of Concavissimisporites, such as Concavissimisporites exquisitus, also do not seem to have any modern analogues.

Palaeoenvironmental implications

The great abundance of verrucate spores in the assemblages suggests the presence of ferns showing a strong affinity with species of the extant genus Lygodium, a pantropical climbing schizaealean, concentrated especially in Malaysia and Central America [START_REF] Kramer | The Families and Genera of Vascular Plants. I. Pteridophytes and Gymnosperms[END_REF]. These ferns are typical of warm, humid environments. Their fossil analogues may well have grown along riverbanks or in understories of forests (Van Konijnenburg-Van Cittert, 2002). The composition of the Angeac palynological assemblages is consistent with the previous interpretation of the depositional environment at this site as a et al., 2012). Nevertheless, this is attested by the relative abundance in unit An2 of brackish ostracods such as Fabanella boloniensis [START_REF] Schudack | Ostracod associations (marine and nonmarine) from the Lower Cretaceous of the Iberian chain (eastern Spain) and their biostratigraphical potential[END_REF] and the presence at different levels (An3-4, An1) of a few benthic foraminifers (Ammocycloloculina sp., Trocholina odukpaniensis) and echinoid or bryozoan debris (Néraudeau et al., 2012 and unpublished data).

No palaeoenvironmental variation has previously been noted for the period of deposition of the sediments comprising units An5-An2. However, the relative composition of Angeac palynological assemblages changes from An4/5 up to An2, the abundance of verrucate spores clearly increasing at the expense of Classopollis, which diminishes greatly (Fig. 7). The verrucate spores recovered from unit An2 appear to be generally larger than the specimens recovered from the older units An3 and An4 (Fig. 8).

From a sedimentological perspective, the succession of green clay (An5), dark clay (An4) and calcareous conglomerate (An3), and then of silty limestone (An2) and fine sand (An1) implies two successive sequences of increasing energy in the depositional environment, which suggests progressive flooding of the swamp by a river nearby, ending with the establishment of a floodplain. This environmental change could explain the increase of the abundance of verrucate spores up-section, ferns being generally more abundant than gymnosperms in a floodplain [START_REF] Coiffard | Early angiosperm ecology: evidence from the Albian-Cenomanian of Europe[END_REF][START_REF] Coiffard | Early Cretaceous angiosperm invasion of Western Europe and major environmental changes[END_REF].

Conclusion

Our taxonomic study of the Angeac palynological assemblages provides a rare insight into French palynofloras of Berriasian-Valanginian age. They have previously only been

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documented from deposits in the Jura mountains, the Vocontian Basin, and on Oléron island [START_REF] Lantz | Etude des spores et pollens d'un échantillon purbeckien de l'île d'Oléron[END_REF][START_REF] Taugourdeau-Lantz | Un aperçu de l'environnement végétal pendant l'épisode régressif du Berriasien terminal dans le Jura méridional (France)[END_REF][START_REF] Médus | Palynologie des niveaux émersifs du Berriasien du Jura. Comparaison avec des spectres de Sardaigne et des Pyrénées[END_REF][START_REF] Kujau | Reconstructing Valanginian (Early Cretaceous) mid-latitude vegetation and climate dynamics based on spore-pollen assemblages[END_REF], but the Angeac assemblages seem more continental not only because of the abundance of verrucate spores but also owing to the scarcity of marine indicators in the preparations.

All of the verrucate spores are attributable to either Concavissimisporites or Trilobosporites, which in turn are believed to have been produced by ferns showing strong affinities with extant Lygodium, a fern genus that typically inhabits warm, humid environments. These ferns could well have grown on the margins of the swamp envisaged by [START_REF] A N U S C R I P T A C C E P T E D Accepted Manuscript | The Hauterivian-Barremian lignitic bone bed of Angeac (Charente, south-west France): stratigraphical, palaeobiological and palaeogeographical implications[END_REF] and been widely dispersed on the developing floodplain.

Despite the important work of [START_REF] Bolchovitina | Fossil and contemporaneous spores of the Family Schizaeaceae[END_REF][START_REF] Bolchovitina | Distribution of Schizaeaceous spores in Jurassic and Lower Cretaceous deposits of Eurasia[END_REF], in the light of recent research on modern representatives of the Lygodiaceae a taxonomic revision of Lower Cretaceous lygodiaceous spores in the Northern Hemisphere is now needed in which both their biological affinities and stratigraphic distribution are taken into account.

  All of the palynological preparations and the megaspores illustrated are housed in the Geological Institute of the University of Rennes 1, under collection numbers IGR-PAL-5735--5753 for the palynological slides, and IGR-PAL-5780-IGR-PAL-5784 for the megaspores illustrated.

