Corentin Bochaton
email: corentin.bochaton@u-bordeaux.fr

David Cochard
email: david.cochard@u-bordeaux.fr

Monica Gala
email: monica.gala@u-bordeaux.fr

Julien Chalifour

Arnaud Lenoble
email: arnaud.lenoble@u-bordeaux.fr.

Éteinte Partiellement

Île De L'

De

Initial observations of the subfossil of partially extinct fauna from Tintamarre Island (Anguilla Bank, Lesser Antilles)

Keywords: Antilles, extinction, island, palaeoontology, zooarchaeology. RÉSUMÉ Antilles, archéozoologie, extinction, île, paléontologie

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

-Introduction

The increasing volume of subfossil material recovered from across the globe provides substantial evidence that human activities have acted as the major driver of faunal biodiversity erosion across the Holocene [START_REF] Ceballos | -Accelerated Modern Human-Induced Species Losses: Entering the Sixth Mass Extinction[END_REF][START_REF] Ceballos | Biological Annihilation via the Ongoing Sixth Mass Extinction Signaled by Vertebrate Population Losses and Declines[END_REF]. It is now clear that human activity has led to dramatic changes in the environment and ultimately to species extinction, and that this impact has been greater and faster than that of natural events. Among all geographic areas, islands are those that are the most impacted by humans, and island fauna often suffers from intense alteration following the initial colonization and subsequent anthropization of their primary environment [START_REF] Olson | Fossil Birds from the Hawaiian Islands: Evidence for Wholesale Extinction by Man before Western Contact[END_REF][START_REF] Steadman | Prehistoric Extinctions of Pacific Island Birds: Biodiversity Meets Zooarchaeology[END_REF][START_REF] Alcover | -The Extent of Extinctions of Mammals on Islands[END_REF]. The Lesser Antillean islands are a good model of the impact of past human colonization on insular environments, including important evidence of a massive extinction event of vertebrate species following the rapid implementation of modern agricultural practices during the colonial period [START_REF] Steadman | Fossil Vertebrates from Antigua, Lesser Antilles: Evidence for Late Holocene Human-Caused Extinctions in the West Indies[END_REF][START_REF] Bochaton | Squamates du Pléistocène supérieur et de l'Holocène de l'archipel guadeloupéen : Évolution de la biodiversité et interactions avec l'Homme[END_REF][START_REF] Boudadi-Maligne M | Evidence for historical human-induced extinctions of vertebrate species on La Désirade (French West Indies)[END_REF]. These islands also demonstrate the predominance of humaninduced extinction over those related to the natural changes in climate since the end of the Pleistocene [START_REF] Bailon | -New data on Pleistocene and Holocene herpetofauna of Marie-Galante (Blanchard Cave, Guadeloupe Islands, French West Indies): Insular faunal turnover and human impact[END_REF][START_REF] Stoetzel | Late Quaternary changes in bat palaeobiodiversity and palaeobiogeography under climatic and anthropogenic pressure: new insights from Marie-Galante, Lesser Antilles[END_REF]. However, despite the continual increase in the knowledge of the Holocene biodiversity of Caribbean islands, information remains localized, concentrated on some islands while other remain poorly investigated. The result is that it is presently difficult to have an accurate understanding of the general processes impacting this region because only a few direct comparisons between islands can be drawn.

For instance, in the Lesser Antillean insular chain that includes the southern part of the West Indies, Guadeloupe [START_REF] Bochaton | Fossil and subfossil herpetofauna from Cadet 2 Cave (Marie-Galante, Guadeloupe Islands, F. W. I.): Evolution of an insular herpetofauna since the Late Pleistocene[END_REF][START_REF] Boudadi-Maligne M | Evidence for historical human-induced extinctions of vertebrate species on La Désirade (French West Indies)[END_REF][START_REF] Stoetzel | Late Quaternary changes in bat palaeobiodiversity and palaeobiogeography under climatic and anthropogenic pressure: new insights from Marie-Galante, Lesser Antilles[END_REF][START_REF] Bochaton | From a thriving past to an uncertain future: Zooarchaeological evidence of two millennia of human impact on a large emblematic lizard (Iguana delicatissima) on the Guadeloupe Islands (French West Indies)[END_REF], Barbuda (Etheridge, 1964 ;[START_REF] Watters | -Vertebrates from archaeological sites on Barbuda, West Indies[END_REF][START_REF] Pregill | Late Quaternary vertebrate faunas of the Lesser Antilles: historical components of Caribbean biogeography[END_REF], Antigua [START_REF] Steadman | Fossil Vertebrates from Antigua, Lesser Antilles: Evidence for Late Holocene Human-Caused Extinctions in the West Indies[END_REF][START_REF] Pregill | Late Holocene fossil vertebrates from Burma Quarry, Antigua, Lesser Antilles[END_REF] and Anguilla [START_REF] Cope | On the Contents of a Bone Cave in the Island of Anguilla (West Indies)[END_REF][START_REF] Pregill | Late Quaternary vertebrate faunas of the Lesser Antilles: historical components of Caribbean biogeography[END_REF][START_REF] Roughgarden | Anolis lizards of the Caribbean: ecology, evolution and plate tectonics[END_REF][START_REF] Kemp | -Early Holocene turnover, followed by stability, in a Caribbean lizard assemblage[END_REF], the islands have been the object of many palaeontological studies, whereas the past biodiversity of most other islands still remain unknown. The Anguilla bank (fig. 1-A) is an unusual case, as one of its main islands (Anguilla) has been heavily studied, whereas the past biodiversity of the Saint Martin (St. Martin) and Saint Bartholomew (St. Barth) islands remain poorly understood. In St.

Martin this is due to the intense destruction of its palaeontological and archaeological cave deposits over the two last centuries. The caves of St Martin and their sedimentary infillings have been mostly destroyed, first from phosphate exploitation, and later from real estate development [START_REF] Auffenberg | West Indies Field Trip[END_REF][START_REF] Hummelinck | -Land and Freshwater Localities[END_REF][START_REF] Mcfarlane | Amblyrhiza and the vertebrate paleontology of anguillean caves[END_REF]. The fact that most limestone caves are near the coast make them especially susceptible to destruction from development related to coastal mass tourism installations and private residences [START_REF] Genoways | Bats of Saint Martin, French West Indies/Sint Maarten, Netherlands Antilles[END_REF]. The effect is that very little palaeontological data has been collected on St. Martin Island and only two sites are known to contain bones preserved in their sedimentary infilling: the caves of Devil's Hole and of Cuckoo Hole near Pelikankey [START_REF] Cope | On the Contents of a Bone Cave in the Island of Anguilla (West Indies)[END_REF][START_REF] Auffenberg | West Indies Field Trip[END_REF]. However, the recovered palaeontological material was mostly composed of remains of the now-extinct giant hutia, Amblyrhiza inundata [START_REF] Cope | Exhibition of bones and teeth of a large rodent from the cave deposits of Anguilla, one of the West Indian Islands[END_REF] with nearly no remains of other vertebrate taxa present [START_REF] Cope | On the Contents of a Bone Cave in the Island of Anguilla (West Indies)[END_REF]. Nevertheless, some open-air archaeological sites do exist on St. Martin [START_REF] Bonnissent | Archéologie précolombienne de l'île de Saint-Martin, Petites Antilles (3300 BP -1600 AD[END_REF] containing vast quantities of fossil remains: Hope Estate [START_REF] Grouard | ), d'après l'analyse des restes des petits vertébrés[END_REF][START_REF] Newsom | On Land and Sea: Native American Uses of Biological Resources in the West Indies[END_REF], Anse des Pères [START_REF] Hofman | Archaeological investigations on St. Martin (Lesser Antilles): The sites of Norman Estate, Anse des Pères and Hope Estate with a contribution to the 'La Hueca problem[END_REF][START_REF] Nokkert | Archaeological investigations on St. Martin (Lesser Antilles). The sites of Norman Estate, Anse de Pères, and Hope Estate, with a contribution to the[END_REF], Norman Estate [START_REF] Hofman | Archaeological investigations on St. Martin (Lesser Antilles): The sites of Norman Estate, Anse des Pères and Hope Estate with a contribution to the 'La Hueca problem[END_REF][START_REF] Nokkert | Archaeological investigations on St. Martin (Lesser Antilles). The sites of Norman Estate, Anse de Pères, and Hope Estate, with a contribution to the[END_REF], and Baie aux Prunes [START_REF] Grouard | Une Population De Pêcheurs-Piégeurs-Collecteurs À Baie Aux Prunes / Plum Bay, Saint-Martin, Petites Antilles[END_REF][START_REF] Bonnissent | Archéologie précolombienne de l'île de Saint-Martin, Petites Antilles (3300 BP -1600 AD[END_REF].

Still, despite having been the object of some zooarchaeological study, these sites were never exhaustively studied from a palaeontological point of view and provide limited information on the past biodiversity of the island. These substantial limitations in our knowledge of the biodiversity of St. Martin also apply to its nearest island neighbor; the Island of Tintamarre less than 3km away from the northeast coast of St. Martin (fig. 1-B).

Tintamarre is a small island of 0.8 km² entirely composed of limestone [START_REF] Dugain | An online database of bird distribution and abundance[END_REF]. This island is part of the Anguilla bank, mainly composed of the islands of St. Martin, Saint-Barthelemy, and Anguilla that are currently separated by areas of sea less than 30m deep, and during the Late Pleistocene formed a single island [START_REF] Christman R | Geology of St. Bartholomew, St. Martin, and Anguilla, Lesser Antilles[END_REF]. Tintamarre is nearly flat with a maximum elevation of 30m and is particularly dry due to a combination of low precipitation and a porous limestone substrate. The vegetation of Tintamarre includes the remnant of a dry deciduous forest with numerous xerophytes and succulent plant species, part of a semi-deciduous forest on the plateau with lignum vitae (Gaiacum officinale) and water mampoo (Pisonia subcordata), and finally salt-tolerant trees like manchineel (Hippomane mancinella) and seagrape (Coccoloba uvifera) near the lagoons and beaches [START_REF] Impact Mer | Évaluation de l'impact du cyclone Irma sur la flore sensible de quatre îlets de la Réserve Naturelle Nationale de Saint-Martin -Rapport final[END_REF]. Nothing is currently known about the past biodiversity of Tintamarre but its modern faunal diversity is extremely poor. Tintamarre is currently occupied only by around 33 vertebrate species, and with no more than seven species of terrestrial mollusks discovered by the collecting missions carried out in the second part of the twentieth century [START_REF] Hovestadt | A critical checklist of the non-marine molluscs of St. Martin, with notes on the terrestrial malacofauna of Anguilla and Saint-Barthélemy, and the description of a new subspecies[END_REF]. Initially only four taxa in the families Gastocoptidae and Pupillidae were known from the material collected in 1949 by A. Hummelinck [START_REF] Hummelinck | -Land and Freshwater Localities[END_REF] and published by [START_REF] Haas | Caribbean Land Molluscs: Vertiginidae[END_REF], before three new taxa were added after the recent review of the collections made in the 1970s by Hummelinck [START_REF] Hovestadt | A critical checklist of the non-marine molluscs of St. Martin, with notes on the terrestrial malacofauna of Anguilla and Saint-Barthélemy, and the description of a new subspecies[END_REF]. Mammals are currently represented by at least three species: goats (Capra hircus), black rats (Rattus rattus), and house mice (Mus musculus) (Brown, 2008, pers. obs.). Brown rats (Rattus norvegicus) may have once been present but recent trapping campaigns have demonstrated they are not currently found on Tintamarre [START_REF] Lorvelec | Compte rendu de mission sur Saint-Martin et les îlets de sa réserve naturelle nationale (1er-15 mars 2013)[END_REF][START_REF] Lorvelec | -Discovery of a population of Spondylurus powelli (Squamata: Mabuyidae) on Île Tintamarre (Saint-Martin, French Antilles) and comments on relationships among skinks of the Anguilla Bank[END_REF]. Mongooses and cats are absent from the Island. In respect to reptiles, the Tintamarre islet is currently occupied by five species of lizards: the Anguilla Bank ameiva (Pholidoscelis plei), the Anguilla Bank tree anole (Anolis gingivinus), the Anguilla Bank skink (Spondylurus powelli), the Sphaerodactyle of St. Martin (Sphaerodactylus parvus), and the green iguana (Iguana iguana) [START_REF] Henderson R | -Island list of West Indian amphibians and reptiles[END_REF][START_REF] Lorvelec | -Discovery of a population of Spondylurus powelli (Squamata: Mabuyidae) on Île Tintamarre (Saint-Martin, French Antilles) and comments on relationships among skinks of the Anguilla Bank[END_REF]. A single tortoise species is also present, the red-footed tortoise (Chelonoidis carbonaria) [START_REF] Henderson R | -Island list of West Indian amphibians and reptiles[END_REF]. The island is currently home to a total of 24 different bird species. Tintamarre hosts several species of marine birds and is classified as an Important Bird Area for breeding populations of Redbilled Tropicbird (Phaethon aethereus), Brown Noddy (Anous stolidus), Audubon's Shearwater (Puffinus lherminieri) and a few other seabirds [START_REF] Collier | Important Bird Areas in the Caribbean: Key Sites for Conservation[END_REF]Brown, 2008, Leblond, 2012). It also hosts a small number of terrestrial bird species including doves (Zenaida aurita), passeriforms (e. g. Elaenia martinica, Tyrannus dominicensis, Coereba flaveola, Loxigilla noctis, and Grassquit (Melanospiza bicolor) [START_REF] Leblond | Les oiseaux marins nicheurs de Guadeloupe et de Saint-Martin et de Saint-Barthélemy[END_REF]eBird, 2020).

