Hui Zhi

Shirley Lee

François Chau

Siden Top

Gérard Jaouen

Anne Vessières

Eric Labbé

Olivier Buriez

New mechanistic insights into osmium-based tamoxifen derivatives

Keywords: Bioorganometallic, Cyclic voltammetry, Osmium, Anti-Cancer, Tamoxifen

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

Introduction.

Recently, there has been a growing demand for organometallic complexes in the development of innovative anticancer therapies [START_REF] Ndagi | Metal complexes in cancer therapy -an update from drug design perspective[END_REF][START_REF] Zhang | Advances in the design of organometallic anticancer complexes[END_REF][START_REF] Barry | Exploration of the medical periodic table: towards new targets[END_REF][START_REF] Gasser | Organometallic Anticancer Compounds[END_REF][START_REF] Hartinger | Challenges and Opportunities in the Development of Organometallic Anticancer Drugs[END_REF][START_REF] Hillard | Bioorganometallics: Future Trends in Drug Discovery[END_REF]. Indeed, organometallic compounds combine as many properties as structural diversity, variable redox states of metals, high lipophilicity and metal-specific reactivity. In this field, recent major contributions have focused on the functionalization of clinically validated purely organic drugs with complexes modified by a metallocene group which exalts the anti-cancer properties of the organic moiety. For instance, grafting a ferrocenyl unit onto chloroquine allowed the preparation of a new molecule called "ferroquine" which possesses a high antiplasmodial activity against the chloroquine resistant FcB1 strain of Plasmodium falciparum [START_REF] Biot | Synthesis and Antimalarial Activity in Vitro and in Vivo of a New Ferrocene-Chloroquine Analogue[END_REF]. Similarly, "ferrocifens" are complexes possessing both a ferrocenyl group and a tamoxifen skeleton, i.e. the current gold standard for endocrine breast cancer therapy [START_REF] Jaouen | Ferrocifen type anti cancer drugs[END_REF][START_REF] Top | Ferrocenyl hydroxytamoxifen: a prototype for a new range of oestradiol receptor site-directed cytotoxics[END_REF][START_REF] Top | Facile route to ferrocifen 1-[4-(2dimethylaminoethoxy)]-1-(phenyl-2-ferrocenyl-but-1-ene), first organometallic analogue of tamoxifen, by the McMurry reaction[END_REF]. The functionalization chemistry achieved on ferrocifens has not only brought more and more efficient molecules [START_REF] Top | Ferrocenyl hydroxytamoxifen: a prototype for a new range of oestradiol receptor site-directed cytotoxics[END_REF], but has also contributed to establish a coherent framework for the oxidative metabolism of these compounds [START_REF] De Paiva | Electrochemically Driven Supramolecular Interaction of Quinones and Ferrocifens: An Example of Redox Activation of Bioactive Compounds[END_REF][START_REF] De Jesús Cázares-Marinero | Synthesis, Characterization, and Antiproliferative Activities of Novel Ferrocenophanic Suberamides against Human Triple-Negative MDA-MB-231 and Hormone-Dependent MCF-7 Breast Cancer Cells[END_REF][START_REF] De Jesús Cázares-Marinero | The effect of protic electron donor aromatic substituents on ferrocenic and [3] ferrocenophanic anilines and anilides: Some aspects of structure-activity relationship studies on organometallic compounds with strong antiproliferative effects[END_REF][START_REF] Tan | Ferrocenyl catechols: synthesis, oxidation chemistry and anti-proliferative effects on MDA-MB-231 breast cancer cells[END_REF][START_REF] Messina | Deciphering the Activation Sequence of Ferrociphenol Anticancer Drug Candidates[END_REF]. The replacement of the ferrocene group by a ruthenocene was recently reported to be of interest for anti-cancer activity [START_REF] Pigeon | Selective Estrogen Receptor Modulators in the Ruthenocene Series. Synthesis and Biological Behavior[END_REF][START_REF] Hillard | Organometallic diphenols: The importance of the organometallic moiety on the expression of a cytotoxic effect on breast cancer cells[END_REF][START_REF] Lee | Oxidative Sequence of a Ruthenocene-Based Anticancer Drug Candidate in a Basic Environment[END_REF]. More recently, several original osmocenebased tamoxifen derivatives have also been prepared and tested in-vitro against breast cancer cells [START_REF] Lee | Synthesis, Characterization, and Biological Properties of Osmium-Based Tamoxifen Derivatives -Comparison with Their Homologues in the Iron and Ruthenium Series[END_REF][START_REF] Scalon | Osmocenyl-tamoxifen derivatives target the thioredoxin system leading to a redox imbalance in Jurkat cells[END_REF][START_REF] Scalon | Tamoxifen-like metallocifens target the thioredoxin system determining mitochondrial impairment leading to apoptosis in Jurkat cells[END_REF]. Interestingly, osmocifen derivatives exhibit similar biological activity than ruthenocifen analogs against MDA-MB231 a hormone-independent breast-cancer cell line (IC 50 : 34 and 29.7 µM for the ruthenociphenol and osmociphenol derivatives, respectively. See Table 1) [START_REF] Jaouen | Ferrocifen type anti cancer drugs[END_REF][START_REF] Lee | Synthesis, Characterization, and Biological Properties of Osmium-Based Tamoxifen Derivatives -Comparison with Their Homologues in the Iron and Ruthenium Series[END_REF]. Yet, this does not mean that the oxidative metabolism of both derivatives is the same. On the basis of our electrochemical investigations performed on both ferrocifens and ruthenocifens, the oxidative metabolism of osmocifen complexes is expected to be rooted on the osmocene/ osmocenium (Oc/Oc + ) electron transfer. The mechanism of osmocene electrochemical oxidation has been far less investigated than ferrocifens [START_REF] Lee | Synthesis, Characterization, and Biological Properties of Osmium-Based Tamoxifen Derivatives -Comparison with Their Homologues in the Iron and Ruthenium Series[END_REF]. In this context, we recently investigated the electrochemical oxidation of osmocene in dichloromethane (DCM) and acetonitrile (MeCN) in the presence of various electrolyte anions ([BF 4 ]  , [PF 6 ]  , and [B(C 6 F 5 ) 4 ]  (TFAB)) [START_REF] Chau | Revisiting the Complex Osmocene Electro-Oxidation Mechanism[END_REF]. The preliminary results showed that the reactivity of the electrogenerated osmocenium cation was dramatically affected by the coordinating properties of the electrolyte anion and the donor strength of the solvent. In the course of our investigation of the reactive sequence of metallocifen drugs, we decided to explore the base-dependent electrochemical behavior of osmociphenol 3 (Scheme 1) in a model environment, i.e. in the presence of a base having a pKa value close to those of peptidic moiety in cells. On the contrary to what was done in the ferrocifen and ruthenocifen series, pyridine could not be used since its nucleophilic character prevails with osmocenium. Instead, the reactivity of the oxidized form of 3 was investigated in the presence of sterically hindered 2,6-lutidine. Under basic conditions, the quinone methide (the final stable complex obtained with ferrocifens) was not formed [START_REF] Amatore | Molecular electrochemistry: A central method to understand the metabolic activation of therapeutic agents. The example of metallocifen anti-cancer drug candidates[END_REF]. Since the anti-cancer properties of ferrocifens were mostly ascribed to this stable oxidized metabolite, we had to revisit the oxidative sequence of osmocifens to explain their anti-proliferative properties. ) was prepared as previously described [START_REF] Chau | Revisiting the Complex Osmocene Electro-Oxidation Mechanism[END_REF] according to the procedure reported by W.E. Geiger [START_REF] Lesuer | Comparison of the Conductivity Properties of the Tetrabutylammonium Salt of Tetrakis(pentafluorophenyl)borate Anion with Those of Traditional Supporting Electrolyte Anions in Nonaqueous Solvents[END_REF].