  ), verrucate spores such as Concavissimisporites montuosus (Döring) Fensome (Fig. 5J, K, M), Concavissimisporites apiverrucatus (Couper) Döring (Fig. 5A, B), Concavissimisporites exquisitus (Singh) Fensome (Fig. 5C, D) and Trilobosporites canadensis Pocock (Fig. 6H), the papillate spore Pilosisporites trichopapillosus (Thiergart) Delcourt et Sprumont (Fig. 4P), the hilate spore Aequitriradites verrucosus (Cookson et Dettmann) Cookson et Dettmann (Fig. 4G), and various smooth walled spores belonging to the genera Cyathidites, Deltoidospora, and Gleicheniidites (Fig. 4H, J). Freshwater algae such as Ovoidites spriggi (Cookson et Dettmann) Zippi (Fig. 4N) are relatively common, but no more dinoflagellate cysts were encountered during the reexamination.An2 yielded an assemblage of 16 species (Fig.3), numerically and taxonomically dominated by verrucate spores (Fig.7). It is the only assemblage in which there are no Classopollis pollen, the gymnosperm component being represented by only two inaperturate pollen grains referable to Araucariacites australis Cookson. Eighteen species were recovered from the An3 unit. Verrucate spores are less diverse than in the An2 unit with eight species, but the gymnosperms are better represented, with numerous specimens referable to M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT Classopollis torosus (Reissinger) Couper, and bisaccates such as Abietineaepollenites sp.

  The sample from the boundary between An3 and An4 (preparation MFP422) also proved to contain a few specimens of a form tentatively recorded here as Minerisporites sp.(Fig.11C-F). Apart from a couple specimens that are currently unidentifiable without recourse to examination under an SEM, all other megaspore remains are too damaged or fragmentary to be positively identified: among these are possible representatives of Paxillitriletes. Associated with the megaspores in MFP422 are isolated, comparatively large specimens of the freshwater algae Schizosporis reticulatus Cookson et Dettmann emend Pierce and Ovoidites spriggii (Cookson et Dettmann) Zippi. Most of the specimens of Erlansonisporites (Figs. 10E, F, 11A, B) are closely similar to

  Concavissimisporites and Trilobosporites. They are morphologically well separated, principally on the account of their size, the relative size of their verrucae, and the variation in the dimensions of verrucae on a specimen. Three species of Trilobosporites, and eight species of Concavissimisporites have been identified, along with several intermediate forms. Except

  fact, wider ranges. In any case, they do not seem to have encountered many verrucate spores, and their species of Trilobosporites are quite different from those recovered from Angeac.Although most of the palynomorphs identified herein indicate a stratigraphic distribution that is not confined to the lowest Cretaceous, previous observations in Néraudeauet al. (2012, p. 5) and our new data suggest that the Angeac assemblage is more likely to have been deposited during the Berriasian-early Valanginian rather than in the Hauterivian-Barremian, as previously stated.

  the absence of marine dinoflagellate cysts apart from the single specimen identified as Odontochitina imparilis, suggests very limited connection to the sea (Néraudeau

Acknowledgements

We are very grateful to E. Masure for making available the palynological preparations for analysis and for her useful insight concerning dinoflagellate identification. We also thank L. Londeix, who processed an additional sample from Angeac unit An2 at Bordeaux University. We are grateful to the Audoin Society for access to the quarry, to R. Allain, who took time to show the Angeac excavation site to FP and discuss the latest discoveries there, and to the referees for their helpful comments on the manuscript.

Figure captions

Soubran is mentioned in the introduction (Section 1), but is located about 50 km south-east of Royan, and is not, therefore, indicated on the map. GPS coordinates of the site: latitude: 45° Erlansonisporites sp. in Batten (1975, pl. 13, fig. 6) Erlansonisporites sp. in Batten (1975, pl. 13, fig. 6)


Fatiha Elhouiti
email: f.elhouiti@lagh-univ.dz

Djilali Tahri

Djalila Takhi

Mohamed Ouinten

Christian Barreau

Marie-Noëlle Verdal-Bonnin

Isabelle Bombarda

Mohamed Yousfi

Variability of composition and effects of essential oils from Rhanterium adpressum Coss. & Durieu against mycotoxinogenic Fusarium strains

Keywords: Essential oils, Rhanterium adpressum, Antifungal activity, Fusarium culmorum, Fusarium graminearum, Mycotoxin production

Flowers extract showed strong inhibitory activity, with very interesting concentrations of IC50 and MIC for both tests on solid and liquid medium. The effect of these oils on the production of type B trichothecenes (TCTBs) was evaluated, showing a significant inhibitory effect on TCTBs production, for both extracts (leaves and flowers). The rates of inhibition were 66-97 and 76-100% of FX, 3-ADON and 15-ADON, respectively. The inhibition of fungal biomass and the production of TCTBs depended on the used concentration of the essential oils. These results suggest that the essential oils from R. adpressum are able to control the growth of the tested strains and their subsequent production of TCTB mycotoxins.

