Quentin Wackenheim

Nicole Limondin-Lozouet

Julie Dabkowski

Larbi Boudad

Jean-François Berger

Reconstitution De

L A Dynamique

Paléoenvironnementale De L'holocène

Moyen Dans

L E Moyen

RECONSTRUCTING MID-HOLOCENE PALAEOENVIRONMENTAL DYNAMIC IN THE MIDDLE ATLAS (MOROCCO) INFERRED FROM NON-MARINE MOLLUSCS SUCCESSION OF THE AÏT SAID OU IDDER TUFA SEQUENCE n

Keywords: non-marine molluscs, palaeoenvironment, calcareous tufa, Morroco, Middle Holocene, Upper Pleistocene

The Mediterranean area is particularly sensitive to environmental changes. In Morocco, research undertaken over the past ten years has shown that continental sedimentary archives are useful to document Holocene environmental and climatic variability. However, palaeoenvironmental reconstruction using non-marine molluscs is in an early stage of research. This paper aims to present a new chronostratigraphical and malacological analysis from a Middle-Holocene and Upper Pleistocene tufa sequence in the Middle Atlas (Aït-Said-ou-Idder). Results highlight that the lower part of the sequence is dated from the Upper Pleistocene which corresponds to a fluvio-paludal model of deposition. Radiocarbon dates assign the upper part of the sequence to the Middle Holocene. During the Pleistocene phase, the environment is a reed swamp that turns to permanent body water. During the Middle Holocene, three main environmental phases have been identified. Between 6600 and 6100 cal BP, a mesophilic environment is characterised by open and dry areas with some paludal zone. Between 6100 and 5600 cal BP, a durable marshland developed. Between 5600 and 4600 cal BP, the paludal zone tended to disappear while an open and sclerophyllous environment developed. The malacological study provides important elements to the construction of a Holocene malacological framework in Morocco initiated at Blirh (Upper Moulouya). Both records identify a drastic switch to environmental aridification at the end of the Middle Holocene. By recording at the same time local evolution and regional trends and events, Aït Said ou Idder results provide material to understand environmental variability in north-eastern Morocco.

Les milieux méditerranéens sont des espaces particulièrement sensibles aux changements environnementaux. Au Maroc, les études réalisées ces dix dernières années ont démontré que les archives sédimentaires continentales sont d'excellents enregistrements de la variabilité des environnements holocènes. Cependant, les reconstitutions paléoenvironnementales à partir des assemblages de mollusques continentaux sont encore très peu développées. Ce travail présente une toute nouvelle étude chronostratigraphique et malacologique d'un tuf de l'Holocène moyen et du Pléistocène supérieur dans le Moyen Atlas (Aït Said ou Idder). Les résultats démontrent que la séquence datée du Pléistocène supérieur correspond à un tuf de type fluvio-palustre. La partie supérieure est un tuf de versant qui se dépose au cours de la seconde moitié de l'Holocène moyen. La séquence débute au cours du Pléistocène supérieur par une zone marécageuse à roselière à laquelle succède un plan d'eau pérenne dont la végétation avoisinante est plutôt ouverte. Au cours de la seconde moitié de l'Holocène moyen, trois grandes étapes paléoenvironnementales sont identifiées. Entre 6600 et 6100 cal BP, l'environnement mixte est dominé par des zones terrestres assez sèches et ouvertes mais comprend aussi un espace palustre. Puis, entre 6100 et 5600 cal BP une zone marécageuse pérenne se développe. Enfin, entre 5600 et 4600 cal BP l'assèchement et la réduction importante du milieu palustre permet l'augmentation de l'emprise des habitats ouverts et sclérophylles. L'étude malacologique apporte de solides éléments de construction du référentiel malacologique holocène au Maroc initié à Blirh (Haute Moulouya). Les deux séquences enregistrent un changement environmental caractérisé par une aridification à la fin de l'Holocène moyen. En enregistrant à la fois des évolutions locales mais aussi des dynamiques et évènement régionaux, les résultats d'Aït Said ou Idder fournissent un support pour comprendre la variabilité des paléoenvironnements du nord-est du Maroc.

Mots-clés : mollusques continentaux, paléoenvironnements, tuf calcaire, Maroc, Holocène moyen, Pléistocène supérieur Laboratoire de Géographie Physique, UMR 8591 CNRS, Université Paris 1 et UPEC, Meudon, France. Email: quentin.wackenheim@cnrs.fr Trajectoires, UMR 8215 CNRS & Univ. Paris 1, Nanterre, France Département de Géologie, Univ. Mohammed V, Rabat, Maroc Environnement, Ville et Société-IRG, UMR 5600 CNRS, Université de Lyon 2, France succession of Aït Said ou Idder to discuss the variability of past Holocene landscapes and to complete regional palaeoenvironmental records.

-STUDY SITE

-LOCATION

The 'Aït Said ou Idder' site is located in the north-east of Morocco, 42 km south from the city of Fez (fig. 1A) in the tabular calcareous Middle Atlas [START_REF] Michard A | Eléments de géologie marocaine[END_REF][START_REF] Martin | Le Moyen Atlas Central : étude géomorphologique[END_REF]. The deposit is developed on a south-facing slope at 1520 m a.s.l. (fig. 1B). The studied sequence appears nearby P5016 road, 2 km from Dayet Aoua Lake (33°38'49.50"N, 4°59'38.33"W). The exploitation of a former quarry exposed a 45 m-long section of about 3 to 4 m-high where a football field has been established (fig. 1C). At Aït Said ou Idder, tufa deposition is likely to form from calciumrich water flow from Sidi Mimoun spring 2 km-east from the site. Today, the drainage pattern and the spring are completely canalised for agricultural needs.

-STRATIGRAPHIC DATA

The 45 m-long tufa locating on a football field and called 'Football section' is divided into three sections: west, central and east (fig. 2). The earliest part of the deposit appears as indurated facies mainly developed in the eastern section of the quarry. Westward, hard levels are less exposed while upper soft deposits thicken. The cumulative stratigraphy of the succession reaches 5 m of thickness and comprises ten consistent lithostratigraphic units (including four grey levels labeled A, B, C and D) detailed below from bottom to top (fig. 2).

-Unit 10: A beige-orange indurated tufa with pluricentimetric oncolithes. This lithostratigraphic unit is visible only in the eastern section and is around 20 cm thick.

-Unit 9: Yellowish indurated layer constituted by encrusted reed reef called 'phytoherm' [START_REF] Pedley | Classification and environmental models of cool freshwater tufas[END_REF]; fig. 3A) filled by a sandy matrix. This unit represents about 50 to 100 cm.

-Unit 8: Yellowish-orange silty tufa alternating with centimetric rounded oncolithes and laminar layer. The scattered laminar structure is constituted of encrusted chironomid larva burrows (fig. 3B). Oncolithic levels show occasionally a graded bedding. This lithostratigraphic unit varies from 50 cm in the central section to 130 cm in the east section.

-Unit 7: White fine indurated and sometimes powdery tufa including disseminated centimetric oncolithes and laminas in a sandy matrix on its basis. The thickness of this unit is constant in the sequence and reaches 40 cm.

-Unit 6: Silty grey level with phytoclasts (i.e. encrusted plant debris). Grey level D is well developed and corresponds to the entire lithostratigraphic unit 6 (i.e. unit 6 = level D). The lower part of the unit is darker, rich in charcoals and contains decimetric fragments of a white indurated tufa (fig. 3C-D). The grey level is more

-INTRODUCTION

Tufas are freshwater carbonated deposits resulting from calcite precipitation and commonly rich in shells. In palaeoenvironmental studies, such formation presents one of the most reliable sedimentary conditions to achieve palaeoenvironmental analysis [START_REF] Dabkowski | High potential of calcareous tufas for integrative multidisciplinary studies and prospects for archaeology in Europe[END_REF][START_REF] Capezzuoli | -Decoding tufa and travertine (fresh water carbonates) in the sedimentary record: The state of the art[END_REF]. Land snails occupy a great variety of environments, but each species has a different tolerance determined by temperature, humidity, vegetation and calcium carbonate availability [START_REF] Puisségur | Mollusques continentaux quaternaires de Bourgogne[END_REF][START_REF] Welter-Schultes F | European non-marine molluscs, a guide for species identification[END_REF]. These observations promote continental mollusc assemblages as a reliable bioindicator of past environmental conditions and freshwater tufa as one of the best ecological context for mollusc development and shell preservation. As well as yielding rich assemblages of molluscs, tufa deposits also provide a high resolution palaeoenvironmental signal by the in situ preservation of land snail communities over thousands of years [START_REF] Preece R | Mapping snails in time: the prospect of elucidating the historical biogeography of the European malacofauna[END_REF].

In north-eastern Morocco, research undertaken over the past ten years has shown that continental sedimentary records are important archives of Holocene landscapes and most of the investigations were carried out on paludal and lacustrine deposits in the Middle Atlas [START_REF] Rhoujjati | Environmental changes over the past 29,000 years in the Middle Atlas (Morocco): a record from Lake Ifrah[END_REF][START_REF] Nourelbaït | -An 18 000 year pollen and sedimentary record from the cedar forests of the Middle Atlas, Morocco[END_REF][START_REF] Cheddadi R | -A History of Human Impact on Moroccan Mountain Landscapes[END_REF][START_REF] Cheddadi R | Human demography changes in in Morocco and environmental imprint during the Holocene[END_REF][START_REF] Campbell | -Environmental Drivers of Holocene Forest Development in the Middle Atlas, Morocco[END_REF][START_REF] Zielhofer | Atlantic forcing of Western Mediterranean winter rain minima during the last 12,000 years[END_REF] and on fluvial sequence in the Moulouya valley [START_REF] Zielhofer | -Flood frequencies reveal Holocene rapid climate changes (Lower Moulouya River, northeastern Morocco)[END_REF]. Calcareous tufas are common in the Mediterranean basin but, as they are often indurated, their fossil contents have scarcely been studied [START_REF] Pentecost | The quaternary travertine deposits of Europe and Asia Minor[END_REF][START_REF] Pedley | Tufas and travertines of the Mediterranean region: a testing ground for freshwater carbonate concepts and developments[END_REF]. The discovery of a non-indurated formation at Aït Said ou Idder in 2016 was then the opportunity to develop a multi-proxy palaeoenvironmental study on a calcareous tufa in Morocco. Here we present the results of the malacological analysis. Our research was integrated into the program PALEOMEX (INSU-INEE -Work Package 'Transect Maghreb'dir. J.-F. Berger), which discusses environmental and climatic processes and their consequence on Mediterranean societies during the Holocene. This period is characterised by abrupt climatic changes and a strong environmental variability (Alley et al. 1997;[START_REF] Mayewski | -Holocene climate variability[END_REF][START_REF] Roberts | -The mid-Holocene climatic transition in the Mediterranean: causes and consequences[END_REF][START_REF] Fletcher | Fragility of Western Mediterranean landscapes during Holocene rapid climate changes[END_REF][START_REF] Finné | Holocene hydro-climatic variability in the Mediterranean: A synthetic multi-proxy reconstruction[END_REF][START_REF] Roberts | -Mediterranean landscape change during the Holocene: Synthesis, comparison and regional trends in population, land cover and climate[END_REF] to which Mediterranean landscapes are particularly sensitive [START_REF] Faust | -The last 15 ka of environmental change in Mediterranean regions interpreting different archives[END_REF][START_REF] Fletcher | Orbital-and sub-orbital-scale climate impacts on vegetation of the western Mediterranean basin over the last 48,000 yr[END_REF][START_REF] Rohling | -Mediterranean climate and oceanography, and the periodic development of anoxic events (sapropels)[END_REF].