Scheme1

Cyclic voltammetry

Cyclic voltammetry measurements were performed by using an Autolab potentiostat (PGSTAT 20). they were carried out at room temperature under an argon atmosphere in a three-electrode cell. The reference electrode was an SCE (saturated calomel electrode; Radiometer), which was separated from the solution by a bridge compartment filled with the same solvent/supporting electrolyte solution as used in the cell. The counter electrode was a platinum wire (1 cm length, Goodfellow). The glass-sealed platinum working electrode disk (0.5 mm diameter, Goodfellow) was homemade.

The reproducibility of electrochemical measurements was ensured by careful polishing of the working electrode before each run. Note that in the SCE reference scale the Fc/Fc + electrochemical wave is observed at E 1/2 = 0.40 V vs SCE.

Synthesis of 1,1-Diphenyl-2-osmocenyl-but-1-ene 2

TiCl 4 (0.20 ml, 1.82 mmol) was added to a suspension of zinc powder (238 mg, 3.64 mmol) in THF (7 ml). The mixture was then heated at reflux for 1 h. A second solution was prepared by dissolving benzophenone (97 mg, 0.52 mmol) and propionyl osmocene (100 mg, 0.26 mmol) in THF (2 ml). This latter solution was added to the first solution and the resulting mixture was heated for 2 hours. After cooling to room temperature, the mixture was hydrolyzed with water (40 ml). After extraction with dichloromethane and solvent removal, the crude product was purified by column chromatography. Hexane/diethyl ether (99/1, v/v) was first used as eluent and after with Hexane/diethyl ether (98/2, v/v). 50 mg of compound was isolated (42 % yield, mp 148°C). 1 Osmocifen 3 was synthesized according to a reported process [START_REF] Lee | Synthesis, Characterization, and Biological Properties of Osmium-Based Tamoxifen Derivatives -Comparison with Their Homologues in the Iron and Ruthenium Series[END_REF] based on the McMurry crosscoupling reaction, as was first reported for the ferrocifen series [START_REF] Top | Synthesis, Biochemical Properties and Molecular Modelling Studies of Organometallic Specific Estrogen Receptor Modulators (SERMs), the Ferrocifens and Hydroxyferrocifens: Evidence for an Antiproliferative Effect of Hydroxyferrocifens on both Hormone-Dependent and Hormone-Independent Breast Cancer Cell Lines[END_REF][START_REF] Jaouen | The First Organometallic Antioestrogens and Their Antiproliferative Effects[END_REF].

3. Results and Discussion.

Lutidine vs. pyridine as a base model

Recently, we showed that the stability of the electrogenerated osmocenium cation 1 + was dramatically affected by the coordinating properties of the electrolyte anion and/or the donor strength of the solvent [START_REF] Chau | Revisiting the Complex Osmocene Electro-Oxidation Mechanism[END_REF]. The cyclic voltammogram of 1 obtained in acetonitrile or in the presence of [BF 4 ]  as the supporting electrolyte anion, led to irreversible oxidation waves in both cases (Figure 1). Conversely, in dichloromethane (DCM), a low-donor solvent and in the presence of [B(C 6 F 5 ) 4 ]  (=[TFAB]) a weakly coordinating supporting electrolyte anion, 1 + was stable on the cyclic voltammetry timescale in agreement with a full reversible wave (Figure 1A, blue curve). Accordingly, we decided to investigate the reactivity of osmium-based tamoxifen derivatives (= "osmocifens") in the same solvent/electrolyte couple, i.e. DCM/[TBA][TFAB]. Conditions: studies performed at a Pt electrode, 0.5 mm in diameter; scan rate 200 mV/s. Adapted from [START_REF] Chau | Revisiting the Complex Osmocene Electro-Oxidation Mechanism[END_REF].