Introduction

Algeria offers an important and unexplored plant potential. In the studies of natural resources, several plant substances have been identified and have been proven to be useful for therapeutic purposes. In this context, the unexplored antifungal and antimycotoxigenic activities of the essential oils (EO) of Rhanterium adpressum Coss. & Durieu, have been studied. This spontaneous Asteraceae, commonly called Aarfadj, is found in different areas in southern Algeria [START_REF] Chehma | Catalogue des plantes spontanées du Sahara septentrional Algérien[END_REF]. It is often used by the local population for tanning and as an antidiuretic in folk medicine [START_REF] Bouheroum | Terpenoids and triynepoxide from the aerial part of Rhantherium adpressum[END_REF].

Many diseases affecting plants are of fungal origin. Among these, and one of the most damaging, is ''Ear Fusariosis''. It is widespread in small-grain cereals such Abstract The antifungal potency of the essential oils of Rhanterium adpressum was evaluated against four mycotoxigenic strains of the genus Fusarium. The essential oils were obtained, separately, by hydro-distillation of the aerial parts of R. adpressum (leaves and flowers). The parts were collected during the period of bloom (3 months) for 3 years. The GC-MS analysis revealed thirty-six compounds for the essential oils, divided into four classes of chemical compounds, with variable percentages according to the month of extraction. The monoterpene hydrocarbons form the main class in these oils. On the other hand, the highest percentages of the oxygenated compounds are observed in the samples collected during the month of May. The direct contact method was used to evaluate the antifungal activity of the essential oils. The activity can be attributed to their relatively high composition of oxygenated monoterpenes.

1 3 as wheat, barley, oats and corn. This disease causes severe symptoms on different parts of the affected plants and can provoke necrosis of the ears. Thus, it generates significant quality and yield losses, and may lead to the loss of all crops [START_REF] Pirgozliev | Chemical composition and the nutritive quality of different wheat cultivars for broiler chickens[END_REF].

The main phytopathogenic agents responsible for this fusariosis are the ascomycete fungi belonging to the genera Fusarium and Microdochium, including Fusarium graminearum and Fusarium culmorum. During the infection, these phytopathogenic fungi, with mycotoxigenic potential, can produce two major families of mycotoxins: trichothecenes B (TCTBs) and zearalenone (ZEN) [START_REF] Merhej | The pH regulatory factor Pac1 regulates Tri gene expression and trichothecene production in Fusarium graminearum[END_REF].

There are few studies on secondary metabolites of Ranthterium species mentioned in scientific literature [START_REF] Bouheroum | Terpenoids and triynepoxide from the aerial part of Rhantherium adpressum[END_REF][START_REF] Hamia | Chemical composition and antioxidant activity of the essential oil and fatty acids of the flowers of Rhanterium adpressum[END_REF][START_REF] Kala | Composition of the essential oil of Rhanterium adpressum Coss. and Durieu from Algeria[END_REF]. Organic extracts (ethyl acetate) from Ranthterium adpressum flowers and aerial parts of Rhanterium suaveolens were found to offer a potential source of antioxidants [START_REF] Boussoussa | Effect of different solvent polarity on extraction of phenolic compounds from Algerian Rhanterium adpressum flowers and their antimicrobial and antioxidant activities[END_REF][START_REF] Bouaziz | Polyphenols content antioxidant and antimicrobial activities of extracts of some wild plants collected from the south of Tunisia[END_REF]. The essential oils extracted by hydro-distillation from the aerial parts of the plant showed antifungal activity against Fusarium oxysporum [START_REF] Houiti | Inhibition of Fusarium oxysporum f. sp. albedinis by essential oils of flowers and stems of Rhanterium adpressum[END_REF].

In this work, the inhibition of fungal biomass using essential oils extracted from R. adpressum, the kinetics of mycelial growth and the rate of inhibition in the presence of different concentrations of EOs were studied. In addition, the inhibition of TCTBs production by the EOs was examined. Eventually, this work will provide solutions for the preservation of food by proposing natural plant molecules that can serve as antifungal agents. These products could replace chemical fungicides that could be used to, partially or totally, inhibit the growth of fungi causing deleterious effects to economically important plant crops such as wheat, barley or corn.

The purpose of this work was to study the chemical composition of the essential oils (leaves and flowers) extracted from plant samples collected over 3 months, during 3 years. On the other hand, the antifungal and mycotoxigenic properties against four phytopathogenic and mycotoxinogenic isolates of the genus Fusarium (two F. graminearum and two F. culmorum) were assessed. The effects of the two EOs were evaluated on three major parameters:

-The kinetics of mycelial growth, in solid medium; -The rate of inhibition of the mycelium, by two methods (by the method of direct contact, and in a liquid medium by, the microdilution technique); -The inhibition of the production of mycotoxins type B trichothecenes by UPLC analysis, to quantify the accumulation of mycotoxins .

Materials and methods

Plant material and extraction of the essential oils

The aerial parts of [START_REF] Hamia | Chemical composition and antioxidant activity of the essential oil and fatty acids of the flowers of Rhanterium adpressum[END_REF]).

