Denis Geraads
email: geraads@mnhn.fr

Musteloid carnivores from the upper Miocene of South-Western Bulgaria, and the phylogeny of the Mephitidae Carnivores mustéloïdes du Miocène supérieur

Keywords:

The upper Miocene sites of Hadjidimovo and Kalimantsi in South-Western Bulgaria have yielded a few fossils of the ailurid Simocyon Wagner, 1858, a skull and associated mandible of Promeles Zittel, 1890 (a relatively well-known member of the Mustelidae), and several skulls of Promephitis Gaudry, 1861, an Eurasian member of the Mephitidae, a family represented today by three American and one South-Asian genera, to which were also ascribed Palaeomephitis Jäger, 1839, Miomephitis Dehm, 1950, and Proputorius Filhol, 1890, from the Middle Miocene of Europe, as well as several American Clarendonian to Hemphillian genera. We show that the mephitid status of Palaeomephitis, Miomephitis, and Proputorius is doubtful, and that the late Miocene Promephitis is at a lower grade of encephalization than modern Mephitidae and could be the sister taxon of Pleistocene and modern forms, which can be called Mephitinae. Within this subfamily, the American Pleistocene and modern taxa form the sister group of the Asian Mydaus Cuvier, 1821, which displays numerous autapomorphies. A major, previously unnoticed characteristic of the Mephitinae and Promephitis is that their P4 lingual cusp is a hypocone, unlike that of the Clarendonian American forms, in which it is a protocone. If the latter taxa really belong to the Mephitidae, they must have split early in its history.

INTRODUCTION

Upper Miocene fossiliferous continental deposits are common in Eastern Mediterranean countries, and Bulgaria is no exception. The sites of Kalimantsi and Hadjidimovo in the valleys of the Struma and Mesta Rivers, which flow southwards in the south-western corner of the country (map in [START_REF] Geraads | -New Chalicotheriidae (Perissodactyla, Mammalia) from Bulgaria[END_REF] have yielded more than 30,000 mammalian fossils. Most of them have been studied, allowing placement of the main Kalimantsi assemblage at about the same biostratigraphic level as Pikermi in Greece, or slightly below it, in zone MN12, Hadjidimovo being probably slightly earlier, perhaps in zone MN 11 [START_REF] Spassov N | The Turolian megafauna of West Bulgaria and the character of the Late Miocene "Pikermian Biome[END_REF][START_REF] Spassov | -Late Neogene stratigraphy, biochronology, faunal diversity and environments of South-West Bulgaria (Struma River valley)[END_REF][START_REF] Geraads | Upper Miocene mammals from Strumyani, South-Western Bulgaria[END_REF]Hristova et al. 2013, and refs therein). Some of the carnivores remain unpublished, and we describe and discuss below those that can be ascribed to the Mephitidae, Mustelidae and Ailuridae, i.e., all members of the Musteloidea, as these sites yielded no Procyonidae.

MATERIAL AND METHODS

The fossils from Kalimantsi and Hadjidimovo described below are part of the rich collections built up under the leadership of the late D. Kovachev, and deposited in the Kovachev Museum of Paleontology (a branch of the National Museum of Natural History, Sofia), Assenovgrad, Bulgaria. We have compared it to modern and fossil material kept in the institutions listed below. Skull HD-9505 was CT-scanned at the AST-RX platform of the MNHN, with a voxel size of 43.7 μm, and reconstructed with Mimics®, allowing visualization of tooth morphology and some internal cranial structures. Members of the Mephitidae, consisting today of the American skunks (Mephitis Geoffroy Saint-Hilaire and Cuvier, 1795, Spilogale Gray, 1865, and Conepatus Gray, 1837) and south-Asian stink badgers (Mydaus Cuvier, 1821), were previously included in the Mustelidae but are now united in a family of their own, which is not more closely related to the Mustelidae than to some other Musteloidea [START_REF] Dragoo | Systematics of mustelid-like carnivores[END_REF][START_REF] Finarelli | A total evidence phylogeny of the Arctoidea (Carnivora: Mammalia): relationships among basal taxa[END_REF][START_REF] Eizirik | Pattern and timing of diversification of the mammalian order Carnivora inferred from multiple nuclear gene sequences[END_REF][START_REF] Nyakatura | Updating the evolutionary history of Carnivora (Mammalia): a new species-level supertree complete with divergence timeestimates[END_REF][START_REF] Catalano | The More, the Better: The Use of Multiple Landmark Configurations to Solve the Phylogenetic Relationships in Musteloids[END_REF]. Their best-known osteologic feature is the lateral expansion of the epitympanic recess into the petrosal and squamosal, forming a large sinus that inflates the ventral part of the lateral side of the braincase, if not its ventral side. In some Mustelidae, such as Taxidea and Melogale, there is also a large sinus in this area, but in the former genus it communicates with the bulla through a wide, caudal opening, whereas in the latter it can be considered as an expansion of the supra-meatal fossa (what Schmidt-Kittler 1981, fig. 12, called the epitympanic recess in "Helictis" is in fact the tensor tympani fossa; the expansion of what he called the "Fossa suprameatale" corresponds to the epitympanic recess). In these latter genera the epitympanic recess, although relatively deep, has no connection with a sinus that is, therefore, not homologous with the mephitid one, although probably playing the same role. The Mephitidae may also be characterized by the extreme shortening of the premolar row. A major feature of the Mephitidae, seemingly previously unnoticed, is that the major cusp of the lingual talon of P4, when present, is a hypocone, because it interlocks within the trigonid of m1, and not a protocone as reported by most authors [START_REF] Pilgrim | A fossil skunk from Samos[END_REF][START_REF] Şenyürek | A study of a skull of Promephitis from the Pontian of Küçükyozgat[END_REF][START_REF] Petter G | Origine, phylogénie et systématique des blaireaux[END_REF][START_REF] Baskin | Evolution of Tertiary Mammals of North America, Volume 1: Terrestrial Carnivores, Ungulates, and Ungulate-like Mammals[END_REF][START_REF] Koufos | The late Miocene vertebrate locality of Perivolaki, Thessaly, Greece. 4. Carnivora[END_REF][START_REF] Carranza-Castañeda Ó | -Earliest hog-nosed skunk, Conepatus (Mephitidae, Carnivora), from the early Pliocene of Guanajuato, Mexico and origin of South American skunks[END_REF][START_REF] Bryant | Phylogenetic relationships within the extant Mustelidae (Carnivora): appraisal of the cladistic status of the Simpsonian subfamilies[END_REF], recognized the presence of a hypocone in Mydaus, but incorrectly denied it in Mephitis). Other diagnostic features, although not exclusive of this family (e.g., [START_REF] Baskin | Evolution of Tertiary Mammals of North America, Volume 1: Terrestrial Carnivores, Ungulates, and Ungulate-like Mammals[END_REF][START_REF] Finarelli | A total evidence phylogeny of the Arctoidea (Carnivora: Mammalia): relationships among basal taxa[END_REF] One skull from Bulgaria, HD-9505, was CT-scanned; it is thus much better known, and we will start with the description of the species to which this specimen belongs. [START_REF] Pilgrim | A fossil skunk from Samos[END_REF] HOLOTYPE. -Well-preserved cranium AMNH 20585, from Quarry 1 in Samos, Upper Miocene, probably close to 7 Ma [START_REF] Koufos | The late Miocene Mammal faunas of the Mytilinii Basin, Samos Island, Greece: new collection. 16. Chronology[END_REF].

Promephitis majori

DIAGNOSIS. -A species slightly smaller than the type-species, P. lartetii. Snout less inflated, occipital relatively higher.