So far, mechanistic investigations involving ferrocene-based tamoxifen derivatives (= "ferrocifens") were performed in the presence of pyridine as base model. Unfortunately, pyridine could not be used to investigate osmocifens due to its nucleophilicity towards oxidized osmocenium Os + . Indeed, and as shown in Figure 2A, the addition of pyridine drastically modified the cyclic voltammogram of osmocene [START_REF] Ndagi | Metal complexes in cancer therapy -an update from drug design perspective[END_REF]. The oxidation wave of 1 became fully irreversible and its peak current intensity doubled in the presence of excess pyridine, suggesting the occurrence of an ECE mechanism. A similar behavior was observed for ruthenocene [START_REF] Lee | Oxidative Sequence of a Ruthenocene-Based Anticancer Drug Candidate in a Basic Environment[END_REF] and was interpreted, considering the reactivity of the electrogenerated 17-electron species towards pyridine, to give the corresponding (Cp 2 Os-Py) + complex, which is immediately oxidized at a similar potential value into the 18-electron dication (Cp 2 Os-Py) 2+ . Accordingly, the susceptibility of (Cp 2 Os) + toward nucleophilic attack can be ascribed to the large ring-ring separation of Cps. Indeed the ring-ring distances for Cp 2 M, where M = Fe, Ru, and Os, were reported as being 3.32 [START_REF] Dunitz | The crystal structure of ferrocene[END_REF], 3.68 [START_REF] Hargrove | The crystal structure of ruthenocene[END_REF], and 3.71 Å [START_REF] Jellinek | Die Struktur des Osmocens[END_REF], respectively.

Lutidine, sterically hindered and expected to preclude the attack of the metal center, was therefore preferred as the base model (pKa = 6.65 vs. 5.22 for pyridine). As shown in Figure 2B (black curve), the cyclic voltammogram of 1 remained fully reversible in the presence of lutidine even used in excess. Under these conditions, and as expected, the addition of low amounts of pyridine was enough to make the cyclic voltammogram irreversible (Figure 2B, red and blue curves).

Electrochemical behavior of 3 in the absence and the presence of lutidine

In the absence of lutidine

In DCM/[TBA][TFAB] and without any added base, the cyclic voltammogram of 3 exhibited an oxidation wave O3 which was irreversible at low scan rates. Under the same conditions, a fully This result differs from what was observed for ferrocifens (E° = 0.39 V [START_REF] Messina | Deciphering the Activation Sequence of Ferrociphenol Anticancer Drug Candidates[END_REF]) and ruthenocifens (E° = 0.76 V [START_REF] Lee | Oxidative Sequence of a Ruthenocene-Based Anticancer Drug Candidate in a Basic Environment[END_REF]) where reversible oxidation waves were obtained in the absence of base [START_REF] Messina | Deciphering the Activation Sequence of Ferrociphenol Anticancer Drug Candidates[END_REF][START_REF] Lee | Oxidative Sequence of a Ruthenocene-Based Anticancer Drug Candidate in a Basic Environment[END_REF]. This suggests that the acidity of the phenol group depends on nature of the metal, the acidity increasing in the order Fe < Ru < Os. By comparison to our previous investigations on iron compounds [START_REF] Tan | Ferrocenyl catechols: synthesis, oxidation chemistry and anti-proliferative effects on MDA-MB-231 breast cancer cells[END_REF][START_REF] Messina | Deciphering the Activation Sequence of Ferrociphenol Anticancer Drug Candidates[END_REF], the phenol on 3 could undergo spontaneous deprotonation after oxidation followed by an intramolecular electron transfer between phenolate and the electrogenerated osmocenium cation through the -conjugated system. Interestingly, the oxidation process of 3 was reversible at high scan rate (Figure 3C). Considering that the intramolecular electron transfer between electrogenerated Os + and the hydroxyl group OH (not discriminated and referred to as 3 + in Scheme 2) is too fast to be kinetically frozen at 250 V/s [START_REF] Amatore | Ferrocenyl oligo(phenylene-vinylene) thiols for the construction of self-assembled monolayers[END_REF], it can be assessed that the irreversibility arises from the subsequent deprotonation of the phenol

I / µA E / V vs. SCE A C B O2 R2 O3 O3 [R3] R3 group (3 + → 3 • in Scheme 2)
. Therefore, the oxidation of 3 yields the corresponding phenoxy radical 3  , even in the absence of base.

Scheme 2. Detailed oxidation sequence of 3 (Oc-OH) in the absence and the presence of a nonnucleophilic base such as lutidine.

In the presence of lutidine

Upon addition of increasing amounts of 2,6-lutidine, the intensity of the irreversible oxidation wave of 3 increased until reaching twice its initial peak current intensity and shifted towards less positive potential values at 200 mV/s (Figure 4A). These changes were less dramatic at higher scan rate (Figure 4B). As for ferrocifens [START_REF] Amatore | Molecular electrochemistry: A central method to understand the metabolic activation of therapeutic agents. The example of metallocifen anti-cancer drug candidates[END_REF], the presence of a base apparently allowed a 2-electron oxidation of 3. However, and contrary to the results reported on ferrocifens, no additional oxidation peak ascribed to a stable quinone methide (3c in Scheme 2) was observed.

