Frédéric Delsuc
email: delsuc@isem.univ-montp2.fr

François M Catzeflis

Michael J Stanhope

Emmanuel J P Douzery

The evolution of armadillos, anteaters, and sloths depicted by nuclear and mitochondrial phylogenies: implications for the status of the enigmatic fossil Eurotamandua

Keywords:

The evolution of armadillos, anteaters and sloths depicted by nuclear and mitochondrial phylogenies:

implications for the status of the enigmatic fossil Eurotamandua.

Frédéric Delsuc, François M. Catzeflis, Michael J. Stanhope, Emmanuel J.P.

Douzery

To cite this version:

Frédéric Delsuc, François M. Catzeflis, Michael J. Stanhope, Emmanuel J.P. Douzery. The evolution of armadillos, anteaters and sloths depicted by nuclear and mitochondrial phylogenies: implications for the status of the enigmatic fossil Eurotamandua.. Proceedings of the Royal Society B: Biological Sciences, 2001Sciences, , 268 (1476Sciences, ), pp.1605Sciences, -15. 10.1098Sciences, /rspb.2001Sciences, .1702. halsde-00192975

INTRODUCTION

The mammalian order Xenarthra (ex Edentata; see [START_REF] Glass | History of classification and nomenclature in Xenarthra (Edentata)[END_REF] is composed of three major lineages corresponding to three distinct living morphotypes: armadillos (Cingulata:

Dasypodidae), anteaters (Vermilingua: Myrmecophagidae) and sloths (Bradypodidae and Megalonychidae). Xenarthrans include 30 living species classified in 13 genera, almost all endemic of South and Central Americas, the nine-banded armadillo (Dasypus novemcinctus)

being the only one to occur in North America. Extant species provide only a glimpse of xenarthran past diversity since 218 fossil genera are recognised [START_REF] Mckenna | Classification of Mammals above the species level[END_REF]. This order was still greatly diversified no later than 10000 years ago, when the majority of genera became extinct possibly because of human impact [START_REF] Patterson | The fossil mammal fauna of South-America[END_REF][START_REF] Fariña | Trophic relationships among Lujanian mammals[END_REF].

The radiation of Xenarthra occurred between Palaeocene and Eocene at an epoch where South America was isolated from other continental masses [START_REF] Patterson | The fossil mammal fauna of South-America[END_REF]. Their fossil record is almost restricted to the American continent (Carrol 1988) with three striking exceptions: a "primitive edentate" (Ernanodon antelios) from the Upper Palaeocene of China [START_REF] Ding | A new Edentate from the Paleocene of Guangdong[END_REF]), a purported anteater (Eurotamandua joresi) discovered in the Middle Eocene of Europe [START_REF] Storch | Eurotamandua joresi, ein Myrmecophagidae aus dem Eozän der "Grubbe Messel" bei Darmstadt (Mammalia, Xenarthra)[END_REF], and an unidentified Eocene sloth from Antarctica [START_REF] Vizcaíno | A Eocene Tardigrade (Mammalia, Xenarthra) from Seymour Island, West Antartica[END_REF]. These unexpected reports complicated the reconstruction of xenarthran biogeographical history and raised doubts on their confinement to the New-World. More specifically, the affinities of Eurotamandua with Xenarthra are still highly debated [START_REF] Gaudin | The morphology of xenarthrous vertebrae (Mammalia: Xenarthra)[END_REF][START_REF] Rose | Eurotamandua and Palaeanodonta: convergent or related? Paläontol[END_REF].

Despite highly specialised and distinct morphologies of the three lineages, the monophyly of the order Xenarthra is classically recognised [START_REF] Mckenna | Classification of Mammals above the species level[END_REF]. All living and fossil xenarthrans exhibit a dental reduction by the loss of enamel, reaching its paroxysm in anteaters which totally lack teeth (Carrol 1988). The name "Edentata" has been used to recall this evolutionary trend, but it leads to confusion because it originally referred to the grouping of New-World xenarthrans and Old-World pholidotans (pangolins) which are also lacking teeth [START_REF] Glass | History of classification and nomenclature in Xenarthra (Edentata)[END_REF]. One exclusive morphological synapomorphy of Xenarthra is the presence of "xenarthry" constituted by additional atypical articulations between vertebrae [START_REF] Engelmann | The phylogeny of the Xenarthra[END_REF][START_REF] Patterson | The ear region in xenarthrans (= Edentata, Mammalia). Part II. Sloths, Anteaters, Palaeanodonts, and a Miscellany[END_REF][START_REF] Rose | Relationships of Xenarthra, Pholidota, and fossil "Edentates": the morphological evidence[END_REF][START_REF] Gaudin | The morphology of xenarthrous vertebrae (Mammalia: Xenarthra)[END_REF]. From the molecular point of view, xenarthran monophyly is strongly supported by a three consecutive amino acids deletion in the eye lens αA-crystallin protein, that is unique among studied eutherian mammals [START_REF] De Jong | Protein sequence analysis applied to xenarthran and pholidote phylogeny[END_REF][START_REF] Van Dijk | The virtues of gaps: Xenarthran (Edentate) monophyly supported by a unique deletion in αA-crystallin[END_REF].

The order Xenarthra is of crucial importance in understanding mammalian phylogenetics. Following [START_REF] Mckenna | Toward a phylogenetic classification of the Mammalia[END_REF] assumption that Xenarthra represents the sistergroup to all other living eutherians, thereby named Epitheria, numerous molecular workers have used its representatives to root eutherian phylogenies (see e.g. [START_REF] Allard | Support for interordinal eutherian relationships with an emphasis on primates and their archontan relatives[END_REF]. [START_REF] Gaudin | Reexamination of the morphological evidence for the Cohort Epitheria (Mammalia, Eutheria)[END_REF], however, emphasised the weakness of the morphological synapomorphies defining epitherians. The comparison of complete mitochondrial genomes suggested a close relationship between Xenarthra and either Ferungulata (Cetartiodactyla, Perissodactyla and Carnivora: [START_REF] Arnason | Phylogenetic analyses of mitochondrial DNA suggest a sister group relationship between Xenarthra (Edentata) and Ferungulates[END_REF] or Afrotheria (Proboscidea, Hyracoidea, Sirenia, Macroscelidea, Chrysochloridae and Tenrecidae; Waddell et al. 1999a,b). Recently, nuclear DNA sequences showed that Xenarthra represents one of the four major clades of placentals [START_REF] Madsen | Parallel adaptative radiations in two major clades of placental mammals[END_REF][START_REF] Murphy | Molecular phylogenetics and the origins of placentals mammals[END_REF].

Within Xenarthra, the intra-ordinal relationships and their timing remain unclear.

Morphological data suggest the grouping of anteaters and sloths into Pilosa, a clade defined by the replacement of the carapace by a coat [START_REF] Engelmann | The phylogeny of the Xenarthra[END_REF][START_REF] Patterson | The ear region in xenarthrans (= Edentata, Mammalia). Part II. Sloths, Anteaters, Palaeanodonts, and a Miscellany[END_REF][START_REF] Mckenna | Classification of Mammals above the species level[END_REF]. However, the possibility of an early emergence of anteaters from the ancestral xenarthran stock is suggested by studies on fossils (Carrol 1988), ear region [START_REF] Guth | La région temporale des Edentés[END_REF], and cephalic arterial pattern [START_REF] Bugge | Cephalic arterial pattern in New World edentates and Old World pangolins with special reference to their phylogenetic relationships and taxonomy[END_REF]. From the molecular perspective, the first immunological [START_REF] Sarich | Xenarthran systematics: albumin immunological evidence[END_REF] and protein [START_REF] De Jong | Protein sequence analysis applied to xenarthran and pholidote phylogeny[END_REF] studies, as well as the analysis of partial mitochondrial 12S and 16S rRNA genes [START_REF] Höss | Molecular phylogeny of the extinct ground sloth Mylodon darwinii[END_REF], did not clarify intraxenarthran relationships. Data from the mitochondrial ND1 favoured the grouping of armadillos and sloths to the exclusion of anteaters [START_REF] Cao | Conflict among individual mitochondrial proteins in resolving the phylogeny of eutherian orders[END_REF], contra the eye lens αAcrystallin which provided support for a monophyletic Pilosa, but suggested the paraphyly of armadillos [START_REF] Van Dijk | The virtues of gaps: Xenarthran (Edentate) monophyly supported by a unique deletion in αA-crystallin[END_REF]. Most recently, [START_REF] Murphy | Molecular phylogenetics and the origins of placentals mammals[END_REF], in an analysis of a data set including five xenarthran genera, present a tree with strong support for Pilosa, but with no statistical analysis of xenarthran issues or estimates of dates for the various splitting events within the order.

To better understand xenarthran evolution, we here present a study encompassing most of the order's diversity, based on three genes already used to explore mammalian phylogenetics: the exon 28 of the von Willebrand Factor (vWF) -a single copy nuclear marker -, and the mitochondrial 12S and 16S rRNAs (e.g. [START_REF] Huchon | Molecular evolution of the nuclear von Willebrand Factor gene in mammals and the phylogeny of rodents[END_REF]Montgelard et al. 1997;[START_REF] Höss | Molecular phylogeny of the extinct ground sloth Mylodon darwinii[END_REF]. The aims of this study are (1) to investigate the intra-ordinal relationships of Xenarthra using a larger taxonomic diversity than in previous molecular studies;

(2) to discuss the evolution of morphological characters in light of the phylogeny obtained; and (3) to specify the chronology of their radiation in relation to South American biogeography, with special reference to the occurrence of the purported fossil anteater Eurotamandua in Europe.