The freshly collected samples were dried at room temperature, away from light and humidity. The plant was then divided into two parts: the flowers and the stems. The essential oil was obtained by hydro-distillation during 6 h, using a Clevenger type apparatus. The obtained oil was treated by anhydrous sodium sulfate, filtrated then stored at +4 °C, until analysis.

Analysis of essential oils

The separation of the compounds of the EO was performed by Agilent technologies 7890A gas chromatograph (GC), equipped with a flame ionization detector (FID), with a HP5 capillary column (30 m × 0.32 mm, film thickness 0.40 μm). The oven temperatures program was as follows: 2 min at 80 °C; from 80 to 200 °C at 5 °C/min; 5 min at 200 °C; then from 200 to 260 °C at 20 °C/min; followed by 5 min at 260 °C. Detector and inlet temperatures were 280 °C. Hydrogen was used as a carrier gas at a constant flow of 1 mL/min with a split ratio 70/1. The injections were 1 μL of essential oil in methylene chloride (50 mg of EO in 1 mL CH 2 Cl 2 ).

The GC-MS analysis was performed using an Agilent technologies gas chromatography 7890A equipped with a HP5MS capillary column (30 m × 0.25 mm × 0.25 μm), and a mass detector MS 5975C VL MSD operated in EI mode. Helium was used as a carrier gas at a flow rate of 1 mL/min, split 50:1. The oven temperature program was as follows: 2 min at 80 °C; from 80 to 200 °C at 5 °C/ min; 5 min at 200 °C; then from 200 to 260 °C at 20 °C/ min; followed by 5 min at 260 °C. Detector and inlet temperatures were 280 °C. Identification of components was based on comparison of their mass spectra with those of WILEY and NIST Libraries, as well as on comparison of their retention indices with literature [START_REF] Adams | Identification of essential oil components by gas chromatography/mass spectrometry[END_REF][START_REF] Benzo | Determination of the threshold odor concentration of main odorants in essential oils using gas chromatography-olfactometry incremental dilution technique[END_REF]).

3

Fungal strains

In this study, four mycotoxinogenic strains of the genus Fusarium were tested for antifungal activity. These strains are known to cause spoilage of cereals. Two isolates of F. graminearum from the INRA-MycSA laboratory (PH-1, NRRL 31084, originally isolated from Michigan) from chemotype and CBS 185.32 (Centraal Bureau voor Shimmelkulturen,Netherlands) from the chemotype DON/15-ADON coded INRA 349. Two isolates of F. culmorum were also tested. These isolates were collected in Oued Smar and Rouiba (North of Algeria), and belong to the Mycothèque Touati-Hattab S. (one isolate of the chemotype NIV/FX coded T5; the other from the chemotype DON/3-ADON-coded BD17). All these strains have a high ability to produce Type B trichothecenes (TCTBs).

Antifungal activity

The EOs from June 2013 samples (leaves and flowers) were chosen for the tests of activity because they presented the highest yields. Various methods exist to access the antifungal activity of essential oils [START_REF] Corp | Aromatic plants of tropical Central Africa. Part XXXII. Chemical composition and antifungal activity of thirteen essential oils from aromatic plants of cameroon[END_REF]. The effects of our EOs were evaluated regarding two major parameters:

-Kinetics of mycelial growth in solid medium.

-Rate of mycelium inhibition, which was evaluated by two methods: in solid medium by the method of direct contact, and in liquid medium by the microdilution technique using 96-well microplates.

To inoculate the solid and liquid culture media, a 5-mm diameter agar disk containing mycelium was transferred onto the center of PDA medium plates (Potato Dextrose Agar), using a pure culture prepared beforehand. Incubation was carried out at 25 ± 2 °C for 6 days [START_REF] Collin | Huiles essentielles et extraits 'micro-ondes[END_REF]. Another liquid medium (CMC, Carboxymethyl cellulose) was used to promote the production of spores which serves as an inoculum for the synthetic liquid culture medium (LSM, liquid synthetic medium).

Kinetics of fungal growth

Different concentrations of the EOs, ranging from 0.5 to 20 μL/mL, were added to the culture medium. Afterwards, the kinetics of the fungal growth was determined by measuring the diameter of the growth daily at the same hour.

Antifungal activity on solid medium

The effect of the EOs on mycelial development was evaluated using the direct contact method by calculating the inhibition percentage of the mycelium after 7 days of incubation at 25 ± 2 °C [START_REF] El Ajjouri | Activité antifongique des huiles essentielles de Thymus bleicherianus Pomel et Thymus capitatus (L.) Hoffm. & Link contre les champignons de pourriture du bois d'oeuvre[END_REF][START_REF] Remmal | Improved method for the determination of antimicrobial activity of essential oils in Agar medium[END_REF][START_REF] Satrani | Composition chimique et activité antimicrobienne des huiles essentielles de Satureja calamintha et Saturija alpina du Maroc[END_REF].