These results led us to analyze by cyclic voltammetry an authentic sample of quinone methide 3c prepared by chemical oxidation with Ag 2 O. As shown in Figure 5, the cyclic voltammogram of 3c exhibited an irreversible ill-defined oxidation wave at low scan rate (200 mV/s). Conversely, a welldefined reversible wave was obtained at high scan rate (250 V/s) indicating that the oxidized species generated from 3c was stable only over few milliseconds. Furthermore and as already established with the ruthenium derivative, 3c appeared to be unstable in basic media. As shown in Figure 5, the initial 3c oxidation wave observed at low scan rate disappeared to the benefit of a new one located at a more positive potential value in the presence of lutidine. As observed with ruthenocifens, this new oxidation wave could be assigned to the oxidation of a cyclic derivative obtained through a base-promoted rearrangement of the 3c [START_REF] Lee | Oxidative Sequence of a Ruthenocene-Based Anticancer Drug Candidate in a Basic Environment[END_REF]. Finally, the electrochemical behavior of 3 revealed a dramatic base dependence: in the absence of lutidine only a monoelectronic oxidation was observed, followed by deprotonation to yield 3 • . When lutidine was present, a 2-electron oxidation was observed, which did not give the quinone methide 3c as the final stable metabolite. This is a striking difference compared to what was observed with ferrocifens (formation of a stable quinone methide) and with ruthenocifens (formation of a transient quinone methide). Accordingly, it was shown that the enzymatic oxidation of 3 did not produce the quinone methide 3c, but the corresponding carbocation 3b + (Scheme 2) [START_REF] Scalon | Osmocenyl-tamoxifen derivatives target the thioredoxin system leading to a redox imbalance in Jurkat cells[END_REF].

Interestingly, it was recently established that osmocenyl-tamoxifen derivatives are prone to target the thioredoxin system [START_REF] Scalon | Osmocenyl-tamoxifen derivatives target the thioredoxin system leading to a redox imbalance in Jurkat cells[END_REF]. More precisely, in vitro assays showed that thioredoxin was inhibited by the carbocation arising from the oxidation of 3 • (3b + in Scheme 2). This result is in agreement with the stabilization sequence of carbocations and radicals in the metallocene triad, in the order Fe < Ru < Os, with osmium being the most stabilized [START_REF] Rybinskaya | On the problem of stabilization of alphacarbocationic centers in metallocene series -related interconversions of permethylated alphametallocenylcarbocations and metallocenium cation-radicals of the iron sub-group[END_REF][START_REF] Rybinskaya | On the problem of the stabilization of alpha-metallocenylcarbocation -synthesis, properties and crystal structure of [C 5 Me 5 OsC 5 Me 4 CH 2 + ]BPh 4 -.CH 2 Cl 2[END_REF]. Actually, in ruthenium compounds, the stabilization of the cation analogous to 3b + results from electron donation of the metal to the Cp ligand -through overlapping of a filled metal d-orbital with the LUMO of the Cp ligand -and this overlapping in turn stabilizes the exocyclic carbon [START_REF] Long | Metallocene: An introduction to sandwich complexes[END_REF]. Such a stabilizing effect could help connecting the oxidative behavior of osmociphenols with their cytotoxic properties.

Conclusions

We have shown that the general oxidation sequence of osmociphenol 3 differs significantly from that of ruthenociphenol. Firstly, 3 underwent spontaneous deprotonation of its phenol function after oxidation. Secondly, the corresponding quinone methide was not the final and stable complex obtained. This feature can be partially explained by the enhanced stability of the intermediate carbocation 3b + . Finally, despite distinct oxidation pathways, the antiproliferative activities of ruthenocifens and osmocifens are similar and lower than that of ferrocifens. As previously discussed [START_REF] Scalon | Tamoxifen-like metallocifens target the thioredoxin system determining mitochondrial impairment leading to apoptosis in Jurkat cells[END_REF], this could be due to the lower oxidation potential value of ferrocene derivatives which could favor in vivo electron transfer [START_REF] Kovacic | Unifying mechanism for anticancer agents involving electron transfer and oxidative stress: Clinical implications[END_REF]. The redox status of these drugs could be investigated in cellulo by using the recently published fluorescence modulation of metallocene-based dyads [START_REF] Čížková | Redox switchable rhodamine-ferrocene dyad: Exploring imaging possibilities in cells[END_REF].

Fig. 1 .

 1 Fig. 1. (A) Cyclic voltammograms of Cp 2 Os (1, 2 mM), in DCM/[TBA][TFAB] (0.1M), in the absence (blue curve) and in the presence of increasing amounts of [TBA][BF 4 ] (0.01 M (red curve) and 0.1 M (green curve) respectively). (B) Cyclic voltammogram of Cp 2 Os (2 mM), in CH 3 CN/[TBA][TFAB] (0.1M).Conditions: studies performed at a Pt electrode, 0.5 mm in diameter; scan rate 200 mV/s. Adapted from[START_REF] Chau | Revisiting the Complex Osmocene Electro-Oxidation Mechanism[END_REF].

Fig. 2 .

 2 Fig. 2. (A) Cyclic voltammograms of Cp 2 Os (1, 2 mM), in the absence (black curve) and in the presence of increasing amounts of pyridine (2, 4, 10, 20 and 40 mM, respectively). (B) Cyclic voltammograms of Cp 2 Os (2 mM), in the presence (black curve) of lutidine (60 mM) then after addition of pyridine (2 mM, red curve and 4 mM, blue curve). Conditions: studies performed in DCM/[TBA][TFAB] (0.1M); Pt electrode, 0.5 mm in diameter; scan rate 200 mV/s.

  was obtained for 2 (E° = 0.71 V) indicating that the irreversibility observed for 3 was related to the presence of the OH group (compare Figures.3A and 3B).