DESCRIPTION

Skull HD-9505 is an almost complete and well-preserved specimen that includes the cranium and attached mandible (Fig. 1A). Being virtually undistorted, it was chosen for CT-scanning but poor contrast between bone and sediment and imperfect internal preservation made observation of fine structures very limited. The description below includes both its externally visible features and those only visible in the 3D reconstruction.

The skull is small (measurements: Table 1), about as large as that of Spilogale putorius, and much more similar, in its short, broad face, to American mephitids than to the south-Asian Mydaus. The facial sutures are not discernible. The nasal opening is slightly higher than broad; the anterior border of the orbit is located above the metacone of P4 but the face is short because the teeth are crowded rostrally. The snout narrows rostrally and is inflated, as in American mephitids, with no concave part, except shallow depressions between the canine and nasal aperture, and at the infra-orbital foramen, which is single and small. On the palate, the incisive foramina are large, and the choanae reach the level of the middle of M1; they are separated by a bony septum. The orbit is small, and limited posteriorly by a blunt but conspicuous post-orbital process of the frontal; the zygomatic arch is broken off. The temporal lines converge slightly behind the post-orbital constriction, where the skull is narrower than across the orbits, to form a long, low sagittal crest that diverges, at the top of the occipital, into almost straight lambdoid crests that border a high, triangular occipital. The paroccipital processes are of moderate size, and antero-posteriorly compressed.

A condyloid canal is present; the condylar (hypoglossal) foramen is located close to the occipital condyle. The jugular foramen is large. The large post-glenoid foramen is located above the anterior part of the long external auditory duct. There is no supra-meatal fossa. The bulla is poorly inflated. In ventral view, postero-laterally to it, the ventral floor of the mastoid sinus forms a smooth, concave area between the paroccipital and mastoid processes. The mastoid processes do not protrude significantly, but the skull is much broader at this level than across the paroccipital processes. Above them, the lateral walls of the braincase are laterally displaced by the expansion of the underlying mastoid sinus. In the endocranium, a distinctive feature is the very deep subarcuate fossa.

Because of bone cracks in the walls of the braincase, the reconstructed parts of the endocranium lack precision, but the main features are discernible. The mastoid sinus is mostly lateral with respect to the brain and labyrinth and extends far laterally; it is roughly pyramidal, with an apex above the mastoid. The frontal sinus is restricted to minute canals.

The brain shape, as reflected by the endocranial reconstruction (Fig. 2A), is remarkably primitive. The olfactory bulbs are not very large and not quite distinct from the rest of the brain. All sulci are shallow; the rhinal sulcus is located high; the sylvian sulcus is not discernible; the supra-sylvian sulcus is moderately arched, and short; the lateral sulcus is barely arched; there is no cruciate sulcus.

The CT-scan allowed examination of the teeth, which are well-preserved (Fig. 3A-B).

The upper incisors are small, with I3 slightly larger than the central ones. The canine bears small but distinct cuspids at the base of the mesiolingual and distal keels. On the left side only, there is a minute P2, displaced lingually by the closeness of C and P3. P3 is a simple tooth, broadest distally. P4 is a short, triangular tooth. There is a minute parastyle; the paracone is much longer and taller than the metacone, and the blade is not notched. A crest descends from the distobuccal angle of the tooth, along its distal margin, towards the main lingual cusp, but is interrupted before reaching it. This cusp, which assumes the shape of a mesiobuccally to distolingually compressed cone along the distolingual margin of the tooth, is a hypocone, because it interlocks with the trigonid of m1. The protocone is indistinct as a cusp, but forms a small shelf instead. M1 is slightly longer than P4, but much broader than long. The paracone and metacone are subequal in size; an extremely strong buccal cingulum greatly increases the width of the tooth; its mesial half forms a high, strong ridge around the paracone, and almost forms a cusp near the middle of the tooth. The protocone is crescentic, and separated from the buccal blade by a narrow valley, but not too much weight should be given to this morphology, because there is much variation in the shape of the postprotocrista in modern forms; the hypocone consists of a long crescent along the distolingual edge of the tooth. Dental measurements are given in Table 2.

The mandibular symphysis has a moderately inclined ventral border. The masseteric fossa is limited ventrally by a sharp crest that descends antero-ventrally from the condyle; ventrally to this ridge, the goniac area is reduced, so that the ventral border of the mandible clearly ascends from below m1 to the angular process, which is located close to the condyle.

The lower incisors are uprightly inserted, and mesiodistally narrow, with i3 slightly larger than i1 and i2. The canine has a strong ridge around its lingual half. There are only two premolars, but on neither side is the area between p3 and the canine well preserved, so that the presence of a p2 cannot definitely be ruled out. The p3 and p4 have two roots and are tall and slender, with a steep mesial edge, a distinct cingulum mesially and a broad distal part, bordered by a robust cingulum on p4.

On m1, the trigonid is slightly longer than the talonid. The protoconid is by far the tallest cuspid; it is unconnected to the metaconid, but a cristid descends towards the hypoconid, from which it is separated by a notch. In connection with the long, oblique hypocone of P4, the metaconid is distinctly more posterior than the protoconid, and the paraconid is long and longitudinally oriented, so that the three cuspids form an angle distinctly larger than 90°. The talonid is deeply basined, the central depression occupying most of the area, and the ridge-like cuspids being marginally located. The hypoconid is taller than the entoconid; in front of the latter is a second lingual cuspid, separated from the metaconid by a notch, deeper in the right m1. The CT-scan shows that the left m1 has a single accessory labial root while the right one also has an additional lingual one; we observed the same variability in CT-scanned Mydaus skulls, with NBC-33685 having a single, accessory lingual root and NBC-14540 having both lingual and buccal accessory roots. The m2 is shorter than broad, and also consists of a central basin circled by ridge-like elements. The latter, K-9504, is very incomplete as it includes only the dorsal part of the braincase and frontal area; HD-9506 is a relatively complete cranium but is somewhat crushed dorsoventrally and the dentition is in poor condition; HD-9507 is much distorted and lacks most of the snout and dentition, except the right P4-M1; HD-9508 is an undistorted skull with attached mandible, but it lacks most of the parietals and occipital. HD-9508 (Fig. 1B) is very similar to HD-9505, assigned above to P. majori, and we will mostly note the differences. It is slightly larger; the difference does not exceed what one would expect within a single species, but the other specimens are dimensionally close to HD-9508 rather than intermediate between it and HD-9505, suggesting bi-modal size distribution (Tables 1 and2). The face of HD-9508 is distinctly broader in the frontal region and the snout is more inflated; the post-orbital processes are slightly better developed, but remain weak. The occipital is relatively lower. A minute P2 is present on the right side at least. The labial cingulum of M1 is less prominent than in HD-9505. A minute p2, displaced lingually, is visible between the canine and p3. The p3 and p4 are similarly tall and slender, with mesial and distal accessory cuspids.

Promephitis lartetii

Skull HD-9506 is similar in size to HD-9508 and has the same broad snout, but no details are observable. HD-9507 has well-preserved P4 and M1; on P4 there is a distinct ridge descending from the hypocone around the mesio-lingual part of the tooth, which is more expanded than in HD-9505, although without forming a distinct protocone. The labial cingulum of M1 is moderate. The skull fragment K-9504 also agrees in size and snout width with HD-9508; its sagittal crest was certainly shorter, if not absent, but whether this is due to individual variation or to the age difference between the sites is unknown.

COMPARISONS

We compared the Bulgarian material with most modern species of Mephitidae and to fossil material from Pikermi (MNHN) and Perivolaki (LPGUT) in Greece. In addition, G. Koufos made available photos and cast of skulls from Samos, whose original specimens are stored in the AMNH and NHMW, and M. Sotnikova provided photos of material from Novo Elisavetovka.