In Morocco, molluscan data have been reported mainly from archaeological contexts [START_REF] Taylor | -The Epipalaeolithic (Iberomaurusian) at Grotte des Pigeons (Taforalt), Morocco: a preliminary study of the land Mollusca[END_REF][START_REF] Hutterer R | Human manipulation of terrestrial gastropods in Neolithic culture groups of NE Morocco[END_REF][START_REF] Taylor | -Land mollusk middens[END_REF] and systematic palaeoenvironmental analysis using malacological assemblages are still in the early stage of research [START_REF] Limondin-Lozouet N | -Holocene molluscan succession from NE Morocco: Palaeoenvironmental reconstruction and biogeographical implications[END_REF]. Analyses of fossil molluscan assemblages are based on a palaeoecological approach that involves a broad understanding of modern malacofauna and its habitat. While most of the encountered species are shared with European faunas, identification and ecology of taxa specific to the area are carefully discussed in order to provide accurate palaeoenvironmental information. This paper aims to present the stratigraphy and the malacological Eastern section

M1 M2 M3 M4 M5 M6 M7 M8 M9 M15 M14 M13 M12 M11 M10 M21 M20 M19 M18 M17 M16 M24 M23 M22 M27 M26 M25 M28 M29 M30 M31 M32 9 8 7 6 4 D C 3 B 2 1
A 5 Reliable species of land snail have been selected to avoid reservoir effect (i.e. consumption of old carbon by the snails) in the shell [START_REF] Pigati | Radiocarbon dating of small terrestrial gastropod shells in North America[END_REF][START_REF] Douka | Radiocarbon dating of marine and terrestrial shells[END_REF]. Raw data are reported as uncalibrated radiocarbon years before 1950 and their equivalent ages expressed in calibrated years before present (BP) using OxCal 4.3 software and IntCal13 calibration curve [START_REF] Reimer | -IntCal13 and Marine13 radiocarbon age calibration curves 0-50,000 years cal B[END_REF] are given in table 1 andfigure 4. The chronological period covered by the age from unit 5 is large, due to the heterogeneity of the sample (a set of microcharcoals). Considering this uncalibrated age (4730 ± 370 BP), the Holocene chronology seems to show a date inversion, but after calibration the result is consistent with the rest of chronological features (fig. 4). The chronology implied gives confidence on the consistency of the measures obtained from shells.

Facies

However, if the ages are regularly distributed along levels A to C, the time interval between levels C and D is much shorter for a similar thickness of deposition. This discrepancy corresponds to the transition from ages obtained on charcoals (level D) to ages obtained on shells (levels C, B, A). However, the rhythmicity of the facies is homogeneous throughout the sequence. Therefore, a possible ageing effect on ages obtained on shells cannot be completely excluded.

-CHRONOSTRATIGRAPHICAL INTERPRETATION

Field observations allow us to identify two main phases of deposition: the lower and the upper part of the sequence, showing specific sedimentary facies and separated by an erosion surface (fig. 4).

The lower tufa comprises four lithostratigraphic units (10 to 7) which represent up to 3-4 m of the stratigraphy, visible in the eastern section of the profile. Several lateral variations from encrusted reed reef to sandy tufa are particularly noticeable (fig. 2: unit 9). Graded bedding and oncolithe layers imply energetic water flow. Sedimentary features indicate fluvial dynamics alternating spatially and temporally with a swampy environment. Indurated fine tufa, sandy oncolithic bed and phytoherm lithofacies correspond to a fluvio-paludal depositional model (sensu [START_REF] Pedley | Classification and environmental models of cool freshwater tufas[END_REF][START_REF] Pedley | Tufas and travertines of the Mediterranean region: a testing ground for freshwater carbonate concepts and developments[END_REF]. This lower part is attributed to the Upper Pleistocene as the lithostratigraphic unit 7 and is dated around 40000 a cal BP (tab. 1, fig. 4). expanded in the western and central sections (30 cm) whereas it is only 10 cm thick in the eastern part.

-Unit 5: Loose beige tufa where lenticular silty sands alternating with oncolithic and phythoclastic lithofacies occur. Decimetric to centimetric fragments of encrusted plant reefs are disseminated. The thickness of this level reaches 1 m in the central section and represents only 40 cm in the eastern section.

-Unit 4: Coarser grey tufa with reworked oncolithes and phytoclasts in a silty-sand matrix. Centimetric to decimetric tubular in situ encrusted tufa are locally present. The upper part of unit 4 corresponds to the silty grey level C (about 10 cm). In the west section, this unit reaches 30 cm whereas in the rest of the profile it is only represented by the grey level C.

-Unit 3: Coarsely stratified oncolithic and phytoclastic slightly indurated tufa beds alternating with silty levels. The light-grey silty level B (10 cm), only visible on the western and central sections, is present at the top of the unit. This unit is about 50-80 cm in thickness.

-Unit 2: Beige-orange granular tufa with phytoclasts (fig. 3E) in a silty-sand loose matrix. Stratification is diffuse and sometimes lenticular. The thickness of unit 2 is constant along the profile (60-70 cm).

-Unit 1: White-beige homogenous silty tufa containing a centimetric phytoclastic bed, scarse tufa debris and a silty grey level in the middle labelled A (10 cm). This unit is only present in the western section and reaches 40 cm of thickness.

-Unit 0: Modern soil, a silty perturbed level resulting of pedogenesis on the underlying tufa bed.. The thickness of the modern soil is quite constant (20-30 cm) except in the western section where it is reduced due to the erosion.

-RADIOCARBON DATING

Nine radiocarbon ages were obtained from charcoals and land snail shells (tab. 1). Three were obtained on three different charcoals sampled from the grey layer D. A set of micro charcoals extracted from a sedimentological sample from unit 5 provides one age. Five ages were obtained on samples of mollusc shells picked up from unit 7 (two samples) and from levels A, B and C (one sample each) . the tufa surface. Radiocarbon ages (layers A, B, C and D) indicate that the upper part of the tufa developed between 6800 and 4800 a cal BP (fig. 4). Consequently, the upper tufa episode formed during the second half of the Middle Holocene (fig. 4).

To sum up, the lower part of the sequence is an Upper Pleistocene fluvio-paludal tufa whereas the upper part is a Middle Holocene slope tufa (fig. 4). Unit overlays an eroded surface corresponding to the top of unit 7. Presence of tufa fragments from unit 7 reworked at the base of unit 6 support the interpretation of a strong erosion event between the two phases of tufa accumulation (fig. 3C). This sedimentary hiatus is also identified thanks to radiocarbon ages and corresponds to a time gap of about 30000 years. On [START_REF] Martin | Le Moyen Atlas Central : étude géomorphologique[END_REF] geomorphological map, a "recent" and an "older" tufa are shown; this partition The upper tufa comprises lithostratigraphic units 6 to 1 (including the four grey levels) which represent 2.50 m of the stratigraphy in the west section and around 2 m in the eastern section. All units have a silty matrix light colour from white beige to grey depending on its enrichment in organic matter and in the degree of postdepositional leaching. The encrusted tufa debris are relatively scattered and mainly distributed in level 5. Phytoclast levels correspond to granular levels of detrital tufa (units 1-3). The grey level D, very rich in charcoals and burnt tufa fragments in a very silty matrix, suggests a fire event. Overall, alternation of detrital tufa beds and silty levels is typical of perched spring-line tufa on a slope [START_REF] Pedley | Sedimentology of Quaternary perched springline and paludal tufas: criteria for recognition, with example from Guadalajara Province, Spain[END_REF]. These sedimentary facies suggest a mosaic of humid environments on the slope and the development of four periods of relative stabilisation of A unique specimen named 'Viviparidae' refers to an apex of Viviparus-like shell (tab. 2, sample M33). However, the genus was never observed in Africa and no argument supports further identification [START_REF] Van Damme | The freshwater Mollusca of northern Africa: distribution, biogeography and palaeoecology[END_REF][START_REF] Brown | Freshwater snails of Africa and their medical importance[END_REF].

Slope tufa

Fluvio-paludal tufa

The family range is chosen to name five similar specimens of the same unidentified Hydrobiidae (fig. 5: 27-28). Moroccan Hydrobids are indeed particularly rich and no extended work on their taxonomy exists [START_REF] Van Damme | -The status and distribution of freshwater molluscs[END_REF].

The generic appellation for Theodoxus sp. is adopted for a unique Neritidae operculum.

Although only Radix labiata (Rossmässler, 1835) occurs nowadays in Morocco [START_REF] Van Damme | The freshwater Mollusca of northern Africa: distribution, biogeography and palaeoecology[END_REF][START_REF] Taybi | -The freshwater malacological composition of Moulouya's watershed and Oriental Morocco[END_REF], the secure identification of this species requires complete shell specimens or anatomy. Radix material from the tufa is always fragmented, consequently, all Radix-like shells are called Radix sp.

Melanopsis species taxonomy stays unclear because of high intraspecific shell variations [START_REF] Glaubrecht | Mapping the diversity: Geographical distribution of the freshwater snail Melanopsis (Gastropoda: ? Cerithioidea: Melanopsidae) with focus on its systematics in the Mediterranean basin[END_REF][START_REF] Heller | -Conchometrics, systematics and distribution of Melanopsis (Mollusca: Gastropoda) in the Levant[END_REF]. Specimens from Aït Said ou Idder tufa are designated by Melanopsis cf. praemorsa (Linnaeus, 1758) because M. praemorsa represents actually a complex of species and also the most common species of the genus Melanopsis [START_REF] Van Damme | -The status and distribution of freshwater molluscs[END_REF].

-ECOLOGICAL CLASSIFICATION

Data are presented on distinct ecological and malacological diagrams [START_REF] Limondin-Lozouet N | Les mollusques continentaux[END_REF]. The ecological classification in Table 2 is elaborated from Puisségur's groups (1976). Taxa identified without ecological affiliation have been distributed in groups based on knowledge about their habitat and field observations. Geomitridae sp. 1 and sp. 3 are similar to modern Geomitridae observed near the site that occupied exposed herbaceous habitat. Furthermore, most of Geomitridae species in Morocco are considered as xerophilous and were formerly designated by the invalid genus "Xerophila". Cochlicella barbara and Rumina decollata are described as typical species of dry and open habitats (Welter-Schultes, 2012; Cadeval & Ozozco 2016). Moreover in Morocco, Rumina decollata is particularly common in open ground places is likely to correspond to the two phases identified in the football profile.

-MALACOLOGICAL SAMPLING AND IDENTIFICATION

Two sampling columns have been made in Aït Said ou Idder tufa to cover the entire sedimentary sequence (fig. 2). A total of 55 malacological samples of a constant volume of 5 litres has been undertaken continuously at 10 cm intervals with local adjustment to stratigraphy (fig. 2). The sediment has been wet sieved on a 500 µm mesh and the residues sorted under a microscope to extract shells. Shells and identifiable fragments have been counted to reach a minimum number of individuals for each sample [START_REF] Evans | Land snails in Archaeology. With special reference to the Bristish Isles[END_REF][START_REF] Limondin-Lozouet N | Les mollusques continentaux[END_REF].