MATERIALS AND METHODS

Taxonomy

Throughout the text, we introduced "Folivora" for the clade containing the sloths, because the usual terms of "Phyllophaga" (e.g. [START_REF] Engelmann | The phylogeny of the Xenarthra[END_REF] or "Tardigrada" (e.g. [START_REF] Patterson | The ear region in xenarthrans (= Edentata, Mammalia). Part II. Sloths, Anteaters, Palaeanodonts, and a Miscellany[END_REF]) both referred to different groups of protostomians. Etymologically, "Folivora" means "leaf eater" and is the Latin correspondence for "Phyllophaga".

Data acquisition

Xenarthran samples preserved in 95% ethanol were stored in the Collection of Mammalian Tissues from the Institut des Sciences de l'Evolution de Montpellier [START_REF] Catzeflis | Animal tissue collections for molecular genetics and systematics[END_REF]. Total DNAs were extracted for Chaetophractus villosus (larger hairy armadillo),

Cabassous unicinctus (southern naked-tailed armadillo), Dasypus novemcinctus (nine-banded armadillo), Cyclopes didactylus (pygmy anteater), Tamandua tetradactyla (collared anteater),

Myrmecophaga tridactyla (giant anteater), Bradypus tridactylus (pale-throated three-toed sloth) and Choloepus didactylus (southern two-toed sloth).

PCR amplifications of the complete 12S rRNA gene were conducted following [START_REF] Hassanin | The tribal radiation of the family Bovidae (Artiodactyla) and the evolution of the mitochondrial cytochrome b gene[END_REF]. The 16S rRNA fragment (722 bp) for Myrmecophaga was amplified with primers R9 = 5'-CGCCTGTTTACCAAAAACATC-3' (direct) and S1 = 5'-TTATGCAATTACCGAGATCTGCCA-3' (reverse), and cloned using the pCR TM 2.1 plasmid vector and Escherichia coli strain INVαF' (Original TA cloning kit, Invitrogen). For the vWF exon 28 of sloths and armadillos, two overlapping fragments (V1/W2: 907 bp, and V2/W1: 962 bp)

were amplified according to [START_REF] Huchon | Molecular evolution of the nuclear von Willebrand Factor gene in mammals and the phylogeny of rodents[END_REF]. Three overlapping fragments (V1/W11: 398 bp, V8/W10: 504 bp, and V9/W12: 523 bp) were amplified for anteaters, with the following direct (V) and reverse (W) primers: V8 = 5'-GGAGCTGCGGCGCRTCGCCAGCCAGGTGAA-3', V9 = 5'-ACGAGATCATCAGCTACCTCTGYGRCCTG-3', W10 = 5'-TGTTGAAGCCGGCCTCGCCGATCCTGTC-3', W11 = 5'-CCGAAGACCTGGAACAGCGTGTAC-3', and W12 = 5'-TGAGGGCCCACRCCRATGGG-3'. PCR products were purified from 1% agarose gels using Amicon Ultrafree-DA columns (Millipore), and sequenced on both strands using manual (Thermo Sequenase cycle kit, Amersham) and automatic sequencing (Big Dye Terminator cycle kit) on an ABI 310 (PE Applied Biosystems). Three distinct clones were sequenced for the partial 16S rRNA of Myrmecophaga. Sequences have been deposited in EMBL under accession numbers AJ278151-AJ278161, and AJ297939 (Electronic Appendix I). Two already published vWF sequences have been updated (Chaetophractus villosus: AF076480 and Bradypus tridactylus: U31603).

Taxonomic sampling

Since species sampling has been shown to have a major impact on phylogenetic reconstruction [START_REF] Douzery | The pitfalls of molecular phylogeny based on four species, as illustrated by the Cetacea/Artiodactyla relationships[END_REF], we chose a data set encompassing a wide range of ordinal mammalian diversity. To reduce potential long branch attraction artefacts, we analysed, whenever possible, at least two species per placental order. The sequences of two marsupials from Australia (Macropus giganteus) and America (Didelphis virginiana) were used to unambiguously root eutherian trees. Thirty five mammalian genera were considered for the three genes. All sequences accession numbers are listed in Electronic Appendix I.

Sequence alignment and phylogenetic analyses

Sequences were manually aligned with the ED editor of the MUST package [START_REF] Philippe | MUST: a computer package of management utilities for sequences and trees[END_REF]. The alignment of vWF exon 28 sequences started at position 38 of the human sequence, was 1233 bp long, and non sequenced positions were coded as missing data. Indels and hypervariable zones of the 12S and 16S rRNAs were removed from subsequent analyses.

Alignments are available upon request to FD.

Phylogenetic reconstructions were conducted with PAUP* 4.0b4a [START_REF] Swofford | PAUP*[END_REF], TREE-PUZZLE 4.02 [START_REF] Strimmer | Quartet puzzling: a quartet maximum-likelihood method for reconstructing tree topologies[END_REF], and PAML 3.0b [START_REF] Yang | Phylogenetic Analysis by Maximum Likelihood (PAML), Version 3.0b[END_REF].

Distance analyses used the Minimum Evolution (ME) criterion with TBR branch swapping on a Neighbor Joining (NJ) tree based on paralinear Log-Det distances with constant site removal. Maximum Parsimony (MP) analyses consisted of heuristic searches using 100 random addition sequence replicates with TBR branch swapping. Maximum Likelihood (ML)

analyses with PAUP* were made in two steps in order to speed up computation times. First, ML parameters were estimated using a heuristic search with nearest neighbour interchange (NNI) branch swapping on a NJ starting tree. Second, a new search was conducted with random addition of sequences and tree bisection-reconnection (TBR) branch swapping, using the previously estimated parameters. Substitution rates were described by the GTR (under PAUP* and PAML) and JTT (under TREE-PUZZLE and PAML) models of sequence evolution for nucleotides and amino acids respectively. Among sites substitution rate heterogeneity for DNA and vWF protein was described by a gamma distribution with eight categories (Γ 8 ) [START_REF] Yang | Among-site rate variation and its impact on phylogenetic analyses[END_REF].

Robustness of the different nodes was estimated by Bootstrap Percentages (BP) after 1000 replications for ME and MP with TBR branch swapping, and after 300 replications using previously estimated parameters for ML, with NJ starting trees and 1000 TBR rearrangements per resampling. Reliability Percentages (RP) for the protein dataset were obtained after 10000 quartet-puzzling (QP) steps with TREE-PUZZLE. Bremer Support Indices, i.e. the number of extra steps required to break the corresponding node under the MP criterion, were computed with PAUP*. Log-likelihoods of alternative topologies were compared by the ML test of [START_REF] Kishino | Evaluation of the maximum likelihood estimate of the evolutionary tree topologies from DNA sequence data, and the branching order in Hominoidea[END_REF] implemented in PAML 3.0b. Three different GTR+Γ 8 models were assumed for the combination of the three genes.

Molecular datings

The presence of local molecular clocks [START_REF] Yoder | Estimation of primate speciation dates using local molecular clocks[END_REF] was tested for different data sets, and molecular dates were calculated by PAML 3.0b using maximum likelihood branch lengths in a locally clocklike tree. Taxa were removed one by one until acceptance by the likelihood ratio test (LRT) of the hypothesis of three local clocks: one for paenungulates, one for perissodactyls, and one for the remaining species. Three independent paleontological calibration points were taken into account to check for molecular datings with crosscalibrations [START_REF] Huchon | Variance of molecular datings, evolution of rodents and the phylogenetic affinities between Ctenodactylidae and Hystricognathi[END_REF]: (1) 55 million years (Myr) for the split between Ceratotherium and Equus (Waddell et al. 1999b) ; (2) 60 Myr for the split between Procavia and Elephas [START_REF] Gheerbrant | A Palaeocene proboscidean from Morocco[END_REF]; and (3) 63 Myr for the Arctocyonia -Mesonychia divergence [START_REF] Gingerich | Likelihood estimation of the time of origin of Cetacea and the time of divergence of Cetacea and Artiodactyla[END_REF], represented here by the Sus (domestic pig) -Physeter (sperm whale) split. To discuss our molecular dates in the light of geochronology, we have used the geological timescale compiled for the Tertiary by [START_REF] Mckenna | Classification of Mammals above the species level[END_REF].

RESULTS

Phylogenetic results

Nuclear exon 28 of vWF

Due to significant base composition heterogeneity (evaluated by a χ 2 test in TREE-PUZZLE) for numerous taxa (including Dasypus, Chaetophractus, Choloepus, Tamandua and Myrmecophaga within Xenarthra), third codon positions were excluded from all subsequent analyses. All phylogenetic reconstructions provided strong support for most nodes involving xenarthrans (Table 1): (i) monophyly of Xenarthra; (ii) monophyly of each of its three lineages: armadillos, anteaters and sloths; (iii) grouping of anteaters and sloths into a clade called Pilosa; and (iv) close relationship within anteaters between Myrmecophaga and Tamandua -the subfamily Myrmecophaginae -to the exclusion of Cyclopes. Within armadillos, the grouping of Cabassous with Chaetophractus to the exclusion of Dasypus received mixed support (Table 1). Phylogenetic analyses on vWF amino acids yielded high support for all nodes within Xenarthra, and provided several exclusive amino acid substitutions supporting them (Table 1 and Electronic Appendix II).