1/5th-1/200th dilutions of the EOs were prepared in an agar solution (2%). In tubes, each containing 13.5 mL of sterile PDA medium (45 °C), 1.5 mL of each essential oil dilution was added to obtain final concentrations ranging from 0.5 to 20 µL/mL. The tubes were then shacked and poured into Petri plates. Witnesses, containing the medium and agar solution without EO were prepared and used as controls. The Petri plates were inoculated by putting an agar disc containing mycelium taken from the periphery of the thallus derived from a Fusarium culture of 7 days on PDA. A Fusarium culture on PDA medium without extract was used as a control. The cultures were incubated in the dark at 25 ± 2 °C for 7 days. The antifungal activity was determined by comparing the mycelial growth after treatment, with the mycelial growth of the control, then by calculating the percentage of mycelial inhibition.

Antifungal activity on liquid culture medium

The antifungal properties of the EOs were accessed by determining the rate of mycelium inhibition on liquid medium by the microdilution technique, using 96-well microplates. 10 μL of inoculum with a concentration of 10 6 spores/μL of each fungal species were diluted in a liquid synthetic culture medium (LSM). After inoculation, the microtiter plates were treated with appropriate doses of the EOs to obtain concentrations ranging from 0.25 to 10 μL/mL per well [START_REF] Fleurat-Lessard | In vitro large screening of bioactive compounds of botanical origin for antifungal activity against two mycotoxigenic fungi of wheat: Aspergillus westerdijkiae and Fusarium graminearum[END_REF]. Each strain is tested on the same microplate for different concentrations, hence the use of a transparent adhesive film to close the wells (avoid mixing due to volatility). Then, the whole is left to grow for 4 days, at 25 °C in total darkness. A series of repetition are made, three wells (repetitions) for each concentration as well as for controls.

The assessment of mycelial growth is determined by comparing the fungal growth with and without EOs (controls). The antifungal activity was evaluated by measuring the absorbance at 630 nm immediately after the period of Incubation by a simple spectrophotometric reading (microplate reader Bio-TEK®instruments ELX 800, Germany).

The IC50 (50% inhibitory concentrations) and MIC (minimal inhibitory concentrations) were determined for both extracts (leaves and flowers) against all tested strains. 1 3

Analysis of mycotoxin production for the treated fungi

The same extracts (June 2013) were chosen for this analysis. The TCTBs were extracted from 4 mL of liquid culture medium, recovered after 4 days of incubation, and centrifuged to remove the mycelium, to which two volumes of ethyl acetate were added. After mixing in a vortex (1 min) and decantation, 2 mL of the supernatant were evaporated under a stream of nitrogen (at 50 °C). The dry residue was recovered in 1 mL of methanol/water mixture (50/50 v/v). Subsequently, it was filtered through a 0.45 μm porosity filter and analyzed by ultra performance liquid chromatography (UPLC), coupled to a detector with diode arrays (DAD).

The quantification of TCTB was performed on a Shimadzu Prominence UPLC chain, equipped with two pumps LC-20 AD, a degasser DGU-20A3R, an auto sampler SIL-30 AC and a diode-array detector SPD-M20A (Shimadzu Scientific Instruments, France). Separation was achieved on a Kinetex 2,6U XB-C18 100 Å column (150 × 4.6 mm, 2.6 µm) (Phenomenex, France) maintained at 45 °C. Mobile phase consisted of water acidified with ortho-phosphoric acid to reach pH 2.6 (solvent A) and acetonitrile (solvent B). The following gradient was used for elution: 7% B for 1 min, 7-30% B for 6 min, 30-90% B for 2 min, 90% B for 2 min, 90-7% B for 1 min, and 6 min post-run equilibration with initial conditions. The flow was kept at 0.7 mL min-1 for a total run time of 18 min. The injection volume was 0.5 μL. The UV-Vis spectra were recorded from 220 to 550 nm and peak areas were measured at 230 nm. Quantification was performed using external calibration ranging from 6.25 to 100 µg/g prepared with standard solutions (Romer Labs, Austria).

Statistical analysis

All experiences were performed in triplicate. Data processing was carried out with Origin b9.3.226. The significant differences between mean values were analyzed by an ANOVA oneway with significant values at P < 0.05. The determination of IC50 and MIC was performed with dose-response curve with variable Hill slope at error probability ≤0.05.

Results and discussion

Variability of the chemical composition of essential oils

The GC/MS analysis of the essential oils allowed the identification of 36 compounds, for both parts leaves and flowers (Fig. 1). These compounds are divided into four classes (Table 1), with varying percentages according to the month of plant collection. Mainly the temperature of the region [START_REF] Holopainen | Ecological functions of terpenoids and climate changes[END_REF], where the flowering period occurs between the months of April and May, could influence this variability. As of June, the temperature increases. Thus, the percentages of the classes are high in the samples collected in May, where the average temperatures are between 21 and 24 °C. Monoterpene hydrocarbons form the main class of compounds in the extracts. As for the oxygenated compounds, their rates are at their maximum in the samples collected in May, especially in the flowers extract.