Fig. 3 .

 3 Fig. 3. Cyclic voltammogram of (A) complexes 2 (2 mM) and (B) 3 (2 mM) at 200 mV/s. (C) cyclic voltammogram of complex 3 (2 mM) at 250 V/s. Conditions: studies performed in DCM/[TBA][TFAB] (0.1M); Pt electrode, 0.5 mm in diameter.

Fig. 4 .

 4 Fig. 4. (A) Cyclic voltammogram of complex 3 (2 mM), at 200 mV/s, in the absence (red curve) and the presence of increasing amounts of lutidine (2, 4, and 20 mM respectively). (B) Cyclic voltammogram of complex 3 (2 mM), at 250 V/s, in the absence (red curve) and the presence of lutidine (2 mM). Conditions: studies performed in DCM/[TBA][TFAB] (0.1M); Pt electrode, 0.5 mm in diameter.

Fig. 5 .

 5 Fig. 5. Cyclic voltammogram of the authentic quinone methide of complex 3 (1.5 mM), in DCM/[TBA][TFAB] (0.1M), at 200 mV/s, in the absence (black curve) and in the presence (blue curve) of lutidine (2 mM). Inset: Cyclic voltammogram of the authentic quinone methide of complex 3 obtained at 250 V/s.

  . Chemical Structures of osmocene, osmocifen, and osmociphenol derivatives investigated in this work.

	2. Experimental
	2.1. Chemicals
	Solvents were purchased from Acros Chemicals and used without further purification.
	Bis(cyclopentadienyl)osmium (Osmocene) was from Strem Chemicals and lithium
	tetrakis(pentafluorophenyl)borate was from TCI Chemicals; all other chemicals were purchased from
	other commercial sources (Sigma-Aldrich and Acros) and used as supplied.
	Tetrabutylammonium tetrakis(pentafluorophenyl)borate [n-Bu 4 N][B(C 6 F 5 ) 4 ] ([TBA][TFAB]
	Table1. Summary of the oxidation pathways for 3 (Oc-OH) and the corresponding Ru and Fe
	derivatives.
	Compound	IC 50 (µM) MDA-MB231	Electrochemical oxidation	Enzymatic oxidation; HRP/H 2 O 2	Chemical oxidation (Ag 2 O)
	Oc-OH	34 [20]	-2 e  / -1 H	-2 e  / -1 H [21]	-2 e  / -2 H [20]
	Rc-OH	29.7 [20]	-2 e  / -2 H [19]	nd	-2 e  / -2 H [20]
	Fc-OH	1.5 [8]	-2 e  / -2 H [16]	-2 e  / -2 H [36]	-2 e  / -2 H [16]

Acknowledgements

This work was supported by the CNRS (UMRs 8640 and 8232), the Ecole Normale Supérieure, Sorbonne Université and PSL University and by a Research Scholarship under the NTU-ParisTech joint PhD Programme (for H. Z. S. L.). The authors also thank Prof. Dr. Christian Amatore for fruitful discussions.


Shaghayegh Nasr
email: nasr@ibrc.ir

Matthias Lutz
email: matthias.lutz@uni-tuebingen.de

Mohammad Ali Amoozegar
email: amoozegar@ut.ac.ir

Véronique Eparvier
email: veronique.eparvier@cnrs.fr

Didier Stien
email: didier.stien@cnrs.fr

Seyed Abolhassan

Shahzadeh Fazeli
email: shfazeli@yahoo.com

Andrey Yurkov
email: andrey.yurkov@dsmz.de

Graphiola fimbriata sp. nov. is the first anamorphic species of Graphiolaceae (Exobasidiales, Basidiomycota)

Keywords: 1 new taxon, Graphiola fimbriata sp. nov., Asexual state, Morphological characterization, Phylogenetic analyses, Yeast

The systematic position of three yeast strains isolated from a plant cell culture, a piece of termite nest, and as a foliar endophyte of Coffea arabica, respectively, is evaluated using morphological, physiological, and phylogenetical analyses. In culture, all three isolates produced white, pale orange to pink colored colonies of cylindrical cells with monopolar budding and pseudohyphae.

Standard phenotypic, biochemical, physiological characterization and phylogenetic analyses of the combined 26S rRNA gene (D1/D2 domains) and ITS region sequences showed the conspecificity of these isolates and suggest their placement within the Exobasidiales (Ustilaginomycotina) as a sister lineage of the sampled and sequenced Graphiola species. Here, we describe this species as Graphiola fimbriata sp. nov. MycoBank MB 825077 (holotype: PC1 T ; ex-type cultures: IBRC-M 30158 T = CBS 13945 T = DSM 104832 T ). This is the first anamorphic saprobic species described in the genus Graphiola.