In Morocco, modern non-marine molluscs are particularly diversified [START_REF] Seddon | Hotspots of Molluscan biodiversity: use of presence/absence data for prioritisation of conservation management in North Africa[END_REF]. Many Holocene taxa are shared with North-West European faunas [START_REF] Kerney | Guide des escargots et limaces d'Europe[END_REF][START_REF] Limondin-Lozouet N | -Holocene molluscan succession from NE Morocco: Palaeoenvironmental reconstruction and biogeographical implications[END_REF] but the taxonomy of some families remains unclear [START_REF] Rour | -Etat actuel des connaissances de la malacofaune terrestre du Maroc[END_REF][START_REF] Holyoak | -Evolution and taxonomy of the populations of Eremina (Gastropoda, Pulmonata, Helicidae) in Morocco[END_REF]. Several malacological guides and atlases have been employed to identify the most common land and freshwater molluscs [START_REF] Van Damme | The freshwater Mollusca of northern Africa: distribution, biogeography and palaeoecology[END_REF][START_REF] Kerney | Guide des escargots et limaces d'Europe[END_REF]Gloër, 2002;[START_REF] Welter-Schultes F | European non-marine molluscs, a guide for species identification[END_REF][START_REF] Cadeval | Caracoles y Babosas de la Peninsula Iberica y Baleares[END_REF]. Former Pallary's work [START_REF] Pallary | Première contribution à l'étude de la faune malacologique du Nord-Ouest de l'Afrique[END_REF][START_REF] Pallary | Deuxième contribution à l'étude de la faune malacologique du Nord-Ouest de l'Afrique. Supplément à 'La faune malacologique du Maroc' d'A. Morelet[END_REF][START_REF] Pallary | Troisième contribution à l'étude de la faune malacologique du Nord-Ouest de l'Afrique[END_REF][START_REF] Pallary | Quatrième contribution à l'étude de la faune malacologique du Nord-Ouest de l'Afrique. Deuxième supplément à la 'Faune malacologique du Maroc' d'A. Morelet[END_REF][START_REF] Pallary | Diagnoses d'une cinquantaine de mollusques terrestres nouveaux du Nord de l'Afrique[END_REF][START_REF] Pallary | Hélicidées nouvelles du Maroc. 2 e Partie[END_REF][START_REF] Pallary | Descriptions d'une nouvelle cinquantaine de mollusques terrestres nouveaux du Nord-Ouest de l'Afrique[END_REF][START_REF] Pallary | Compléments à la faune malacologique de la Berbérie[END_REF][START_REF] Pallary | -Complément à la faune malacologique de la Berbérie (suite)[END_REF] and recent literature have been consulted to identify specifically Moroccan species (Gittenberger & Ripkens, 1987;[START_REF] Seddon | -Taxonomy of five species of Helicopsis (Helicopsis) from Morocco (Gastropoda: Helicoidea)[END_REF][START_REF] Seddon | Problematic taxa from Morocco-the status of 'Helix argonautula' Webb & Berthelot, 1833, 'Helix renati' Dautzenberg, 1894 and 'Xeroleuca antoinei' Pallary[END_REF][START_REF] Van Damme | -The status and distribution of freshwater molluscs[END_REF][START_REF] Holyoak | -A Revision of the landsnail genera Otala and Eobania in Morocco and Algeria[END_REF][START_REF] Holyoak | -Evolution and taxonomy of the populations of Eremina (Gastropoda, Pulmonata, Helicidae) in Morocco[END_REF][START_REF] Kneubühler | Anatomical and phylogenetic investigation of the genera Alabastrina Kobelt, 1904, Siretia Pallary[END_REF]. Furthermore, modern shells have been collected by handpicking near the site to support the identification and ecological interpretation of fossil material.

-MOLLUSCAN ANALYSIS

In total 10169 shells have been extracted from the tufa succession. It comprises 42 taxa, including 26 terrestrial snails (tab. 2).

-IDENTIFICATION COMMENTS

A big Helicid species was found in six samples of the Holocene tufa (fig. 5: 1-4). These specimens fit with modern shells of Otala tingitana (Paladhile, 1875) collected near the site (fig. 5678) and present all morphological features given in the recent revision of the genus (Holyoak & Holyaok, 2017).

The Geomitridae family is the most problematic to identify because of the high number of species described during the 19 th and 20 th centuries and the entire lack of systematic revision [START_REF] Rour | -Etat actuel des connaissances de la malacofaune terrestre du Maroc[END_REF]. At Aït Said ou Idder, the abundant Geomitridae shells were separated in three morphologically homogeneous groups, respectively referred as Geomitridae sp. 1, Geomitridae sp. 2 and Geomitridae sp. 3. Geomitridae sp. 1 is the most 1 0 0 0 8 0 0 6 0 0 4 0 0 0 2 0 0 7 0 0 6 0 0 4 0 0 3 0 0 1 0 0 5 0 0

M32 M31 M30 M29 M28 M27 M26 M25 M24 M23 M22 M21 M20 M19 M18 M17 M16 M15 M14 M13 M12 M11 M10 M9 M8 M7 M6 M5 M4 M3 M2 M1 M32 M31 M30 M29 M28 M27 M26 M25 M24 M23 M22 M21 M20 M19 M18 M17 M16 M15 M14 M13 M12 M11 M10 M9 M8 M7 M6 M5 M4 M3 M2

Pisidium amnicum

• Pisidium sp. Fig. 7 : Diagramme de fréquence spécifique, profil 2. Les astérisques correspondent à des échantillons qui sont en-dessous du seuil de représentativité de 50 individus. two steps are identifiable in the profile 2 : the first seems to be drier (M33 & M34) while the second records the marsh expansion (M35 & M36).

Cochlicopa lubrica

• • • • Euconulus fulvus • • • • • Vitrea contracta • • • Otala tingitana • Vallonia pulchella * * * Truncatellina cylindrica * * * • • Vertigo pygmaea * • Lauria cylindracea * * * • • • Punctum pygmaeum * Oxyloma elegans * * * • • • • Vertigo moulinsiana
0 100 200 300 • Rumina decollata • Cochlicella barbara • • Geomitridae sp.1 Geomitridae sp.3 • • • • • • Truncatellina cylindrica • Vallonia costata • • • • • Vallonia pulchella • • • Otala tingitana • • Cochlicopa lubrica • • Lauria cylindracea • • • • • • • • Euconulus fulvus • • • Punctum pygmaeum • Vitrea contracta • • • • • • • Slugs Oxyloma elegans • • • • Vertigo antivertigo • • • • • • • • Vertigo moulinsiana • • Vallonia enniensis • • • Zonitoides nitidus • Sucinella oblonga • Vertigo angustior • Theodoxus sp. Melanopsis cf. praemorsa • • • • • • • Galba truncatula • • • Radix sp. • • • • • Anisus spirorbis • • Ancylus fluviatilis • Pisidium amnicum • • • Pisidium casertanum • • • Pisidium personatum • Pisidium subtruncatum • • • • • • Carychium minimum Samples Depth (cm) M32 M31 M30 M29 M28 M27 M26 M25 M24 M23 M22 M21 M20 M19 M18 M17 M16 M15 M14 M13 M12 M11 M10 M9 M8 M7 M6 M5 M4 M3 M2

-Malacozone 4: M4 to M10 (Unit 5)

In malacozone 4, the proportion of paludal and aquatic species increases at the expense of xerothermic taxa (fig. 6). Swampy taxa dominate (30 to 50 % of the species diagram). All swampy taxa found at Aït Said ou Idder are registered in this malacozone (fig. 8). Aquatics vary between 20 and 30 % of the species assemblages. The two hygrophiles Succinella oblonga and Vertigo angustior are absent. The slug occurrence is constant and reflects wet conditions (fig. 8). The xerothermic taxa Geomitridae sp. 1 and Cochlicella barbara decline and then disappear whereas aquatic bivalves Pisidium casertanum and Pisidium personatum appear. The occurrence of Vallonia pulchella, an open area taxa which likes humid conditions, is constant in the zone.

The malacological composition of this environmental phase reflects the expansion of a marsh environment with a water supply. In the upper part of the zone, this environment tends to be drained and transformed in a humid grassland.

-Malacozone 5: M11 to M16 (Top of Unit 5 + Unit 4)

In Malacozone 5, marsh and aquatic groups remain important, representing about 50 % and 30 % of the specific assemblage (fig. 6). This trend is particularly visible in the diagram of individuals where the sum of aquatic and marshes exceeds 90 % (fig. 6). All xerothermic, mesophilic and open environment taxa disappeared except the mesophilic taxon Euconulus fulvus, a tolerant snail that appreciates moist conditions (fig. 8). Despite its preference for wet meadows Vallonia pulchella which was well developed in the associations of the previous area (fig. 8), completely desappears. This temporary extinction implies the significant development of a swampy habitat. The slug proportion remains constant in the zone.

The environmental conditions deduced from the malacological assemblages highlight the optimum of a permanent marshy area. Malacological assemblages of this third zone particularly contrast with the previous one. They are characterised by the abundance in shells, high diversity and predominance of terrestrial molluscs (tab. 2 and fig. 6). Aquatic species, dominated by the ubiquitous Melanopsis cf. praemorsa, represent up to 40 % at the base of the zone but quickly decrease to 10-15 % of the assemblage (fig. 6). Xerothermic, open area and mesophilic groups comprise respectively 10 to 15 % of the assemblage diversity. In terms of individuals, xerothermic taxa reach more than 60 % of the assemblage (e.g. sample M36) mainly because of the expansion of Geomitridae sp. 1 (figs. 7 & 8). The occurrence of Geomitridae sp. 3 and Otala tingitana is a distinctive feature of the zone indicating the diversification of the xerophilous group. In total, eight species are present within hygrophilous and marshy groups and reach around 40 % of the diversity of the assemblages. Among those, Vertigo angustior, Vertigo moulinsiana and Vallonia enniensis are strong paludal demanding species, which suggest high humidity conditions and a well-developed marshy vegetation [START_REF] Kerney | Guide des escargots et limaces d'Europe[END_REF]. Their occurrence thus suggests the development of a small marsh.

Malacofauna is typical of an open and xerophilous vegetation with a permanent humid zone nearby. Following the ratio between aquatic/hygrophile/paludal and xerothermic species on ecological diagrams (fig. 6),

The malacological succession of zone 9 highlights a much drier terrestrial environment where vegetation is rather open and where sclerophyllous develop at the expense of swampy habitats.

-DISCUSSION

-AÏT SAID OU IDDER PALAEOENVIRON-MENTAL RECONSTRUCTION

Aït Said ou Idder tufa has been constructed during two main phases. This feature is supported by stratigraphic data, radiocarbon ages and by palaeoenvironmental reconstruction from malacofauna (fig. 9).

According to two radiocarbon ages obtained on shells from the lithostratigraphic unit 7, the first phase (units 10 to 7) is assigned with confidence to the Upper Pleistocene around 40 000 a cal BP (fig. 9). The absence of shells or of other datable material in units 10, 9 and 8 makes it difficult to assess a precise duration and start of this deposition phase. All the units of this phase show coherent lithofacies, rich in oncoliths and phytoherms, indicating a common fluvio-paludal environment. Additionally, no major hiatus is identified within this phase. It may thus correspond to a single stage of deposition. Such a fluviopaludal model is consistent with the geometry and extent of 'old travertine' mapped by [START_REF] Martin | Le Moyen Atlas Central : étude géomorphologique[END_REF]. Mollusc shells are preserved only in the upper part of this phase (fig. 8: Malacozones 1 and 2 in units 8 and 7 respectively) and point to an open environment with a persistent body of water. These data are correlated with the presence of large phytoherm reef and graded bedding in the oncolithic levels (units 8, 9 10). Finally, the Pleistocene record can be divided into two environmental steps: first, a rather dynamic fluvial environment (unit 10), then the development of a swampy reedbed area (units 9-8) which gradually opens up into a permanent body of water where the nearby environment is also open (fig. 8). Malacological and chronological Pleistocene data are not precise enough to allow a reliable comparison with contemporaneous regional records. Consequently, further discussion will focus only on the Holocene data.