Compounds known as important antimicrobials [START_REF] Bajpai | Essential oils as antimicrobial agents[END_REF][START_REF] Dorman | Antimicrobial agents from plants: antibacterial activity of plant volatile oils[END_REF] are present in the extracts with more or less high concentrations, such as: α-Pinene (11.54%: leaves, 15.64%: flowers), β-Myrcene (21.59%: leaves, 12.81%: flowers), α-Thujene (0.33%: leaves, 0.22%: flowers), β-Pinene (13.91%: leaves, 9.92%: flowers), δ-Cadinene (1.16%: leaves, 1.20%: flowers), Linalool (6.04%: leaves, 6.07%: flowers), Geraniol (1.02%: leaves, 1.19%: flowers), α-Eudesmol (2.64%: leaves, 3.01%: flowers) [START_REF] Dorman | Antimicrobial agents from plants: antibacterial activity of plant volatile oils[END_REF][START_REF] Özek | Composition and antimicrobial activity of the oils of Ferula szowitsiana DC. from Turkey[END_REF][START_REF] Kim | Antibacterial activity of carvacrol, citral, and geraniol against Salmonella typhimurium in culture medium and on fish cubes[END_REF][START_REF] Böhme | Antibacterial, antiviral and antifungal activity of essential oils: mechanisms and applications[END_REF]). Antimicrobial activity is influenced even more by the high percentages of oxygenated compounds [START_REF] Bajpai | Essential oils as antimicrobial agents[END_REF]. Extracts from June showed a good antifungal activity, compared to the extracts of May and April, where the percentage of compounds is lower (Fig. 2).

Kinetics of fungal growth

The kinetics of the fungal growth of the selected strains (Fig. 3) in the presence of different concentrations of the EOs showed variable susceptibilities (P ≤ 0.05). The effect of leaves essential oil on mycelial growth is observed from a concentration of 2 μL/mL. The strains INRA 349 and INRA 812 of Fusarium graminearum were less affected than the Fusarium culmorum strains.

A remarkable sensitivity of all strains to the EO of flowers was observed. Starting from the concentration of 1 μL/ mL, the inhibition of growth was very important. As for leaves extract, the inhibition was at its maximum at the concentration of 10 μL/mL, and a total inhibition was recorded at 20 μL/mL. On the other hand, the extract of the flowers inhibited totally the growth of all strains at 10 and 20 μL/ mL, while the concentration of 4 μL/mL induced a strong inhibition of mycelial growth.

The two strains of F. graminearum appeared to be much more sensitive to the flowers extract, particularly the INRA 812 strain, where the concentration of 4 μL/ mL showed a very similar effect to those obtained with 10 and 20 μL/mL. Some of the results of [START_REF] Velluti | Impact of essential oils on growth rate, zearalenone and deoxynivalenol production by Fusarium graminearum under different temperature and water activity conditions in maize grain[END_REF], on the effect of essential oils of five plants on the growth of F. graminearum, showed a more or less significant effect at the concentration of 1 μL/mL. Similarly, 1 3 The two strains of F. culmorum also exhibited a high sensitivity towards the flowers extract starting from the concentration of 4 μL/mL. In previous studies, [START_REF] Terzi | In vitro antifungal activity of the tea tree (Melaleuca alternifolia) essential oil and its major components against plant pathogens[END_REF] also observed a high inhibition level at the concentration of 5 μL/mL with tea tree essential oil, terpinen-4-ol, 1.8-cineole and γ-terpinen. On the other hand, at the concentration of 2 μL/mL, the essential oil of Cinnamomum zeylanicum exhibited an important inhibition of the growth of F. culmorum [START_REF] Sumalan | Assessment of inhibitory potential of essential oils on natural mycoflora and Fusarium mycotoxins production in wheat[END_REF]. [START_REF] Kordali | Antifungal and herbicidal properties of essential oils and n-hexane extracts of Achillea gypsicola Hub-Mor. and Achillea biebersteinii Afan. (Asteraceae)[END_REF] reported low susceptibility of F. graminearum and F. culmorum towards essential oils of Achillea gypsicola and Achillea biebersteinii, despite the richness of these oils in oxygenated monoterpenes (94.42 and 83.98%).