Mitochondrial 12S and 16S rRNAs

All nodes involving xenarthrans were retrieved by phylogenetic analyses using 12S and 16S rRNAs (Table 1), with high support (monophyly of Cingulata, Vermilingua, Folivora, and Myrmecophaginae), moderate support (grouping of anteaters and sloths), or low support (monophyly of the order). One conflict arose from 12S rRNA relationships within armadillos (Table 1): ME favoured the grouping of Cabassous with Chaetophractus, contra MP and ML which suggested a sister-group relationship between Cabassous and Dasypus.

Combination of the three genes

The ML phylogram obtained with the combined nuclear and mitochondrial data sets (2107 bp) is presented in Figure 1. Concatenation resulted in a general increase of robustness indices (Table 1). Xenarthra, Pilosa, Cingulata, Folivora, Vermilingua and Myrmecophaginae were clearly monophyletic (BP ML > 97), and the grouping of the armadillos Cabassous and Chaetophractus to the exclusion of Dasypus was favoured (BP ML = 60).

Tests of alternative hypotheses

The three alternative branching patterns connecting armadillos, anteaters, and sloths were compared by [START_REF] Kishino | Evaluation of the maximum likelihood estimate of the evolutionary tree topologies from DNA sequence data, and the branching order in Hominoidea[END_REF] likelihood tests. Pilosa appeared to be the most likely hypothesis in all cases. Alternative hypotheses placing either anteaters or sloths as sister-group to the remaining xenarthrans involved a drop in log-likelihoods which is significant when the three markers are combined (Table 2). With regard to Xenarthra sister-group relationships, their emergence second amongst placentals -after Afrotheria -was the most likely hypothesis in the vWF and combination cases.

Alternative hypotheses, i.e. Xenarthra sister-group of either remaining placentals (Epitheria), or Afrotheria, or Ferungulata, all appeared less likely when the three markers were combined as they involved a drop in log-likelihood by ca. 1 standard error (Table 2).

The morphological hypothesis of "Edentata" (Xenarthra + Pholidota) was significantly rejected in all cases (Table 2).

Molecular timings

Local clocks have been used in the ML datings because they represent a compromise between setting a single substitution rate among all lineages (global clock constrained) and independent rates for each branch (no clock constrained). Amino acid substitutions in vWF behaved locally clocklike for a reduced data set of 18 taxa, including Xenarthra, Paenungulata, Perissodactyla, and Cetartiodactyla (Lama excluded due to a fast rate of evolution) with the two marsupials as outgroup (LRT between non-clock and locally clocklike tree: 2∆lnL = 27.2 with 19 degrees of freedom; P = 0.10). Using respectively the perissodactyl, cetartiodactyl, and paenungulate calibration points, we obtained average substitution rates of 0.160, 0.194, and 0.205 % amino acid / Myr / lineage in the vWF protein (Figure 2). Cross-calibration comparisons between these three points showed that the cetartiodactyl and paenungulate points were compatible, with the latter yielding the youngest ages, whereas the perissodactyl point provided the oldest dates.

Within placentals, the separation between Afrotheria (here represented by two paenungulates: Figure 2) and the remaining groups was estimated to have occurred 122 Myr Datings were also conducted with the three concatenated markers at the DNA level, assuming three independent ML models, and using the same set of 18 species (LRT: 2∆lnL = 28.8; P = 0.07). The paenungulate and the cetartiodactyl points yielded similar dating estimates between them (data not shown), and also similar relative to vWF amino acids. For example, within Xenarthra, the first (armadillos vs. others) and second (anteaters vs. sloths) splits are estimated to be respectively 57-69 Myr and 50-60 Myr old at the DNA level, against 59-63 Myr and 51-54 Myr for vWF amino acids (Figure 2). The perissodactyl calibration point provided the oldest dating estimates, and was not compatible with the two other points:

it estimated the Hyracoidea / Proboscidea split to have occurred 118 Myr, against 60 Myr for palaeontology.

DISCUSSION

Molecular systematics and character evolution in Xenarthra

Xenarthra monophyly

The Xenarthra monophyly is unambiguously supported in all reconstructions by the vWF alone or in combination with the 12S and 16S rRNAs (Table 1). The single common ancestry of the eight xenarthrans here studied is also defined by three exclusive vWF amino acid replacements (Table1 and Electronic Appendix II), which should be added to the unique derived deletion in the αA-crystallin known for seven xenarthrans [START_REF] Van Dijk | The virtues of gaps: Xenarthran (Edentate) monophyly supported by a unique deletion in αA-crystallin[END_REF]). The monophyly of Xenarthra is also supported by morphological characters like accessory articulations between vertebrae ("xenarthry"), ischiosacral fusion -with a secondary reversal in Cyclopes likely related to its strictly arboreal life style [START_REF] Gaudin | The phylogeny of the Myrmecophagidae (Mammalia, Xenarthra, Vermilingua) and the relationship of Eurotamandua to the Vermilingua[END_REF]) -and dental simplification by the loss of enamel [START_REF] Engelmann | The phylogeny of the Xenarthra[END_REF]. These characters are generally thought to reflect adaptation towards fossoriality and myrmecophagy [START_REF] Carroll | Vertebrate paleontology and evolution[END_REF][START_REF] Gaudin | The morphology of xenarthrous vertebrae (Mammalia: Xenarthra)[END_REF].

Xenarthra intra-ordinal relationships

The monophyly of Cingulata, Vermilingua, and Folivora finds strong support from vWF and mitochondrial rRNAs sequence comparisons. It is also corroborated by numerous morphological characters like, for example, the modification of dermal ossicles into articulate plates for Cingulata, the total absence of teeth for Vermilingua, and the presence of paired perforations of the centra in lumbar vertebrae for Folivora [START_REF] Engelmann | The phylogeny of the Xenarthra[END_REF].

Our data, including all anteater and sloth genera, provide strong support for the clade Pilosa (Tables 1,2), extending the results of [START_REF] Madsen | Parallel adaptative radiations in two major clades of placental mammals[END_REF] and [START_REF] Murphy | Molecular phylogenetics and the origins of placentals mammals[END_REF].

This arrangement is in agreement with studies of the ear region [START_REF] Patterson | The ear region in xenarthrans (= Edentata, Mammalia). Part II. Sloths, Anteaters, Palaeanodonts, and a Miscellany[END_REF]) and other morphological characters which provided remarkable synapomorphies for Pilosa like, e.g. the interruption of the zygomatic arch or the intra-pelvian location of the testes [START_REF] Engelmann | The phylogeny of the Xenarthra[END_REF]. These results contrast with those of [START_REF] Guth | La région temporale des Edentés[END_REF], [START_REF] Bugge | Cephalic arterial pattern in New World edentates and Old World pangolins with special reference to their phylogenetic relationships and taxonomy[END_REF]), Carrol (1988[START_REF] Cao | Conflict among individual mitochondrial proteins in resolving the phylogeny of eutherian orders[END_REF] who claimed that anteaters are the sister-group of the remaining xenarthrans. In the light of our results, it is likely that earlier studies of the ear region [START_REF] Guth | La région temporale des Edentés[END_REF]) and cephalic arterial pattern [START_REF] Bugge | Cephalic arterial pattern in New World edentates and Old World pangolins with special reference to their phylogenetic relationships and taxonomy[END_REF]) may have been misled by the extreme specialisation of the skull towards myrmecophagy in anteaters. Reanalysis of ear characters [START_REF] Patterson | The ear region in xenarthrans (= Edentata, Mammalia). Part II. Sloths, Anteaters, Palaeanodonts, and a Miscellany[END_REF]) supported the Pilosa hypothesis, and the character pointed by [START_REF] Bugge | Cephalic arterial pattern in New World edentates and Old World pangolins with special reference to their phylogenetic relationships and taxonomy[END_REF] for the grouping of armadillos and sloths (i.e. the median course of the internal carotid artery) is likely to be symplesiomorphic. Carrol (1988) suggested an early emergence of anteaters by emphasising the fact that no fossil anteaters were found to have retained dermal ossicles, unlike some mylodontid sloths which possess these relicts of the cingulate-like xenarthrans' ancestor. The fossil record of anteaters is particularly scarce [START_REF] Gaudin | The phylogeny of the Myrmecophagidae (Mammalia, Xenarthra, Vermilingua) and the relationship of Eurotamandua to the Vermilingua[END_REF], and our results showing that anteaters are the sister-group of sloths suggest that fossil anteaters with armour relicts remain to be found. Thus, if fossils are taken under consideration, the replacement of the ancestral carapace by a coat could not be considered as a synapomorphy defining Pilosa. However, this evolutionary trend has led to a discrete pattern in living xenarthrans where armoured xenarthrans (armadillos) have retained the ancestral carapace and hairy xenarthrans (anteaters and sloths) share the possession of a true coat. Our conclusions also contradict those of [START_REF] Cao | Conflict among individual mitochondrial proteins in resolving the phylogeny of eutherian orders[END_REF] based on the mitochondrial NADH dehydrogenase 1 gene (ND1) which moderately supports a close relationship between the armadillo Cabassous and the sloth Bradypus to the exclusion of the anteater Tamandua. This result may be due to the reduced taxonomic sampling and/or to the particular behaviour of this fast evolving molecule which seems to be subject to convergence [START_REF] Cao | Conflict among individual mitochondrial proteins in resolving the phylogeny of eutherian orders[END_REF].