During the second main phase of tufa edification, seven radiocarbon ages related to the second half of the Middle Holocene provide a reliable chronological framework for the upper part of the deposit. The erosion surface at the top of unit 7 and fragments of reworked tufa (from unit 7) in the base of unit 6 support an indisputable sedimentary hiatus. Considering the radiocarbon ages, this hiatus between the two phases of tufa deposition lasts ca. 30000 years (fig. 8). The seven rich malacozones defined (MZ 3 to MZ 9) in the Holocene part allow identifying three main palaeoenvironmental steps that are consistent with the different lithostratigraphic units and involved deposition processes (fig. 8).

The first step corresponds to unit 6 malacozone 3 and is characterised by a mosaic landscape composed of marshy and xerophilous open areas. According to radiocarbon Zone 6 malacological assemblage indicates the permanence of the marsh and the reappearance of wet grassland habitat on its margins, environmental conditions are similar to the malacozone 4. 4.3.2.5 -Malacozone 7: M20 & M21 (Unit 3) The Malacozone 7 highlights a dramatic palaeoenvironmental rupture in the succession showing the decrease and disappearance of aquatic and marsh species and the development of xerothermic taxa (fig. 6). This trend is particularly noticeable in the spectra of individuals where xerothermic species represent nearly 70 % of the population (fig. 6). They are mainly represented by Geomitridae sp. 1 and Cochlicella barbara, both typical of dry, exposed habitats.

Results from Malacozone 7 illustrate the disappearance of the marsh and the significant development of a drier habitat. 4.3. 8).

Low species diversity and the fact that 80 % of the malacological population is represented by a quasimono-specific group of aquatic molluscs suggest a phase of environmental instability that could correspond to a flooding episode. 4.3.2.7 -Malacozone 9: M28 to M32 (Unit 1) Malacozone 9 is characterised by rich malacological assemblages that show the progression of xerothermic and open area taxa. The sum of xerothermic, open and mesophilic species reaches 30 to 50 % of the assemblages (fig. 6). This malacozone is particularly rich in shells belonging to xerothermic species (i.e. Geomitridae sp. 1) which represent the largest part of the population. The reappearance of Geomitridae sp. 3 and Rumina decollata in the top part of the zone is noticeable of drier habitat development. These trends are characteristics of xerophilous vegetation. Swampy taxa are present (Oxyloma elegans, Vertigo antivertigo, Carychium minimum, Vertigo antivertigo) but their frequency, although more regular, remains low (fig. 8). Malacozone 9 is also richer in terms of aquatic species (7 taxa) but they comprise a small number of individuals representing about 20 % of the population (fig. 6). Slugs are reappearing with a low frequency.

succession shows an alternation between detrital tufa layers and grey levels.

The second environmental step concerns the development of a permanent wetland area (units 5 to 3) between at least 6100 and 5600 a cal BP. (MZ4 to MZ6, lasting about 600 years) according to radiocarbon ages from level B (fig. 8). The sediment thickness between level D and B represents 1.65 m. While the stratigraphy comprises a well-identified grey level (C) interstratified between two silty detrital tufa beds, the swampy environment recorded in the malacological succession is constant. Local variations on the extension of marshy habitats exist but the optimum of the swampy area is reached during the malacozone 5 which indicates particularly humid environmental conditions. dating, at the beginning and at the end of the level, this phase lasted about 500 years and corresponds to a period between 6209 cal BP and 6740 a cal BP. representing around 30 cm of thickness. This carbonaceous silty level (unit 6) is particularly rich in shells and shows a good species diversity. The xerophilous environment deduced from this step is consistent with the occurrence of a possible fire event recorded at the base of unit 6. Lithological observation and environmental reconstruction deduced from molluscs indicate a phase of stabilisation characterised by the development of terrestrial habitat. More arid climatic conditions could have stimulated the occurence of fires which may have contributed to opening up the vegetation.

During the next two environmental steps, lithological environmental Aït Said ou Idder phase (6700-6200 cal BP). During this period of the Middle Holocene, at Iffer and Sidi Ali, sedimentary data show a low level of the lakes explained by a low water input [START_REF] Rhoujjati | Significations paléoenvironnementales des dépôts du remplissage holocène du lac Iffer (Moyen Atlas, Maroc)[END_REF][START_REF] Zielhofer | Atlantic forcing of Western Mediterranean winter rain minima during the last 12,000 years[END_REF]. Furthermore, these elements are corroborated both by peaks of Poaceous pollen at Sidi Ali and Tiguelmamine, that imply an episode of rapid environmental opening [START_REF] Lamb | Vegetational response to Holocene climatic change: pollen and palaeolimnological data from the Middle Atlas, Morocco[END_REF][START_REF] Campbell | -Environmental Drivers of Holocene Forest Development in the Middle Atlas, Morocco[END_REF][START_REF] Zielhofer | Atlantic forcing of Western Mediterranean winter rain minima during the last 12,000 years[END_REF] and by an increase of temperatures modeled from Tiguelmamine pollen data (Chedaddi et al., 1998). The sum of these different regional records supports a period of aridity around 6500-6400 a cal BP which is also identified by an increase of semi-arid taxa in the pollen diagram of the Alboran Sea marine core (Fletcher & Sanchez-Goni, 2008). Aït Said ou Idder fire event which may have contributed to opening up the vegetation as recorded by the mollusc succession, can be explained by regional arid climatic conditions. During the Aït Said ou Idder phase of marshy expansion (6100-5600 a cal BP), more humid climatic conditions based on variations in oxygen isotopes of ostracod shells and high level of the lake are indicated at Sidi Ali [START_REF] Zielhofer | Atlantic forcing of Western Mediterranean winter rain minima during the last 12,000 years[END_REF] as well as cooler temperatures from modelisation at Tiguelmamine (Chedaddi et al., 1998). This period is also well recorded in the Ras el Ma peat sequence [START_REF] Nourelbaït | -An 18 000 year pollen and sedimentary record from the cedar forests of the Middle Atlas, Morocco[END_REF]. In terms of pollen records at Ras el Ma, Tiguelmamine and Sidi Ali, this period is marked by a slight reduction of tree and shrubs taxa that imply an opening of the landscape [START_REF] Lamb | Vegetational response to Holocene climatic change: pollen and palaeolimnological data from the Middle Atlas, Morocco[END_REF][START_REF] Nourelbaït | -An 18 000 year pollen and sedimentary record from the cedar forests of the Middle Atlas, Morocco[END_REF][START_REF] Campbell | -Environmental Drivers of Holocene Forest Development in the Middle Atlas, Morocco[END_REF]. Thus regional environmental conditions related to differents records appear consistent with malacological results.

Between 5600 and 4600 a cal BP, when drier conditions are emphasized by the mollusc succession at Aït Said ou Idder, in Tiguelmamine and Sidi Ali sequences, pollen indicate variations in the vegetation composition: some aridification evidence are identified but forest and shrub taxa persist [START_REF] Lamb | Vegetational response to Holocene climatic change: pollen and palaeolimnological data from the Middle Atlas, Morocco[END_REF][START_REF] Campbell | -Environmental Drivers of Holocene Forest Development in the Middle Atlas, Morocco[END_REF]. Geochemical results on ostracod shells show a quite drier climate without demonstrating an evident pattern of aridification [START_REF] Zielhofer | Atlantic forcing of Western Mediterranean winter rain minima during the last 12,000 years[END_REF]. During this period a strong climatic and environmental variability is noticeable, for instance, a cold event recorded between 5000 and 5500 a cal BP in Dayet Hachlaff sequence [START_REF] Nourelbaït | Climate change and ecosystems dynamics over the last 6000 years in the Middle Atlas, Morocco[END_REF]. From 5000 a cal BP, evidence of landscape aridification is identified in Dayet Hachlaff's pollen sequence ten kilometers south of the site by a slight reduction of arboreal vegetation (Nourelbait et al. 2016). During this period, palaeoenvironmental records differ locally and identifying regional trends remains difficult.

-CONCLUSIONS

The chronostratigraphic study of the tufa sequence identified two main deposition phases corresponding to a basal fluvio-paludal tufa allocated

The last environmental step (units 2 & 1; MZ7 to MZ9) corresponds to the end of the Middle Holocene and records at least 1000 years of environmental history (5600-4600 a cal BP) according to ages from levels B and A for a thickness of 1 m (fig. 8). This period is characterised by a significant reduction of the marsh, which is replaced by open and sclerophyllous habitats. A record of a flooding event in the beginning of the period is correlated with the silty-sand unit 2 where the mollusc diversity is poor (fig. 6) and mostly represented by the tolerant taxa Melanopsis cf. praemorsa (fig. 8). The development of xerophilous snail populations, an indicator of dry habitats, is linked to grey levels B and A.

-AÏT SAID OU IDDER HOLOCENE RECORD IN THE REGIONAL CONTEXT

In North Morocco, the Blirh succession in Moulouya fluvial sediments provides a detailed malacological succession for almost all the Holocene except the first part of the Middle Holocene [START_REF] Limondin-Lozouet N | -Holocene molluscan succession from NE Morocco: Palaeoenvironmental reconstruction and biogeographical implications[END_REF]. Aït Said ou Idder sequence thus partly fills the time hiatus of Blirh and overlaps the record of the second half of the Middle Holocene. At Blirh, the malacofauna assemblages show a drastic environmental rupture around 5100 a cal BP from aquatic and swampy environments to open and dry landscapes [START_REF] Limondin-Lozouet N | -Holocene molluscan succession from NE Morocco: Palaeoenvironmental reconstruction and biogeographical implications[END_REF]. A similar environmental pattern is observed at Aït Said ou Idder around 5600 a cal BP when the permanent marshland is replaced by well-drained and open habitats (fig. 9). The 500-year difference could be explained by the radiocarbon dating based on shells at Aït Said ou Idder while on charcoals at Blirh: Aït Said ou Idder dating on shells may give ages older than the real one for this phase of deposition. Further analyses and accurate dating are needed to improve the comparison between these malacological records but both sites provide consistent reconstructions of major environmental events at a regional scale. The environmental rupture recorded by both malacological successions is likely to be contemporaneous with a global climatic change at the end of the Middle Holocene which corresponds to a switch to dry conditions in the Mediterranean region [START_REF] Roberts | -The mid-Holocene climatic transition in the Mediterranean: causes and consequences[END_REF]Bini et al., 2019). This environmental shift appears during an interval of forest decline (Fletcher et al., 2013) and corresponds to the end of the African Humid Period [START_REF] Demenocal | Abrupt onset and termination of the African Humid period: rapid climate responses to gradual insolation forcing[END_REF] and to a period of Rapid Climate Change [START_REF] Mayewski | -Holocene climate variability[END_REF][START_REF] Fletcher | Fragility of Western Mediterranean landscapes during Holocene rapid climate changes[END_REF].

Additionally, in North-East Morocco, considering the set of available ages, Middle Atlas lakes and peat sequence provide consistent Holocene records to be compared with the Aït Said ou Idder results [START_REF] Lamb | Vegetational response to Holocene climatic change: pollen and palaeolimnological data from the Middle Atlas, Morocco[END_REF]Chedaddi et al., 1998;[START_REF] Rhoujjati | Significations paléoenvironnementales des dépôts du remplissage holocène du lac Iffer (Moyen Atlas, Maroc)[END_REF][START_REF] Nourelbaït | -An 18 000 year pollen and sedimentary record from the cedar forests of the Middle Atlas, Morocco[END_REF][START_REF] Nourelbaït | Climate change and ecosystems dynamics over the last 6000 years in the Middle Atlas, Morocco[END_REF][START_REF] Campbell | -Environmental Drivers of Holocene Forest Development in the Middle Atlas, Morocco[END_REF][START_REF] Zielhofer | Atlantic forcing of Western Mediterranean winter rain minima during the last 12,000 years[END_REF].