All skulls of Promephitis from Hadjidimovo and Kalimantsi are very similar, and the presence of two species is not obvious at first sight. However, of the two best preserved skulls, HD-9508 is distinctly larger than HD-9505, and some of the few dental measurements are also larger. The main difference is in the greater inflation of the fronto-maxillary area, especially in the rostral part; the width of the maxilla over canine roots, in front of the infraorbital foramina, is 18.9 mm in HD-9508, in contrast to only 13.6 mm in HD-9505. Given the documented co-occurrence of two different species of Promephitis in the late Miocene of China (Wang and Qiu 2004), and of modern species of both Mustela and Martes in Europe [START_REF] Geptner | Mammals of the Soviet Union. T. 2 (Part 1). Manatees and carnivores[END_REF]Wolsan 1999a;Salek et al. 2013), we have no hesitation in referring these two skulls to different species, but it is not so easy to clarify the systematics of European and Eastern Mediterranean forms of the genus.

Few characters are observable on the type-specimen of the type-species, P. lartetii from Pikermi, because of its extreme crushing, and poor preservation of the teeth. The few cranial measurements provided by Gaudry of the specimen that he figured (1862, pl.6), repeated by several authors since then, are either grossly incorrect or unreliable. Neither the few dental measurements that can be taken nor the morphology differ from those of the two Hadjidimovo species. Pilgrim (1931:53) diagnosed P. lartetii in having a "metaconid of m1 somewhat in front of the protoconid", but this is clearly incorrect. However, the mandible of the holotype is distinctly longer from condyle to most rostral point of the dentary (44.5 mm, slightly overestimated by crushing) than that of HD-9505 (36 mm), and closer to that of HD-9508 (42.5 mm), so that there is no reason to distinguish HD-9508 from P. lartetii, implying that HD-9505 must belong to another, smaller species.

The holotype and only specimen of Promephitis gaudryi [START_REF] Schlosser M | Beiträge zur Kenntniss der Säugethierreste aus den süddeutschen Bohnerzen[END_REF], an m1 from the Vallesian (?) of Melchingen, Germany, obviously does not belong to this genus, as noted by [START_REF] Pilgrim | A fossil skunk from Samos[END_REF] and later authors, because of its small talonid, low trigonid cuspids, and lack of accessory roots.

The next species to be described was P. maeotica from the Turolian of Novo Elisavetovka in Ukraine [START_REF] Alexejew | Animaux fossiles du village Novo-Elisavetovka[END_REF]). The skull is clearly distinct from those of the Bulgarian and Pikermi forms in its larger size, gently converging temporal lines, and especially in its narrow P4 with a shallow buccal depression, short hypocone located centrally instead of more distally, small but distinct protocone that bulges mesially, and extremely twisted M1. P. maeotica is clearly distinct from the Balkan forms. Zdansky (1937) described some remains from Baode, China, as P. cf. maeotica, with which they agree in size, but [START_REF] Qiu Z.-X | -Late Miocene Promephitis (Carnivora, Mephitidae) from China[END_REF] referred them to another species, P. hootoni.

Promephitis alexejewi [START_REF] Schlosser | Tertiary Vertebrates from Mongolia[END_REF] was erected for scrappy remains from Ertemte, China. [START_REF] Qiu Z.-X | -Late Miocene Promephitis (Carnivora, Mephitidae) from China[END_REF], on the basis of Schlosser's measurements, assumed that this species had a long P4 compared to M1 but the difference is less than stated by Schlosser (Table 2), and the two teeth might well be from two different individuals. Besides large size, probably due to the young geological age of the localities (the species was also reported from the uppermost Miocene of Venta del Moro in Spain by [START_REF] Montoya | -Musteloidea (Carnivora, Mammalia) from the Late Miocene of Venta del Moro (Valencia, Spain)[END_REF], P. alexejewi lacks distinctive features.

Promephitis malustenensis Simionescu, 1930 (this name was chosen by [START_REF] Qiu Z.-X | -Late Miocene Promephitis (Carnivora, Mephitidae) from China[END_REF], in preference to P. rumanus, erected by Simionescu for the same type-specimen), is based upon a mandible that has far too long a premolar row to belong to this genus. [START_REF] Pilgrim | A fossil skunk from Samos[END_REF] erected a new species from Samos, P. majori. Cranial, dental and mandibular features do not differ from the few ones that can be observed in P. lartetii. The brain, observable on a cast of the holotype AMNH 20585 (Fig. 2G), is very similar to that of HD9505. Concerning dental features, in contrast to Pilgrim's claims, there is no evidence that: (1) M1 is shorter than P4 in P. lartetii; (2) a parastyle was present in the P4 of P. majori, as the missing mesial part of P4 may just be the paracone; (3) a P1 and/or a P2 was present in P. lartetii. The clearest difference is of size: the teeth are slightly smaller in P. majori (Table 2), and so is the length of the mandible (35 mm according to [START_REF] Pilgrim | A fossil skunk from Samos[END_REF], vs. 44.5 in the type of P. lartetii). In addition, P. majori differs from specimens assigned to P. lartetii (other than the distorted type) in its relatively high occipital, a feature also present in HD-9505. Thus, P. majori differs from P. lartetii in the same way as HD-9505 differs from HD-9508, and we refer HD-9505 to P. majori and HD-9508 to P. lartetii. Şenyürek (1954) erected P. hootoni for a partial skull and associated mandible from the Turolian of Küçükyozgat (Western Turkey); listed differences from previously named species are either subtle or non-existent, being based on Gaudry's (1862) incorrect drawings and measurements. In size and inflation of the snout, the type of P. hootoni matches P. lartetii and HD-9508, so that there is no reason to separate it from this species, as also suggested by [START_REF] Koufos | The late Miocene vertebrate locality of Perivolaki, Thessaly, Greece. 4. Carnivora[END_REF]. [START_REF] Bonis | -Carnivora (Mammalia) from the late Miocene of Akkaşdağı, Turkey[END_REF] described as P. hootoni a mandible from Akkaşdağı (Turkey); assuming that the scale [START_REF] Bonis | -Carnivora (Mammalia) from the late Miocene of Akkaşdağı, Turkey[END_REF], fig. 14) is correct, this mandible is indeed likely to represent P. lartetii.

Promephitis pristinidens Petter, 1963, from the Vallesian of Spain, is based on a maxilla fragment in which P4 has a distinct parastyle and a weak lingual talon with no distinct cusp, while M1 has a poorly inflated hypocone and weak buccal cingulum. These are primitive features, but no derived character links this form with later Promephitis, and we consider the generic assignment unsupported. [START_REF] Meladze | Hipparion fauna of Arkneti and Bazaleti[END_REF] from Bazalethi in Georgia is so poorly known that it is better to restrict the name to the type-series. The loss of one pair of incisors may have occurred post-mortem.

Promephitis brevirostris

Among other significant remains of Promephitis in the Eastern Mediterranean, there is a skull from Samos NHMW A4798, discussed by [START_REF] Koufos | The late Miocene vertebrate locality of Perivolaki, Thessaly, Greece. 4. Carnivora[END_REF], who concluded that it suggests the presence of a larger species at Samos, in addition to the smaller P. majori. We agree with him; although the occipital is dorsoventrally compressed, in its broad, inflated snout and overall size, A4798 matches HD-9507 and HD-9508 and we assign it to P. lartetii.

The material from Perivolaki in Greece is crushed and distorted, but the mandible length of PER-1278 (42.2 mm) is close to those of HD-9508 and of the type of P. lartetii, a species to which [START_REF] Koufos | The late Miocene vertebrate locality of Perivolaki, Thessaly, Greece. 4. Carnivora[END_REF] ascribed it. We agree with his identification, even though P3, p3, and p4 are larger than usual in this species.