INTRODUCTION

Fungi with yeast and yeast-like morphology are present within all three main lineages of Basidiomycota, namely the subphyla Agaricomycotina, Pucciniomycotina, and Ustilaginomycotina [START_REF] Sampaio | Diversity, phylogeny and classification of basidiomycetous yeasts[END_REF], Boekhout et al. 2011[START_REF] Kurtzman | Yeasts as Distinct Life Forms of Fungi[END_REF]. Yeast and filamentous taxa are often intermixed as revealed by recent phylogenetic studies (Liu et al. 2015a, 2015b, Wang et al. 2015a, 2015b[START_REF] Wang | Phylogenetic classification of yeasts and related taxa within Pucciniomycotina[END_REF]. With a few exceptions, a transition from filamentous to yeast stage is often associated with the change from a parasitic to saprobic life style [START_REF] Begerow | Parasitism in Yeasts[END_REF]. The subphylum Ustilaginomycotina comprises a highly diverse assemblage of fungi, including teliosporic plant pathogens, non-teliosporic plant pathogens, anamorphic plant pathogens, as well as endophytic, lipophilic, saprobic, and zoophilic yeasts [START_REF] Begerow | The Mycota, systematics and evolution[END_REF]). The orders Malasseziales, Monilielalles, and Violaceomycetales include exclusively yeast species of the genera Malassezia, Moniliella, and Violaceomyces, respectively [START_REF] Wang | Moniliellomycetes and Malasseziomycetes, two new classes in Ustilaginomycotina[END_REF][START_REF] Albu | Violaceomyces palustris gen. et sp. nov. and a new monotypic lineage, Violaceomycetales ord. nov. in Ustilaginomycetes[END_REF], while the remaining yeasts and yeast-like fungi are scattered amongst the remaining orders of Ustilaginomycotina, with the exception of the members of Doassansiales, Tilletiales and Uleiellales for which no yeasts have been discovered so far.

The anamorphic saprobic members of Ustilaginomycotina have previously been classified mainly in the polyphyletic genera Pseudozyma and Tilletiopsis [START_REF] Begerow | Phylogenetic placements of ustilaginomycetous anamorphs as deduced from nuclear LSU rDNA sequences[END_REF][START_REF] Sampaio | Diversity, phylogeny and classification of basidiomycetous yeasts[END_REF], [START_REF] Kurtzman | The yeasts, a taxonomic study, 5th edn[END_REF][START_REF] Kurtzman | Advances in yeast systematics and phylogeny and their use as predictors of biotechnologically important metabolic pathways[END_REF], and later in several monophyletic genera Acaromyces, Farysizyma, Fereydounia, Jaminaea, Meira, and Sympodiomycopsis (Sugiyama et al. 1991[START_REF] Boekhout | Novel anamorphic mite-associated fungi belonging to the Ustilaginomycetes: Meira geulakonigii gen. nov., sp. nov., Meira argovae sp. nov. and Acaromyces ingoldii gen. nov., sp. nov[END_REF][START_REF] Inácio | Farysizyma gen. nov., an anamorphic genus in the Ustilaginales to accommodate three novel epiphytic basidiomycetous yeast species from America, Europe and Asia[END_REF][START_REF] Sipiczki | Jaminaea angkorensis gen. nov., sp. nov., a novel anamorphic fungus containing an S943 nuclear small-subunit rRNA group IB intron represents a basal branch of Microstromatales[END_REF][START_REF] Nasr | Expanding evolutionary diversity in the Ustilaginomycotina: Fereydouniaceae fam. nov. and Fereydounia gen. nov., the first urocystidalean yeast lineage[END_REF]. Sexual and asexual morphs in Ustilaginomycotina were recently grouped together in order to unify the taxonomy of plant parasites and species known only from their yeast states [START_REF] Piątek | A molecular phylogenetic framework for Anthracocystis (Ustilaginales), including five new combinations (inter alia for the asexual Pseudozyma flocculosa), and description of Anthracocystis grodzinskae sp[END_REF], Wang et al. 2015a[START_REF] Kijpornyongpan | Rare or rarely detected? Ceraceosorus guamensis sp. nov.: a second described species of Ceraceosorales and the potential for underdetection of rare lineages with common sampling techniques[END_REF]. As the result, several monophyletic genera (Dirkmeia, Golubevia, Kalmanozyma, and Robbauera) were erected to accommodate species previously classified in the genera Pseudozyma and Tilletiopsis (Wang et al. 2015a). The current classification scheme recommends that each novel lineage not linked with a teleomorphic genus or recognized anamorphic genus should be assigned to a new genus. The subphylum Ustilaginomycotina comprises the least number of yeast and yeast-like species (71) compared to the two other subphyla of Basidiomycota (Wang et al. 2015a). However, studies that involved environmental sequencing suggest a much greater, unexplored diversity of fungi in this group (e.g., [START_REF] Richards | Marine fungi: their ecology and molecular diversity[END_REF][START_REF] Nasanit | The assessment of epiphytic yeast diversity in sugarcane phyllosphere in Thailand by culture-independent method[END_REF][START_REF] Dunthorn | Yeasts dominate soil fungal communities in three lowland Neotropical rainforests[END_REF][START_REF] Jimu | Illumina DNA metabarcoding of Eucalyptus plantation soil reveals the presence of mycorrhizal and pathogenic fungi[END_REF]).

In the course of independent studies two yeast strains have been isolated. The first culture (PC1) was isolated in the Iranian Biological Resource Center (IBRC) from a contaminated plant culture, and the second isolate (SNB-CN72) was obtained from a nest of the termite species Nasutitermes corniger harvested in Rémire-Montjoly, French Guiana. Phylogenetic analyses of the ITS region and the D1/D2 domains of the LSU rRNA gene placed the strains within Graphiolaceae (Exobasidiomycetes, Exobasidiales) close to plant parasites of the genus Graphiola, and revealed relatedness to a third yeast isolate (IBL 03150) reported by [START_REF] Posada | Inoculation of coffee plants with the fungal entomopathogen Beauveria bassiana (Ascomycota: Hypocreales)[END_REF] and deposited in the CBS yeast collection of the Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands. Species in the plant pathogenic genus Graphiola are traditionally circumscribed based on their host spectrum and symptoms, and nucleotide sequence data for these fungi are rare (Wang et al. 2015a).

The aim of the study was to characterise the yeast-like isolates and determine their phylogenetic placement, incorporating morphological, physiological, and molecular data yet available for members of the genus Graphiola.