The bottom of Iffer, a part of Tiguelmamine and Sidi Ali sequences are also contemporaneous to the first to the Upper Pleistocene and a tufa slope dated from the Middle Holocene. This study leads to the following conclusions:

-the Upper Pleistocene fluvio-paludal phase dated around 40000 a cal BP records three main environmental stages: first, an aquatic environment, then a swampy area with reed beds, finally followed by a permanent body of water whose neighbouring vegetation is rather open;

-three major phases of environmental history have been identified from the Holocene slope tufa malacological succession. The first step corresponds to a mixed environment comprising a marsh and a betterdrained grassland area. The second step indicates a durable marshy area. The last step presents the drying out and a significant reduction of the swampy environment, which is being replaced by open and sclerophyllous habitats;

-the malacological study provides important elements to the construction of a Holocene malacological referential in Morocco initiated by the malacological succession of Blirh. Both records identify a drastic switch to environmental aridification at the end of the Middle Holocene. Mollusc successions provide a reliable reconstruction of local environmental evolution and also allow identifying regional and supra-regional environmental trends and events;

-the succession of Aït Said or Idder completes other regional paleoenvironmental data, particularly during the Middle Holocene, and provides material to understand landscape variability in northeastern Morocco. The first phase of mosaic of environment and fire events is consistent with the aridification of climatic conditions recorded in the area (low lake level and pollen reconstruction). More humid climatic conditions registered by oxygen isotopes from ostracod shells at Sidi Ali are contemporaneous with the Aït Said ou Idder marshy expansion. The final switch to drier environments is not evidenced by other regional biological proxy studies because of a climatic and environmental variability but is echoed in Dayet Hachlaff sequence.

Fig. 1 :

 1 Fig. 1: A Location of Aït Said ou Idder tufa site; B) Digital elevation model of the site surrounding (SRTM 30 m); C) Aït Said ou Idder football section. Fig. 1 : A Localisation du site; B) Modèle numérique de terrain des environs du site (SRTM 30 m); C) Coupe du terrain de football à Aït Said ou Idder.

Fig. 2 :Fig. 2 :

 22 Fig. 2: Stratigraphy of the 'football section' and detailed sampling profiles in Aït Said ou Idder tufa. Fig. 2 : Présentation de la coupe du terrain de football d'Aït Said ou Idder, de sa stratigraphie et des profils d'échantillonnage.

Fig. 3 :

 3 Fig. 3: Plate of the lihofacies observed in Aït Said ou Idder tufa, football section. A) Phytoherm at the base of the profile (Unit 9); B) Oncolithic levels alternating with encrusted lamine i.e. Chironomid burrow; C) Erosional transition between the lower and the upper part of the tufa; D) Zoom of the Unit 6 showing the carbonaceous level; E) Phytoclastic lithofaciès of Unit 5. Fig. 3 : Planche des lithofaciès observés sur la coupe football du tuf d'Aït Said ou Idder A) Phytoherme dans la base de coupe (Unité 9) ; B) Exemple de l'aternance entre des niveaux sableux, à oncolithes et lamines encroutées dans l'unité 8, vue de détail de terrier de chironomid encrouté en coupe ; C) Photo commentée de la zone de transition entre la partie inférieure et supérieure ; D) Vue de détail de l'unité 6 montrant le niveau riche en charbons ; E) Lithofaciès à phytoclastes dans l'unité 5.

Tab. 2 :Fig. 5 :

 25 Fig. 5: Modern and Holocene molluscs from Aït Said ou Idder and nearby. 1-4: Modern Otala tingitana (Paladhile, 1875) from Dayet Aoua; 5-8: Otala tingitana (Paladhile, 1875) fossil (unit 6) ; 9-11: Modern Geomitridae sp., Aït Said ou Idder; 12-14; Geomitridae sp.1 fossil (unit 1); 15-17: Geomitridae sp.2 fossil (unit 7); 18-20: Geomitrida sp.3 fossil (unit 1); 21-23: Juvenile of modern Geomitridae sp., Aït Said ou Idder; 24-26: Adult of modern Geomitridae sp., Aït Said ou Idder; 27-28: SEM view of fossil Hydrobiidae (unit 7). All scale bars = 5 mm except 27-28 scale bar = 0.5 mm & 0.1 mm. Fig. 5 : Mollusques continentaux actuels et holocènes d'Aït Said ou Idder et des environs. 1-4 : Otala tingitana (Paladhile, 1875) actuel, Dayet Aoua ; 5-8 : Otala tingitana (Paladhile, 1875) fossile (unité 6) ; 9-11 : Geomitridae sp. actuel, Aït Said ou Idder ; 12-14 : Geomitridae sp.1 fossile (unité 1) ; 15-17 : Geomitridae sp.2 fossile (unité 7) ; 18-20 : Geomitrida sp.3 fossile (unité 1) ; 21-23 : Juvénile de Geomitridae sp. actuel, Aït Said ou Idder ; 24-26 : Adulte de Geomitridae sp. actuel, Aït Said ou Idder ; 27-28 : Hydrobiidae fossile (unité 7). Toutes les barres d'échelle font 5 mm en dehors de 27-28 (barre d'échelle = 0.5 mm & 0.1 mm).

Fig. 6 :

 6 Fig. 6: Malaco-ecological ecological diagrams of the two profiles by species and by individuals. Curves represents number of species and shell abundance in each sample. Hatched diagrams correspond to non-significant sample where individuals are less than 50. Fig. 6 : Spectres écologiques des deux profils exprimés par espèces et par individus. La richesse spécifique et l'abondance de chaque échantillon sont représentées par les courbes noires. Les spectres hachurés correspondent aux échantillons non représentatifs dont les effectifs sont inférieurs à 50 individus.

Fig. 7 :

 7 Fig. 7: Percentage frequency histogram of profile 2.Asterisks represent samples under the threshold of 50 individuals.

Fig. 8 :

 8 Fig. 8: Percentage frequency histogram of profile 1.Asterisks represent samples under the threshold of 50 individuals.Fig.8: Diagramme de fréquence spécifique, profil 1. Les astérisques correspondent à des échantillons qui sont en dessous du seuil de représentativité de 50 individus.

  4.3.2.4 -Malacozone 6: M17 to M19 (bottom of Unit 3) The proportion and diversity of marshy species are very similar to malacozone 5 (figs. 6 & 8). However, in malacozone 6, the decrease in aquatic taxa is balanced by the reappearance and increase of xerothermic, open and mesophilic species (fig. 8): Geomitridae sp. 1, Cochlicella barbara, Vallonia pulchella, Truncatellina cylindrica, Vallonia costata, Punctum pygmaeum and Euconulus fulvus. The sum of xerothermic, open and mesophilic taxa reaches 40 % (fig. 8). The freshwater species are only 20 % of assemblages and are mainly represented by Melanopsis cf. praemorsa and Pisidium casertanum, the occurrence of Galba truncatula, Anisus spirorbis and Radix sp. is occasional (fig. 8).

Further

  investigation will be necessary to draw a precise palaeoecological reconstruction as the threshold of 50 individuals is not reached in any of the sample allocated to MZ 1. Nonetheless, this malacozone suggests a humid environment with patches of open vegetation. 4.3.1.2 -Malacozone 2: M51 to M45 (Unit 7) The malacozone 2 is characterised by a constant increase in aquatic taxa that represents 40 to 50 % of the assemblage (fig. 6). Malacofaunas show a diversity of 20 freshwater molluscs. Several species arrived such as Hippeutis complanatus, Ancylus fluviatilis, Anisus spirorbis, Radix and Bathyomphalus contortus (fig. 7). These elements imply the establishment of a durable aquatic habitat. Paludal taxa are mostly represented by Oxyloma elegans, a common hygrophilous and marshy Succineidae. The number of xerothermic, mesophilic and open area snails represents 10-15 % of the assemblages. The appearance of Cochlicopa lubrica, Euconulus fulvus and Vitrea contracta indicate an expansion of mesophilic environment. The contribution of xerophilous taxa is minor but Geomitridae sp. 1 and Cochlicella barbara slightly increase. In this part of the succession, some samples show a low number of shells but the whole malacozone presents a reliable data set. Results indicate the development of a durable body of clear water. The adjacent terrestrial open herbaceous environment is quite humid.4.3.2 -The Mid-Holocene slope tufa 4.3.2.1 -Malacozone 3: M33 to M34 + M1 to M3 (Unit 6 and base of Unit 5)

Fig. 9 :

 9 Fig. 9: Synthesis of chronostratigraphical and malacological results. Fig. 9 : Synthèse des résultats malacologiques, stratigraphiques et de la chronologie.

  . It is a 5-6 whorls globular little shell (6-9 mm) with relatively deep sutures(fig. 5: 9-11). The umbilicus is tiny and the mouth rounded with sometimes a slight callus. The shell is white with a brown band, regularly and strongly striated. A modern population showing similar features was found near the site(fig. 5: 12-14).

	Modern soil	Land snail shell
		Charcoal Geomitridae sp. 2 (10 mm) has a bigger umbilicus and
	Silty white-beige tufa with a grey phytoclastic silty level (A)	a smoother shell (fig. 5: 15-17). It is represented by a unique shell from the Pleistocene part (tab. 2; sample
		M49). Geomitridae sp. 3 is a little white keeled shell
	White-beige granular tufa with encrusted plant remains in a	(3-4 mm) with fine irregular striations and a medium umbilicus (fig. 5: 18-20). In the fossil assemblage, this
	beige-orange silty-sand matrix	taxon is represented in nine samples and only by juvenile
		specimens. Geomitridae sp. 3 is morphologically close to
	White-beige indurated tufa alterning with silty phytoclastic level (B) and	an unidentified modern Geomitrid found in Aït Said ou Idder area (fig. 5: 21-26).
	granular oncolithic layer
	Coarsed tufa in a silty sand matrix
	which upper level is grey (C)
		4730 ± 370
	Silty grey layer with disseminated phytoclast. The lower part is darker, coarser and carbonaceous	30 kyr sedimentary
		hiatus
	White indurated tuf with lamination
	and oncolithes in the lower part
	Coarsed orange tufa with oncolithic
	layer alterning with sand
	Indurated yellowish tufa bed
	with phytoherm
	Indurated beige-orange tufa with
	oncolithes

Fig. 4: Lithochronostratigraphical synthesis of the tufa sequence at Aït Said ou Idder. Fig. 4 : Synthèse lithochronostratigraphique du tuf d'Aït Said ou Idder.

abundant

Individual distribution in ecological groups for assemblages of Profile 1(%) Species richness Abundance

  Carychium minimum and Melanopsis cf. praemorsa occur continually in the zone (fig.7). such as cultivated areas, grasslands and semi-deserts (Welter-Schultes, 2012). Both species were observed in the surroundings of the Aït Said ou Idder living in xerothermic environments. Consequently, Geomitridae sp. 1, sp. 2, sp. 3, Cochlicella barbara and Rumina decollata have been assigned to the xerothermic group. The lack of reliable data on Otala tingitana's habitat and its wide range of living environments[START_REF] Holyoak | -A Revision of the landsnail genera Otala and Eobania in Morocco and Algeria[END_REF] support the allocation of this taxon to the mesophilic group.