Our results confirm the classical arrangement within Vermilingua by favouring the grouping of the strictly terrestrial Myrmecophaga and semi-arboreal Tamandua into the subfamily Myrmecophaginae, to the exclusion of the strictly arboreal Cyclopes. Such a relationship is corroborated by numerous myological [START_REF] Reiss | Myology of the feeding apparatus of myrmecophagid anteaters (Xenarthra: Myrmecophagidae)[END_REF]) and morphological [START_REF] Gaudin | The phylogeny of the Myrmecophagidae (Mammalia, Xenarthra, Vermilingua) and the relationship of Eurotamandua to the Vermilingua[END_REF] characters.

The phylogeny of Cingulata is still poorly understood from a morphological point of view [START_REF] Patterson | The ear region in xenarthrans ( = Edentata, Mammalia). Part I. Cingulates[END_REF]. Our results suggest the grouping of Cabassous (Priodontini) and Chaetophractus (Euphractini) to the exclusion of Dasypus (Dasypodini), contra [START_REF] Engelmann | The phylogeny of the Xenarthra[END_REF] who claims a close relationship between Dasypus and Cabassous. Our results are in agreement with a recent study of sperm morphology and morphometry [START_REF] Cetica | Evolutionary sperm morphology and morphometry in armadillos[END_REF] showing that Cabassous and Chaetophractus have rather similar spermatozoa. However, the discrepancies existing between vWF, 12S, and 16S rRNA suggest that additional genes for a greater taxonomic diversity are needed to further resolve relationships within armadillos.

Searching for the Xenarthra sister-group

The vWF and mitochondrial rRNA genes do not provide a clear response to the difficult question of Xenarthra affinities within Eutheria. However, our analyses including eight armadillos, anteaters and sloths genera suggest that Xenarthra may constitute the second offshoot of the placental tree, Afrotheria being the first one to branch off (Figure 1 and Table 2). This is in agreement with the results obtained by [START_REF] Madsen | Parallel adaptative radiations in two major clades of placental mammals[END_REF] and [START_REF] Murphy | Molecular phylogenetics and the origins of placentals mammals[END_REF] on respectively four and five genera. Alternative affinities of Xenarthra with either epitherians, afrotherians, or ferungulates appear less likely (Table 2), but cannot be significantly rejected. Sequencing of additional molecular markers to provide an increased number of phylogenetically informative positions concomitant with adequate taxonomic diversity of Xenarthra is needed to further resolve their position within the eutherian tree. Interesting to note, the classical hypothesis of "Edentata" [START_REF] Novacek | Mammalian phylogeny: shaking the tree[END_REF]) is significantly rejected in all cases (Table 2). Together with other molecular results (de Jong 1998; [START_REF] Madsen | Parallel adaptative radiations in two major clades of placental mammals[END_REF][START_REF] Murphy | Molecular phylogenetics and the origins of placentals mammals[END_REF], this confirms that morphological similarities between xenarthrans and pangolins are a spectacular example of convergence in relation to myrmecophagy [START_REF] Bugge | Cephalic arterial pattern in New World edentates and Old World pangolins with special reference to their phylogenetic relationships and taxonomy[END_REF][START_REF] Rose | Relationships of Xenarthra, Pholidota, and fossil "Edentates": the morphological evidence[END_REF].

Molecular timing of the xenarthran radiation

With vWF amino acids, the three calibration points -cetartiodactyls, perissodactyls, paenungulates -are reciprocally compatible in the local molecular clocks analysis (Figure 2).

One should note that setting 63 Myr for the split between Bos (rather than Sus) and Physeter would involve 85 Myr for the split between Procavia and Elephas, i.e. a divergence time 25

Myr older than the paleontological one. This is the reason why our molecular datings tend to yield younger dates than previously published for the Hippopotamus / Physeter split: 41-53

Myr (Figure 2) vs. 53-54 Myr [START_REF] Montgelard | Phylogenetic relationships of artiodactyls and ceteacans as deduced from the comparison of cytochrome b and 12S rRNA mitochondrial sequences[END_REF]Ursing & Arnason 1998). However, our datings of deeper splits become more compatible with other proposed dates because of increasing intervals between the dates obtained from the three different calibration points. For example, our estimates of the xenarthran separation from other placentals (Figure 2: 100-128

Myr) are in the range of those of Springer (1997) and Waddell et al. (1999b).

The xenarthran radiation, corresponding to the emergence of armadillos, is here estimated to have occurred close to the K/T boundary (59-76 Myr) and the separation between anteaters and sloths during the Palaeocene (51-65 Myr). These results are younger than other molecular estimations yielding about 80 Myr for the radiation of xenarthran families (review in [START_REF] Bromham | Growing up with dinosaurs: molecular dates and the mammalian radiation[END_REF]. Indeed, immunological comparisons suggest ca. 80 Myr [START_REF] Sarich | Xenarthran systematics: albumin immunological evidence[END_REF], partial 12S and 16S rRNA gave the same result [START_REF] Höss | Molecular phylogeny of the extinct ground sloth Mylodon darwinii[END_REF] Eocene of Antarctica (Vizcaíno & Scillatto-Yané 1995), and the first apparition of undoubted myrmecophagid anteaters is from the Early Miocene of Patagonia [START_REF] Carlini | Un singular Myrmecophagidae (Xenarthra, Vermilingua) de Edad Colhuehuapense (Oligoceno Tardio-Miocene Temprano) de Patagonia, Argentina[END_REF]). Long gaps in the fossil record are clearly not unusual, and our dating of the origin of anteaters and sloths (around the Palaeocene/Eocene limit) suggests a gap of about 30 million years in the South American fossil record of anteaters, and emphasises its incompleteness. Regarding Myrmecophagidae, the very ancient origin of the pygmy anteater (Cyclopes) explains its very divergent morphology shaped by ca. 30 Myr of arboreal life-style, since arboreality is considered ancestral for anteaters [START_REF] Gaudin | The phylogeny of the Myrmecophagidae (Mammalia, Xenarthra, Vermilingua) and the relationship of Eurotamandua to the Vermilingua[END_REF]. The separation between the two modern sloths, unknown as fossils [START_REF] Patterson | The fossil mammal fauna of South-America[END_REF], is dated by our data at ca. 18

Myr (end of Early Miocene). Two-toed (Choloepus) and three-toed sloths (Bradypus) have been placed into two distinct families (respectively Megalonychidae and Bradypodidae) on the basis of their numerous morphological differences and a presumably diphyletic origin [START_REF] Webb | The interrelationships of tree sloths and ground sloths[END_REF]. Our estimation is slightly younger than the 25 Myr of [START_REF] Sarich | Xenarthran systematics: albumin immunological evidence[END_REF] but confirms their considerable divergence. It suggests that their apparent external similarities are the result of paralellism and that arboreality may have evolved at least twice within Folivora [START_REF] Höss | Molecular phylogeny of the extinct ground sloth Mylodon darwinii[END_REF][START_REF] Greenwood | A molecular phylogeny of two extinct sloths[END_REF]. The quite ancient dates obtained for the armadillos radiation strengthen the fact that Cingulata contains strikingly divergent taxa, a result coherent with marked differences observed in the structure of their spermatozoa [START_REF] Cetica | Evolutionary sperm morphology and morphometry in armadillos[END_REF]). Finally, it is noteworthy that numerous cladogenic events within Xenarthra seem to occur close to transitions between Epochs of the chronostratigraphic scale (Figure 2). Since those transitions are defined by dramatic environmental and climatic changes [START_REF] Pascual | Evolving climates and mammal faunas in Cenozoic South America[END_REF], such a synchrony suggests a major role of paleobiogeographic changes in the diversification of xenarthrans.

Eurotamandua: helping to solve the enigma?

Our molecular estimates of divergence dates for armadillos, anteaters and sloths raise the question of the occurrence of purported fossil xenarthrans outside of South America.