No amphibians are currently present on the island.

The low levels of biodiversity on Tintamarre are contrasted by those of the nearby St. Martin Island [START_REF] Genoways | Bats of Saint Martin, French West Indies/Sint Maarten, Netherlands Antilles[END_REF]Yokoyama, 2013 ;[START_REF] De | -Liste taxinomique de l'herpétofaune dans l'outre-mer français : I. collectivité de Saint-Martin[END_REF]. This difference may be due to the species-area relationship (e.g. [START_REF] Ricklefs R | -The Roles of Island Area per Se and Habitat Diversity in the Species-Area Relationships of Four Lesser Antillean Faunal Groups[END_REF], but may also be a result of human impact, including intensive environmental modification. The latter hypothesis matches the historical data of the past use of Tintamarre Island, which despite being currently uninhabited has been subject to occupation and exploitation. The first evidence of occupation dates back to the beginning of the 18th century for the production of lime, as well as a high likelihood of forest exploitation for fuel [START_REF] Lafleur | Saint-Martin (XVIIIe -XIX siècles), Carrefour des Antilles. Saint-Martin, FeniXX réédition numérique[END_REF]. Between 1902 and1931 following the installment of Diederik Christian as the "King of Tintamarre", the small island hosted around 150 people and was subject to an intense level of agricultural activity in comparison to its size [START_REF] Hartog | History of Sint Maarten and Saint Martin[END_REF]. At that time the island was home to about 60-70 cattle and 540 sheep, in addition to cotton being cultivated [START_REF] Hartog | History of Sint Maarten and Saint Martin[END_REF]. In 1931 the island was sold to Louis Fleming, whose family still owns the inner part of the island. The island was first rented to be used as an airport by the "Compagnie Aérienne Antillaise" until 1947 [START_REF] Casius | Compagnie Aérienne Antillaise[END_REF]. At that time the island was only occupied by some employees of the company, and is since free of all permanent human occupation although tourists still frequent the beach. There is also some sparse evidence of an occupation of the island during the pre-Columbian era, but this needs to be described with greater precision [START_REF] Hénocq | Prospection diachronique post-Irma, Saint-Martin, Phase 2. Rapport final d'opération[END_REF]. The impact of each different phases of occupation has remained until now completely unknown, as there was no data available on any subfossil material from Tintamarre. The present study begins to start filling this gap in knowledge by providing the first data on the past biodiversity of Tintamarre Island based on the study of subfossil remains collected from two fossil-bearing cave deposits on the island.

-MATERIALS AND METHODS

In order to survey the caves of St Martin, members of our team visited Tintamarre Island in March 2016 and described several caves that included two sites identified as being of palaeontological interest: the caves of Trou de Souris 1 and Trou de Souris 4 [START_REF] Lenoble | Potentiel archéologique et paléontologique des cavités naturelles de Saint Martin[END_REF]. During this first visit samples of bone remains were collected from the floor of each of these two cavities. The preliminary study of these samples revealed they were likely to be quite ancient as well as containing several species that are no longer present on Tintamarre. It was therefore of interest to conduct excavations in order to investigate the composition of the past biodiversity of Tintamarre within a stratigraphic and chronological framework. However, because of administration related to the ownership of the island it has been impossible to obtain an excavation permit. We visited the island again in 2019 to collect further surface samples at the two sites and decided to publish the study of these assemblages pending future excavations. The material we describe here is thus probably a minor fraction of the full palaeontological content of these sites but it still provides some interesting preliminary data on the past biodiversity of Tintamarre.

The site of the Trou de Souris 1 (18°07'22.26''N, 62°58' 31.368''O) (TS1) is the first of a group of five caves located on Tintamarre in the east part of the Island, near the Mice Hole locality (fig. 1-C). This small cave is the lateral chamber of a banana hole that lies between one and two meters below the topographic surface; the chamber is a dozen meters long, and two to three meters wide (fig. 1-D) . Its floor is composed of loose clay sediment covered by many vertebrate bones. The bedrock of the cavity is not visible which suggests the existence of a sedimentary infilling of unknown depth. The site of the Trou de Souris 4 Cave (18°07'22.728''N, 62°58' 31.62''O) (TS4) is located nearby TS1 (fig. 1-C). The cave is at a depth of six to seven meters and is accessible through a narrow shaft. It is six to seven meters long and four meters wide (fig. 1-E). The cave floor is similar to that of Trou de Souris 1, including the scattered small vertebrate bones on the surface. These two caves are currently unoccupied by bats as evidenced by the absence of decaying organic remains on the floor, and overall seem to present all the suitable characteristics for the preservation of subfossil assemblages. At both sites the bone and shell samples were collected by hand from the floor and no sieving was conducted. The collected remains were not sampled for isotopic Bone and terrestrial snail shell remains recovered in Tintamarre and from the Trou de Souris caves were identified using the West Indian landsnail shell and osteological collections of the PACEA laboratory (Bordeaux University, France), the comparative anatomy collection of the Muséum national d'Histoire naturelle (Paris, France) and appropriate published identification criteria cited throughout the text. Vertebrate bone remains were quantified; terrestrial snail shells were recorded as only presence/absence for each locality.

-RESULTS

During the two surveys of Tintamarre and the sites of Trou de Souris 1 and 4, 519 vertebrate bone remains and many fragmented or complete shells of terrestrial snails were recovered (Tabs. 1 & 2).

-VERTEBRATE BONE REMAINS (TAB. 1)

Most of the mammal bones collected at both sites were attributed to unidentified rodents (N=76) but we still were able to identify some cranial, mandibular, and dental remains with more precision. Among these remains is a nearly complete right mandible (fig. 2-B) identified as Rattus rattus (Linnaeus, 1758). Some rodent post-cranial remains were also identified as Mus musculus (Linnaeus, 1758) on the basis of their very small size. This is further confirmed by the presence of a mandible bearing two embedded molars (m1 and m2). In addition to these two fairly common rodents, four dental remains were attributed to an endemic Lesser Antillean rodent, Pennatomys nivalis Turvey, Weksler, Morris, and Nokkert, 2010 (fig. D,E), from the criteria described by [START_REF] Turvey | -Taxonomy, Phylogeny, and Diversity of the Extinct Lesser Antillean Rice Rats (Sigmodontinae: Oryzomyini), with Description of a New Genus and Species[END_REF]. The two M1 (complete with little wear) may be identified by: the presence of three alveolar roots, the absence of or a much reduced accessory root, an undivided anterocone (anteromedian flexus absent see fig. 2-D), a well-developed mesoloph (see fig. 2-C), and a very small posteroflexus (notch at posterior margin of metacone see fig. 2-D). In addition, the position of the posterior border of the zygomatic arch is slightly anterior to the most anterior alveolus of the M1. The M2 was broken but its three roots were visible and the mesoflexus comprised a single internal fossette (see fig. 2-E). The final rodent remain of interest was a mandible bearing a much worn M1 with two large roots and two accessory roots. The material was too poorly preserved to take precise measurements of the dental rows, however, the complete mandibular and maxillary rows probably would not have exceeded 7mm in length. This would be close in size to Pennatomys nivalis (respectively 6.61 and 6.12mm; [START_REF] Turvey | -Taxonomy, Phylogeny, and Diversity of the Extinct Lesser Antillean Rice Rats (Sigmodontinae: Oryzomyini), with Description of a New Genus and Species[END_REF] and quite unlike the other forms of native rodents present in the north of the Lesser Antilles, namely Antillomys rayi (> 9mm; [START_REF] Brace | Unexpected Evolutionary Diversity in a Recently Extinct Caribbean Mammal Radiation[END_REF][START_REF] Goedert | Isotopic ecology and regional extirpation chronology of the extinct Lesser Antillean rat Antillomys rayi[END_REF].

Among the other mammal taxa, 28 remains were identified as Oryctolagus cuniculus (Linnaeus, 1758) including a complete mandible (fig. 2-A). Some remains of Caprinae (phalanx and petrosal bones) were also identified but no diagnostic elements were present that would have allowed a specific identification as either Capra or Ovis. An unidentified Phyllostomid bat was represented by a single clavicle, and a cat canine (Felis catus;

(Linnaeus, 1758)) and a probable lower incisor of Agouti (Dasyprocta leporina; Linnaeus, 1758) was also present.

Squamates were represented by four taxa; at least one snake and three lizards. The snake remains comprise an assemblage of 109 colubrid snake vertebrae (fig. 2-F) plus a few cranial remains. The morphology of the identified trunk vertebrae are similar to those of Alsophis rijgersmaei Cope, 1869 (see Bochaton et al., 2019a) but the diverse morphological variability observed in the assemblage precludes a definitive identification and limits attribution to the level of the genus Alsophis. Vertebrae with morphological criteria of the genus Erythrolamprus (see Bochaton et al., 2019a) are however absent from the assemblage. It is possible that the morphological variability of the fossil vertebrae of Tintamarre may correspond to several species, as has been proposed for assemblages in Guadeloupe (Bochaton et al., 2019, a) but further study of the Antillean colubrid snakes vertebral morphology should be carried out to better understand the intraspecific and ontogenetic variability. Lizard remains are mostly identified as anoles (N=90), the remains primarily comprising dentaries of a size similar to the St Martin anole currently present on Tintamarre, Anolis gingivinus Cope, 1864. These bones bear a series of pleurodont teeth slightly labialolingually flattened with tricuspid posterior teeth and monocuspid anterior teeth. Ground lizards of the genus Pholidoscelis Fitzinger, 1843 are represented by 17 cranial remains including several dentaries (fig. 2-H). The Meckel groove is fully open on these bones, and the dental region is slightly arched and bears a heterodont dentition with teeth increasing posteriorly in size (Bochaton et al., 2019, b). A sample of 62 gecko bones were identified to the genus Thecadactylus Goldfuss, 1820. The dentaries of this gecko (fig. 2-G) are recognized by their homodont dentition composed of a high number (30-50) of monocuspid, slender, slightly blunt teeth; a ratio inferior to 4 between the dental length (in mm) and the number of teeth; and the extension of the lateral coronoid facet that reaches the level of the last dental position [START_REF] Bochaton | Identifying gecko species from Lesser Antillean paleontological assemblages: Intraspecific osteological variation within and interspecific osteological differences between Thecadactylus rapicauda (Houttuyn, 1782) (Phyllodactylidae) and Hemidactylus mabouia (Moreau de Jonnès, 1818) (Gekkonidae)[END_REF]. The gecko represented by these remains is now absent from Tintamarre. Four fragments of tortoise shell (costal and neural plates) have been identified but do not bear any distinctive criteria. We propose a tentative attribution to Chelonoidis carbonaria (Spix, 1824) as this tortoise is still present on the island. A single remain (a tibiofibula) of a small amphibian whose size and overall morphology is similar to members of the genus Eleutherodactylus Duméril and Bibron, 1841 was also identified.