MATERIALS AND METHODS

Sample collection and isolation

A plant cell culture of an unidentified plant species mixed with a yeast strain was obtained from the plant bank section of the Iranian Biological Resource Center (IBRC) in 2013 (strain designation: PC1). Another strain was isolated from a piece of Nasutitermes corniger nest collected in Rémire-Montjoly, (Cayenne, French Guiana) in July 2011 (strain designation: SNB-CN72; [START_REF] Nirma | Antifungal agents from Pseudallescheria boydii SNB-CN73 isolated from a Nasutitermes sp. termite[END_REF][START_REF] Nirma | Antibacterial ilicicolinic Acids C and D and ilicicolinal from Neonectria discophora SNB-CN63 isolated from a termite nest[END_REF]. A third strain was obtained from the CBS yeast collection of the Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands (original strain designation: IBL 03150). It was isolated as foliar endophyte of Coffea Arabica [START_REF] Posada | Inoculation of coffee plants with the fungal entomopathogen Beauveria bassiana (Ascomycota: Hypocreales)[END_REF].

Designations and GenBank accession numbers of the yeast strains used in this study are given in Table 1.

Cultures were maintained on YPG agar medium (0.5% yeast extract, 1% peptone, 2% glucose, 2% agar, w/v) at 25 °C during experiments.

To expand the ITS sequence sampling for molecular phylogenetic analyses a specimen of the plant parasitic genus Graphiola was additionally used and newly sequenced: Graphiola phoenicis (Moug. ex Fr.) Poit. on Phoenix reclinata Jacq., South Africa, Kwa Zulu-Natal, Durban, Yellowwood Park, Kenneth Stainbank Nature Reserve, 14 Feb. 2012, leg. A.R. Wood 883, KR-M-0042315.

Morphological examination

Colony morphology images were taken using a stereo microscope coupled with the Nikon zoom digital camera. In addition, scanning electron microscopy was performed on the isolate using a VEGA3-TESCAN SEM instrument [START_REF] Van Wyk | Ultrastructure of ascus arrangement and ascospore development in Ophiostoma seticolle[END_REF]. Briefly, the cells were fixed in 3.0%, 0.1 M, pH 7.0 sodium phosphate-buffered glutaraldehyde for 3 h at room temperature, followed by 1 h fixation in 2% osmium tetroxide. The cells were dehydrated by increasing ethanol concentrations (30%, 50%, 70%, 90%, and 96%) for 30 min and two 30 min washes in 100% ethanol. The standard characterization of the yeast isolates was performed according to methods described earlier [START_REF] Barnett | Yeasts: Characteristics and Identification[END_REF][START_REF] Kurtzman | The yeasts, a taxonomic study, 5th edn[END_REF]. Assimilation of carbon and nitrogen sources was carried out on solid and in liquid media, respectively.

DNA extraction, PCR, and sequencing

Nuclear DNA was extracted by the method of Hanna & Xio (2006). For the Graphiola phoenicis specimen, the genomic DNA was isolated directly from the herbarium specimen. The 5'-end (D1/D2 region) of the nuclear large subunit ribosomal DNA (LSU) and the ITS 1 and ITS 2 regions of the nuclear rDNA including the 5.8S rDNA (ITS) were amplified and sequenced using primer pairs NL1 (5'GCATATCAATAAGCGGAGGAAAAG-3') and NL4 (5'-GGTCCGTGTTTCAAGACGG3') [START_REF] Kurtzman | Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences[END_REF] andITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') (White et al. 1990), respectively. DNA sequences determined for this study were deposited in GenBank (accession numbers are given in Figs 1 & 2 and in Table 1. Additional sequences of Graphiola cylindrica and G. phoenicis were obtained from GenBank and the public catalogue of the NITE Biological Resource Center collection (NBRC), Japan.

Phylogenetic analyses

To elucidate the phylogenetic position of the isolated strains their sequences were analysed within a LSU and a concatenated ITS + LSU dataset. Since preliminary analyses and Blast searches [START_REF] Altschul | Gapped BLAST and PSI-BLAST: a new generation of protein database search programs[END_REF]) revealed an affinity of the stains to the order Exobasidiales (class Exobasidiomycetes), the LSU dataset was reduced to members of the Exobasidiales and some representatives of the Ustilaginomycetes were used as an outgroup. Isolates and clones, for which only ITS sequences were available, were analysed in the LSU + ITS dataset. If present in GenBank sequences of the respective type species were used. Additionally sequences of all available brachybasidiaceous species and all available sequences clustering within the Graphiolaceae including sequences from the Biological Resource Center, NITE, Japan were added.