Antifungal activity

Solid medium culture

The analysis of the antifungal activity of the extracts on solid medium revealed a variability between the effect of the EOs of the leaves and those of the flowers (Fig. 4). The concentration of 20 μL/mL inhibited completely fungal growth for both extracts, if it exceeds the MIC (Table 2). However, the significant difference (P ≤ 0.05) between the two EOs started from the concentration of 0.5-10 μL/mL. The concentration of 10 μL/mL of flowers extract presented the same effect as the concentration 20 μL/mL, against all strains, regarding the inhibition rate (94.04%). This was not the case with leaves extract, whose inhibition rate was less important than the previous one, and varied between strains. The effect of concentrations 0.5, 1, 2 and 4 μL/mL differed significantly between the two extracts. As in the case of flowers extract, the lowest concentration (0.5 μL/mL) generated high levels of inhibition, varying between 52 and 67%, for all strains. However, the inhibition levels of the same concentration for leaves extract did not exceed 50% (13-47%).

Liquid culture medium

The antifungal activity in liquid medium also allowed to note the variability of the inhibitory effect of the extracts (Fig. 5). The strains were more sensitive to the EO of flowers than the EO of leaves. The lowest concentration of the EO derived from flowers (0.25 μL/mL) was capable of inhibiting more than 70% of the fungal growth. However, the effect of this concentration was about half as much effective as the leaves essential oil. The concentration of 10 μL/mL for all extracts was found sufficient to inhibit completely the growth of the tested strains.

In both culture media, the T5 and INRA 812 strains showed great sensitivity towards the essential oil of flowers (inhibition rate >60% in solid medium, >70% in liquid medium from lower concentration).

The variability of antifungal potency of EOs on solid and liquid medium, and their effects on mycelial growth, depended on the used concentrations, and on the variation in the chemical composition of the tested extracts.

IC50 and MIC of the extracts

The susceptibility of the strains INRA 349 and 812 of F. graminearum to the extract of flowers is also visible by the calculation of IC50 and MIC (Table 2). The values of these two parameters are significantly different (P ≤ 0.05), between extracts of leaves and flowers on solid medium.

The IC50 and MIC presented interesting values in liquid medium with the extract of flowers, but not with a significant difference compared to those of the extract of leaves. According to [START_REF] Dobre | Preliminary research to develop active packaging for bakery products using essential oils[END_REF], F. culmorum and F. graminearum were highly sensitive to essential oils of clove, white thyme and oregano at the concentrations of 1 and 1.5 μL/mL PCMs. However, several studies discussed the ineffectiveness of fungicides in controlling the production of mycotoxins, where they are considered sometimes as additional stressors that rather stimulate the production of mycotoxins [START_REF] Magan | Relationship between growth and mycotoxin production by Fusarium species, biocides and environment[END_REF][START_REF] Jf | Pesticide use and mycotoxin production in Fusarium and Aspergillus phytopathogens[END_REF][START_REF] Milus | Evaluation of foliar fungicides for controlling Fusarium head blight of wheat[END_REF].

Effect of essential oils on TCTBs trichothecenes production

The TCTBs trichothecenes are considered as major determinants in the aggressiveness of F. culmorum and F. graminearum [START_REF] Wagacha | Fusarium culmorum: infection process, mechanisms of mycotoxin production and their role in pathogenesis in wheat[END_REF][START_REF] Muthomi | Characterization of Fusarium culmorum isolates by mycotoxin production and aggressiveness to winter wheat[END_REF][START_REF] Eudes | Impact of trichothecenes on Fusarium head blight [Fusarium graminearum] development in spring wheat (Triticum aestivum)[END_REF]. According to their production, the strains of F. culmorum and F. graminearum were divided into two chemotypes: the NIV chemotype, which includes isolates producing nivalenol and fusarenone X, and DON chemotype, including isolates producing DON and acetyldeoxynivalenol [START_REF] Wagacha | Fusarium culmorum: infection process, mechanisms of mycotoxin production and their role in pathogenesis in wheat[END_REF][START_REF] Bakan | Toxigenic potential of Fusarium culmorum strains isolated from French wheat[END_REF]. [START_REF] Bakan | Toxigenic potential of Fusarium culmorum strains isolated from French wheat[END_REF] have found that total trichothecenes range from 0.052 to 68.1 mg/kg in sixty strains of F. culmorum. Specimens of Polish wheat ears infected with F. culmorum and F. graminearum were contaminated with DON (100%) and 3-ADON (80%) at very high concentrations (30.4 and 29.54 mg/kg, respectively [START_REF] Visconti | Deoxynivalenol and 3-acetyldeoxynivalenol-mycotoxins associated with wheat head fusariosis in Poland[END_REF][START_REF] Bottalico | Toxigenic Fusarium species and mycotoxins associated with head blight in small-grain cereals in Europe[END_REF]. However, 140 specimens of Bulgarian wheat ears, infected with F. graminearum 3-ADON and 15-ADON, contaminated 2.1 and 0.7% of the total with concentrations in excess of 0.1 mg/kg [START_REF] Bottalico | Toxigenic Fusarium species and mycotoxins associated with head blight in small-grain cereals in Europe[END_REF][START_REF] Vrabcheva | First survey on the natural occurrence of Fusarium mycotoxins in Bulgarian wheat[END_REF]. Another study conducted by [START_REF] Touati-Hattab | Pathogenicity and trichothecenes production of Fusarium culmorum strains causing head blight on wheat and evaluation of resistance of the varieties cultivated in Algeria[END_REF], on Algerian populations of Oued Smar and Rouiba, revealed a correlation between the level of F. culmorum invasion and the amount of accumulated TCTB toxins. These authors recorded a production of DON varying between 89 and 340.5 μg/g and 3-ADON between 52.6 and 284.2 μg/g.