Nine bird remains representing two species of Columbidae (Columbina passerina (Linnaeus, 1758), Zenaida cf. aurita (Temminck, 1809), and the passeriform Margarops fuscatus (Vieillot, 1808) have been collected across the two sites (Tab. 1). These bones were identified from their morphological similarity to modern specimens. Zenaida cf. aurita is represented by a single fragment of ulna and was identified from the morphology of the condylus dorsalis ulnae (sensu [START_REF] Baumel | Handbook of Avian Anatomy: Nomina Anatomica Avium[END_REF] which matches that of modern Zenaida aurita. The latter species is the only Zenaida species to have been observed within the last few years on Tintamarre (eBird, 2020).

-TERRESTRIAL SNAIL SHELLS (TAB. 2)

Twelve morphotypes corresponding to at least eleven species of terrestrial snail have been identified from all samples collected in both the caves and outdoor localities. Among these eleven species, seven were found in the Trou de Souris caves, and three were found exclusively in the cave contexts (Tab. 2).

Annulariidae shells were collected at the majority of collection points but were only represented by old or fossil (fully mineralized) specimens. Two species are represented; most specimens are of a medium to small-size, ranging from an elongate conic shape (fig. 3-A) to a short conic shape (fig. 3-B), both displaying equally developed spiral rounded cords and axial erected lamellae fusing to form tufts in the sutures of the whorls. These features agree with identification as Diplopoma crenulatum (Potiez & Michaud, 1835) (see also criteria in [START_REF] Watters | -A Preliminary Review of the Annulariidae (Gastropoda: Littorinoidea) of the Lesser Antilles[END_REF], the elongate morph being similar to the specimen from St. Martin illustrated by Watters (op. cit. fig. 100). The short conic morph is characterized by a smaller length, lower number of whorls (up to 4 ½), more prominent spiral cords, and very developed sutural tufts that are frequently broken off. All these characteristics make this morph similar to the Diplopoma sulculosum species (Pfeiffer, 1852) known from the neighboring island of Anguilla, although D. sulculosum has more pronounced features (see criteria in [START_REF] Watters | -A Preliminary Review of the Annulariidae (Gastropoda: Littorinoidea) of the Lesser Antilles[END_REF]. This short morph is also indistinguishable from the St. Barth specimens assigned to D. crenulatum by [START_REF] Questel | Les escargots terrestres de Saint-Barthélemy[END_REF] and D. sulculosum by [START_REF] Hovestadt | A critical checklist of the non-marine molluscs of St. Martin, with notes on the terrestrial malacofauna of Anguilla and Saint-Barthélemy, and the description of a new subspecies[END_REF]. This morph and Tintamarre's general population of D. crenulatum therefore present characteristics that are intermediary between D. crenulatum and D. sulculosum, which raises taxonomic questions beyond the scope of this paper. We therefore cautiously classify it here as a D. crenulatum subspecies. The second species of Annulariidae is represented by a few specimens collected only in Trou de Souris 1 and 4. These are medium-sized specimens characterized by a teardrop-shaped aperture, a dominant axial sculpture of numerous and close lamellae and minute sutural tufts formed by 1-2 expanded axial lamellae (fig. 3-C), all characters referring to the species Chondropoma pupiforme (Sowerby, 1843) following [START_REF] Watters | -A Preliminary Review of the Annulariidae (Gastropoda: Littorinoidea) of the Lesser Antilles[END_REF].

A perforated Sagdidae shell displaying depressed helicoids, a subcircular aperture, rounded whorls, and a very fine axial striation [START_REF] Shuttleworth | Beiträge zur näheren Kenntniss der Land-und Süsswasser-Mollusken der Insel Portorico[END_REF] (fig. 3-D) is identified as Hyalosagda subaquilla [START_REF] Shuttleworth | Beiträge zur näheren Kenntniss der Land-und Süsswasser-Mollusken der Insel Portorico[END_REF]. This species was collected both in the caves and the outdoor locations and is represented by a small number of old shells. A unique fragmented shell of Oleacinidae is identified as Melaniella gracillima (L. Pfeiffer, 1839) (fig. 3-E), based on its slender form and flattened whorls, its sub-triangular aperture displaying a right margin impressed outward, and some twenty axial cords on the last whorl [START_REF] Pilsbry | Manual of Conchology, Structural and Systematic. Second Series: Pulmonata[END_REF]. The number of intercalated striae (a dozen) permits further identification to the variety M. g. sanctithomensis [START_REF] Pilsbry | Manual of Conchology, Structural and Systematic. Second Series: Pulmonata[END_REF] that is present in the northern Lesser Antilles [START_REF] Pilsbry | Manual of Conchology, Structural and Systematic. Second Series: Pulmonata[END_REF][START_REF] Haas | Caribbean Land Molluscs: Subulinidae and Oleacinidae[END_REF]. Two old shells of a small Bulimulidae were found in Trou de Souris 4 and at one outdoor locality. The occurrence of well-marked irregular axial ribs, a shiny surface and almost straight sides unambiguously enable identification to the species Bulimulus lehmani (Fischer, 1857) (fig. 3-F) known from Anguilla and Dog Island [START_REF] Breure | -Caribbean land molluscs: Bulimulidae, I. Bulimulus[END_REF]. Associated with these specimens were fragments of a very large and thin Bullimulidae shell. Because of the fragmented nature of this material we were unable to identify it to a given genera or species. Among the most frequently encountered shells are those identified as Gastropotidae.

This taxon was recovered in larger quantities at the localities where the litter was sampled, which facilitated the recovery of the very small shells of these snails. Three species were identified in the recovered samples, the most abundant being Gastrocopta bardadensis (Pfeiffer, 1852) (fig. 3-G). The shells of this species were found associated with larger shells (Length ≈ 2.5 mm) with a non-bifid parietal tooth that were identified as Gastrocopta servilis (Gould, 1843). Smaller shells (length < 2 mm) were also found in the same contexts, with a poorly developed bifid angulo-parietal tooth and lacking any infraparietal nodule, both characteristics present in the species Gastrocopta pellucida (Pfeiffer, 1840) (see Characters in [START_REF] Pilsbry | Manual of Conchology, Structural and Systematic. Second Series: Pulmonata[END_REF]Pilsbry, -1918)). Pseudopineria viequensis (Pfeiffer, 1856) is represented by both old and recent shells (fig. 3-H) in the samples collected from both caves and outdoors locations. This species is characterized by its relative moderate size (Length ≈ 5 mm), its conical oval imperforate shape and the strong oblique striations that give a spiny shape to its keel [START_REF] Pilsbry | Manual of Conchology, Structural and Systematic. Second Series: Pulmonata[END_REF]. The shells collected on Tintamarre resemble the elongated variety of Pseudopineria viequensis that is common in the northern Lesser Antilles [START_REF] Pilsbry | Manual of Conchology, Structural and Systematic. Second Series: Pulmonata[END_REF]. A single old shell of a large pupoid, Pupoides albilabris (Adams, 1841) characterized by its reflected peristome (fig. 3-I) [START_REF] Pilsbry | Land Mollusca of North America (North of Mexico)[END_REF] was found in the cave of Trou de Souris 4.

-DISCUSSION

The faunal assemblages recovered from the caves of Trou de Souris 1 and 4 provide evidence of the past occurrence of at least six vertebrate taxa that are no longer present on Tintamarre Island. The timing of the extinction of these taxa is difficult to establish as the studied remains were recovered on the surface and are not dated. However, the faunal composition of the subfossil assemblage, rich in non-native species introduced by Europeans, suggests at least that these assemblages mostly document relatively recent time periods, maybe the few last centuries.

The vertebrate assemblages of Trou de Souris 1 and 4 are mostly composed of rodent remains the majority of which (black rat and house mouse) are taxa that arrived in the Caribbean during the colonial period along with other taxa such as goats. These taxa are still present nowadays on Tintamarre and the fossils assemblages do not provide further clues about the specific date of their introduction on the island. More interesting is the case of two other European mammals that are present in the Trou de Souris bone assemblage but nowadays are absent on Tintamarre: the rabbit and the cat. Both of these taxa were introduced to the Lesser Antilles as early as the 17 th century and were likely present on Tintamarre during the first third of the 20 th century when the island was intensively cultivated. The occurrence of cats near human settlements is the likely association of this species, and a journal article published in the "Journal de Paris" the 23 rd of August 1913 describing the daily life of the "King of Tintamarre" mentions that he was hunting wild rabbits on the island. The disappearance of the rabbit on Tintamarre could be easily explained by the absence of agricultural activities on Tintamarre after the island was sold in 1931 and by the presence of rats on the Island. In general rabbits tend to fail to establish feral populations in the West Indies because juveniles are intensively predated by mongooses and rats [START_REF] Du Tertre J.-B. (r | Histoire générale des isles de S. Christophe, de la Guadeloupe, de la Martinique, et autres dans l'Amérique[END_REF][START_REF] Flux | World Distribution of the Rabbit Oryctolagus Funiculus on Islands[END_REF][START_REF] Lorvelec | Les mammifères terrestres non volants des Antilles françaises et l'introduction récente d'un écureuil[END_REF]. Also, from a more global point of view, rabbits are difficult to breed in tropical areas [START_REF] Lebas | Rabbit production in tropical zones[END_REF]. The disappearance of cats is more difficult to explain as Muridae (mice and rats) are still present on the island and would represent an important food source; but there is no evidence that any population of feral cats ever existed on Tintamarre and this species may have left the island at the same time as the last farmers. The final mammal of probable introduced origin present in these fossil assemblages is the agouti. These rodents are known to have been introduced by Amerindian populations to the Lesser Antilles [START_REF] Newsom | On Land and Sea: Native American Uses of Biological Resources in the West Indies[END_REF][START_REF] Giovas | -Sr and Pb isotopic investigation of mammal introductions: Pre-Columbian zoogeographic records from the Lesser Antilles, West Indies[END_REF] and have not been previously mentioned in reports on Tintamarre. The arrival date of this species on Tintamarre remains very enigmatic as well as the reason for its local extinction. It might have been predated by humans and dogs but more data would be needed to discuss this question further.

There are two wild mammals present in the assemblages of Trou de Souris 1 and 4 that are now absent on Tintamarre (Pennatomys nivalis and Phyllostomidae sp.) along with one snake (Alsophis sp.), one lizard (Thecadactylus sp.), and one amphibian (Eleutherodactylus sp.).