													of misinterpretation of poorly abundant assemblages.
													When the total number of individuals in a sample failed
													to reach 50, results are moderated and represented by
													hatched areas (fig. 6) or asterisks (figs. 7 & 8). The
													molluscan succession can be divided into nine biozones
													based on ecological patterns and on the occurrence and
													development of taxa. Malacozones 1 and 2 are from the
													Upper Pleistocene whereas Malacozones 3 to 9 are from
													the Mid-Holocene tufa.
													4.3.1 -The Pleistocene fluvio-paludal tufa
	4.3.1.1 -Malacozone 1: M55 to M52 (Unit 8) This malacozone is composed of an assemblage of mesophilic/open area and paludal/hygrophile/ aquatic taxa (fig. 6). Vallonia pulchella, Lauria cylindracea, 4.3 -THE MOLLUSCAN SUCCESSiON Malacological data have been plotted as percentages per group in ecological diagrams (fig. 6) and percentages per taxa in frequency histograms (figs. 7 & 8). A threshold
	of 50 individuals has been used to minimize the risk
		Species distribution assemblage of Profile 1 according
				to ecological groups (%)
	0	10	20	30	40	50	60	70	80	90	100
													Species distribution assemblage of Profile 2 according
													to ecological groups (%)
													0	10	20	30	40	50	60	70	80	90	100	Species richness
													M36
													M35
													M34
													M33
											0	5	10	15	20	M45 M46
													M47
													M48
													M49
													M50
													M51
													M52
													M53
													M54
													M55
													0	5	10	15	20
	0	10	20	30	40	50	60	70	80	90	100
													Individual distribution in ecological groups
													for assemblages of Profile 2 (%)
													0	10	20	30	40	50	60	70	80	90	100	Abundance
													M36
													M35
													M34
													M33
													M45
											0 2 0 0		M46 M47
													M48
													M49
													M50
													M51
													M52
													M53
													M54
													M55

  2.6 -Malacozone 8: M22 to M27 (Unit 2) Malacozone 8 shows a decrease in species diversity and is mainly composed of aquatic, marshy and xerothermic molluscs. Freshwater taxa represent about 40 to 50 %, swamp molluscs up to 20 % and xerothermic species about 20 to 30 % of the assemblages (fig. 6). Mesophilic and open ground species show few occurrences (fig. 8: Vallonia pulchella, Truncatellina cylindrica and Euconulus fulvus). Slugs disappear during this environmental phase. Marshy taxa are only represented by Oxyloma elegans, Vertigo antivertigo and a few Vertigo moulinsiana. Aquatic species are more diversified but their large individual proportion relies almost exclusively on Melanopsis cf. praemorsa shells (fig.

ACKNOWLEDGEMENTS

Moroccan fieldwork was funded by PALEOMEX (INSU-INEE) Transect Maghreb (dir. J.F. Berger) research program. Radiocarbon ages have been supported by LMC14 (CEA-GIF-SUR-YVETTE) Artemis program and by the Czech Science Foundation (P504/17-05696S). Q.Wackenheim is grateful to P. Lozouet (MNHN Paris) who provided SEM photograph of the fossil Hydrobiidae, to D. Holyoak and E. Neubert

for their helpfull discussion on Moroccan land snails and to T. Piau for his help with GIS. I aslo thank the two reviewers whose comments served to improve the manuscript. Finally, the authors are thankful to Dayet Aoua guest house who provides suitable conditions for sieving operation.


Marc Presset

Michel Rajzmann
email: michel.rajzmann@univ-amu.fr

Guillaume Dauvergne
email: guillaume.dauvergne@univ-amu.fr

Jean Rodriguez
email: jean.rodriguez@univ-amu.fr

Yoann Coquerel
email: yoann.coquerel@univ-amu.fr

Periselectivity in the Aza-Diels-Alder Reaction of 1-Azadienes with α-Oxoketenes: A Combined Experimental and Theoretical Study

Keywords: hetero-Diels-Alder, ketenes, imines, periselectivity, density functional theory

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Introduction

↵-Oxoketenes [1][2][3][4][5][6] were first employed in organic synthesis in 1902 with the pioneering work of Ludwig Wol↵ on 2-diazo-1,3-dicarbonyl compounds [7]. Since, ↵-oxoketenes have revealed as versatile chemical intermediates for synthetic applications, ranging from the preparation of simple -oxocarbonyl derivatives [8] to the total synthesis of complex natural products [4,5]. However, and with few exceptions [9,10], ↵-oxoketenes are not stable species because of their propensity to react with a number of chemicals, including themselves [11], at significant rates at ambient temperature; thus, they must be generated in situ immediately before use. The most common and practical methods for the generation of ↵-oxoketenes are the thermal decomposition of dioxinones with concomitant extrusion of acetone [12], and the thermal or photochemical Wol↵ rearrangement of 2-diazo-1,3-dicarbonyl compounds extruding nitrogen gas [13,14]. ↵-Oxoketenes 1 are electrophilic species that most commonly react with imines through formal aza-Diels-Alder cycloadditions involving the ↵-oxoketenes as 4⇡ reaction partners (1-oxadienes) and the imines as the 2⇡ reaction partners to give 1,3-oxazin-4-ones of type 4 (Scheme 1) [1][2][3][4][5][6]15]. However, when ↵, -unsaturated imines 2 are employed, another formal aza-Diels-Alder process becomes possible, involving the ↵, -unsaturated imines as 4⇡ reaction partners (1-azadienes) and the ↵-oxoketenes as the 2⇡ reaction partners to give hydropyridin-2-ones of type 3 [16]. Periselectivity, the selectivity in formation of the pericyclic products, in the formal aza-Diels-Alder cycloadditions between ↵-oxoketenes and some electron rich 2-azadienes was recently examined and found to be dependent on kinetic and thermodynamic factors, as well as the nature of the ↵-oxoketene employed [11]. For the present study, the periselectivity in the reactions of ↵-oxoketenes with ↵, -unsaturated imines as prototypical 1-azadienes was further investigated and rationalized by a combination of experimental and computational studies.

Scheme 1. Periselectivity in the formal aza-Diels-Alder cycloadditions of ↵-oxoketenes with ↵, -unsaturated imines.

Results and Discussion

We previously reported the formal aza-Diels-Alder cycloaddition between the ↵, -unsaturated imine 2a and the five-membered cyclic ↵-oxoketene 1a derived from the thermal Wol↵ rearrangement of 2-diazodimedone to give the spiro hydropyridin-2-one 3a in 24% yield after 15 min of reaction at 160 C (Scheme 2a) [16]. Re-examination of the temperature dependence of the reaction outcome revealed that the other pericyclic product, namely the 1,3-oxazin-4-one 4a, is actually formed when the reaction is performed at 140 C with a 2:1 3a/4a ratio after 15 min, which allowed isolating 3a in 38% yield and 4a in 21% yield. A similar reaction conducted at 150 C for 30 min a↵orded only 3a in 22% isolated yield (3a/4a > 25:1). Stimulated by these observations, some complementary experiments were performed to further illustrate the possible change in periselectivity in the aza-Diels-Alder cycloadditions between ↵-oxoketenes and 1-azadienes as a function of the reaction temperature (Scheme 2b-e). For instance, the reaction between ↵-oxoketene 1a and the enantiopure 1-azadiene 2b derived from (-)-perillaldehyde and allylamine at 140 C for 15 min a↵orded the corresponding diastereomeric 1,3-oxazin-4-ones 4b in 59% (dr = 1:1) while it was previously shown to a↵ord the spiro hydropyridin-2-one 3b after 15 min at 200 C [16]. Similarly, it was found earlier that spiro hydropyridin-2-one 3c could be obtained e ciently from the 2-methyl-1-azadiene 2c from a reaction at 160 C [16], while a mixture of the two possible pericyclic products 3c and 4c was obtained at 140 C. Some degree of thermodynamic control of the reaction could be demonstrated experimentally in this case: heating a toluene solution of 4c at 160 C for 15 min produced 25% of 3c together with 20% of 2-methylcinnamaldehyde and unidentified products. Surprisingly, the reactions of ↵-oxoketene 1a with the 3-chloro 1-azadiene 2d, led exclusively to the corresponding 1,3-oxazin-4-one 4d with no detectable amount of 3d, at 140 C or 200 C. The influence of the nature (phenyl vs. methyl) of the substituent at position 4 of the 1-azadiene on periselectivity was briefly evaluated through the reaction of ↵-oxoketene 1a with 1-azadienes 2e (R = Ph) and 2f (R = Me) at 140 C for 15 min. It was earlier found that the reaction with 1-azadiene 2e provided the spiro hydropyridin-2-one 3e in 70% yield [16]. The same reaction conducted with azadiene 2f provided only the 1,3-oxazin-4-one 4f albeit in low yield.

The influence of the nature of the ↵-oxoketene on periselectivity was also evaluated experimentally. The six-membered cyclic ↵-oxoketene 1b derived from the thermal Wol↵ rearrangement of 2-diazocycloheptan-1,3-dione was reacted with 1-azadiene 2g at 160 C to a↵ord cleanly the 1,3-oxazin-4-one 4g, while the same reaction mixture at 200 C for a prolonged time previously a↵orded the spiro hydropyridin-2-one 3g in 53% yield (Scheme 3a) [16]. The aza-Diels-Alder reaction of the acyclic ↵-oxoketene 1c with the 1-azadiene 2h was also examined and found to lead to the 1,3-oxazin-4-one 4h when conducted at 140 C for 5 min, while the same reaction performed at 200 C for 15 min resulted in the exclusive formation of iso-3h (Scheme 3b). Significantly, the relative stereochemistry in iso-3h is opposite to the stereochemistry that has been described so far in this and other series of cycloadditions of ↵-oxoketenes with 4⇡ reaction partners [11,16,17]. Ultimately, it was shown that ↵-oxoketene 1d derived from the Wol↵ rearrangement of methyl 2-diazoacetylacetate reacted with the 1-azadiene 2i at 160 C to give the 1,3-oxazin-4-one 4i in 43% yield, and no reaction at higher temperature was attempted.

Scheme 2. Temperature dependence of the periselectivity in the formal aza-Diels-Alder cycloadditions between 1-azadienes 2a-f and ↵-oxoketene 1a. Scheme 3. Temperature dependence of the periselectivity in the formal aza-Diels-Alder cycloadditions between 1-azadienes 2g-i and ↵-oxoketenes 1b-d.

These new experimental data indicate that periselectivity in the aza-Diels-Alder cycloadditions between ↵-oxoketenes and 1-azadienes is generally controlled by competitive kinetic and thermodynamic factors. However, there are some marked di↵erences in reactivity within relatively homogeneous series of substrates, which called for rationalization to enable further developments and applications. To do so, the problem was tackled through computational modeling using density functional theory (DFT) methods. The ↵-oxoketenes 1b,c and 1e and the model 1-azadienes 2j-m were selected for this part of the work. All calculations were performed using the B3LYP-D3/6-311++G(d,p) level of theory. Free Gibbs energies were generally computed at 298 K corresponding to the temperature of isolation of the products, and additionally at the reaction temperature for kinetically meaningful steps (see the Supplementary Materials for details of the calculations).