We conclude that, in the Turolian of Western Eurasia, Promephitis includes three species, in decreasing size order P. maeotica from Novo Elisavetovka, P. lartetii from Pikermi, Samos, Perivolaki, Küçükyozgat, Akkaşdağı, Hadjidimovo and Kalimantsi, and P. majori from Samos and Hadjidimovo. Thus, in these two latter sites (although the precise origin of the Samos fossils is unknown), both species co-exist, the smaller form being rarer.

From the Pliocene of China, He and Huang (1991) described P. maxima; it is much larger than Upper Miocene forms but there is reason to doubt the generic attribution, although the illustrations are too imperfect for detailed comparisons.

Wang and Qiu ( 2004) described rich collections from the Upper Miocene of China, and named two species P. parvus and P. qinensis, in addition to what they called P. hootoni but is, in our opinion, identical with P. lartetii (the teeth are very similar to those of HD-9507). We have not seen this material, but the coexistence of two species of different sizes, as in some Eastern European sites, is worth noting.

CONTENTS OF THE FAMILY MEPHITIDAE

The distinction of the Mephitidae from the Mustelidae is now generally accepted, and the inclusion of the South-Asian Mydaus (including Suillotaxus) in the former family, first suggested by [START_REF] Pocock | The auditory bulla and other cranial characters in the Mustelidae[END_REF], and more recently by [START_REF] Petter G | Origine, phylogénie et systématique des blaireaux[END_REF] and [START_REF] Radinsky | Are stink badgers skunks? Implications of neuroanatomy for mustelid phylogeny[END_REF], is also beyond doubt; it is confirmed by recent molecular analyses (e.g., [START_REF] Flynn | Molecular phylogeny of the Carnivora (Mammalia): assessing the impact of increased sampling on resolving enigmatic relationships[END_REF][START_REF] Sato | Evolutionary and biogeographic history of weasel-like carnivorans (Musteloidea)[END_REF][START_REF] Nyakatura | Updating the evolutionary history of Carnivora (Mammalia): a new species-level supertree complete with divergence timeestimates[END_REF]. All recent analyses [START_REF] Bryant | Phylogenetic relationships within the extant Mustelidae (Carnivora): appraisal of the cladistic status of the Simpsonian subfamilies[END_REF][START_REF] Carranza-Castañeda Ó | -Earliest hog-nosed skunk, Conepatus (Mephitidae, Carnivora), from the early Pliocene of Guanajuato, Mexico and origin of South American skunks[END_REF][START_REF] Finarelli | A total evidence phylogeny of the Arctoidea (Carnivora: Mammalia): relationships among basal taxa[END_REF][START_REF] Eizirik | Pattern and timing of diversification of the mammalian order Carnivora inferred from multiple nuclear gene sequences[END_REF] Wolsan (1999b). [START_REF] Helbing H | Die Carnivoren des Steinheimer Beckens. A. Mustelidae[END_REF] noted that it differs from Conepatus and Promephitis majori in that the condylar foramen is distinct from the posterior lacerate foramen, but this is incorrect, as they are also separated in Conepatus and in HD-9505, while [START_REF] Pilgrim | A fossil skunk from Samos[END_REF] made no positive observation on the type of P. majori. [START_REF] Whistler | -A new basal skunk Martinogale (Carnivora, Mephitinae) from Late Miocene Dove Spring Formation, California, and origin of New World Mephitines[END_REF] accepted P. steinheimensis as a member of the Mephitidae. However, this family attribution solely rests on the assumption that the enlarged mastoid sinus is homologous with the mephitid one, i.e., that it is an expanded epitympanic recess, but the large opening to which Wolsan (1999b) gave this name faces ventrally instead of medially in the Mephitidae, showing that this area is imperfectly preserved. Given that this partial cranium is probably (Wolsan 1999b) of the same taxon as the highly derived teeth called Trochotherium cyamoides, and that it further differs from the Mephitidae in the presence of a supra-meatal fossa and large caudal entotympanic, we believe that the affinities of Palaeomephitis remain doubtful. What is known of the still earlier Miomephitis Dehm, 1950 also lacks clear mephitid characters and is best left aside. Proputorius Filhol, 1890, is known only by dental and mandibular remains that display no exclusive mephitid features. In the best known species, P. sansaniensis Filhol, 1890, from the Middle Miocene of Sansan, the P4 has a very small and mesially located protocone (Peigné 2012, figs. 88-89), and is quite unlike those of modern Mephitidae; the seven isolated m1s have no accessory roots; pending cranial evidence, we do not include this genus in the Mephitidae. The m1 of the Vallesian type-species of the poorly known Mesomephitis [START_REF] Petter G | Mustélidés nouveaux du Vallésien de Catalogne[END_REF], M. medius (Petter, 1963), differs from that of the Mephitidae in its low lingual talonid margin and lack of accessory roots, and inclusion in this family is poorly supported. We conclude that Promephitis is the only definite fossil Old World member of the Mephitidae. American Forms. [START_REF] Carranza-Castañeda Ó | -Earliest hog-nosed skunk, Conepatus (Mephitidae, Carnivora), from the early Pliocene of Guanajuato, Mexico and origin of South American skunks[END_REF] included in one clade, together with what we have called Mephitini above but to the exclusion of Mydaus and Promephitis, the Miocene to Pliocene Martinogale [START_REF] Hall | Three new genera of Mustelidae from the later Tertiary of North America[END_REF]Buisnictis Hibbard, 1950. They defined this clade by: (1) the presence of a parastyle on P4; (2) a hypoconid taller than the entoconid on m1; and (3) a cingulum not surrounding the protocone on M1. However, characters ( 1) and ( 2) are also present in Bulgarian Promephitis, and character ( 3) is quite variable, as we have observed many Mephitini in which the cingulum surrounds the protocone of M1.

In fact, a previously unnoticed sharp difference separates modern and Plio-Pleistocene forms from earlier ones: in the latter, the P4 lingual cusp is clearly a protocone, because it is located mesially or very mesially (Hibbard 1950, fig.21, in Buisnictis meadensis;Stevens and Stevens 2003, fig.9.8, in Buisnictis chisoensis;Wang et al. 2005, fig.5, in Martinogale faulli;Wang et al. 2014, fig.5A, in Buisnictis metabatos; we know no figure of the P4 of M. alveodens, but Baskin [1998:159] stated that it has "a very small, anteriorly placed protocone"). This sharply contrasts with the condition in modern and Pleistocene American Mephitini, also found in Mydaus and Promephitis, in which the lingual cusp interlocks with the trigonid of m1 and is therefore a hypocone. We regard this difference in homology of the lingual cusp of P4 as fundamental, and sufficient to exclude Martinogale and Buisnictis from the clade containing Promephitis and modern Mephitidae. It echoes to the suggestion of [START_REF] Baskin | Evolution of Tertiary Mammals of North America, Volume 1: Terrestrial Carnivores, Ungulates, and Ungulate-like Mammals[END_REF], followed by [START_REF] Stevens | Carnivora (Mammalia, Felidae, Canidae, and Mustelidae) from the earliest Hemphillian Screw Bean Local Fauna, Big Bend National Park, Brewster County, Texas[END_REF], that the American "mephitids" were the result of two migration waves, one in the Clarendonian giving rise to Martinogale and Buisnictis, and a second one in the early Blancan giving rise to modern and closely related genera. If the inflated mastoid sinus of Martinogale and Buisnictis is homologous of the one found in modern Mephitidae, these genera could be a basal branch of this family, but they are not part of the clade in which the P4 lingual cusp is a hypocone. As a consequence, we will restrict the following discussion to these latter forms.