The occurrence of Pennatomys nivalis on Tintamarre is worth noting as it is the first identification of this taxon outside of the St. Kitts bank [START_REF] Turvey | -Taxonomy, Phylogeny, and Diversity of the Extinct Lesser Antillean Rice Rats (Sigmodontinae: Oryzomyini), with Description of a New Genus and Species[END_REF][START_REF] Brace | Unexpected Evolutionary Diversity in a Recently Extinct Caribbean Mammal Radiation[END_REF]. One of us (D. C.) also identified this taxon in the archaeological assemblage of Hope Estate in St. Martin, which would possibly extend the distribution of this taxon to the whole Anguilla bank. This taxon may also have been introduced from the nearby St. Kitts bank but its native or introduced status on Tintamarre and the whole Anguilla bank cannot be tested with the currently available data. Lesser Antillean rice rats (Antillomys rayi et Megalomys ssp.) all became extinct after the European arrival, between the XVI th and the XX th century probably following the destruction of their native environments and the introduction of predators, and competitors such as the black rat [START_REF] Harris | Review of Negative Effects of Introduced Rodents on Small Mammals on Islands[END_REF][START_REF] Goedert | Isotopic ecology and regional extirpation chronology of the extinct Lesser Antillean rat Antillomys rayi[END_REF]. The presence of several introduced taxa and intensive agricultural activities on Tintamarre across the last few centuries could also potentially have led to the extinction of Pennatomys nivalis on the island, an explanation also viable for the four other now-extirpated taxa discovered in the Trou de Souris bone collections. There is no available record regarding the modern diversity of bats on Tintamarre but we did not observe any during our exploration of the caves of the island and it is possible that phyllostomid bats are no longer present. The three species of birds identified in the Trou de Souris sites are relatively common birds on the neighboring island of St. Martin. The Pearly-eyed Thrasher (Margarops fuscatus) and the Common Ground Dove (Columbina passerina) have not been observed on the islet for the last decade. They are however listed as present on the island by older studies [START_REF] Collier | Important Bird Areas in the Caribbean: Key Sites for Conservation[END_REF]. These birds could be either occasional occupants of the Island, or have disappeared relatively recently. In respect to reptiles and amphibians, the extirpation of Alsophis on Tintamarre is not surprising as these snakes are known to be very vulnerable to human impact and have suffered many local extinctions in the Lesser Antilles including on the nearby island of St. Martin where the native species of these snakes is supposed to have become extinct very recently [START_REF] Breuil | Histoire naturelle des amphibiens et reptiles terrestres de l'archipel guadeloupéen: Guadeloupe, Saint-Martin, Saint-Barthélémy[END_REF][START_REF] Henderson R | -Island list of West Indian amphibians and reptiles[END_REF]. The extirpation of Thecadactylus is more unusual as this taxon generally resists human disturbance quite well, and there is no clear explanation for its disappearance on Tintamarre. This gecko genus is represented by an endemic species on the Anguilla bank, Thecadactylus oskrobapreinorum Köhler & Vesely, 2011 and so is likely to have been present for a long time in the bank and also on Tintamarre. There is no evidence for a similarly endemic species frog of the genus Eleutherodactylus on the Anguilla banc. Such a species may have become extinct and replaced by two other lesser Antillean species: E. martinicensis (Tschudi, 1838) and E.

johnstonei Barbour, 1914. As it is not possible to identify the two frog remains found in the Trou de Souris caves we cannot state if they belong to an introduced species or a native one.

The extinction of this unidentified amphibian is however surprising and could be potentially explained by aridification following the removal of the primary forest for agricultural purpose, or by the final abandonment of the agricultural activity of the island.

The other non-mammal taxa represented by bone remains collected in the fossil deposits are still present on the island today. Two of them are lizard genera present on most islands in the Anguilla bank (Anolis sp. and Pholidoscelis sp.) where they are typically represented by endemic species [START_REF] Henderson R | -Island list of West Indian amphibians and reptiles[END_REF]. The remaining taxon is the red-footed tortoise, a non-native species that is today present on all major islands of the Anguilla bank and whose introduction date in the West Indies is subject to much debate. Tortoises of the genus Chelonoidis were present on the Anguilla bank since the Pleistocene [START_REF] Lazell | Tortoise, cf. Geochelone carbonaria, from the Pleistocene of Anguilla, Nothern Lesser Antilles[END_REF] but the specific attribution of these remains to Chelonoidis carbonaria remain doubtful, and this species is absent in the Amerindian archaeological deposits of the region. An alternative hypothesis regarding the introduction of this turtle species to the Lesser Antilles were recently reviewed by [START_REF] De | -Liste taxinomique de l'herpétofaune dans l'outre-mer français : II. Collectivité de Saint-Barthélemy[END_REF], and it appears that they were most likely introduced to most regions during the 19 th century.

From a general point of view, the data collected from the Trou de Souris bone assemblages indicates that the fauna of Tintamarre was probably strongly impacted by human activities during the last few centuries although the lack of isotopic dating of the material prevents any definitive conclusions. The biodiversity profile of this island is however unusual, as although it has suffered from the same extinctions that have occurred in many if not all of Lesser Antillean islands (rabbit, rice rat, and the Alsophis snake) other unusual extinction events (Thecadactylus and Eleutherodactylus) seem to point towards particularly strong environmental modifications of the island. However, conversely, the endemic Anguilla Bank skink (Spondylurus powelli) is still extant on the island despite skinks being nowadays extinct on most of the Lesser Antillean islands [START_REF] Hedges | -A new skink fauna from Caribbean islands (Squamata, Mabuyidae, Mabuyinae)[END_REF]. This could be related to the fact that the mongoose was never introduced to Tintamarre, which seems to be confirmed by our observations, and that the stone walls constructed by farmers on the island represent a very suitable and protective environment for these lizards, as suggested by [START_REF] Lorvelec | -Discovery of a population of Spondylurus powelli (Squamata: Mabuyidae) on Île Tintamarre (Saint-Martin, French Antilles) and comments on relationships among skinks of the Anguilla Bank[END_REF] Regarding terrestrial snails, our newly collected data enriches our knowledge of the biodiversity of these organisms on Tintamarre by doubling the number of taxa listed for this island. With one exception, all the species collected on Tintamarre are known from the neighboring island of St. Martin, to which this island was attached during the periods of low sea levels during the Pleistocene. The number of species recorded on Tintamarre is still however much lower, as no fewer than 40 species of snails have been recorded on St. Martin [START_REF] Hovestadt | A critical checklist of the non-marine molluscs of St. Martin, with notes on the terrestrial malacofauna of Anguilla and Saint-Barthélemy, and the description of a new subspecies[END_REF]. The terrestrial mollusk population of Tintamarre thus forms a sub-sample of the fauna of St. Martin, including calcicole (e.g. Pupoides albilabris, Diplopoma crenulatum), xeric (e.g., Gastocopta spp.), and coastal (e.g., Pseudopineria viequensis) species.

The only Tintamarre species absent from St. Martin is Bulimulus lehmani, a species present on the island of Anguilla, while the genus Bulimulus is currently represented on St Martin by Bulimulus diaphanus and B. guadalupensis [START_REF] Breure | -Caribbean land molluscs: Bulimulidae, I. Bulimulus[END_REF]. Bulimulus lehnmani was formerly observed on the island of St. Martin [START_REF] Coomans | The Non-marine Mollusca of St. Martin (Lesser Antilles)[END_REF][START_REF] Van Den Berge | Notes préliminaires sur les mollusques terrestres de Guadeloupe[END_REF], but these records are unreliable as they are not associated with a locality and this species has not been found during the systematic surveys carried out during the last few decades [START_REF] Hovestadt | A critical checklist of the non-marine molluscs of St. Martin, with notes on the terrestrial malacofauna of Anguilla and Saint-Barthélemy, and the description of a new subspecies[END_REF]. In this study, we demonstrate the past presence of B. lehmani on Tintamarre but this species is only represented by few old shells. The status of this species on the island still has to be demonstrated and it could have been unintentionally introduced from Anguilla during historical times as many Tintamarre workers were Anguillan inhabitants.

The biogeographic distribution of Annulariidae on the Anguilla Bank is complex. One species is endemic to the northern island of Sombrero (Chondropoma julieni (L. Pfeiffer, 1866)), another is known to be endemic to Anguilla and the satellite islets (Diplopoma sulculosum), a third species is found in Anguilla and the limestone terrain of the southern part of St. Martin (Chondropoma pupiforme), and a final species (Diploploma crenulatum) is known only from the northern part of St. Martin but from the more southern islands of the Lesser Antilles below Martinique. Meanwhile, the specimens found in St. Bartholomew are identified as either D. sulculosom or D. crenulatum depending on the authors [START_REF] Watters | -A Preliminary Review of the Annulariidae (Gastropoda: Littorinoidea) of the Lesser Antilles[END_REF][START_REF] Questel | Les escargots terrestres de Saint-Barthélemy[END_REF][START_REF] Hovestadt | A critical checklist of the non-marine molluscs of St. Martin, with notes on the terrestrial malacofauna of Anguilla and Saint-Barthélemy, and the description of a new subspecies[END_REF]. In this study the species of interest are the two Annulariid land snails Diplopoma crenulatum and Chondropoma pupiforme. The recovery of C. pupiforme shells from the Tintamarre caves and its absence in outdoor localities indicates that this species is currently very rare on the islet, if not already extinct. Furthermore, this record fills the gap of the distribution of this species between Anguilla and the island south of 1849); and some Caribbean species such as the Cuban Zachrysia provisoria (Pfeiffer, 1858) or the even more widespread Bulimulus guadalupensis (Bruguière, 1789). These absences show that the initial land snail community of Tintamarre is potentially well preserved, which could reflect restrictive environmental conditions and/or the limited frequentation of the island by human populations. The character profile of a preserved fauna is, however, counterbalanced by the high proportion of species represented only by old shells in the collected material, such as the abundant Diplopoma crenulatum, but also the rare shells of Chondropoma pupiforme, Melaniella gracillima, Pupoides albilabris, Hyalosagda subaquilla, and Bulimulus lehmani for which only a single or a very small number of specimens were found. The fact that the collected remains are rare and do not present the characteristic appearance of recent shells suggests that these species may no longer be extant on Tintamarre. However, the period during which the collecting was carried out, at the end of the dry season, and only a year and a half after a major cyclone, may have impacted the diversity we observed. A better assessment of the current mollusk fauna of the island, including surveys carried out under better climatic conditions, is needed to resolve this issue.

Our data also demonstrates the value of cave deposits in documenting the malacological community of the island. All identified species were found in the Trou de Souris caves, with the exception of the very small Gastrocoptidae and the fragments of Oleacinidae (see Tab. 2) that were difficult to collect without the sampling and washing of sediment. Moreover, several species that were not observed in outdoors locations were collected in the caves (Chondropoma pupiforme, Pupoides albilabris, Bulimulidae sp.). This is probably related to the better preservation condition of the shells trapped in caves.

-CONCLUSION

Our study of the remains collected on the surface of two fossiliferous deposits on Tintamarre Island demonstrates the past occurrence of at least six vertebrate taxa as well as potentially six terrestrial snail species that are no longer present on the island. The lack of data regarding the age of the studied material prevents the association of these extinctions with any specific events but, based on events observed on several other islands in the Lesser Antilles, we propose several hypotheses linking these extinctions to a probable anthropogenic impact across the last few centuries.

It would be very interesting to build on these initial results concerning the past biodiversity of Tintamarre with an additional survey aiming to document its current terrestrial malacofaune and, of course, a proper excavation of the Trou de Souris caves. Such an excavation would provide an opportunity for a major improvement in our current knowledge and would enable (1) the addition of a chronological framework to our observations, (2) more precise identification of the taxa by obtaining more material and (3) a better characterization of the evolution of the biodiversity on Tintamarre. Finally, this island has proved to be very interesting, as the trajectory of biodiversity seems to deviate from the usual trends observed and could therefore be very useful in testing several hypotheses regarding past and present human impact on the Lesser Antillean fauna. [START_REF] Reig | A Proposed Unified Nomenclature for the Enamelled Components of the Molar Teeth of the Cricetidae (Rodentia)[END_REF]. [START_REF] Haas | Caribbean Land Molluscs: Vertiginidae[END_REF], and those with a dagger by [START_REF] Hovestadt | A critical checklist of the non-marine molluscs of St. Martin, with notes on the terrestrial malacofauna of Anguilla and Saint-Barthélemy, and the description of a new subspecies[END_REF].