Indeed, if the presence of a sloth in the Eocene of Seymour Island is compatible with land connections between Antarctica and the Patagonian province until the Oligocene (Vizcaíno & Scillatto-Yané 1995), the occurrence of Ernanodon in the Palaeocene of China [START_REF] Ding | A new Edentate from the Paleocene of Guangdong[END_REF] and Eurotamandua in the Middle Eocene (45 Myr) of Europe [START_REF] Storch | Eurotamandua joresi, ein Myrmecophagidae aus dem Eozän der "Grubbe Messel" bei Darmstadt (Mammalia, Xenarthra)[END_REF]) are more difficult to explain since South America was an island at these epochs. The true belonging of Ernanodon and Eurotamandua to Xenarthra have been widely debated [START_REF] Rose | Relationships of Xenarthra, Pholidota, and fossil "Edentates": the morphological evidence[END_REF][START_REF] Gaudin | The morphology of xenarthrous vertebrae (Mammalia: Xenarthra)[END_REF]). Actually, Ernanodon antelios seems likely to be assigned to a group of endemic Chinese mammals whose morphological similarities with Xenarthra would reflect convergent adaptations to fossorial habits [START_REF] Rose | Relationships of Xenarthra, Pholidota, and fossil "Edentates": the morphological evidence[END_REF][START_REF] Gaudin | The morphology of xenarthrous vertebrae (Mammalia: Xenarthra)[END_REF]. However, there is no consensus about the status of Eurotamandua joresi [START_REF] Storch | Eurotamandua joresi, ein Myrmecophagidae aus dem Eozän der "Grubbe Messel" bei Darmstadt (Mammalia, Xenarthra)[END_REF]. Vermilinguan affinities of Eurotamandua have been suggested [START_REF] Storch | Eurotamandua joresi, ein Myrmecophagidae aus dem Eozän der "Grubbe Messel" bei Darmstadt (Mammalia, Xenarthra)[END_REF][START_REF] Storch | Rückverlagerte Choanen und akzessorische Bulla tympanica bei rezenten Vermilingua und Eurotamandua aus dem Eozän von Messel (Mammalia: Xenarthra)[END_REF], but several of the most crucial features for assessing its phylogenetic position remain equivocal [START_REF] Gaudin | The phylogeny of the Myrmecophagidae (Mammalia, Xenarthra, Vermilingua) and the relationship of Eurotamandua to the Vermilingua[END_REF][START_REF] Gaudin | The morphology of xenarthrous vertebrae (Mammalia: Xenarthra)[END_REF][START_REF] Rose | Eurotamandua and Palaeanodonta: convergent or related? Paläontol[END_REF]. If Eurotamandua belongs to Vermilingua, our results dating the origin of Vermilingua well nested in the Tertiary (between 54 and 38 Myr:

Figure 2) raise again the question of how an anteater reached Europe at a time where South America was isolated from all other continental masses? The only possible response is dispersal which seems to be very unlikely in the present state of our knowledge of paleobiogeography based on plate tectonic models. Thus, our results, contra [START_REF] Höss | Molecular phylogeny of the extinct ground sloth Mylodon darwinii[END_REF], cast doubt on the true belonging of Eurotamandua to Vermilingua. They suggest that the striking morphological resemblances between this taxon and Myrmecophagidae might be the result of adaptative convergence towards fossoriality and ant-feeding. This example underlines, once again, how morphological adaptation to similar ecological niches could be positively misleading in terms of phylogenetic signal.

The xenarthran status of Eurotamandua was also widely questioned and this fossil was interpreted as either an endemic Old World xenarthran sister-group of New World xenarthrans [START_REF] Szalay | Middle Eocene Eurotamandua and the early differentiation of the Edentata[END_REF], or a Pholidota [START_REF] Shoshani | Eurotamandua is a pholidotan not a xenarthran[END_REF], or a sister-group to Pilosa [START_REF] Gaudin | The phylogeny of the Myrmecophagidae (Mammalia, Xenarthra, Vermilingua) and the relationship of Eurotamandua to the Vermilingua[END_REF]. However, the evidence for the presence of true xenarthry in Eurotamandua is far from convincing [START_REF] Rose | Relationships of Xenarthra, Pholidota, and fossil "Edentates": the morphological evidence[END_REF][START_REF] Gaudin | The phylogeny of the Myrmecophagidae (Mammalia, Xenarthra, Vermilingua) and the relationship of Eurotamandua to the Vermilingua[END_REF][START_REF] Rose | Eurotamandua and Palaeanodonta: convergent or related? Paläontol[END_REF]. Based on an exhaustive and detailed study of the evolution of xenarthrous vertebrae, [START_REF] Gaudin | The morphology of xenarthrous vertebrae (Mammalia: Xenarthra)[END_REF] concluded that there is at present little evidence of a close phylogenetic relationship between true xenarthrans and Eurotamandua. Moreover, on the study of forelimbs from a second specimen, [START_REF] Rose | Eurotamandua and Palaeanodonta: convergent or related? Paläontol[END_REF] argued for a close relationship of Eurotamandua and Palaeanodonta (a group of extinct fossorial mammals with reduced dentitions). Our results placing the origin of Pilosa in the Early Tertiary contradict those of [START_REF] Gaudin | The phylogeny of the Myrmecophagidae (Mammalia, Xenarthra, Vermilingua) and the relationship of Eurotamandua to the Vermilingua[END_REF], but our dating of the xenarthrans origin into Cretaceous (between 106 and 63 Myr:

Figure 2) leave opened the possibility that Eurotamandua could be a basal member of Xenarthra. Only the discovering of additional specimens will provide us with a better understanding on the evolutionary affinities of this enigmatic fossil.

FIGURE CAPTIONS

(

  range of datings depending on the choice of calibration points: 115-148 Myr), whereas Xenarthra diverged from Cetartiodactyla + Perissodactyla some 106 Myr (range: 100-128 Myr). Our estimates then placed the xenarthran radiation -corresponding to the split between Cingulata and Pilosa -at ca. 63 Myr (Figure 2), around the Cretaceous/Tertiary (K/T) boundary (range: 59-76 Myr). Within Xenarthra, the following splits have been dated: (i) Vermilingua vs. Folivora: 54 Myr in Early Eocene (range: 51-65 Myr) ; (ii) Dasypus vs. other armadillos: 33 Myr in Early Oligocene (31-40 Myr), followed by Chaetophractus vs. Cabassous: 21 Myr in Early Miocene (20-25 Myr) ; (iii) pygmy anteater vs. other anteaters: 38 Myr in Late Eocene (35-45 Myr), followed by giant vs. collared anteaters: 13 Myr in Middle Miocene (12-15 Myr) ; (iv) Bradypus vs. Choloepus: 18 Myr in Early Miocene (16-21 Myr).

  , and Cao et al. (1998) even suggest an interval of 65 to 130 Myr based on the deep divergences they observed in ND1. These different molecular dates suggest that breaking long isolated branches through increased taxonomic sampling within Xenarthra might have modified the divergence time estimates. Our estimates obtained after extensive taxonomic sampling within Xenarthra are compatible with paleontological data. The first armadillo scutes are from the late Palaeocene (ca. 58 Myr) of Brazil (Scillato-Yané 1976), the first sloth remains are from the Middle

Figure 1 :

 1 Figure 1: Maximum Likelihood phylogram obtained using the combination of complete

Figure 2 :

 2 Figure 2: Molecular estimates of divergence dates for the xenarthran radiation based on a

Table 1 .

 1 TABLES Indices of robustness for the nodes of the Xenarthra subtree, obtained for 12S rRNA, partial 16S rRNA, vWF exon 28 (codon positions 1 + 2), and combination of the three genes, with three methods of phylogenetic reconstruction (ME: distance, MP: maximum parsimony, ML: maximum likelihood). Reliability percentages (RP) after Quartet Puzzling and number of Exclusive (non homoplasic) Synapomorphies (ExS) observed for the vWF exon 28 amino acid data set are also indicated for each node. Position and nature of these exclusive synapomorphies are indicated in Electronic Appendix II.Notes: BSI = Bremer Support Indices. ME = bootstrap percentages obtained after 1000 replications of minimum evolution on paralinear Log-Det distances. MP = bootstrap percentages obtained after 1000 replications of standard parsimony. ML = bootstrap percentages obtained after 100 replications of maximum likelihood. RP = reliability percentages obtained with TREE-PUZZLE after 10000 QP steps. A dash indicates that the node does not appear in the corresponding majority-rule consensus bootstrap tree. Electronic Appendix II. Position and nature of the exclusive Amino-Acids synapomorphies observed in the vWF exon 28 protein sequences alignment supporting nodes of the Xenarthra subtree.

			12S rRNA		Partial 16S rRNA		vWF (1 + 2)	Combination	vWF a.a.
		BSI ME MP ML	BSI ME MP ML	BSI ME MP ML	BSI ME MP ML	RP ExS
	Cingulata (armadillos)	+ 25 100 100 100	+ 4 100 94 96	+ 10 100 99 99	+ 36 100 100 100	99	2
	Cabassous + Chaetophractus	0	75	-	-	+ 1 85 61 48	+ 2	92 67 50	+ 1 83 61 60	90	2
	Vermilingua (anteaters)	+ 3 88 73 96	+ 4 99 80 84	+ 11 100 99 100	+ 16 97 100 100	98	3
	Tamandua + Myrmecophaga	+ 22 100 100 100	+ 3 100 92 87	+ 32 100 100 100	+ 44 100 100 100	99	5
	Folivora (sloths)	+ 15 100 99 100	+ 5 100 95 95	+ 34 100 100 100	+ 33 100 100 100	99	6
	Pilosa (anteaters + sloths)	+ 3 78 53 75	+ 2 73 43 59	+ 4	97 89 93	+ 6 87 88 97	93	2
	Xenarthra	+ 3 43 29 48	0	50 24 62	+ 17 100 100 100	+ 27 100 100 100	93	3

ACKNOWLEDGMENTS

This study would not have been feasible without the invaluable contribution of several people who kindly provided us with xenarthran samples: Danny Devillier, Eric Hansen, Jesus Mavarez, Jean-Christophe Vié and his team "Faune Sauvage" at Petit-Saut (French Guiana).