PHYLOGENY OF THE MEPHITIDAE

Parsimony analysis. The main distinctive characters of Promephitis, of modern genera, and of the purported American Clarendonian mephitids are listed in Appendix A. A parsimony analysis conducted with TNT [START_REF] Goloboff | TNT, a free program for phylogenetic analysis[END_REF], using any of the latter as outgroup, yields a single shortest tree shown in Appendix B, of which Fig. 4 would be a simplified version. Promephitis appears as the sister group of a clade, including all modern forms, defined by several characters reflecting increased brain complexity and a stronger superficial masseter muscle. This might look surprising given the overall skull similarity between Promephitis and modern American forms, but the cladogram suggests that reluctance to admit Mydaus within the Mephitinae is based upon features that are in fact autapomorphic. It could be argued that the derived brain characters that unite Mydaus with the American Mephitini are merely the consequence of the well-known complexification of carnivore brains over time, and could have in fact occurred in parallel, as also suggested by the very unlikely reversal that is inferred for Mephitis. Still, this cladogram is in agreement with chronology, and it is also informative in highlighting the numerous autapomorphies of Promephitis and Mydaus, but we certainly do not take it at face value.

Pleistocene and modern American forms (Mephitini) share small incisor foramina, a larger P4 parastyle, complete loss of P4 protocone, and a shelf-like hypocone more mesially located than in Eurasian forms, a mandible with expanded angular area, and loss of the entepicondylar foramen in the humerus; their monophyly matches geography, and there is no reason to question it. Some further characters were said by [START_REF] Finarelli | A total evidence phylogeny of the Arctoidea (Carnivora: Mammalia): relationships among basal taxa[END_REF] to define the American clade but, in contrast to his figure 3, their mastoid process is not weaker than in Promephitis, the metacone-metaconule crest is not better indicated on M1, and the m1 is not more basined, so that the only remaining additional synapomorphy of the Mephitini would be the reduction of the buccal cingulum of M1; however, this cingulum is so strong in Promephitis that it is more probably its condition that is derived.

A Eurasian Clade ? [START_REF] Carranza-Castañeda Ó | -Earliest hog-nosed skunk, Conepatus (Mephitidae, Carnivora), from the early Pliocene of Guanajuato, Mexico and origin of South American skunks[END_REF] recognized the American and Eurasian Mephitidae, fossil and recent, as sister-groups. Of the characters that they used to define the Eurasian clade, the reduction of P2 and the lengthening of M1 are also found in most American forms, and the exclusion of the maxillae from the border of the incisive foraminae is hard to document because these bones are fused with the premaxillae (even the CT-scans failed to visualize their limits). Wang et al. ( 2005) also considered as a derived feature of Eurasian forms the lack of a notch between entoconid and metaconid in m1, but this notch is present in Mydaus and Promephitis. The only important feature shared by Mydaus and Promephitis is the shape of the hypocone of P4.

The Position of Promephitis. Compared to other Mephitidae, Promephitis has both more primitive and more derived characters. Among the latter are some features of the dentition, such as the marked basal cingulum on canines, the extremely short premolar series, and the very large M1 with a prominent buccal cingulum; Promephitis brought these trends further than other mephitids. However, it also retains primitive traits that are lost in Mydaus and American Mephitini. The brain remains at a very primitive stage of folding. The zygomatic arch is of normal size, which is probably related to a deep masseteric fossa in the mandible, which lacks the ventral flat extension of the Mephitinae; in the latter, the very slender zygomatic arch, shallow fossa and ventrally expanded angular region are probably related to the greater importance of the superficial masseter, which might, as in Procyon [START_REF] Gorniak | Architecture of the masticatory apparatus in eastern raccoons (Procyon lotor lotor)[END_REF]), originate on an aponeurosis and not on the arch itself. Last, the subarcuate fossa is deep; the polarity of this character is debatable, but the resemblance of Promephitis with Mustela is striking.

The Position of Mydaus. [START_REF] Finarelli | A total evidence phylogeny of the Arctoidea (Carnivora: Mammalia): relationships among basal taxa[END_REF] excluded Mydaus from a clade consisting of Promephitis and the Mephitini, defined by: (1) more anterior choanae; (2) lack of a condyloid canal; (3) M1 hypocone formed by swelling of the cingular ridge; (4) parastyle stronger than metastyle on M1; and (5) M1 parastyle oriented mesiobuccally. In fact, the very caudal choanae of Mydaus are obviously derived, a condyloid canal is present in Promephitis, and there is no reason to distinguish the origin of the M1 hypocones in all these taxa, so that the only remaining difference is the absence of styles in the M1 of Mydaus. It is more likely, however, that the latter feature is derived, like many other characters: long skull and face creating a long mandibular diastema, very posterior choanae, ventrally inflated mastoid wall, indistinct tensor tympani fossa, complex post-cruciate sulcus in the brain, loss of parastyle on P4, procumbent ventral incisors, shape of m1 trigonid with a mesially located protoconid and short paraconid, and a proximal femur with a weak trochanter and flat caudal side with almost no trochanteric fossa. Mydaus lacks the characters, listed above, that define the Mephitini.

Conclusion.

The monophyly of modern and Pleistocene American forms is not in doubt, and they can be called Mephitini. The identity of their sister taxon is more debatable. Overall skull shape suggests Promephitis, but this similarity does not hold if the skull shape of Mydaus is autapomorphic, as are many of its characters. We believe instead that the more derived brain morphology, slender zygomatic arch and related changes in masticatory musculature, and shallow subarcuate fossa, unite Mydaus with the Mephitini, as Mephitinae (Fig. 4). Regarding Promephitis as the sister-group of the Mephitini implies convergent brain evolution between the latter and Mydaus. Because of the retention of a large protocone and absence of hypocone, we exclude Buisnictis and Martinogale from the Mephitidae s.str.; they might, however, represent early offshoot(s) of the family.

Family MUSTELIDAE Fischer, 1817 Genus Promeles Zittel, 1890 TYPE SPECIES. -Promeles palaeattica [START_REF] Weithofer | Beiträge zur Kenntnis der Fauna von Pikermi bei Athen[END_REF].

DIAGNOSIS. -Skull profile regularly curved, frontals rather broad, sagittal crest usually present but weak, choanae located very posteriorly. I3 much larger than I1-I2, canine conical, upper premolars much increasing in size from P1 to P4, the latter with a parastyle and a strong protocone. M1 with a robust buccal cingulum along the paracone, a metacone close to the paracone, and a long, crescent-shape protocone that ends distally in a prominent metaconule. Trigonid of m1 long, paraconid long and directed forwards, talonid about as long as the trigonid, central basin entirely surrounded by a ridge consisting of numerous cuspids and cusplets.

Promeles palaeattica [START_REF] Weithofer | Beiträge zur Kenntnis der Fauna von Pikermi bei Athen[END_REF] HOLOTYPE. -unnumbered cranium and associated mandible, in the Institut für Paläontologie, University of Wien, Austria (Weithofer 1888, pl. 10, figs. 1-11).

DIAGNOSIS. -As for genus. DESCRIPTION K-9502 (Fig. 3C) is an incomplete cranium and K-9503 an incomplete mandible; they are probably of the same individual but, like many specimens from Kalimantsi, they are imperfectly preserved. In general shape and size, the skull does not differ from other specimens of P. palaeattica. The only visible cranial characters are that the front border of the orbit is above P4, and that its anterior rim overhangs the infra-orbital foramen. The incisors are inserted in a shallow arch, with I3 distinctly larger than the subequal I1 and I2.

The canines are broken off; their basal outline is a poorly compressed oval. A noticeable feature is the absence of P1 on both sides, P2 coming almost in contact with the canine. The right P3 is triangular in lateral view, with minute mesial and distal cingular accessory cusps; the left cheek-teeth are missing. The P4 is short, with a thin blade; its protocone reaches farther mesially than the paracone. The very large M1 is incomplete bucally; lingually, the cingulum is widely expanded, increasing both the width and length of the tooth.