First, we examined the reactions between the ↵-oxoketene 1e and the 1-azadiene 2j to a↵ord either the spiro hydropyridin-2-one 3j or the 1,3-oxazin-4-one 4j (Figure 1a,b). As expected, formation of 3j was found largely thermodynamically favored when compared to the formation of 4j [D(DG) = 56.1 kJ/mol] with barriers computed at D(DG , ) = 12.1 kJ/mol at 298 K and 18.9 kJ/mol at 413 K, which is fully consistent with the experimental results in Scheme 2a. However, the actual aza-Diels-Alder thermodynamic product of the reaction was computationally identified as iso-3j (the diastereomer of 3j) with a relative free Gibbs energy determined at 75.8 kJ/mol, that is more stable than 3j by a tiny but meaningful 2.4 kJ/mol; modeling its formation indicated a relative activation energy of DG , = 73.9 kJ/mol at 298 K and 100.5 kJ/mol at 413 K plausible with its formation under the actual reaction conditions (Figure 1c). Because the diastereoselectivity in the model molecule iso-3j has never been reported experimentally in cycloadditions of ↵-oxoketene 1e with any 4⇡ reaction partner [11,16,17], a competing process is certainly at hand. Actually, the formation of iso-3j through TS6 was found kinetically outcompeted by the cyclodimerization of ↵-oxoketene 1e (Figure 1c) that occurs with a significantly lower relative activation energy through TS7 to give dimer-1e, a molecule itself amenable to further thermodynamically driven transformations under the reaction conditions [11]. 311++G(d,p)) of the aza-Diels Alder reactions between the ↵-oxoketene 1e and the 1-azadiene 2j, and cyclodimerization of ↵-oxoketene 1e. Free Gibbs energies noted between parentheses were computed at 413 K.

The aza-Diels-Alder reactions of ↵-oxoketene 1e with the 3-substituted 1-azadienes 2k (Figure 2) and 2l (Figure 3) were next examined in silico for comparison with the experimental results in Scheme 2c,d. As above, formation of the 1,3-oxazin-4-ones 4k and 4l was found kinetically preferred to the formation of the corresponding spiro hydropyridin-2-ones of 3k and 3l, with two competing reaction paths identified for the formation of 4l (Figure 3a). This is in agreement with the experimentally observed formation of both 1,3-oxazin-4-ones 4c and 4d from reactions at 140 C. However, at 160 C, the methyl-substituted spiro hydropyridin-2-one 3c (Scheme 2c) corresponding to the model molecule 3k (Figure 2b) was obtained experimentally, while somehow unexpectedly the chloro-substituted spiro hydropyridin-2-one 3d (Scheme 2d) corresponding to the model molecule 3l (Figure 3b) could not be obtained even at 200 C. The rationalization of this experimental fact came from the comparison of the relative activation energies for the formation of 3k, 3l, and dimer-1e at 200 C: TS11 leading to 3k competes favorably with the formation of dimer-1e through TS7, though not by much with D(DG , ) = 3.5 kJ/mol at 473 K, while TS17 leading to 3l competes unfavorably with the formation of dimer-1e with D(DG , ) = 3.4 kJ/mol at 473 K. 311++G(d,p)) of the aza-Diels Alder reactions between the ↵-oxoketene 1e and the 1-azadiene 2k, and cyclodimerization of ↵-oxoketene 1e. Free Gibbs energies noted between parentheses were computed at 473 K.

To conclude with the modeling of reactions of the cyclic five-membered ↵-oxoketene 1e with 1-azadienes, the influence of a 4-phenyl or a 4-methyl substituent on the 1-azadiene was investigated to rationalize the results in Scheme 2e. Comparison of the energy profiles of the reactions ↵-oxoketene 1e with the model 1-azadienes 2j (Figure 1) and 2m (Figure 4) essentially shows that the formation of the model spiro hydropyridin-2-one 3m bearing a methyl substituent is significantly slower than the reaction leading to the model spiro hydropyridin-2-one 3j bearing a phenyl substituent with D(DG , ) = 12.4 kJ/mol at 298 K and 34.8 kJ/mol at 413 K (compare TS4 with TS21). The relative increase in the activation energy induced by the 4-methyl substituent is significant and precludes the formation of the corresponding spiro hydropyridin-2-one 3m at the benefit of the ↵-oxoketene cyclodimer dimer-1e for kinetic reasons. Altogether, the selected DFT model allowed a faultless rational of the periselectivity in the reactions of ↵-oxoketene 1e with 1-azadienes, which was found to be governed by a combination of kinetic and thermodynamic factors. 311++G(d,p)) of the aza-Diels Alder reactions between the ↵-oxoketene 1e and the 1-azadiene 2l. Free Gibbs energies noted between parentheses were computed at 473 K. For 1-azadiene 2l, the s-cis/s-trans denomination refers to the conformation of the 4⇡ system of the 1-azadiene, not to the regular CIP nomenclature.

Calculations with the cyclic six-membered ↵-oxoketene 1b (Figure 5) paralleled the previous findings and were also found in full agreement with the experimental results, with the actual thermodynamic diastereomer of the spiro hydropyridin-2-one product being computationally identified as iso-3n, that is not the model molecule corresponding to the experimentally observed diastereomer 3g from the reaction at 200 C (Scheme 3a). At 200 C, there are two orders of magnitude between the kinetic constants estimated for the formation of 3n via TS25 and iso-3n via TS27 (circa k = 60 s 1 and 0.56 s 1 , respectively), but the formation of iso-3n remains plausible after 45 min at 200 C. It should, however, be considered that the ratio Int10/Int11 is higher than 10 4 :1 according to the model, which precludes the formation of iso-3n at a significant rate under the examined experimental conditions (relative rates in the magnitude of 10 6 :1). Additionally, the cyclodimerization of ↵-oxoketene 1b to give dimer-1b through TS28 is also in competition with the formation of iso-3n at 200 C. On this occasion, again, the selected DFT model allowed for a complete rationalization of the periselectivity in the reactions of ↵-oxoketene 1b with 1-azadienes, which was found to be governed by a combination of kinetic and thermodynamic factors, as for ↵-oxoketene 1e. The calculations in the acyclic series were complicated by the existence of the additional s-trans conformer of the ↵-oxoketene 1c (Figure 6a) but again were in full agreement with the experiments. As expected, 4o was identified as the kinetic aza-Diels-Alder product in the model reaction between ↵-oxoketene 1c and 1-azadiene 2j with DG , = 70.7 kJ/mol at 298 K and 102.5 kJ/mol at 473 K. As above, the true thermodynamic cycloadduct was identified as iso-3o with notably a more pronounced di↵erence in stabilization energy between the two diastereomers 3o/iso-3o than in the previous cases for 3j/iso-3j (Figure 1) and 3n/iso-3n (Figure 5). In addition, the di↵erence between the activation energies leading to one or the other diastereomer is reduced in this series with D(DG , ) = 10.3 kJ/mol at 298 K and 10.9 kJ/mol at 473 K for TS33/TS35. This is to be compared with D(DG , ) = 17.0 kJ/mol at 298 K and 17.1 kJ/mol at 473 K for TS4/TS6, and D(DG , ) = 18.8 kJ/mol at 298 K and 18.4 kJ/mol at 473 K for TS25/TS27. The di↵erence between the estimated kinetic constants corresponding to TS33 and TS35 is now reduced to a single order of magnitude (circa k = 0.49 s 1 and 0.031 s 1 for TS33 and TS35 at 200 C, respectively), allowing for both processes to compete at 200 C. In this case, the cyclodimerization of the ↵-oxoketene 1c (via TS36) was found not to compete kinetically with the aza-Diels-Alder processes examined. Altogether, the calculations indicate that in the case of the acyclic ↵-oxoketene 1c, and only in this case, the actual thermodynamic product of the reaction iso-3o can be obtained, which was demonstrated experimentally with the isolation of iso-3h (Scheme 3b). 311++G(d,p)) of the aza-Diels Alder reactions between the ↵-oxoketene 1b and the 1-azadiene 2j. Free Gibbs energies noted between parentheses were computed at 473 K.

All the aza-Diels-Alder processes described herein are stepwise processes, occurring first with the formation of the N-C bond to produce a zwitterionic intermediate and then cyclization to the product with formation of the C-C or O-C bond. These processes should thus preferably be referred to as formal aza-Diels-Alder cycloadditions. The physical reason at the origin of this behavior is that the LUMO of all the ↵-oxoketenes are essentially located over the ketene carbonyl groups, not over the two or four sp 2 hybridized atoms involved in the cycloaddition, and directed in the mean plane of the ↵-oxoketenes, not perpendicular to it as in standard Diels-Alder cycloadditions (Figure 7). Actually, the LUMO+1 of the ↵-oxoketenes are mostly distributed over the ↵-oxo carbonyl groups and the ketene C=C bonds and oriented perpendicular to the molecular plane [11], but with a significant LUMO/LUMO+1 gap of 0.01453 Ha or 38.1 kJ/mol on average, and no transition states accounting for the symmetry-allowed concerted transformations were located computationally during this work. 311++G(d,p)) of the aza-Diels Alder reactions between the ↵-oxoketene 1c and the 1-azadiene 2j. Free Gibbs energies noted between parentheses were computed at 473 K.

In the course of the computational investigations summarized herein, some alternative kinetically, and for the most also thermodynamically, disfavored reaction paths leading to other cycloadducts were identified; this includes -lactams resulting from formal [2+2] cycloadditions between the C=C bond of the ketene and the C=N bond of the 1-azadiene, and 1,3-oxazin-2-ylidenes resulting from a formal [4+2] cycloaddition between the C=O bond of the ketene and the 4⇡ system of the 1-azadiene.

Experimental Section

All reagents were purchased from commercial sources and used without further purification unless otherwise noted. All compounds were weighed and handled in air at room temperature. Microwave-assisted heating was performed using a professional Anton-Paar (Austria) Monowave 300 system in specific sealed tubular reaction vessels. The reactions were monitored by thin layer chromatography (TLC, 60 F 254 ) visualized by UV lamp (254 nm or 365 nm). Flash chromatography was performed on silica gel (particle size 40 63 µm). Petroleum ether refers to the fraction with bp = 40-60 C. NMR spectra were recorded at 300 MHz ( 1 H) and 75 MHz ( 13 C) at 298 K in CDCl 3 using as internal standards the residual non-deuterated signal for 1 H-NMR ( = 7.26 ppm) and the deuterated solvent signal for 13 C-NMR spectroscopy ( = 77.16 ppm). DEPT-135 experiments were used to determine the multiplicity of the 13 C resonances. Chemical shifts ( ) are given in ppm, coupling constants (J) are given in Hz, and the classical abbreviations are used to describe the multiplicity of the 1 H resonances. Copies of all NMR spectra are provided as Supplementary Materials for this article. High-resolution mass spectra were recorded in triplicate at the Spectropole (https://fr-chimie.univ-amu.fr/spectropole/). 2-Diazo-1,3-dicarbonyls were prepared as previously reported in similar yields and purity [18].

General procedure for the aza-Diels-Alder reactions: a solution of a primary amine (1 equiv) and an ↵, -unsaturated aldehyde (1 equiv) in 2-3 mL of anhydrous toluene (ca. 0.4 M) under an argon atmosphere in a microwave dedicated sealed tube containing a Teflon coated magnetic stirring bar was irradiated at 140 C for 15 min (ramp up time = 2 min), cooled down to 55 C by an air flow over 5 6 min, and concentrated directly in the reaction vessel to a↵ord the corresponding imine quantitatively as verified by 1 H-NMR analysis. To this material placed under an argon atmosphere was added the diazo compound (1 equiv) and anhydrous toluene (2-3 mL, ca. 0.4 M), the reaction vessel was sealed, and this mixture was irradiated at the set temperature for the time indicated (after a temperature ramp-up time of ca. 2 min). The resulting reaction mixture was cooled down to 55 C by an air flow over 5 6 min, concentrated, analyzed by NMR to determine the products ratio, and directly purified by flash chromatography when isolation was attempted.