Tab. 2 : Données d'occurrence des escargots terrestres dans les deux sites de Trou de Souris

1 et 4 ainsi que dans les lieux de prélèvement en plein-air. Les espèces marquées d'un astérisque sont celle mentionnées précédemment sur Tintamarre par [START_REF] Haas | Caribbean Land Molluscs: Vertiginidae[END_REF], et celle marquée d'un obèle les espèces listées par [START_REF] Hovestadt | A critical checklist of the non-marine molluscs of St. Martin, with notes on the terrestrial malacofauna of Anguilla and Saint-Barthélemy, and the description of a new subspecies[END_REF].

[

  radiometric] dating as their probable historic/colonial origins (see Discussion) would render 14C dating impossible. Additional snail shells were hand-collected from the crevices in the limestone pavement on the rock surface near Trou de Souris 1 (SXM-009: 18°07'22.26''N, 62°58' 31.368''O) and in several other outdoor localities: near the northeastern point of the island (SXM-012: 18°07'22.296''N, 62°58'27.047''O), above the northern cliffs (where a soil sample was also taken and sorted under the microscope) (SXM-013 : 18°07'25.536''N,62°58' 37.92''O; 62°58' 47.459''O), close to the old sheepfold (SXM-015 : 18°07'04.476''N, 62°59'03.227''O), and from Baie Blanche beach (SXM-016 : 18°07'0.48''N, 62°59' 14.892''O).

  St. Martin. The population of D. crenulatum collected at Tintamarre, with intermediate characteristics between the St. Martin form and the Anguilla Island species (D. sulculosum), also suggests the presence of phenotypic and geographic intergrades between species of the Diplopoma genus within the Anguilla Bank. The distribution of Annulariidae therefore is, or would have been, much less fragmented than previous data suggests. It is interesting to observe that the many invasive species currently present on St. Martin and more widely in the West Indies are absent from Tintamarre. These species includes: Achatinidae, e.g., Subulina octona (Bruguière, 1789), and Allopeas gracile (Hutton, 1834); Polygyridae, e.g., Polygyra cereolus (Megerle von Muhlfeld, 1816); various species of Streptaxidae, including Streptartemon deplanchei (Drouët, 1859) and S. glaber (L. Pfeiffer,

Fig. 1 :

 1 Fig. 1: Maps. (A) Map of the West Indies with the location of the Anguilla island bank. (B)

Fig. 1 :Fig. 2 :

 12 Fig. 1: Cartes et plans. (A) Carte des Antilles avec la localisation du banc d'Anguilla. (B)

Fig. 2 :

 2 Fig. 2 : Quelques-uns des ossements de vertébrés les plus caractéristiques identifiés dans les

Fig. 3 :

 3 Fig. 3: Lateral views of some of the terrestrial snail shells collected on Tintamarre

Fig. 3 :

 3 Fig. 3 : Vues latérales de certaines des coquilles d'escargots terrestres collectées sur l'îlet

ACKNOWLEDGMENTS

This study was conducted as a part of the ECSIT Project: « Écosystèmes insulaires tropicaux, réponse de la faune indigène terrestre de Guadeloupe à 6 000 ans d'anthropisation du milieu », conducted by the CNRS with a financial support from the European PO-FEDER program (grant n° 2016-FED-503), the Guadeloupe Regional Council, and the DAC of Guadeloupe.

The authors are grateful to the Réserve naturelle de Saint-Martin which provided vital support to the carrying out of this project. We are also grateful to Alain Queffelec (CNRS) and Christian Stouvenot (DAC Guadeloupe) who also took part in the survey. We also thank Christophe Henocq (Community of St. Martin) who provided useful insights and comments regarding the historical and archaeological information available on Tintamarre.


Andrei Corbu

Maurizio Aquino

Manuel Perez

Zoila Gandara

Siméon Arseniyadis
email: simeon.arseniyadis@icsn.cnrs-gif.fr

Natural and Unnatural A-seco Terpenes from Pulegone: Synthesis of Galbanic Acid and Marneral Revisited

Keywords: Terpenoids, Natural products, Chiral pool, Mitsunobu etherification, Microwave chemistry

We describe herein an improved chiral-pool strategy for assembling the core structure 3 (both antipodes) of various Aseco terpenoids that have allowed us to prepare natural galbanic acid [(-)-1] from (S)-(-)-pulegone [(-)-2]. Furthermore, we were able to increase both the yield and step efficiency [

Introduction

A range of secondary metabolites contain a trans or cis vic-dimethyl-substituted six-membered ring along with two side-chains in various stereochemical combinations. We initiated a program based on a chiral-pool approach to the synthesis of selected targets (1 and 7-10, Scheme 1) that represent several families of naturally occurring A-seco terpenes that have in common a central B-ring nucleus with three adjacent stereogenic centers, one of which is a quaternary carbon atom, and a three-carbon oxygenated sidechain and an exocyclic dimethyl olefin. Galbanic acid [(-)-1], [START_REF] Bagirov | For the structure and stereochemistry of galbanic acid from galbanum[END_REF] a sesquiterpene coumarin ether, shows a range of biological activities, such as antibiotic, [2] antibacterial, antithrombotic, [START_REF]During in vitro experiments galbanic acid suppressed the development of platelet aggregation induced by ADP, thrombin, histamine, adrenaline, acetylcholine, and collagen at concentrations of 50, 100, and 200 µg/mL[END_REF] and hepatoprotective activities. [4] The compound is a potent antifertility and hormonal lead and it has also been demonstrated to synergize the activity of various antibiotics, [5] opening up promising applications for the treatment of MRSA (methicillin-resistant Staphylococcus aureus) infections. [6] Very recently, methyl galbanate [(-)-18] and galbanic acid [(-)-1] were screened in an in vitro antiinfluenza A viral (H 1 N 1 ) assay with amantadine as a positive control. They are reported to show great potency against influenza A virus H 1 N 1 (IC 50 = 0.26 and 0.45 µg/ mL, respectively), with the C-3 methyl ester showing better potency than the corresponding free (galbanic) acid. [7] The sesquiterpene-isofraxidin ether secodrial (8) [8] differs from 1 only by the presence of two methoxy groups on the aro-1 of the synthesis of marneral [(+) -7] as well as to access higher homologues of ent-galbanic acid [(+)-4], ent-secodrial [(+)-5], and bis-epi-secochiliotrin ( 6) from (R)-(+)-pulegone [(+)-2]. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009) matic ring and the oxidation state at C-3. The indole isoprenoid suaveolindole (9), which shows activity against the Gram-positive bacteria Bacillus subtilis and methicillinresistant Staphylococcus aureus, [9] and the hitherto not synthesized secochiliotrin (10) [10] bear an indole-and a furancontaining side-chain, respectively, the latter belonging to the diterpene family (Scheme 1). Marneral [START_REF] Matsuda | named this compound marneral (and its corresponding alcohol 3b marnerol) in recognition of the pioneering work of Marner on iridals. Compound 3 has never been isolated but was hypothesized to be the first carbocyclic precursor in the biosynthesis of iridals[END_REF] [(+)-7], a monocyclic triterpene, which was hypothesized by Marner and co-workers to be the first carbocyclic precursor in the biosynthesis of triterpenoid iridals, [12] shares the same relative B-ring stereochemistry as suaveolindole [(+)-9], galbanic acid [(-)-1], and secodrial [(-)-8] (though antipodal). Recently we published the first synthesis of two structurally similar A-seco terpenes, ent-galbanic acid [(+)-1] and marneral [(+)-7] from (R)-(+)-pulegone. [13] The key feature of the synthetic route was a highly selective Claisen rearrangement that secured the stereochemical arrangement of both targets. This work allowed the determination of galbanic acid's absolute structure by comparison of the final product with natural galbanic acid, [START_REF] Thank | Università del Piemonte Orientale, Novara, for supplying natural galbanic acid and for helpful discussion[END_REF] thereby resolving the controversy regarding its relative stereochemistry. [15] Because the absolute stereochemistry of natural galbanic acid [(-)-1] has not been proven before, we first carried out the total synthesis by using the more accessible enantiomer (R)pulegone. Starting from the key intermediate (+)-3, common to both targets and prepared in nine steps from (R)-(+)-pulegone, the targeted products (+)-1 and (+)-7 could be accessed by using the appropriate sequence of steps. The retrosynthetic scheme to which we were attracted involved the disconnection of the strategic bonds indicated in Scheme 2. Despite the inefficient segment coupling, the synthesis of the antipodal 1 in this work secured both the relative and absolute stereochemistry attributed to natural galbanic acid.

Although the quantities of ent-(+)-galbanic acid prepared by the above sequence were sufficient to complete the characterization of the target molecule, we were disappointed with the inefficiency of both the elaboration of the quaternary carbon center at C-9 and the segment coupling. We detail herein a full account of our results of the revised chiral-pool strategy that partially overcomes these problems while still permitting large-scale synthesis of the requisite trisubstituted cyclohexane framework 3 (both antipodes) for coupling with the aromatic partners and farnesyl chain precursors. In this work, the B-ring chiral fragment gains its asymmetry from (S)-(-)-pulegone for galbanic acid and from (R)-(+)-pulegone for an improved synthesis of marneral as well as galbanic acid, secodrial, and secochiliotrin analogues.