On the mandible, i2 and i3 are larger than i1; the canine is conical, separated by a very short diastema from p2; p4 has no clear distal accessory cuspid. On the incomplete m1, the protoconid is not much taller than the paraconid; the talonid is deeply basined, and bordered by a sharp rim of which the hypoconid is the tallest and most distinct cuspid. Dental measurements are provided in Table 4.

COMPARISONS

Besides the type-locality, Pikermi, Promeles palaeattica has been reported from Samos, Maragha and Dorn-Dürkheim (Kaya et al. 2005, and refs therein), and Perivolaki [START_REF] Koufos | The late Miocene vertebrate locality of Perivolaki, Thessaly, Greece. 4. Carnivora[END_REF] while new species names have been erected for the specimens from Maramena in Greece (Promeles macedonicus Schmidt-Kittler, 1995) and Esendere in Turkey (Promeles smyrnensis [START_REF] Kaya | -A new late Miocene mammalian fauna in the Karaburun Peninsula (W. Turkey)[END_REF] on the basis of some differences in the proportions of P4, the position of its protocone, the thickness of its blade, the size of the lingual expansion of M1, the shape of its buccal side, and the proportions of m1. Given the observed variations in the Pikermi population, it may well be that all these forms can be accommodated within P. palaeattica. For instance, the M1 of NHML M9028 is distinctly more expanded distally than that of M9029, both from Pikermi, and on M9028 the labial cingulum is much stronger along the paracone than along the metacone, whereas it is of regular thickness on M9029. However, we refrain from synonymizing all these names because at least the specimens from Samos (MCGL S272) and Esendere seem to have less expanded M1s than at Pikermi. In any case, K-9502/K-9503 is more like the Pikermi fossils, and we ascribe it to P. palaeattica with confidence.

Family AILURIDAE Gray, 1843

Genus Simocyon Wagner, 1858

Simocyon primigenius (Roth and Wagner, 1854) This species has recently been discussed by [START_REF] Spassov | A skull of Simocyon primigenius (Roth & Wagner, 1854) from the late Miocene of Karaslari (Republic of Macedonia), with remarks on the systematics and evolution of the genus[END_REF] who confirmed the identification by [START_REF] Bakalov P | Les fossiles de Bulgarie. X. Mammifères tertiaires[END_REF] of two incomplete mandibles from Kalimantsi.

An isolated, unnumbered M1 from Hadjidimovo (Fig. 3D), badly worn and damaged, could be of the same species. It is broader mesially than distally. The central basin is open mesially, and a distinct crest descends lingually from the paracone, but the metacone and protocone are worn out. Its labial length (16.5 mm) is close to the upper end of the range of S. primigenius, to which we tentatively refer it, pending revision of the early Turolian forms of Central Europe.

CONCLUSIONS

Three species of Promephitis (P. lartetii, P. majori, and P. maeotica) can be distinguished in the late Miocene of Southeastern Europe and Turkey, two species co-existing in some sites, as in China. Promephitis appears as an early offshoot of the Mephitidae; its sister-group (Mephitinae) includes all modern forms, with the American (and Plio-Pleistocene) species (Mephitini) being the sister-group of the Asian Mydaus that displays numerous autapomorphies, but the mephitid status of several Middle Miocene European genera is doubtful.

In addition to the well-known expansion of the epitympanic recess into the squamosal and lateral part of the petrosal, the Mephitidae can be defined by the presence of a large hypocone on P4; this character is definitely present in Promephitis, Mydaus, and in Pleistocene and modern American Mephitini, but not in Clarendonian forms, which should therefore be excluded from the Mephitidae s.str.

The only Bulgarian late Miocene member of the Mustelidae is Promeles; we tentatively assign all late Miocene Balkano-Iranian forms to P. palaeattica; the genus may include more than one species, but their boundaries are unclear. A few remains represent the ailurid Simocyon.

  of Geology and Paleontology, University of Thessaloniki, Greece MCGL, Musée Cantonal de Géologie, Lausanne, Switzerland MNHN, Muséum National d'Histoire Naturelle, Paris NBC, Naturalis Biodiversity Center, Leiden, Netherlands NHMW, Naturhistorisches Museum Wien, Austria NMNHA, Kovachev Museum of Paleontology (branch of the National Museum of Natural History, Sofia), Assenovgrad, Bulgaria.

  are: the palatine foramina open far rostrally; the choanae are separated by a bony septum; there is no supra-meatal fossa, and no osseous tentorium; M1 has a strong labial cingulum and an expanded hypocone; on m1 the paraconid is long, the metaconid robust, the talonid large and basined, with its buccal and lingual cristids separated by notches from the trigonid; m1 has accessory roots. By far the best known and most widespread fossil form is Promephitis, to which genus the Bulgarian fossils belong. Genus Promephitis Gaudry, 1861 TYPE SPECIES. -Promephitis lartetii Gaudry, 1861 DIAGNOSIS. -A mephitid ranging in size from that of Spilogale to that of Mephitis. Skull and face short and broad; muzzle inflated; zygomatic arch robust; goniac area of the mandible unexpanded; mastoid sinus moderately inflated; cerebral features primitive (frontal sinus absent, sylvian sulcus not distinct, supra-sylvian short, lateral sulcus almost straight, cruciate sulcus and post-cruciate complex virtually absent); premolar series extremely shortened; P4 hypocone long, tall, and distally located; buccal cingulum of M1 very strong.

  Gaudry, 1861 1954 Promephitis hootoni sp. nov. -Şenyürek 1954: 281, figs 1-14. HOLOTYPE. -Much distorted skull and attached mandible from Pikermi, PIK-3019 in the MNHN. DIAGNOSIS. -A species of Promephitis slightly larger than P. majori; snout more inflated, broader over the canines; occipital lower. DESCRIPTION There are three skulls of Promephitis lartetii from Hadjidimovo, and one from Kalimantsi.

  FIG. 1. -Promephitis from Hadjidimovo. A, Promephitis majori HD-9505 in lateral (A1), dorsal (A2), rostral (A3), and occipital (A4) views. B, Promephitis lartetii, HD-9508 in lateral (B1) and dorsal (B2) views. Scale bar equals 5 cm

  recognize the American modern Mephitidae, together with the closely related Pleistocene genera Osmotherium Cope, 1896 and Brachyprotoma Brown, 1908 but to the exclusion of Eurasian Mydaus, Promephitis and Palaeomephitis, as a monophyletic group, and they can be called Mephitini. Relationships of other purported Mephitidae with this core group are discussed below.

	Miocene Eurasian Forms. Palaeomephitis steinheimensis is based upon the posterior half of
	a Middle Miocene juvenile skull described by Helbing (1936) as Trocharion albanense, and
	further discussed by

TABLE 1 .

 1 -Cranial measurements of Promephitis (in mm)

		condylo-	width	width	width over	post	length
		basal	over	across	post-orbital	orbital	C-M1
		length	mastoids	orbits	processes	width
	P. majori:
	HD-9505	53.8	33.7	15.5	16.2	12.9	16.9
	AMNH 20585 (type)	53.6	33	16.6	17.3	14.7	17.1
	P. lartetii:
	K-9504	-	c. 39.7	19.9	21.6	16.7	-
	HD-9506	59.4	38.2	18.2	20.0	16	18.5
	HD-9508	c. 58	39.5	19.5	20.8	15.4	17.5
	NHMW A4798 1	c.59	-	-	20.6	-	-
	P. maeotica	67.5	44	22	24	16.6	21.4
	1 Koufos 2006

TABLE 2 .