4a: Following the general procedure, the reaction between ortho-nitrocinnamaldehyde (172 mg, 0.97 mmol), furfurylamine (86 µL, 0.97 mmol), and 5,5-dimethyl-2-diazo-cyclohexan-1,3-dione (161 mg, 0.97 mmol) at 140 C for 15 min a↵orded the spiro hydropyridin-2-one 3a (147 mg, 38%) as a yellow oil and the 1,3-oxazin-4-one 4a (80 mg, 21%) as a yellow oil. Rf (AcOEt/petroleum ether 4:6) = 0.45. ), 36.3 (C), 30.0 (CH 3 ), 29.9 (CH 3 ). 1 H-NMR (300 MHz, ppm/CDCl 3 ): 7.99 (dd, J = 1.6, 7.8 Hz, 1H), 7.59 (ddd,J = 1.6,7.4,7.4 Hz,1H),2H),7.33 (dd,J = 0.9,1.5 Hz,1H),7.15 (d,J = 15.7 Hz,1H),3H),5.85 (dd,J = 0.9,6.7 Hz,1H),4.90 (d,J = 15.8 Hz,1H),4.40 (d,J = 15.8 Hz,1H),4H),1.15 (s,3H),1.13 (s,3H). 4b: Following the general procedure, the reaction between (-)-perillaldehyde (155 µL, 1.0 mmol), allylamine (75 µL, 1.0 mmol), and 5,5-dimethyl-2-diazo-cyclohexan-1,3-dione (166 mg, 1.0 mmol) at 140 C for 15 min a↵orded the 1,3-oxazin-4-one 4b (193 mg, 59%, dr = 1:1) as a yellow oil. 4c: Following the general procedure, the reaction between ↵-methyl-cinnamaldehyde (140 µL, 1.0 mmol), benzylamine (109 µL, 1.0 mmol), and 5,5-dimethyl-2-diazo-cyclohexan-1,3-dione (167 mg, 1.0 mmol) at 140 C for 15 min a↵orded a 2:1 mixture of the 1,3-oxazin-4-one 3c and the 1,3-oxazin-4-one 4c (311 mg, 82% total) that we failed at separating by flash chromatography. Some amount of 1,3-oxazin-4-one 4c could be isolated by treatment of the 3c/4c 2:1 mixture by 0.75 equiv NaBH 4d: Following the general procedure, the reaction between ↵-chloro-cinnamaldehyde (144 mg, 0.86 mmol), benzylamine (94 µL, 0.86 mmol), and 5,5-dimethyl-2-diazo-cyclohexan-1,3-dione (144 mg, 0.86 mmol) at 140 C for 15 min a↵orded the 1,3-oxazin-4-one 4d (210 mg, 61%) as a colorless oil. Rf (Et 2 O/petroleum ether 4:6) = 0.35. MS (ESI+) m/z: 316 [M+Na] + , 332 [M+K] + . 13 C{ 1 H}-NMR (75 MHz, ppm/CDCl 3 ): 164.8 (C), 161.9 (C), 136.5 (C), 132.9 (C), 129.5 (CH), 129.5 (CH), 129.1 (CH), 128.9 (CH), 128.7 (CH), 128.7 (CH), 128.3 (CH), 128.3 (CH), 128.0 (CH), 128.0 (CH), 127.6 (CH), 127.1 (C), 108.1 (C), 90.3 (CH), 46.2 (CH 2 ), 46.1 (CH 2 ), 41.0 (CH 2 ), 36.3 (C), 29.9 (CH 3 ), 29.1 (CH 3 ). 1 H-NMR (300 MHz, ppm/CDCl 3 ): 7.58 (dd, J = 1.7, 7.3 Hz, 2H), 7.41-7.29 (m, 7H), 6.62 (s, 1H), 5.76 (s, 1H), 5.44 (d, J = 15.4 Hz, 1H), 3.99 (d, J = 15.4 Hz, 1H), 2.53-2.29 (m, 5H), 1.19 (s, 3H), 1.13 (s, 3H). 4f: Following the general procedure, the reaction between crotonaldehyde (76 µL, 0.91 mmol), furfurylamine (81 µL, 0.91 mmol), and 5,5-dimethyl-2-diazo-cyclohexan-1,3-dione (152 mg, 0.91 mmol) at 140 C for 15 min a↵orded the 1,3-oxazin-4-one 4f (53 mg, 20%) as a colorless oil. Rf (Et 2 O/petroleum ether 2:8) = 0.41. HRMS (ESI+) m/z: [M+H] + calcd for C 17 H 22 NO 3 + 288.1594, found 288.1591. 13 C{ 1 H}-NMR (75 MHz, ppm/CDCl 3 ): 165.9 (C), 162.5 (C), 151.0 (C), 142.0 (CH), 133.4 (CH), 124.4 (CH), 110.3 (CH), 108.1 (CH), 107.9 (C), 89.7 (CH), 46.1 (CH 2 ), 41.1 (CH 2 ), 38.2 (CH 2 ), 36.2 (C), 30.0 (CH 3 ), 29.9 (CH 3 ), 17.5 (CH 3 ). 1 H NMR (300 MHz, ppm/CDCl 3 ): 7. 1H),2H),2H),5.56 (d,J = 6.4 Hz,1H),4.87 (d,J = 15.7 Hz,1H),4.18 (d,J = 15.7 Hz,1H),4H), 1.73 (d, J = 6.4 Hz, 3H), 1.13 (s, 3H), 1.12 (s, 3H).

Figure 1 .

 1 Figure 1. Free Gibbs energy profiles computed at 298 K (DFT, B3LYP-D3/6-311++G(d,p)) of the aza-Diels Alder reactions between the ↵-oxoketene 1e and the 1-azadiene 2j, and cyclodimerization of ↵-oxoketene 1e. Free Gibbs energies noted between parentheses were computed at 413 K.

Figure 2 .

 2 Figure2. Free Gibbs energy profiles computed at 298 (DFT, B3LYP-D3/6-311++G(d,p)) of the aza-Diels Alder reactions between the ↵-oxoketene 1e and the 1-azadiene 2k, and cyclodimerization of ↵-oxoketene 1e. Free Gibbs energies noted between parentheses were computed at 473 K.

Figure 3 .

 3 Figure3. Free Gibbs energy profiles computed at 298 K (DFT, B3LYP-D3/6-311++G(d,p)) of the aza-Diels Alder reactions between the ↵-oxoketene 1e and the 1-azadiene 2l. Free Gibbs energies noted between parentheses were computed at 473 K. For 1-azadiene 2l, the s-cis/s-trans denomination refers to the conformation of the 4⇡ system of the 1-azadiene, not to the regular CIP nomenclature.

Figure 4 .

 4 Figure 4. Free Gibbs energy profiles computed at 298 K (DFT, B3LYP-D3/6-311++G(d,p)) of the aza-Diels Alder reactions between the ↵-oxoketene 1e and the 1-azadiene 2m. Free Gibbs energies noted between parentheses were computed at 413 K.

Figure 5 .

 5 Figure 5. Free Gibbs energy profiles computed at 298 K (DFT, B3LYP-D3/6-311++G(d,p)) of the aza-Diels Alder reactions between the ↵-oxoketene 1b and the 1-azadiene 2j. Free Gibbs energies noted between parentheses were computed at 473 K.

Figure 6 .

 6 Figure 6. Free Gibbs energy profiles computed at 298 K (DFT, B3LYP-D3/6-311++G(d,p)) of the aza-Diels Alder reactions between the ↵-oxoketene 1c and the 1-azadiene 2j. Free Gibbs energies noted between parentheses were computed at 473 K.

Figure 7 .

 7 Figure 7. Contour plots of the lowest unoccupied molecular orbitals (LUMOs) in the ↵-oxoketenes 1e, 1b, scis-1c, and strans-1c, and those of the highest occupied molecular orbitals (HOMOs) of the model 2-azadienes strans-2j, strans-2k, strans-2l, scis-2l, and strans-2m.

  4 in ethanol followed by standard work-up and flash chromatography eluted with Et 2 O/petroleum ether 2:8 because 4c is chemically inert under these conditions. Rf (Et 2 O/PE 2:8) = 0.21. HRMS (ESI+) m/z: [M+H] + calcd for C 25 H 28 NO 2 + 374.2115, found 374.2111. 13 C{ 1 H}-NMR (75 MHz, ppm/CDCl 3 ): 165.5 (C), 163.2 (C), 137.4 (C), 135.8 (C), 131.4 (CH), 131.3 (C), 129.0 (CH), 129.0 (CH), 128.5 (CH), 128.5 (CH), 128.3 (CH), 128.3 (CH), 127.8 (CH), 127.8 (CH), 127.3 (CH), 127.2 (CH), 107.7 (C), 93.5 (CH), 46.2 (CH 2 ), 45.7 (CH 2 ), 41.1 (CH 2 ), 36.2 (C), 30.0 (CH 3 ), 29.8 (CH 3 ), 13.9 (CH 3 ). 1 H-NMR (300 MHz, ppm/CDCl 3 ): 7.38-7.19 (m, 10H), 6.37 (s, 1H), 5.65 (s, 1H), 5.32 (d, J = 15.4 Hz, 1H), 3.96 (d, J = 15.4 Hz, 1H), 2.46-2.33 (m, 4H), 1.90 (d, J = 1.2 Hz, 3H), 1.20 (s, 3H), 1.16 (s, 3H).

  HRMS (ESI+) m/z: [M+H] + calcd for C 22 H 23 N 2 O 5 + 395.1601, found 395.1604. 13 C{ 1 H}-NMR (75 MHz,

	ppm/CDCl 3 ): 165.8 (C), 161.8 (C), 150.4 (C), 147.8 (C), 142.3 (CH), 133.4 (CH), 131.3 (C), 131.0 (CH),
	129.1 (CH), 128.9 (CH), 127.2 (CH), 124.7 (CH), 110.5 (CH), 108.8 (CH), 108.3 (C), 88.7 (CH), 46.2 (CH 2 ),
	41.0 (CH 2 ), 38.6 (CH 2

  Rf (Et 2 O/petroleum ether 2:8) = 0.18. HRMS (ESI+) m/z: [M+H] + calcd for C 21 H 30 NO 2

	+ 328.2271,
	found 328.2271. 13 C{ 1 H}-NMR (75 MHz, ppm/CDCl 3 ): 165.3 (C), 164.9 (C), 163.0 (C), 162.7 (C),
	148.8 (C), 148.6 (C), 133.3 (CH), 133.3 (CH), 132.0 (C), 131.8 (C), 128.9 (CH), 128.2 (CH), 117.0 (CH 2 ),
	116.9 (CH 2 ), 109.0 (CH 2 ), 109.0 (CH 2 ), 107.9 (C), 107.7 (C), 91.7 (CH), 91.3 (CH), 46.0 (CH 2 ), 46.0 (CH 2 ),
	44.8 (CH 2 ), 44.6 (CH 2 ), 41.0 (CH 2 ), 40.9 (CH 2 ), 40.6 (CH), 40.2 (CH), 36.1 (C), 36.0 (C), 30.2 (CH 2 ),
	30.0 (CH 2 ), 29.9 (CH 3 ), 29.9 (CH 3 ), 29.8 (CH 3 ), 29.7 (CH 3 ), 27.0 (CH 2 ), 26.7 (CH 2 ), 24.6 (CH 2 ), 23.6
	(CH 2 ), 20.7 (CH 3 ), 20.6 (CH 3 ). 1 H-NMR (300 MHz, ppm/CDCl 3 ): 5.79-5.66 (m, 4H), 5.46 (br, 2H),
	5.15-5.07 (m, 4H), 4.10-4.65 (m, 4H), 4.59-4.45 (m, 2H), 3.31-3.20 (m, 2H), 2.32-2.25 (m, 8H), 2.16-1.90
	(m, 8H), 1.86-1.80 (m, 2H), 1.69 (s, 6H), 1.50-1.36 (m, 2H), 1.21-1.14 (m, 2H), 1.10 (s, 6H), 1.08 (s, 6H).