Results and Discussion

As described in our previous article, [13] (-)-14 was obtained from (-)-2, but despite extensive experiments, the yield-decreasing enol carbonate formation during the first step could not be avoided. We therefore studied other alternatives with the hope that the C-9 quaternary center could be installed more efficiently. After further study, key intermediate 14 was more conveniently obtained by an alternative route simply by using ethyl cyanoformate instead of ethyl chloroformate as the C-9 side-chain precursor. Functional group manipulation of diol 14 derived from the three-step reaction sequence cleanly afforded the conveniently substituted cyclohexane skeleton found in target sesquiterpene frameworks. The three-step transformation of allyl alcohol 14 into the desired γ,δ-unsaturated aldehyde 16 was achieved by a Claisen rearrangement [START_REF] Ireland | The construction of a functionalized six-membered ring by conversion of an allylic alcohol into an allyl-vinyl-substituted ether followed by subsequent Claisen rearrangement was first reported by Ireland et al. in the early eighties[END_REF] and was followed by homologation of the aldehyde to furnish enol ether 17 in 90 % yield as a mixture of E/Z isomers, which was subsequently converted into the required ester 3 by using the Piancatelli protocol (Scheme 3). [17] The targeted ester alcohol 3 (both antipodes) is a potentially flexible compound because it contains the requisite absolute chirality at C-8, C-9, and C-10, as well as appropriate functional handles for introducing the remaining substituents to complete the side-chains of sesquiterpene-coumarin ethers galbanic acid, secodriol, and higher homologues, as well as the A-seco triterpene marneral (7) and the diterpene bisepi-secochiliotrin (6). In our previous synthesis, coupling, though in very low yield, was carried out by treatment of commercially available umbelliferone (11) with the neopentylic tosylate obtained from (+)-3. Applying the Reymond protocol [18] to the etherification in the presence of 18-crown-6 under microwave irradiation gave (+)-18. Much effort was expended to generate the desired coumarin ether 18 in better yields by using different conditions (triflate instead of tosylate, higher temperatures, and longer microwave irradiation), but all failed. In the hope of overcoming the aforementioned difficulty, several other options were envisaged, and finally the Mitsunobu route (Scheme 4) was adopted. As expected, coupling of the precursor alcohol 3 with umbelliferone (11) under conventional Mitsunobu conditions (DIAD, PPh 3 , and ROH) [19] proved ineffective due to steric crowding, yielding only the starting materials. [20] However, microwaveassisted [START_REF]For reviews on the use of microwaves in organic synthesis[END_REF] coupling applying the Mitsunobu etherification conditions resulted in a clean transformation, affording the desired coupling of 11 and 3, although in a modest yield (28 %, along with 40 % of recovered starting material). Nevertheless, this concise route allows for a rapid entry into coumarin sesquiterpene ether (-)-1 and constitutes the first synthesis of methyl galbanate [(-)-18], which is also a natural product and shows greater potency against influenza A virus H 1 N 1 than galbanic acid [(-)-1] and three-fold increased potency relative to that of amantadine. [7] Note that the improved method reported here allows all three stereogenic centers of the precursor molecule 3 to be assembled with complete stereocontrol from pulegone in nine straightforward steps and, moreover, introduces chemoselectivity at the two desired sites (C-3/C-11). Although the preparation of the stereo-defined cyclic backbone was a simple undertaking, the most difficult part of the venture was the late-stage manipulation required to produce segment coupling α to a quaternary center. [START_REF]This phenomenon is quite general as numerous examples are reported[END_REF] In view of the relevance of iridals as biologically active compounds, there still exists a need for the design of a practical route to marneral, the biogenetic precursor of all iridal representatives, with the ultimate goal to search for a chemobiological approach towards several members of the iridal family, mimicking Nature, and maximize flexibility with respect to analogue preparation. The common inter-Scheme 5. Improved synthesis of marneral [(+)-3] (Farn represents the C13-C30 farnesyl chain, squalene numbering). mediate (+)-3, prepared on a large-scale using our improved route (this work), was elaborated to the alkylborane precursor (+)-13 by Swern oxidation and subsequent Wittig olefination. Having reached this stage it was expected that exposure of (+)-13 first to 9-BBN then to vinyl iodide 12 in the presence of the bis(diphenylphosphanyl)ferrocenepalladium(II) catalyst with the required co-ligand triphenylarsine and base would lead to the desired formation of marneric acid methyl ester (19) along with its reduced counterpart marnerol (20). Indeed, the B-alkyl Suzuki-Miyaura coupling, [23] carried out according to the Johnson-Braun modification [24] Our previously published approach was four steps longer, hampered by a sequence of protecting group modifications required by the series of operations that effected the conversion of (+)-21 into (+)-22 (Scheme 6). Apart from providing a highly convergent synthetic plan, the current protocol also offers the added benefit of sequence flexibility in the final stages of the synthesis. For example, a suitably functionalized B-ring segment could first be converted into (+)-22 or (+)-24 and then joined to the remaining farnesyl portion should problems be encountered in the original sequence of events. Through the success achieved in improving the marneral [(+)-7] synthesis, the stage was set to test the planned bioconversion to the iridal parent molecule (+)-25 and several representatives of the iridal family (Scheme 7) with the ultimate goal to shorten our domino-based route [25] by more than 10 linear steps before coupling. The characterization of cytochrome P450 in Iris rhizomes [26] and the known reactivity of this monooxygenase towards the iridal parent molecule leading to 16-hydroxy-iridal, [27] along with the recently reported bioinspired rigid iron catalyst [Fe(pdp)], [28] Scheme 7. Projected combined chiral-pool/chemobiological approach mimicking Nature (dashed arrows indicate desired oxygenation sites at any stage and the plain arrow refers to P450-mediated hydroxylation, as described in ref. [26] ). which could be used as a mimic of P450 enzymes, provides an insight that should help to define the guidelines for the possible routes to follow.

The availability of (+)-7 is encouraging, however, several of its precursor molecules of the type (+)-15x,y and (+)-11x could well be substrates that bypass competition issues arising from the farnesyl side-chain. [START_REF]both in chemical and microbial oxidations, is how to divert oxidation away from the olefin to the desired unactivated sp 3 C-H position[END_REF]

Chiral-Pool-Derived Analogues

With an effective preparation of (+)-13 in hand, it was a straightforward matter to convert it into the one-carbon homologated coupling partner 24 (Scheme 6) and assess its ability to meet our requirements. Thus, lithium aluminium hydride reduction followed by TBS protection and subsequent hydroboration/oxidation of the terminal olefin (+)-13 afforded the C-11 homologated alcohol (+)-24, which served as a common intermediate for the synthesis of targeted analogues 4, 5, and 6. A variety of segment coupling protocols were screened as coupling proved to be more difficult than had been expected. Williamson etherification using the Reymond protocol [18] with coumarin 11 and the tosylate corresponding to the precursor alcohol 24 was first tried as shown in Scheme 8, but proved fruitless. Most unexpected was the finding that the side-product 26 predominated, in contrast to the C-10 epimeric analogue, which un- der the same conditions smoothly gave the corresponding coumarin ether (conversion of 25 to 26 in Scheme 7 of ref. [13] ). As a result, the conventional Mitsunobu etherification was investigated, which afforded the desired adduct in good yields. We then completed the synthesis of the analogue (+)-4 by a fluoride-promoted deprotection and adjustment of the oxidation state at C-3 by using standard literature conditions.

Coupling of the homologated alcohol (+)-24 with the commercially available isofraxidin ( 29 In summary, although the congested Mitsunobu etherification of 3 with umbelliferone ( 11) (Scheme 4) needs further effort, both etherifications with isofraxidine ( 29) and umbelliferone (11) proceed nearly quantitatively to give the higher homologues of galbanic acid (1), secodriol (31), and secodrial (5), respectively.

Alongside the work just described, we also pushed towards a bis-epi-secochiliotrin analogue 6, which contains two adjacent stereogenic centers on the six-membered ring in the epimeric 9R,10S disposition [required: 9S,10R for natural secochiliotrin (10)], as summarized in Scheme 10. The assembly of the diastereomeric monocyclic diterpene secochiliotrin skeleton proceeds through the lithiation of 3bromofuran followed by addition to the aldehyde derived from the common B-ring unit 24. [START_REF] Bock | [END_REF] The remaining steps consist of deoxygenation at C-12 and readjustment of the oxidation state at C-3.

The synthesis of (+)-6 was completed by the oxidation of the primary alcohol 33 with Dess-Martin periodinane (DMP) to yield the corresponding aldehyde, which in turn was converted into the required carboxylic acid in 65 % yield from the alcohol. The synthetic scheme described herein represents a straightforward route to these compounds and the nonoptimized yields of the target molecules are good to high. Furthermore, the insignificant level of cytotoxicity (see Exp. Sect. in the Supporting Information) shown by all the synthesized products makes them potentially useful as biological probes.

Conclusions

The modified protocol reported herein allows the largescale synthesis of the key B-ring building block of various A-seco terpene frameworks in a step-efficient and stereocontrolled fashion. Our initial approach to introduce the C-9 quaternary center was considerably improved by replacing ethyl chloroformate by ethyl cyanoformate. Even though the original obstacles associated with the segment coupling for the construction of galbanic acid still leave room for improvement, the synthetic scheme is general and broadens the scope of the chiral-pool-based sequence as a tool for the preparation of various natural and unnatural terpenoid ring systems in substantial enough amounts to support biological assays. Finally, the significant improvements in yield and step efficiency for the marnerol/marneral synthesis opens the way for a search aimed at raising the oxidation states on the ring (C-10, iridal numbering) and at C-26 and the olefinic methyl through hydroxylating enzymes.

Experimental Section

General: For general methods and standard procedures see the Supporting Information. Experimental descriptions for the synthesis of compounds (+)-13-(+)-18, (+)-1, and (+)-3 are available in the Supporting Information of our article on the ent-galbanic acid synthesis. [13] Spectral data are identical to those obtained in the preparation of the unnatural enantiomers from (R)-pulegone.

Synthesis of Natural Galbanic Acid [(-)-1]:

A microwave tube was charged with umbelliferone (11; 60 mg, 0.37 mmol), (-)-3 (50 mg, 0.18 mmol), DIAD (75 mg, 0.37 mmol), and PPh 3 (98 mg, 0.37 mmol) in toluene (0.5 mL) and the tube was inserted inside the microwave cavity. The reaction mixture was magnetically stirred FULL PAPER for 30 min (P = 100 W, T = 130 °C). The reaction mixture was evaporated and the resulting crude was purified by silica gel flash chromatography (heptane/EtOAc, 9:1) to yield 20 mg (28 %) of methyl galbanate (-)-18, which was used in the saponification reaction directly. LiOH (6 mg, 0.23 mmol) was added to a solution of methyl galbanate [(-)-18; 19 mg. 0.046 mmol] in THF/H 2 O (0.5 mL/0.1 mL). After stirring for 24 h, the reaction was acidified to pH 2 with 1  HCl, extracted five times with EtOAc, and worked up as usual. The crude was purified by silica gel column chromatography (heptane/EtOAc, 1:1) to yield 12 mg (69 %) of (-)-galbanic acid [(-)-1]. This compound was spectroscopically identical to the synthesized enantiomer, only the optical rotation was of the opposite sign. [α] D = -25 (c = 1.0, CHCl 3 ). [13] Methyl Marnerate (19): A solution of 9-BBN (2.6 mL, 0.5  in THF) was added to the ester olefin 13 (111 mg, 0.42 mmol) and the solution was stirred at 25 °C for 2 h. This solution was transferred by cannula to a separate flask containing a mixture of the vinyl iodide 12 (165 mg, 0.52 mmol), [Pd(dppf)Cl 2 ] (34 mg, 0.04 mmol), AsPh 3 (19 mg, 0.06 mmol), Cs 2 CO 3 (410 mg, 1.26 mmol), and water (0.3 mL, 15.12 mmol) in DMF (6 mL). After 12 h the brown reaction mixture was diluted with water and extracted three times with diethyl ether. The organic layer was worked up as usual. The crude was purified by silica gel flash chromatography (heptane/ EtOAc, 99:1) to yield 74 mg (41 %) of (+)-20 and 65 mg (34 %) of (+)-19 as a colorless oil. [α] D = 85 (c = 1.0, CHCl 3 ). IR (film): ν ˜= 2960, 2916, 2857, 1739, 1434, 1372, 1255, 1166, 726, 696 cm -1 . [START_REF] Bagirov | For the structure and stereochemistry of galbanic acid from galbanum[END_REF] H NMR (500 MHz, CDCl 3 ): δ = 0.71 (d, J = 6.7 Hz, 3 H), 0.86 (s, 3 H), 0.98-1.05 (m, 1 H), 1.17-1.31 (m, 2 H), 1.37-1.43 (m, 1 H), 1.51 (s, 3 H), 1.54 (s, 6 H), 1.60 (s, 3 H), 1.62 (s, 3 H), 1.68 (s, 3 H), 1.52 (d, J = 1.9 Hz, 3 H), 1.61 (d, J = 1.1 Hz, 3 H), 1.69-1.82 (m, 6 H), 1.86-2.02 (m, 4 H), 2.39 (m, 1 H), 2.50 (dd, J = 4.8, 11.1 Hz, 1 H), 3.58 (s, 3 H), 4.85 (m, 3 H) ppm. 13 C NMR (125 MHz, CDCl 3 ): δ = 15. 6, 15.9, 16.0, 17.6, 20.2, 20.7, 22.3, 22.5, 24.2, 24.7, 25.7, 26.6, 26.7, 29.7, 30.9, 32.2, 36.1, 39.0, 39.7, 43.8, 51.3, 124.2, 124.4, 125.5, 130.8, 131.1, 134.2, 133.7, 134.2, 134.8, 174.7 Homogalbanic Acid (4): Oxalyl chloride (0.17 mL, 2  in DCM, 0.34 mmol) was added to a solution of dry DMSO (0.05 mL, 0.68 mmol) in dry DCM (3 mL) at -60 °C under argon. The mixture was stirred for 30 min and a solution of the primary alcohol (+)-28 (45 mg, 0.11 mmol) in dry DCM (1 mL) was added. Upon 1 h of additional stirring at -60 °C, Et 3 N (0.15 mL) was added and the mixture was warmed to 0 °C, stirred for a further 20 min, and poured into ice-cold water, diluted with DCM, washed with 1  HCl and aqueous saturated NaHCO 3 , and worked up as usual. The crude obtained was used directly in the next oxidation step. 2-Methylbut-2-ene (0.12 mL, 1.13 mmol) and a solution of NaClO 2 (13 mg, 0.15 mmol) and KH 2 PO 4 (20 mg, 0.15 mmol) in water (0.5 mL) were added to a solution of the previously obtained aldehyde in tBuOH (1 mL). The reaction mixture was stirred for 12 h at 25 °C and was finally quenched with brine and extracted four times with EtOAc. The organic layer was worked up as usual. The crude obtained was purified by silica gel flash chromatography (heptane/EtOAc, 2:1) to yield 29 mg (63 % over two steps) of the acid (+)-4 as a glassy solid. [α] D = 7 (c = 1.0, CHCl 3 ). IR (film): ν = 3210, 1734, 1702, 1610, 1278, 1229, 1121, 1011, 833, 753 cm -1 . 1 H NMR (500 MHz, CDCl 3 ): δ = 0.83 (d, J = 6.9 Hz, 3 H), 1.03 (s, 3 H), 1.20-1.24 (m, 1 H), 1.45-1.50 (m, 1 H), 1.60 (dt, J = 7.6, 14.0 Hz, 1 H), 1.66 (d, J = 1.5 Hz, 3 H), 1.70 (s, 3 H), 1.76-1.90 (m, 5 H), 2.12-2.26 (m, 2 H), 2.51 (br. d, J = 14.0 Hz, 1 H), 2.59 (dd, J = 4.61, 11.5 Hz, 1 H), 4.03 (dd, J = 7.1, 8.1 Hz, 2 H), 6.24 (d, J = 9.4 Hz, 1 H), 6.79-6.82 (m, 2 H), 7.35 (d, J = 8.5 Hz, 1 H), 7.63 (d, J = 9.5 Hz, 1 H) ppm. 13 C NMR (125 MHz, CDCl 3 ): δ = 15. 6, 20.2, 20.7, 22.2, 24.1, 24.6, 30.8, 30.9, 31.9, 36.0, 38.8, 44.9, 66.3, 101.3, 112.4, 112.9, 113.0, 125.6, 128.7, 130.3, 143.4, 155.9, 161.3, 162.4, 179.7 Bis-epi-secochiliotrin [(+)-6]: Dess-Martin periodinane (DMP, 136 mg, 0.32 mmol) was added to a solution of the alcohol (+)-33 (33 mg, 0.11 mmol) in DCM (0.5 mL) and pyridine (0.1 mL) and the reaction mixture was stirred for 1 h. The solution was diluted with heptane (white precipitate appeared) and the suspension was subjected directly to column chromatography (heptane/EtOAc, 14:1) to yield 30 mg (91 %) of the desired aldehyde. 2-Methylbut-2ene (0.11 mL, 0.99 mmol) and a solution of NaClO 2 (12 mg, 0.13 mmol) and KH 2 PO 4 (18 mg, 0.13 mmol) in water (0.5 mL) were added to a solution of the aldehyde thus obtained in tBuOH (0.2 mL). The reaction mixture was stirred for 12 h at 25 °C and was finally quenched with brine and extracted four times with EtOAc. The organic layer was worked up as usual. The crude obtained was purified by silica gel flash chromatography (heptane/ EtOAc, 4:1) to yield 22 mg (72 %) of the acid (+)-6 as a colorless oil. [α] D = 38 (c = 1.0, CHCl 3 ). IR (film): ν ˜= 3391 (b), 1706, 1452, 1374, 1259, 1025, 873, 801 cm -1 . 1 H NMR (500 MHz, CHCl 3 ): δ = 0.79 (d, J = 6.6 Hz, 3 H), 0.97 (s, 3 H), 1.20 (qd, J = 4.5, 13.2 Hz, 1 H), 1.28-1.33 (m, 1 H), 1.39-1.44 (m, 1 H), 1.48-1.55 (m, 1 H), 1.68 (br. s, 6 H), 1.75-1.81 (m, 2 H), 1.83-1.90 (m, 2 H), 2.12-2.24 (m, 2 H), 2. 27-2.31 (m, 2 H), 2.47 (br. d, J = 14.5 Hz, 1 H), 2.66 (dd, J = 4.8, 11.5 Hz, 1 H), 6.21 (s, 1 H), 7.17 (s, 1 H), 7.33 (s, 1 H) ppm. 13 C NMR (125 MHz, CHCl 3 ): δ = 15.56, 19.3, 20.2, 20.8, 22.3, 24.0, 24.7, 30.9, 31.9, 32.5, 36.1, 39.2, 43.8, 111.0, 124.9, 126.1, 130.6, 138.4, 142.6, 178. Supporting Information (see also the footnote on the first page of this article): Complete characterization data and 1 H and 13 C NMR spectra for compounds 3, 13, 6, and 19-33.

Scheme 1 .

 1 Scheme 1. Structures of representative A-seco terpenes bearing structurally similar B-ring moieties.

  Scheme 2. Divergent synthesis of ent-galbanic acid and Marneral from the common intermediate (+)-3.

Scheme 3 .

 3 Scheme 3. Synthesis of the key intermediate (-)-3.

  Scheme 4. Coumarin appendage by Mitsunobu etherification.

  using 3 equiv. of 9-BBN provided marnerol (20) along with the corresponding carboxylic ester 19 in a 1.2:1 ratio, respectively. Attempts to increase the marnerol content of the reaction mixture by increasing the amount of 9-BBN or extending the reaction time were unsuccessful because a nearly unchanged ratio of ester 19 and alcohol 20 was reproducibly obtained. In the end, the ester was easily transformed into marnerol [(+)-20] by a lithium aluminium hydride reduction. As portrayed in Scheme 5, direct coupling of the olefin ester (+)-13, obtained in 11 straightforward steps from (R)-(+)-pulegone, with the farnesyl chain precursor 12, paved the way for an improved enantioselective synthesis of marnerol [(+)-20] and marneral [(+)-7].

Scheme 6 .

 6 Scheme 6. Preparation of the coupling partners for iridals, homogalbanic acid, and homo-secodrial.

Scheme 8 .

 8 Scheme 8. Initial attempt to synthesize (+)-4 by Williamson etherification and the successful Mitsunobu coupling.

  Scheme 9. Completion of the synthesis of the secodrial analogue (+)-26.

  ) under conventional Mitsunobu conditions (DIAD, PPh 3 , and ROH) proceeded well, yielding a nearly quantitative yield of homo-secodriol precursor (+)-30. Deprotection of the TBS ether as above provided homo-secodriol [(+)-31] in 80 % yield, which was converted into homo-secodrial [(+)-5] by Swern oxidation (Scheme 9).

Scheme 10 .

 10 Scheme 10. Completion of the synthesis of diterpenoid bis-epi-secochiliotrin [(+)-6].

  9 ppm. MS (ESI, MeOH): m/z (%) = 317.2 (100) [M -H] + . HRMS (ESI, MeOH): calcd. for C 20 H 29 O 3 317.2117; found 317.2113.

  ppm. MS (ESI, MeOH + CH 2 Cl 2 ): m/z (%) = 435.2 (100) [M + Na] + . HRMS (ESI): calcd. for C 25 H 32 O 5 Na 435.2147; found 435.2153.

www.eurjoc.org © 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Eur. J. Org. Chem. 0000, 0-0

Eur. J. Org. Chem. 0000, 0-0 © 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.eurjoc.org

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Acknowledgments

Financial support from the Centre National de la Recherche Scientifique (CNRS) and fellowship awards from the Institut de Chimie des Substances Naturelles (ICSN) are gratefully acknowledged.


Margaux Blanchard

Chloé Gervès-Pinquié

Mathieu Feuilloy

Marc Le Vaillant

Wojciech Trzepizur

Nicole Meslier

François Goupil

Thierry Pigeanne

Frédéric Balusson

Emmanuel Oger

Abdelkebir Sabil

Jean-Marc Girault

Frédéric Gagnadoux
email: frgagnadoux@chu-angers.fr

Hypoxic burden and heart-rate variability predict stroke incidence in sleep apnoea

Keywords: Obstructive sleep apnoea; hypoxic burden, heart rate variability, stroke

take home" message: Indices of sleep apnoea-related hypoxic burden and heart rate variability derived from full-night polysomnography might be useful for identifying sleep apnoea patients at risk for stroke.

Obstructive sleep apnoea (OSA) is increasingly recognized as a risk factor for stroke [START_REF] Bassetti | EAN/ERS/ESO/ESRS statement on the impact of sleep disorders on risk and outcome of stroke[END_REF]. However, the incidence of stroke in patients investigated for OSA has been assessed in a limited number of studies reporting conflicting results on the association between the apnoea-hypopnea index (AHI) and stroke incidence [START_REF] Bassetti | EAN/ERS/ESO/ESRS statement on the impact of sleep disorders on risk and outcome of stroke[END_REF][START_REF] Campos-Rodriguez | Role of sleep apnea and continuous positive airway pressure therapy in the incidence of stroke or coronary heart disease in women[END_REF][START_REF] Kendzerska | Obstructive Sleep Apnea and Risk of Cardiovascular Events and All-Cause Mortality: A Decade-Long Historical Cohort Study[END_REF]. As OSA is a very heterogeneous condition, the identification of subgroups of patients at high risk for stroke would be clinically desirable, in order to implement preventive actions [START_REF] Bassetti | EAN/ERS/ESO/ESRS statement on the impact of sleep disorders on risk and outcome of stroke[END_REF]. Population-based studies have demonstrated that the sleep apnoea specific hypoxic burden (SASHB), an easily derived signal from polysomnography (PSG), predicts cardiovascular mortality and incident heart failure [START_REF] Azarbarzin | The hypoxic burden of sleep apnoea predicts cardiovascular disease-related mortality: the Osteoporotic Fractures in Men Study and the Sleep Heart Health Study[END_REF][START_REF] Azarbarzin | The Sleep Apnea-Specific Hypoxic Burden Predicts Incident Heart Failure[END_REF]. A recent study has demonstrated that night-time heart rate variability (HRV) might play an important role in the association between OSA and the cerebral small vessel disease, which is responsible for a substantial proportion of strokes [START_REF] Brutto | Effect of Heart Rate Variability on the Association Between the Apnea-Hypopnea Index and Cerebral Small Vessel Disease[END_REF]. Whether physiological markers of SASHB and HRV might be useful in clinical setting for identifying those patients with OSA at risk for stroke remains to be determined. Within a large multicentre clinic-based cohort of patients investigated for OSA, we hypothesized that PSG-derived indices of SASHB and HRV could predict stroke incidence.