In our study, the inhibition of the production of the mycotoxins was analyzed in the presence of 0.25 μL/mL of each of our extracts (Fig. 6), where it was the average

Conclusion

In the present study, the variation of the composition of the essential oils of leaves and flowers of Rhanterium adpressum was noticed, as it was shown in the results of the composition. This variability could have been influenced by, among other elements, the edaphoclimatic factors (drought and rainfall). This difference has also been shown to be important in the antifungal activity against the four studied mycotoxigenic strains of the genus Fusarium. In addition, the activity of the flowers essential oil is evident in the results of the tests on solid and liquid media according to its very interesting concentrations of IC50 and MIC.

The two extracts exhibited a remarkable inhibition on the production of mycotoxins, where a 100% inhibition was recorded for the flowers extract on the three strains: F. culmorum T5, BD17 and F. graminearum INRA 812. In conclusion, the results of the antifungal power of the essential oils of Rhanterium adpressum and their inhibitory effect on the production of mycotoxins are very interesting. Other complementary work must be carried out on the different chemotypes characterizing the populations of this plant. In addition, the inhibitory compounds should be characterized, and their action levels localized, thus mechanisms of action for these essential oils could be proposed.

Fig. 1

 1 Fig. 1 Chromatogram of Rhanterium adpressum Coss. & Durieu essential oil using a HP-5 capillary column (leaves, above and flowers, below)

Fig. 2 Fig. 3 3 Fig. 4

 2334 Fig. 2 Examples of the effect of Rhanterium adpressum essential oils from different plant samples on the growth of Fusarium culmorum (T5 strain)

Fig. 5

 5 Fig. 5 Antifungal activity in liquid medium in the presence of different concentrations of the two extracts (leaves, on the left, and flowers, on the right)

Table 1

 1 The variation of the percentages of the chemical compounds classes according to the month of extraction for the two parts of the plant (leaves or flowers)

		Ap 11 Ma 11 Ju 11	Ap 12 Ma 12 Ju 12	Ma 13 Ju 13
	Leaves
	Monoterpene hydrocarbons	54.49	63.77	67.17 57.77	69.71	64.21 44.35	22.50
	Oxygenated monoterpenes	13.32	6.25	6.06	4.24	5.04	7.42	6.32	7.12
	Total monoterpenes	67.80	70.01	73.23 62.01	74.75	71.63 50.67	29.62
	Sesquiterpene hydrocarbons	3.70	4.10	2.61	7.29	5.14	1.58	5.50	5.30
	Oxygenated sesquiterpenes	6.42	5.09	4.91	5.30	2.42	4.66	1.97	2.38
	Total sesquiterpenes	10.12	9.20	7.52 12.58	7.56	6.24	7.47	7.68
	Flowers
	Monoterpene hydrocarbons	44.69	49.28	39.85 44.80	43.21	39.78 34.11	36.21
	Oxygenated monoterpenes	10.82	10.20	11.74	4.05	8.36	12.64 10.12	7.66
	Total monoterpenes	55.51	59.48	51.59 48.84	51.58	52.43 44.23	43.87
	Sesquiterpene hydrocarbons	3.96	3.95	4.98	2.54	3.37	1.46	2.55	3.14
	Oxygenated sesquiterpenes	7.06	6.53	8.57	6.30	7.93	6.01	2.93	1.64
	Total sesquiterpenes	11.02	10.48	13.55	8.83	11.30	7.47	5.49	4.79

Table 2

 2 The IC50 and MIC values in the presence of different concentrations of the two extracts for the strains of F. culmorum and F. graminearum

		Leaves		Flowers
		IC50 (µL/	MIC (µL/	IC50 (µL/	MIC (µL/
		mL)	mL)	mL)	mL)
	Solid culture medium
	BD17	0.70	12.81	0.27	9.30
	T5	0.90	13.41	0.32	6.71
	INRA 349	1.54	12.73	0.49	9.17
	INRA 812	1.42	10.41	0.28	5.23
	Liquid culture medium
	BD17	0.27	7.79	0.23	10.37
	T5	0.24	6.69	0.20	8.84
	INRA 349	0.26	8.05	0.24	5.85
	INRA 812	0.37	10.40	0.22	5.90

Acknowledgements

The authors would like to thank Touati Hattab S., Fleurat-Lessard, F. and all the members of the research team of MycSA unit, UR1264, National Institute of Agronomic Research, INRA, Bordeaux, France for their assistance in the realization of this study.