We wish to thank Claudine Montgelard and Mark S. Springer for giving access to unpublished sequences, Ziheng Yang for PAML advices, and Jeff Mauffrey for help with figures. Sergio F. Vizcaíno and three anonymous referees are thanked for helpful comments. This work has been supported by the "Mammalian phylogeny" TMR Network (FMRX-CT98-022) of the European Community, and by the Génopôle Languedoc-Roussillon. Research on French Guiana mammals was funded by EDF-CNEH (contract GP-0850) and Ministère de l'Environnement (contract SOFT-Guyane). F. D. is supported by a M.E.N.R.T. grant (99075) associated with the D.E.A. "Biologie de l'Evolution et Ecologie" (Montpellier, France). This is the contribution ISEM 2001-040 of the Institut des Sciences de l'Evolution de Montpellier (UMR 5554 -CNRS).

Table 2. Results of [START_REF] Kishino | Evaluation of the maximum likelihood estimate of the evolutionary tree topologies from DNA sequence data, and the branching order in Hominoidea[END_REF] likelihood tests of alternative topologies computed for the nuclear vWF (codon positions 1 + 2), the mitochondrial 12S and 16S rRNAs, and their combination. δ = ∆lnL / S. E. refers to the ratio between the difference in log-likelihood (∆lnL) of two phylogenetic hypotheses and its standard error (S. E.). P refers to the one-tailed significance level of the Kishino


Wanli Gao

Hubert Perrot
email: hubert.perrot@upmc.fr

Ozlem Sel
email: ozlem.sel@upmc.fr

Tracking interfacial charge transfer behavior of hydrothermally synthesized ZnO nanostructures via complementary electrogravimetric methods

Keywords: Zinc oxide, Supercapacitor, EQCM, Ac-electrogravimetry, Ionic transfer dynamics

The mechanism of species fluxes during charge-discharge process in nanostructured ZnO electrode was studied by a combined methodology of electrochemical quartz-crystal microbalance (EQCM) and ac-electrogravimetry. Under the conditions of this study, anions (SO 4 2-) possess the highest kinetics to be transferred at the electrode/electrolyte interface in charge balance while cations (identified as Na + .5H 2 O and Na + ) play the major part as charge carriers. Free H 2 O molecules present a sluggish behavior and their interfacial transfer occurs at low scan rate or low frequencies. These findings shed light on the nature of ions and solvent participation in charge balance of hydrothermally synthesized ZnO nanostructures directly grown on the QCM device. The combined methodology proposed herein provides dynamic and gravimetric analysis of interfacial charge transfer and can be extended to investigate other nanostructured metal oxide-based electrodes for energy storage.

Introduction

Supercapacitors (SCs) are a class of electrochemical energy storage devices well suited to the rapid storage and release of energy. 1 The last four decades have seen a tremendous burgeoning of scientific and industrial interest into the potential applications of SCs, mainly due to their high power density and long cycle life. 2,3 Numerous research efforts have been dedicated to investigating the electrode materials, which play an essential role in optimizing the electrochemical performance of SCs.

Transition metal oxides have been intensively studied as electrode materials due to their fast and reversible redox reactions occurring at or near the electrode surface, which can provide significantly higher capacitances. [4][5][6][7] Generally, metal oxides can offer higher energy density than conventional carbon materials and better electrochemical stability than conducting polymers. 2 To date, transitional metal oxides, such as ruthenium oxide (RuO 2 ), 8 manganese dioxide (MnO 2 ), 9 zinc oxide (ZnO) 10 and nickel oxide (NiO) 11 have been intensively studied as electrode materials, which can offer additional pseudocapacitances during electrochemical performance. Among these candidates, ZnO nanomaterials have been widely used due to its low cost, easy fabrication, morphological diversity and electrochemical activities. [12][13][14][15] ZnO nanomaterials can be solely deposited on the substrate 10 or composited with other metal oxides, 14 conducting polymers 16 and carbon-based materials 17 to serve as energy storage electrodes. However, of great fundamental importance but almost barely touched is the mechanism of ionic/non-ionic fluxes during charge-discharge process in ZnO-based electrodes, which plays an essential role in the design of supercapacitor electrodes with high efficiency.

Electrochemical quartz-crystal microbalance (EQCM) has gained wide applicability to evaluate the electrochemical behaviour of electrodes, [18][19][20][21][22] particularly in situ capturing of the global transfer of the species at the electrode/electrolyte interface. Mass and charge variations measured simultaneously during the electrode cycling allow the derivation of the global mass per mole of electrons (MPE) that is exchanged between the electrode and the electrolyte according to the equation:

∆ ∆ ∆ /∆ ∆ /∆ ∆ /∆
with F is the Faraday number, ∆ and ∆ are the mass and charge variations, respectively. If only one species is exchanged, then the MPE corresponds to its molar mass. In the cases where multiple ion transfer occurs, using Faraday's law to interpret classical EQCM data reaches its limitations. To discriminate between the involved species, Donnan-type electrical double layer models were incorporated into the gravimetric EQCM equations. 23 Besides, the complex character of the electrode mass changes as well as the quantification of viscoelastic properties of the electrodes (and also possibly formed solid electrolyte interface (SEI) layer) can be investigated using multiharmonic EQCM with dissipation monitoring (EQCM-D). 24 Additionally, it has been demonstrated that coupling the QCM with electrochemical impedance spectroscopy (the so-called ac-electrogravimetry) contributes to disentangle the subtleties of global charge compensation process involving multiple species, offering a quantitative picture of each participant with their transfer kinetics and identifying them by their molar masses.

The ac-electrogravimetric measurements can detect interfacial processes occurring in the frequency range from ~ 100 Hz to as low as ~ 1 mHz, and separates multi-species contribution with different kinetics, reaching a higher selectivity than slow scan-rate cyclic voltammetry. 25 In ac-electrogravimetry, under a fixed potential with a small sinusoidal perturbation, frequency dependant mass and charge variations are obtained and used to generate both the classical EIS transfer function (TF), ∆E/∆I(ω) and the mass/potential TF, ∆m/∆E(ω). These TFs are then fitted with an appropriate model to obtain detailed identification of the species involved in the charge transfer mechanism, along with the kinetics and the relative concentrations associated with each species. 26 In the present work, this non-classical but emerging methodology in the energy storage domain [27][28][29] is adopted to characterize the electrochemical energy storage behaviour of ZnO-based electrodes and elucidate the subtleties in transfer of species during charge balance. To the best of our knowledge, this is the first time that ZnO is grown on QCM devices for energy storage purposes and investigated by the classical EQCM and its complementary counterpart ac-electrogravimetry for understanding its charge storage mechanism.

Experimental

A precursor solution was prepared with 0.02 M zinc nitrate hexahydrate and 0.02 M hexamethylenetetramine (HMTA). A quartz resonator (9 MHz-AWS, Valencia, Spain) was immersed in the solution, which was transferred to a Teflon-lined stainless steel autoclave and maintained at 120 °C for 12 h. The resonator is placed at the bottom of the autoclave. By this way, the back side is protected and only the upper side of the crystal was found to be deposited with ZnO nanostructures. After the hydrothermal process, ZnO nanostructures generated on the resonators were rinsed several times with deionized water.

EQCM and ac-electrogravimetry measurements were performed in 0.25 M Na 2 SO 4 under nitrogen atmosphere. ZnO nanostructures grown on the quartz resonator were used as the working electrode, with Ag/AgCl (3 M KCl saturated with AgCl) and platinum gauze as the reference and counter electrode, respectively. The potential window for EQCM was confined between 0 V and 0.6 V vs. Ag/AgCl in cyclic voltammetry (CV) (the OCV was at 0.15 V vs. Ag/AgCl) and 0 V and 0.8 V vs. Ag/AgCl in galvanostatic charge/discharge (GCD). A lab-made QCM device (Miller oscillator) was used to measure frequency shift (∆f) of the quartz crystals. The mass change (Δm) of ZnO electrode on the quartz crystals during electrochemical process can be estimated by the microbalance frequency shift (Δf) through Sauerbrey equation, 30 i.e., Δf = -C f ×Δm, where C f is the experimental sensitivity factor of the quartz crystal resonator (C f =16.3×10 7 Hz.g -1 .cm 2 ). The details of the estimation of the experimental sensitivity factor are previously given. 31 (FRA, Solartron 1254) and a lab-made potentiostat (SOTELEM-PGSTAT) were used. The QCM was performed under dynamic regime, and the modified working electrode was polarized at selected potentials to which a sinusoidal small amplitude potential perturbation was superimposed. The frequency range was between 63 KHz and 10 were sent to a four-channel FRA, which led to the electrogravimetric TF (∆m/∆E) and the electrical TF (∆E/∆I) to be obtained simultaneously at a given potential and frequency modulation, f (pulsation, f).

Theoretical Part

Under the effect of a sinusoidal potential perturbation with low amplitude, E, imposed to the electrode/film/electrolyte system, sinusoidal fluctuations of concentration, C i are observed. The theoretical TFs are calculated through the following equations: 28,32 ∆ ∆ (eq. 1)

∆ ∆ ∑ (eq. 2) ∆ ∆ F • ∑ (eq. 3) ∆ ∆ ∑ (eq. 4)
where ΔC i presents concentration variation for each species, is the pulsation, d f is the film thickness, M i depicts the molar mass of involved species, K i and G i are the partial derivatives of the flux (J i ) with respect to the concentration and the potential, respectively. K i represents the transfer kinetics of each species while G i is the reciprocal of the transfer resistance (Rt i =1/FG i ), exhibiting the ease or difficulty in the species transfer at the film/electrolyte interface (i: ions for eq. 1-3, and i: ions and non-charged species for eq. 4).