 2 -Tooth measurements of Promephitis (in mm). L = length; W = width; Wmax =

	oblique maximum width
	UPPER TEETH	C		P3		P4			M1
		L	W	L	W	L	W	L	W Wmax
	P. lartetii :
	PIK-3019 (type)	-	-	-	-

? c.4.2

Acknowledgements

We dedicate this paper to the late D. Kovachev, who collected the fossils described here, and many more. We thank M. García Sanz (MNHN) for the CT-scans of HD-9505 and of modern American Mephitini, F. Goussard (MNHN) for his help with 3D image processing, P. Kamminga (NBC) for the lent of Mydaus specimens, J.-J. Hublin and H. Temming (Max Planck Institute, Leipzig) for their CT-scans, G.D. Koufos for making available the material from Perivolaki and for casts and photos of several other specimens, C. Argot, A. Verguin, and G. Véron (MNHN), D. Berthet (CCEC), A. Currant (NHML), and R. Marchant (MCGL) for giving access to fossil and modern specimens, M. Sotnikova and J.O. Ebbestad for photos of fossils in their care, M. Böhme and R. Ziegler for giving access to the type of Palaeomephitis steinheimensis, DigiMorph.org and NSF grant IIS-9874781 to T. Rowe for Taxidea and Melogale CT-scans, L. Cazes for the photos of the Bulgarian specimens, and A. Barr for polishing the English. An earlier version of this manuscript benefited from helpful comments by X. Wang and M. Wolsan.

HD-9505 3.1 1.9 2.0 1.4 5.5 4.2 5.4 7.5 7.9 P. hootoni type 4 4.0 3.1 2.8 1.9 6.1 5.1 5.7 7.6 -P. maeotica type 5 4.8 3.3 2.5 2.0 7.1 5.3 5.7 6 9.1 -P. alexejewi type 7 7.16 -6.23 -- 4.2 2.9 2.6 2 3.9 2.9 8.5 3.9 --P. majori type 2 3.5 1.8 2.0 1.4 3.0 2.0 7.9 3.5 2.4 2.4 2 4 ,2 6 ,2 8 ,3 7 1 1 ,1 2 ,2 8 0 ,1 6 ,1 8 ,3 7 2 7 ,3 2 2 6 2 3 ,3 2 ,3 4 0 ,1 2 1 0 ,1 5 ,1 6 ,1 7 1 ,2 ,3 ,6 ,7 ,2 0 ,2 1 ,2 2 ,2 4 ,2 5 ,3 0 ,3 1 2 1 ,2 9 ,3 0 ,3 2 ,3 3 ,3 2 3 , 3 2 , 3 4 0 , 1 2 1 0 , 1 5 , 1 6 , 1 7 1 , 2 , 3 , 6 , 7 , 2 0 , 2 1 , 2 2 , 2 4 , 2 5 , 3 0 , 3 1 2 1 , 2 9 , 3 0 , 3 2 , 3 3 , 3 6 2 3 , 3 2 , 3 4 0 , 1 2 1 0 , 1 5 , 1 6 , 1 7 1 , 2 , 3 , 6 , 7 , 2 0 , 2 1 , 2 2 , 2 4 , 2 5 , 3 0 , 3 1 2 1 , 2 9 , 3 0 , 3 2 , 3 3 , 3 6 1 , 7

1 2


IMPORTANT NOTE : Please be aware that slight modifications occurring after Proof correction may occur between this version of the manuscript and the version on the Publisher's website-

 ---------------------------------------------------------------------------------------Core-shell effects of functionalized oxide nanoparticles inside long-range meso-ordered spray-dried silica spheres.

Niki Baccile a,b , Anna Fischer a,b , Beatriz Julián-López a,c , David Grosso a and Clément Sanchez a,* a LCMCP,UPMC-CNRS, 4, Place Jussieu, T54 E5 75252 Paris Cedex 05, France ; E-mail : clement.sanchez@upmc.fr b Present address: Max-Planck Institute Colloid department, MPI Campus Golm,14476 Golm, Deutschland c Present address: Dpto. Química Inorgánica y Orgánica, Universitat Jaume I, Avda. Sos Baynat s/n, 12071, Castellón, Spain Core-shell and homogeneous distributions of functionalized cerium oxide nanoparticles within spray-dried mesostructured silica spheres are achieved by modification of synthesis parameters such as the templating agent and nanoparticle capping functions.

In the past fifteen years a large number of studies has been focused on long-range ordered mesoporous materials obtained in solution through the self-assembly of a variety of amphiphilic templating agents with a growing inorganic phase [1]. Recently, several works showed that their well known structural characteristics (high surface area, tunable geometry of the porous network, narrow pore size distribution) [START_REF] Lu | [END_REF] are preserved and enhanced by mean of a spray drying process, which revealed to be interesting for its ease and possibility to obtain spherical particles. In ref. 2c, 3, experimental conditions as well as different structuring agents were succesfully tested while in ref. 4 SAXS, TEM and mainly solid state NMR were used to better understand the process of particle and mesophase formation with time. As far as the last point is concerned, Boissière et al. [5] used time-resolved in-situ SAXS to determine the required conditions for the mesophase to form. Regarding applications, spherical shaping and possibility to embed maghemite nanoparticles within a mesoporous silica matrix readily showed an interest in combined applications like drug delivery and magnetic resonance imaging [6] while catalysis was rather explored in presence of Pd nanoparticles [7] and nanowires [8]. In these cases, the way in which nanoparticles are distributed inside the silica particles is of primary importance according to the final application. For instance, in catalytic application, one could hope to enhance interactions between external fluids and embedded nanoparticles by placing them in the silica surface rather than in its bulk. In this way, diffusion paths are limited to the external crust. On the contrary, if the application involves magnetic or optical properties, an homogenous distribution within the silica bulk is required in order to avoid destructive interactions (magnetic coupling, optical quenching…) origined from clustering of nanoparticles.

For these reasons, we want to show the possibility of tuning the spatial distribution of plain and functionalized cerium oxide nanoparticles inside spherical mesostructured silica particles produced by the spray-driyng technique. The goal is double: firstly we would like to show some tricky interactions effects between functionalized nanoparticles and surfactants employed to structure silica; secondly, we would like to open some perspectives as far as possibilities of easy controlling the spatial distribution of embedded nanoparticles.

Surface modification of cerium oxide, CeO2 ‡ , nanoparticles (used as received) was accomplished according to the procedure developped in ref. 9. Calculated ceria mass with respect to TEOS is about 10%. Surface modification is performed by addition of phenyl phosphonic acid (PPA) and 3-amino propyl phosphonic acid. When PPA is used, homogenous dispersion of nanoparticles can only be obtained by addition of THF while no THF is employed with amino-propyl phosphonic acid. Ceria solution is then mixed with a CTAB (Cetyltrimethylammonium bromide -Aldrich) /water/HCl/Ethanol solution to which TEOS (tetraethoxysilane -Aldrich) is finally added. Final mass (in grams) of the spray-dried sol are the following: Functionalization was checked by FT-IR spectroscopy for both phenyl and amino modified systems, as shown, respectively, in Fig. S1 and Fig. S2 in electronic supplementary information (ESI). Disappearing of FTIR bands of free P-O vibration at 2300 cm -1 region and lack of free P=O band in the 1200 cm -1 region suggest a tridentate binding of phosphonate group. For the amino modification (Fig. S2, ESI), additional peaks at 1047 and 1090 cm -1 suggest the presence of asymetric stretching vibrations of P-O as detected in PO3 2-group, though C-N vibrations also resonate in this region. Unfortunately, these same vibrations are masked by the strong peak at 1070 cm -1 and belonging to THF. FT-IR gives a primary evidence of functionalization but exact mechanism and quantification is far from being determined, being this a very hard task to put in evidence [10]. Solution of functionalized cerium oxide nanoparticles is finally added to precursor sol mixture and eventually set in the spraying chamber. Experimental apparatus was described elsewhere 2c ; here, we have set the drying chamber temperature to 150°C, the furnace temperature to 350°C while the relative humidity was set to 20%. X-ray diffraction (XRD) was performed in a θ-2θ geometry (Cu Kα, λ ~ 0.154 nm) (Philips PW 1820) while Transmission Electron Microscopy (TEM) was done on a Philips CM20 (200 kV) microscope coupled with a spectrometer for Energy Dispersion of Xray (EDX) analysis (CME, Université d'Orléans, France). Nitrogen adsorption/desorption isotherms were realized on a Micromeritics ASAP 2100 machine.