The study was conducted on the Pays de la Loire Sleep Cohort, linked with data from the French administrative health care database (SNDS) (see ref [START_REF] Justeau | u s c r i p t[END_REF] for details on the Pays de la Loire Sleep Cohort and the process linking it with the SNDS). We included stroke-free patients with available SNDS data investigated by PSG (CID102L8DTM, CIDELEC, Sainte-Gemmes-sur-Loire, France) for OSA between May 15, 2007 and December 31, 2017. Respiratory events were scored manually using A c c e p t e d m a n u s c r i p t recommended criteria [START_REF] Berry | Rules for scoring respiratory events in sleep: update of the 2007 AASM Manual for the Scoring of Sleep and Associated Events. Deliberations of the Sleep Apnea Definitions Task Force of the American Academy of Sleep Medicine[END_REF]. As previously described, the SASHB was defined as the total area under the respiratory event-related desaturation curve [START_REF] Azarbarzin | The hypoxic burden of sleep apnoea predicts cardiovascular disease-related mortality: the Osteoporotic Fractures in Men Study and the Sleep Heart Health Study[END_REF]. Using standard recommendations [START_REF]Heart rate variability. Standards of measurement, physiological interpretation, and clinical use. Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology[END_REF], HRV was computed on 5 minute segments of continuous non-overlapping PSG-derived ECG without ectopy or artefact. Time domain HRV measurements included the standard deviation for the mean value of all normal-to-normal R-R intervals (SDNN) and the root mean square of successive differences in normal-to-normal R-R intervals (RMSSD). Frequency-domain measurements included the normalized low frequency (LF, from 0.04 to 0.15Hz) and high frequency power (HF, from 0.15 to 0.4Hz). The LF to HF ratio (LF/HF) estimated the sympathetic/parasympathetic tone.

The study endpoint was the first episode of stroke at any time between the PSG recording date and the end of December 2018. The first occurrence of stroke was identified from the national hospital discharge database (PMSI) and defined as the entry date of the first hospitalization with a discharge diagnosis G45, G46, I60-I64, or I69 [START_REF] Giroud | Positive Predictive Value of French Hospitalization Discharge Codes for Stroke and Transient Ischemic Attack[END_REF]. The accuracy of PMSI-based algorithm for stroke has been previously demonstrated with a positive predictive value at 90% [START_REF] Giroud | Positive Predictive Value of French Hospitalization Discharge Codes for Stroke and Transient Ischemic Attack[END_REF]. As all physicians routinely contribute to PMSI data collection with annual quality control of coding, the accuracy increases with time [START_REF] Aboa-Eboulé | How accurate is the reporting of stroke in hospital discharge data? A pilot validation study using a population-based stroke registry as control[END_REF].

Cox proportional hazard models were used to evaluate the association of stroke incidence with natural log transformed indices of OSA severity and HRV. Missing values were imputed using a multiple imputation method (MI procedure from SAS) [START_REF] Justeau | u s c r i p t[END_REF]. Associations were considered statistically significant for a p value <0.05. All statistical analyses were performed with SAS 9.4 software (SAS Institute, Cary, NC).

The study population consisted of 3,597 patients (median [interquartile range, IQR] age: 58 [48-67] years), predominantly male (63%), obese or overweight (median [IQR] body-mass index [BMI] = 28 [25-32] kg/m 2 ), frequently presenting cardiovascular and metabolic comorbidities (hypertension, 28.6%; diabetes, 10.4%; cardiac diseases, 8.7%), 85.4% of whom had mild-to-severe OSA (median [IQR] AHI = 20 events/h). During follow-up, 1,159 patients were positive airway pressure (PAP)

A c c e p t e d m a n u s c r i p t adherent (mean daily PAP use≥4 h). After a median follow-up of 5.9 [3.5-8.4] years, 83 patients had been diagnosed with a stroke including 70 ischemic (29 transient ischemic attack, TIA) and 13 haemorrhagic strokes (stroke incidence density rate = 3.9 per 1,000 person-years). Cox proportional hazard models (Table 1) demonstrated an association between natural log transformed indices of OSA severity and stroke incidence (Model 1), which remained significant after adjusting for confounding risk factors (Model 2) for SASHB and the percentage of sleep time with oxygen saturation <90% (T90) (p=0.02 for both). Among natural log transformed indices of HRV, stroke incidence was negatively associated with LF and LF/HF ratio (p=0.01 and 0.008 respectively) and positively associated with HF (p=0.01) after adjusting for confounders (Model 2). The magnitude of the associations was unchanged after adjusting for PAP adherence (Model 3) and controlling for a competing risk of death (not shown).

When the analysis was restricted to 70 ischemic cerebrovascular events, stroke incidence remained significantly associated with natural log transformed SASHB (adjusted hazard ratio, HR [95% confidence interval]: 1.30 [1.05-1.61]; p=0.02) and LF/HF ratio (adjusted HR: 0.66 [0.47-0.93]; p=0.02) in the fully adjusted model (Model 3). After exclusion of 29 TIA from the analysis, stroke incidence remained significantly associated with natural log transformed LF/HF ratio (adjusted HR: 0.60 [0.41-0.90]; p=0.01) but not with SASHB (adjusted HR: 1.17 [0.92-0.1.48]; p=0.2).

Adding interaction terms in the analyses showed no significant interaction of gender and PAP adherence in the relationship between natural log transformed PSG-derived indices and stroke incidence. However, stroke incidence was more strongly associated with SASHB and HF among nonobese subjects compared with those with obesity. The association of stroke incidence with SASHB and LF/HF was also stronger in patients aged ≥ 60 years compared with those aged <60 years (all p values for interactions <0.05).

In a large multicentre, clinic-based cohort, we demonstrated an association of stroke incidence with PSG-derived indices of OSA-related hypoxaemia and HRV. Patients with higher SASHB and lower A c c e p t e d m a n u s c r i p t sympathetic/parasympathetic tone (LF/HF ratio) were at higher risk of stroke after adjusting for confounding risk factors and PAP adherence.

A strong association between untreated OSA and incident strokes (n=17 events) have been previously reported in women [START_REF] Campos-Rodriguez | Role of sleep apnea and continuous positive airway pressure therapy in the incidence of stroke or coronary heart disease in women[END_REF]. Conversely, Kendzerska et al. [START_REF] Kendzerska | Obstructive Sleep Apnea and Risk of Cardiovascular Events and All-Cause Mortality: A Decade-Long Historical Cohort Study[END_REF] reported that stroke in OSA was not associated with AHI. In the present study, we found no relationship between stroke incidence and common metrics of OSA severity excepted for T90 which characterizes not only OSA-related intermittent hypoxaemia, but also persistent hypoxaemia, such as that due to obesity hypoventilation or heart failure. Conversely, we demonstrated a dose-response relationship between stroke incidence and SASHB. Altogether, our findings and those from recent reports [START_REF] Azarbarzin | The hypoxic burden of sleep apnoea predicts cardiovascular disease-related mortality: the Osteoporotic Fractures in Men Study and the Sleep Heart Health Study[END_REF][START_REF] Azarbarzin | The Sleep Apnea-Specific Hypoxic Burden Predicts Incident Heart Failure[END_REF] suggest that SASHB might predicts stroke and cardiovascular morbidity more consistently than AHI, ODI and T90.

Previous studies have demonstrated a relationship between HRV and stroke [START_REF] Lees | Heart Rate Variability as a Biomarker for Predicting Stroke, Post-stroke Complications and Functionality[END_REF]. In subjects with OSA, the sympathetic activation that occurs toward the end of obstructive events is accompanied by vagally mediated bradycardia due to activation of the diving reflex [13]. In older men with OSA, sleep-related reduced sympathetic/parasympathetic tone is associated with a higher risk of atrial fibrillation [14], which has been consistently associated with stroke risk. In the present study, a oneunit increase in natural log transformed LF/HF ratio was associated with a 44% decrease in stroke risk, suggesting a contribution of low sympathetic/parasympathetic tone to stroke occurrence.

The strength of the current study includes a multicentre design, the adjustment for multiple stroke risk factors, and the assessment of different OSA severity and HRV indices. Our study also has some limitations. Its observational design does not allow any conclusions to be drawn regarding the causal pathway of the associations. Potential unmeasured confounding factors cannot be excluded. The use of a composite outcome combining different types of cerebrovascular events is also a potential limitation. We acknowledge that our study did not have sufficient statistical power to examine the association of OSA severity and HRV indices with the different stroke subtypes. All patients were

A

  c c e p t e d m a n u s c r i p t prevalence of OSA has led to an increasing use of simplified home sleep apnoea testing, with no ECG signal[15]. Whether oximetry-derived indices of pulse rate variability could provide an accurate estimation of HRV and predict stroke incidence should be also evaluated.In conclusion, within a large clinic-based cohort of patients with suspected OSA, we demonstrated an association of SASHB and nigh-time sympathetic/parasympathetic tone with stroke risk. PSGderived indices of hypoxic burden and heart rate variability may provide an opportunity to allow for stroke risk stratification in patients with OSA.

Table 1 :

 1 Cox proportional hazard models assessing the association of indices of obstructive sleep apnoea (OSA) severity and heart rate variability (HRV) with stroke incidence.Abbreviations: ln, natural log transformed; AHI, apnoea-hypopnea index; MAI, micro-arousal index; ODI, oxygen desaturation index; T90, percentage of sleep time with oxygen saturation <90%; SDNN, standard deviation for the mean of value of all normal-to-normal R-R intervals; RMSSD, root mean square of successive differences in normal-to-normal R-R intervals; LF, normalized low frequency power; HF, normalized high frequency power. Model 1: unadjusted Model 2: adjusted for age, gender, body mass index, alcohol intake, smoking status, diabetes, hypertension, history of cardiac disease and study site. Model 3: Model 2 + adjusted for positive airway pressure adherence * p<0.05; † p<0.01; ‡ p<0.001

		Hazard ratio [95% confidence interval]
		Model 1	Model 2	Model 3
	Indices of OSA severity
	ln AHI ln SASHB ln MAI ln 3%ODI ln T90 Indices of HRV ln SDNN ln RMSSD	1.45 [1.15-1.84] † 1.46 [1.24-1.72] ‡ 1.44 [1.01-2.05] * 1.30 [1.09-1.55] † 1.11 [1.06-1.16] ‡ 0.97 [0.59-1.61] 1.36 [0.93-1.98] m 1.18 [0.93-1.50] 1.25 [1.04-1.51] * 1.20 [0.86-1.68] 1.13 [0.95-1.33] 1.06 [1.01-1.12] * 1.19 [0.73-1.95] 1.43 [0.99-2.07] a n u s c r i p t 1.20 [0.93-1.55] 1.28 [1.05-1.57] * 1.21 [0.86-1.71] 1.13 [0.95-1.35] 1.06 [1.01-1.12] * 1.23 [0.75-2.04] 1.43 [0.99-2.07]
	ln LF ln HF ln LF/HF ratio A c c e p t e d 0.36 [0.21-0.64] ‡ 3.35 [1.48-7.61] † 2.78 [1.25-6.17] * 2.77 [1.25-6.16] * 0.47 [0.26-0.85] * 0.47[0.26-0.86] * 0.59 [0.43-0.81] † 0.66 [0.48-0.90] † 0.66 [0.48-0.90] †

Acknowledgments

We thank the French National Health Insurance for giving us access to the French administrative health care database. We thank the ERMES study group: Centre Hospitalier Universitaire, Angers: Christine Person, Pascaline Priou; Centre Hospitalier, Le Mans: Olivier Molinier, Audrey Paris. We thank Christelle Gosselin and Jean-Louis Racineux from the Institut de Recherche en Santé Respiratoire des Pays de le Loire We thank Julien Godey, Laetitia Moreno and Marion Vincent, sleep technicians in the Department of Respiratory and Sleep Medicine of Angers University Hospital Sources of Funding This study was supported by a grant from the Pays de la Loire Respiratory Health Research Institute (IRSR), Beaucouzé, France.

A c c e p t e d m a n u s c r i p t

Associations of Obstructive Sleep Apnea With Atrial Fibrillation and Continuous Positive Airway