The theoretical expressions (eq. 2-4) were used to fit the experimental responses of the electrochemical impedance, ∆E/∆I, the charge/potential TF, Δq/ΔEand the electrogravimetric TF, ∆m/∆E, which provided the key parameters (M i , K i and G i ) related to each species (where i can be a cation, an anion or solvent) to be extracted.

With the same set of parameters, all the TFs including cross and partial TFs (detailed in Ref. 26-29) were fitted where a good agreement between the experimental and the theoretical functions in terms of both the shape and the frequencies should be achieved, to consider that these parameters (M i , K i and G i ) are unique.

Results and Discussion

The synthesis of the nanostructures were performed directly on the quartz resonators by hydrothermal method. As shown in Fig. 1a, ZnO nanostructures are densely grown on the quartz resonator, formed by flower-like bundles of individual nanorods with a diameter of ~300-800 nm and length of ~2-7 µm. The discrete ZnO nanorods may facilitate the exposure to the electrolyte and thus offer a large surface area for the electrochemical processes. XRD patterns are recorded (Fig. 1b) and the peaks present between 32° and 69° demonstrate that the synthesized ZnO nanorods exhibit the crystalline nature of wurtzite hexagonal structure, which is in good agreement with standard ZnO peaks (JCPDS 36-1451). 33,34 Cyclic ZnO + Na + + e-↔ (ZnO) -surface Na + However, this simplified scheme does not take into account of (i) the ion solvation effect, (ii) the possible presence of more than one ionic species which may participate in the charge compensation process and affect the charge/discharge rates, and (iii) the influence of free electrolyte molecules that can interact, indirectly, with the electrodes.

Utilizing the simultaneous QCM measurements permits the corresponding mass responses (Δm-E) to be obtained during CV scans (Fig. 2b). Mass response mainly presents ingress/egress during cathodic/anodic sweep, which is indicative of cation participation in charge compensation process. 32 GCD curves were measured to further evaluate the electrochemical performance of ZnO-based electrode.

The corresponding mass-time response proceeds in an inverse pace with potential-time response, i.e., mass decrease during charging and mass increase during discharging, which indicates that the cation plays a major role in charge balance.

To obtain indications on the nature of the transferred species, the MPE=FΔm/Δq was calculated from the EQCM data (Fig. S1, Supporting information). If only one species is exchanged, then the MPE corresponds to its molar mass. Positive and negative values of the MPE correspond to a major contribution to the energy storage mechanism by anions and cations, respectively. Here, we obtain a MPE of ~-100 g.mol -1 below 0.3 V and ~-50 g.mol -1 above 0.4 V for the cathodic sweep and anodic sweep, respectively. The higher values for the cathodic sweep suggest that the cations are hydrated and/or accompanied by free solvent molecules. This value lowers during the anodic sweep (~-50 g.mol -1 ), indicating that multiple species are exchanged and interfacial behaviour is complex.

QCM-coupled CV and GCD measurements are performed at a certain scan rate or current density, therefore they require either the incorporation of Donnan-type models into the gravimetric EQCM equations 23 or complementary methods to elucidate if there is a multiple species contribution to the charge compensation process. 35 To get a deeper insight into the charge compensation behaviour occurring in the ZnO-based electrode, from both gravimetrical and kinetic point of view, the ac-electrogravimetry was suggested. The different scenarios of the charge compensation process for ZnO-based electrodes are scrutinized by this coupled method emerging in the field of energy storage. 27,29 QCM-coupled to Electrochemical Impedance (Ac-electrogravimetry): Ac-electrogravimetry measurements were performed in the same potential range used for CV measurements, at various states-of-polarization from 0 V to 0.6V vs. Ag/AgCl with an interval of 100 mV. Fig. 3a and b present Δq/ΔE(calculated from the electrochemical impedance, theoretical part in eq. 2 and 3) and Δm/ΔETFs at 0.3V, where the experimental data were fitted using theoretical functions in eq. 3 and 4. It is important to note that for the Δm/ΔETF, the experimental data were not visualized at high frequency region in Fig. 3b beyond ~ 100 Hz due to the instrumental limitations, i.e., the validity region of the frequency-voltage convertor is ~100 Hz to ~ 1 mHz. This may indicate that there is a fast species contributing to the charge storage but cannot be detected by ac-electrogravimetry with the current frequency-voltage convertor. Theoretically, anions and cations share the same response behaviour in Δq/ΔETF, but anions characteristically appear in the quadrant I (Cartesian system) in Δm/ΔEwhile cations locate in the quadrant III, as shown in Fig. 3c andd. Therefore, in the present work, the mass response at high frequency emerging in the quadrant I can originate from either anions or free H 2 O molecules with the same transfer direction as anions. 26 Herein, four species based on different kinetics, i.e., anions (SO 4 2-), hydrated cations (Na + .5H 2 O), cations (Na + ) and H 2 O molecules were proposed which led to a good agreement between the experimental and the theoretical functions in terms of both the shape and the frequencies (Fig. 3). It is highlighted that the identification of the species can be achieved thanks to the electrogravimetric TF (Δm/ΔEwhich involves the molar mass (M i ) of the species involved in the charge compensations (eq. 4). This configuration was also verified by the so-called partial TFs (obtained by removing the contribution of one of the species contribution, and analysing the residual response) (not shown).

Then, the ac-electrogravimetry data obtained at various states-of-polarization from 0 V to 0.6V vs. Ag/AgCl were analysed and it was observed that the presence of several species persists. This finding is further supported by comparing the ac-electrogravimetry data with that of EQCM (see the section: Comparison of the EQCM and Ac-electrogravimerty mass responses).

The transfer kinetics of each species, K i , are summarized in Fig. 4a [36][37][38] and also in our recent work on SWCNT based electrodes. 27 The charge storage mechanism of another transitional metal oxide, MnO 2 , has been widely investigated, and it was proposed that the charge could be stored by either i) the surface adsorption of alkali metal cations (C + ) 39 or ii) the intercalation of both protons (H + ) and alkali metal cations (C + ) 40,41 in the electrolyte. However, direct analogy of the charge storage mechanism proposed for the other metal oxides may not be applicable to ZnO. In the present work, SO 4 2-and Na + .5H 2 O present a quick response upon potential perturbation, which is presumably ascribed to their fast electroadsorption behaviour. Dehydrated Na + is also considered to be electroadsorbed but in the ZnO bulk, on to the less accessible sites, due to a relatively slow transfer kinetics (Fig. 5). Furthermore, free H 2 O molecule exhibits a sluggish behaviour and participates in charge balance at lowest frequencies. It is supposed to be electrodragged by Na + , 27 as evidenced by the same flux direction of H 2 O and Na + .

Actually, it is speculated that there exist at least two different electroactive sites for charge storage in synthesized ZnO nanostructures. The first is on the surface of ZnO, where the charge storage occurs (under the conditions of this study) either by the electroadsorption and electrodesorption of SO 4 2-and Na + .5H 2 O, respectively (Fig. 5). After the surficial electroactive sites are saturated with SO 4 2-and Na + .5H 2 O by a fast electroadsorption behaviour, contributing to the responses at high frequency (HF) in ac-electrogravimetry (Fig. 3b), the second electroactive sites in the bulk of ZnO may begin to come into use. Dehydrated Na + .5H 2 O (i.e., Na + ) may electrodrag free H 2 O molecules into/out of ZnO bulk and, consequently, bring about a response from free H 2 O molecules at lowest frequency (LF) in acelectrogravimetry (Fig. 3b).

Comparison of the EQCM and Ac-electrogravimerty mass responses:

In order to validate our hypothesis involving multi-species contribution to the charge storage and also to confirm the presence of the anionic species appearing at the HF region (see theoretical curve in Fig. 3b), a methodology benefiting from the complementarity of the EQCM and ac-electrogravimetry is developed as follows: 27 From ac-electrogravimetry, the relative concentration changes of each species (ΔC i ) with respect to the potential variation can be estimated by using eq. 5;

specifically, the estimation of the relative concentration change with respect to the individual species ( can be obtained after integration of eq. 5:

∆ ∆ →

(eq. 5)

The concentration variation of individual species ( ) obtained from acelectrogravimetry is shown in Fig. 6a. These values are transformed into corresponding mass variations of each species (Fig. 6b). Subsequently, the total mass change (total Δm from ac-electrogravimetry in Fig. 6c) is reconstructed by the addition of individual mass contribution from all species concerned in ac-electrogravimetry. A good agreement between the total mass change from ac-electrogravimetry and the EQCM response obtained at 10 mV/s emerges. It is further highlighted that ac-electrogravimetry results are obtained from individual measurements at stationary potentials and the reconstructed mass response is in good agreement with the EQCM data from cyclic voltammetry at the lowest scan rate measured. This is highly significant since it evidences that the ac-electrogravimetry result in (100 mV/s) (Fig. 6d) is also achieved. This implies that all of these 4 species detected including the free H 2 O molecules can be transferred at the electrode/electrolyte interface at low scan rate (10 mV/s). However, it hardly occurs for H 2 O to be transferred at the electrode/electrolyte interface at a higher scan rate (100 mV/s). These comparisons present further evidence for the fitting hypothesis of the ac-electrogravimetry data that the missing responses at HF are from anion contributions. It is evident that if the contribution of the anions is neglected, a good agreement between the EQCM and ac-electrogravimetry would not be achieved (Fig. 6). These results highlight the complementarity of the EQCM and acelectrogravimetry, the limitations of either of which can be compensated by the other to unveil the subtleties of the charge storage mechanisms.