CeO2
Figure 1 shows the X-ray diffraction patterns of as-synthesized mesostructured silica materials and in presence of aminomodified and phenyl-modified cerium oxide particles. All patterns show three low-angle diffraction peaks which can be safely indexed as the (10), (11) and (20) reflections of a long-range ordered 2D-hexagonal mesophase, that is a hexagonal close packing of micellar cylinders, in complete agreement with previous observation [2c]. Addition of the nanoparticles does not modify the long-range order within the material, both when hydrophilic and hydrophobic functions are used, but slight decrease of hexagonal cell parameter from 47 to 40 Å is observed when ceria is functionalized with amino and phenyl group.

Figure 1

A possibility exists that nanoparticles with hydrophobic functions are embedded within micelles but this is excluded by the nitrogen adsorption/desorption isotherms shown in Figure 2, where adsorbed volumes are of the order of 450 cm 3 /g in both calcined samples and BET surface areas range about 1100 m 2 /g. The capillary condensation occurs at values below P/P0= 0.4, as typically observed for CTAB-templated materials. In addition, no real difference in the isotherms is observed meaning that the presence of nanoparticles does not interfere with mesostructuring process.

Figure 2

Electron microscopy images of all materials are shown in Figure 3. In all cases one remarks both the dispersion of ceria nanoparticles inside the silica matrix as well as the good quality of the long-range order of CTAB micelles. Embedding of cerium oxide was verified by the presence of CeL peak obtained by EDX measurements shown in Figure 3i and taken during TEM measurements, as shown by the presence of the CuK belonging to the copper grid support used for measuring.

When no modification is performed (a,b), ceria nanoparticles are quite well dispersed throughout the mesostructured silica matrix. No preferential site seems to be preferred by nanoceria to settle during the mesostructuring process. When the phenyl group is used as surface modifier, cerium oxide aggregates are also homogeously dispersed within the silica matrix, as figure 3 (c,d) shows. Dispersion is very similar to system without modification. When zooming out on figure 3 (e), an estimation of nanoceria dimension hands out values between 5 and 8 nm, which makes these objects too large to fit within CTAB templated pores, whose average pore diameter from N2 adsorption/desorption data is 2.3 nm. Reasons of quite homogenous dispersion could be probably found in the interactions between silica and functionalized nanoaggregates. An interesting discussion on silica surface chemistry on similar materials is lead by Alonso et al. [4a], they observe a high percentage of unreacted ethoxy groups when initial sols is not aged enough time, as it occurs in our case. As far as hydroxy groups are concerned, they attributed the low OH NMR signal to both water and silanols. It is known that hydrophilicity of silica surface in mesostructured powders vary very much between very acidic and very basic conditions [11]. Here, due to the mild initial pH and to the important presence of ethanol, it would be difficult to extrapolate a clear picture. Nevertheless, it would not be surprising that low percentages of silanols and adsorbed water combined to the existence of unreacted ethoxy groups may render an overall hydrophobicity of the material core. In addition, the homogenously dispersed and generalized presence of CTAB could definitely contribute to exclude possible hydrophilic areas and enhance hydrophobic interaction with phenyl-functionalized cerium particles.

Figure 3

Figure 3 (f) shows the amino modified nanoaggregates embedded in mesostructured silica particle of about 190 nm in diameter. Here, nanoceria mainly settle at the external silica/air interface. Such a radial distribution is also kept in larger silica spheres whose diameter is around 500 nm. In figure 3 (g,h), meso-ordered domains surrounded by nanoceria form the core of the main silica particle indicating that coagulation among primary silica spheres whose diameter ranges between 170-200 nm during spray-drying occurred [START_REF] Kodas | Aerosol Processing of Materials[END_REF]. This observation, though trivial, shows that nanoceria actually migrates to the silica/air interface before silica condensation takes place and not after, since coagulation between droplets occurs in the first stages of the spraying process. More precisely, then, we should consider the migration of nanoceria taking place from the inner core of the liquid droplet towards the liquid/air rather than a solid/air boundary.

Which parameters drive the migration of amino-functionalized ceria nanoparticles towards the outer droplet boundary? The localization of nanoparticles at the air/droplet interface has already been described [13]. In their case, solvent evaporation rate was set to be the main parameter influencing the migration and aggregation of nanoparticles in a corona fashion. Parameters playing a crucial role seem to be the viscosity of the medium, evaporation rate of solvent and time needed to pass from liquid droplet to solid particle. In our system, all experimental conditions remain unchanged between non-functionalized and functionalized ceria embedding. The difference in the distribution behavior should be related to the departure of ethanol during drying and presence of the amino function. Since the synthesis occurs at low pH, it is very likely that grafted amino groups are positively charged making the surface of nanoparticles virtually similar to the surfactant polar heads. Binks [14] has recently shown that nanoparticles and surfactants may behave similarly as far as segregation at interfaces is concerned but self-assembly only occurs for real amphiphiles. In our system, the high quantity of ethanol stabilizes CTAB molecules and ammonium-grafted nanoparticles before spraying. When ethanol evaporates, self-assembly into micelles occurs for CTAB molecules but not for functionalized nanoparticles, which segregate at the liquid/gas interface. The driving force for the particle migration can be found in the repulsion that may take place between positively charged amino functions and headgroups belonging to CTAB surfactant micelles. Verification of such an argument was tested by using neutral surfactants (results not shown), Brij58 and Pluronic P123 under the same synthesis conditions employed for CTAB. In any case a structured silica powder is obtained but surface modification did not influence at all the spatial distribution of ceria particles, which were always homogenously distributed inside silica particles.

In conclusion, we show that spatial distribution of functionalized nanoparticles within mesostructured CTAB-templated silica spheres obtained by spray drying can be varied by adjusting the type of surface function. When ceria nanoparticles are covered with hydrophobic phenylphosphonic acid or when they are used as-such, homogenous distribution inside mesostructured silica is obtained. On the contrary, when an amino-containing function is introduced at low pH, migration towards the air/droplet interface and a final core-shell type of the nanoparticles is eventually obtained.

Notes ‡ CeO2 nanoparticles are obtained from the hydrate CeO2(HNO3)0,5(H2O)4 delivered by Rhodia.

Aknowledgements

B. Julián thanks the finantial support of the "Ramon y Cajal" Program (Spanish governement) and the Bancaixa Foundation-Universitat Jaume I (P1 1B2007-47) project. N. Baccile thanks Prof. P. Innocenzi (LSMN, University of Sassari, Italy) and the EU Erasmus project for intra-European mobility.

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Figure 1 -Figure 2 -

 12 Figure 1 -X-ray diffraction patterns of spray-dried mesostructured silica particles embedding cerium oxide nanoparticles with (a) no functionalization and (b) amino and (c) phenyl functionalizations.

Figure 3 -Figure

 3 Figure 3 -Microtomed TEM images of mesostructured silica embedding: (a,b) non-modified, (c-e) phenylmodified and (f-h) amino-modified cerium oxide nanoparticles. i) EDX spectrum of ceria-containing silica obtained during TEM measurements.

Electronic Supplementary Information (ESI)