Conclusions

The complementary combination of EQCM and ac-electrogravimetry was proposed herein to study the charge storage mechanism of hydrothermally synthesized ZnO nanostructures.

EQCM provides a global mass response from cations in charge storage/delivery process, while ac-electrogravimetry offers a gravimetric and dynamic picture on the subtleties during this process: four different species (SO

Notes

There are no conflicts to declare.

  mHz. The mass change, Δm, of the working electrode was measured simultaneously with the ac response, ΔI, of the electrochemical system. Frequency/voltage converter is the key component of the ac-electrogravimetry set-up which translates the QCM frequency response (Δf) to a continuous voltage change (ΔV f ) with the aim of obtaining a transfer function (TF) via a frequency response analyzer (FRA) (which cannot directly analyze the frequency response). Finally, the resulting signals ∆V f /∆V(ω) and ∆V/∆I(ω)

  Electrogravimetry (EQCM) and QCM-coupled GCD: The electrochemical performance of ZnO nanorods was characterized by cyclic voltammetry (CV) and galvonastatic chargedischarge (GCD) techniques. The QCM was coupled with both measurements to track the simultaneous frequency shifts of the ZnO-based electrode during cycling, which can be converted into mass responses through Sauerbrey equation. 30 Fig. 2a exhibits approximately rectangular CV curves, with a slight contribution of a cathodic peak around 0.1 V and an anodic peak around 0.2 V.This electrochemical behaviour due to the dominant capacitive response in the CV curve can be attributed to an electroadsorption process of Na + ions occurring at different electroactive sites of the ZnO electrode:

Fig. 6b is

  Fig. 6b is indeed a deconvolution of the global EQCM mass response into distinct

Fig. 1 (FIGURESFig. 2 Fig. 3

 123 Fig. 1 (a) FEG-SEM image and (b) XRD spectra of ZnO nanostructures hydrothermally

Fig. 4

 4 Fig. 4 Transfer kinetics, K i (cm·s -1 ) (a) and corresponding transfer resistances, Rt i (Ω·cm 2 ) (b)

Fig. 5 A

 5 Fig.5 A schematic presentation of ions participating in the charge balance, where cations with

Fig. 6

 6 Fig. 6 Relative concentration change (a) and the corresponding masschange (b) of each

  4 2-, Na + .5H 2 O, Na + , and H 2 O) were detected to participate in charge balance with different kinetics to transfer the electrode/electrolyte interface. This study further emphasizes the complementarity of the EQCM and ac-electrogravimetry where certain instrumental limitations that challenge the detection of fast ion dynamics can be overcome by exploiting the two methods. This combined methodology can be extended to investigate the charge storage mechanism and species fluxes in other nanostructured electrodes, facilitating the design of optimized SC electrodes.

Acknowledgements

Wanli Gao acknowledges China Scholarship Council-Sorbonne University scholarship.

The authors thank Ms. Françoise Pillier and Stéphanie Delbrel for the FEG-SEM measurements, and Mr. Cyrille Bazin for XRD measurements.

Supporting Information

MPE=FΔm/Δq calculation from the EQCM data of the ZnO modified quartz resonators measured in 0.25M Na 2 SO 4 .

Supporting Information

Tracking interfacial charge transfer behavior of hydrothermally synthesized ZnO nanostructures via complementary electrogravimetric methods

Wanli Gao a , Hubert Perrot a, *, Ozlem Sel a, * a Sorbonne Université, CNRS, UMR 8235, LISE, F-75005, Paris, France. E-mail address: hubert.perrot@upmc.fr (H. Perrot). E-mail address: ozlem.sel@upmc.fr (O. Sel).


Éric Lichtfouse
email: eric.lichtfouse@dijon.inra.fr

Claire Chenu

François Baudin

Resistant ultralaminae in soils

Keywords:

Observation of a chemically resistant organic residue from maize crop soils by transmission electron microscopy reveals the occurrence of thin laminae with 10-50 nm thickness, similar to resistant, highly aliphatic biopolymers found in kerogens and microbes. This discovery shows evidence for a novel and alternative mode of formation of soil organic matter by selective preservation of resistant biopolymers from soil microbes, in addition to the classical recondensation pathway proposed by the French chemist Maillard in 1917.

INTRODUCTION

Soil organic matter is one of the major pool of carbon on Earth. It has a key position in the global carbon cycle, for instance as a sink of atmospheric CO 2 and a source of sedimentary debris. The main part of soil organic matter is composed of complex, partly macromolecular, brown-yellow components called humic substances [START_REF] Schnitzer | Soil organic matter. The next 75 years[END_REF]. The precise biological origin, mode of formation and transformation of humic substances are so far poorly understood. As early as 1917, when he observed similar elemental compositions of natural humic substances and of condensation products of sugars and amino acids, the French chemist Maillard postulated that natural humic substances could be formed by recondensation of small molecules. More recently, numerous investigations in sediments have shown the existence of an alternative pathway of formation of sedimentary organic matter via selective preservation of resistant biopolymers [START_REF] Tegelaar | A reappraisal of kerogen formation[END_REF][START_REF] Largeau | Occurrence and origin of "ultralaminar" structures in "amorphous" kerogens of various source rocks and oil shales[END_REF][START_REF] Derenne | Chemical evidence of kerogen formation in source rocks and oil shales via selective preservation of thin resistant outer walls of microalgae : origin of ultralaminae[END_REF]. In a study of forest profiles, [START_REF] Kögel-Knabner | Nature and distribution of alkyl carbon in forest soil profiles : implications for the origin and humification of aliphatic biomacromolecules[END_REF] further suggested that aliphatic biopolymers appear to be selectively preserved with increassing depth. Moreover, we have shown recently on isotopic grounds that the bulk of crop organic matter must be formed in situ, either by selective preservation of resistant biopolymers from soil microbes or by recondensation of small molecules from exudates and degradation products [START_REF] Lichtfouse | Unexpected 13 C-enrichment of organic components from wheat crop soils : evidence for the in situ origin of soil organic matter[END_REF]. Here, we provide microscopic evidence for the occurrence of resistant biopolymers in soil. thoroughly extracted with CHCl 3 -MeOH 3/1 v/v (3 x) to remove free lipids, then with NaOH 0.1 M to remove humic and fulvic acids. The residue (humin and minerals) was then treated for classical kerogen extraction : 24 h. with 1050 ml HF (48%)/700 ml HCl (36%), 24 h. with 700 ml HF, 24 h. with 700 ml HCl, and then washed with H 2 O to pH 6 to yield 3.9 g of residue. The removal of main soil silicates and Fe minerals by acid hydrolysis was checked by the very low abundance of Si and Fe relative to C and O by X-Ray diffraction. Elemental analysis gave 31% of C, 3 % of H, 2.5 % of N and 40% of uncombusted matter. Several attempts to reduce the uncombusted matter content by further drastic acid hydrolysis (HCl 36%, reflux 48 h.) resulted in the total degradation/solubilization of organic C, as observed by Schulten and Schnitzer for humic acids (1993). An other attempt to perform a strong alkaline hydrolysis (KOH-MeOH 5 M, reflux 24 h.) gave similar results. The residue obtained by our procedure thus represents the most chemically resistant carbon pool that can be isolated in substantial amounts from a soil. The residue was embedded in epoxy resin, sliced in ultrathin sections, stained with KMnO 4 then observed by transmission electron microscopy (TEM). Observation of the soil residue by TEM showed a large contribution of amorphous organic matter, estimated to more than 60%, along with recognizable wall debris either from plants or fungi (0.2-2 µm thickness). In addition, very thin laminae were observed as shown on Figure 1. Laminae were 10 to 50 nm thick and few µm long. They were composed of dark and light alternate zones, strongly then weakly KMnO 4 -stained, respectively. Since KMnO 4 reacts rather specifically with unsaturated molecules such as aromatic rings from lignin debris [START_REF] Ruel | Ultrastructural aspects of wood degradation by Sporotrichum pulverulentum. Observation on spruce wood impregnated with glucose[END_REF], the light zones are enriched in saturated carbon. A major part of this soil ultralaminae is therefore composed of saturated carbon. These morphological features are very similar to very thin resistant walls that have been recently discovered in various microbes and sediments, and termed 'ultralaminae' [START_REF] Largeau | Occurrence and origin of "ultralaminar" structures in "amorphous" kerogens of various source rocks and oil shales[END_REF][START_REF] Derenne | Chemical evidence of kerogen formation in source rocks and oil shales via selective preservation of thin resistant outer walls of microalgae : origin of ultralaminae[END_REF]. Recognition of resistant ultralaminae in soils imply that the formation and subsequent stabilization of soil organic carbon is not only performed by the classical recondensation of small molecules such as amino acids and carbohydrates [START_REF] Maillard | Identité des matières humiques de synthèse avec les matières humiques naturelles[END_REF], but also by selective preservation of resistant biopolymers from soil microorganisms. Further structural elucidation studies of these novel components are in progress.

Figure 1 .

 1 Figure 1. Transmission electron microscopy of a resistant residue, 'humin' or 'kerogen', from a maize crop soil, showing the occurrence of a resistant soil ultralaminae with thichnesses ranging from 10 to 50 nm (x 17 000).