GenBank accession numbers of the sequences used for both the LSU and ITS+LSU dataset [START_REF] Begerow | Phylogenetic studies on nuclear LSU rDNA sequences of smut fungi and related taxa[END_REF], 2001[START_REF] Begerow | The Exobasidiales: An evolutionary hypothesis[END_REF][START_REF] Guo | Detection and taxonomic placement of endophytic fungi within frond tissues of Livistona chinensis based on rDNA sequences[END_REF][START_REF] Boekhout | Novel anamorphic mite-associated fungi belonging to the Ustilaginomycetes: Meira geulakonigii gen. nov., sp. nov., Meira argovae sp. nov. and Acaromyces ingoldii gen. nov., sp. nov[END_REF][START_REF] Castlebury | Phylogenetic analysis of Tilletia and allied genera in order Tilletiales (Ustilaginomycetes; Exobasidiomycetidae) based on large subunit nuclear rDNA sequences[END_REF][START_REF] Stoll | Molecular phylogeny of Ustilago, Sporisorium, and related taxa based on combined analyses of rDNA sequences[END_REF], Yasuda et al. 2005, 2006[START_REF] Posada | Inoculation of coffee plants with the fungal entomopathogen Beauveria bassiana (Ascomycota: Hypocreales)[END_REF][START_REF] Tanaka | Isolation of basidiomycetous anamorphic yeast-like fungus Meira argovae found on Japanese bamboo[END_REF][START_REF] Piepenbring | New records, host plants, morphological and molecular data of Exobasidiales (Basidiomycota) from Panama[END_REF], 2012, Yuan et al. 2011[START_REF] Jusino | Heart rot hotel: fungal communities in red-cockaded woodpecker excavations[END_REF][START_REF] Urbina | DNA-metabarcoding uncovers the diversity of soil-inhabiting fungi in the tropical island of Puerto Rico[END_REF] Sequence alignment was obtained independently for both the LSU dataset and the ITS and LSU part of the ITS+LSU dataset using MAFFT 7.313 [START_REF] Katoh | MAFFT multiple sequence alignment software version 7: improvements in performance and usability[END_REF] using the L-INS-i option. To obtain reproducible results, manipulation of the alignments by hand as well as manual exclusion of ambiguous sites were avoided as suggested by [START_REF] Gatesy | Alignment-ambiguous nucleotide sites and the exclusion of systematic data[END_REF] and [START_REF] Giribet | On gaps[END_REF], respectively. Instead, highly divergent portions of the alignments were omitted using GBlocks 0.91b [START_REF] Castresana | Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis[END_REF] with the following options for the LSU dataset: For the LSU dataset trees were rooted with the ustilaginomycetous species Urocystis ficariae and Ustilago hordei, for the ITS+LSU dataset trees were rooted with the brachybasidiaceous species Dicellomyces gloeosporus and Meira geulakonigii.

Metabolite profiling analysis was carried out for the three yeast strains and the results are presented as supplementary data.

RESULTS

Morphological examination

Morphology of the strains IBL 03150, PC1, and SNB-CN72 showed no significant differences, results are included in the species description.

Metabolite profiling with UHPLC-MS was carried out for the three yeast strains and the results are provided in supplementary data. In the absence of metabolic data from closely related species, the analysis of UHPLC-MS profiles was restricted to the tree strains. We observed that the culture medium impacted more significantly on the profiles than the nature of the strain (Supplementary data). Hierarchical clustering analyses (HCA) conducted on the 3 (N) × 29 (X) matrix of merged profiles showed that strains PC1 and IBL 03150 are more alike than SNB-CN72 (Supplementary data).

Phylogenetic analyses

The different runs of the BA that were performed and the ML analyses yielded consistent topologies. To illustrate the results the consensus trees of one run of the BA of the LSU and the concatenated ITS+LSU dataset are presented (Figs 1 &2). Using the ustilaginomycetous species as outgroup in the LSU analysis, the clades in the phylogenetic tree were congruent to the families discussed in [START_REF] Begerow | The Mycota, systematics and evolution[END_REF]. In all analyses the sequences of the yeast isolates clustered within the Exobasidiales together with the sequence MF334501 from an uncultured fungus clone forming the sister lineage of the sampled Graphiola spp. of which the G. phoenicis cluster included several sequences of yeast isolates and uncultured fungus clones, respectively.

The clade comprising Graphiola cylindrica, G. geonomae, G. phoenicis, and the yeast isolates received good statistical support (BA: 100%, ML: 93%). The yeast isolates branched first in the Graphiola clade and were placed sister to G. cylindrica, G. geonomae, and G. phoenicis.

Combined ITS+LSU analyses (Fig. 2) revealed the same groups and relations including more sequences from Graphiola spp. as well as from the studied yeast isolates and uncultured fungus clones. The three strains shared identical LSU sequences and showed 5 variable positions (2 substitutions and 3 indels) in the ITS region. Description: After three days on YPG agar at 25 ºC, the cells are cylindrical, 1-2.5 × 4-8.5 µm, and occur single or in small chains. Budding is monopolar. After five days, the colony is white, convex, and the margin is filiform. Pellicles are formed on liquid media. After one week at 25 ºC on MEA and Cornmeal agar, the slide culture undifferentiated pseudohyphae are formed.

TAXONOMY

Fermentation is negative. The following compounds are assimilated: glucose, sucrose, maltose, trehalose, melezitose, D-xylose, raffinose, L-arabinose, D-mannitol, myo-inositol, D-ribose and arbutin; assimilation of salicin, glycerol and cellobiose is weak. No growth occurs on galactose, lactose, L-rhamnose, melibiose, inulin, D-arabinose, soluble starch, ethanol, methanol, DLlactate, succinate, citrate and n-hexadecane. Sodium nitrate, potassium nitrate, L-lysine, Ethylamine hydrochloride (variable) and cadaverine dihydrochloride (variable) are assimilated.

No growth occurs on glucosamine, imidazole, creatine or creatinine. Growth in vitamin-free medium is positive. Growth at 15 °C, 25 °C, 30 °C, and 34 °C is positive but not at 4 °C, 37 °C and 40 °C. Growth occurs on YM agar supplemented with 5% (w/v) NaCl, 10 % (w/v) NaCl but not on YM agar with 16% (w/v) NaCl. Starch-like compounds are not produced. No growth occurs on media supplemented with 0.01% and 0.1% cycloheximide, and 1% acetic acid. The diazonium blue B reaction is weakly positive. Urease activity is positive. We also emend the description of the genus Graphiola to allow the inclusion of species known from saprobic state only (see Taxonomy section).

The ecology and distribution range of the novel species is unknown. All described Graphiola species are plant pathogens on palms (Arecaceae). We hypothesize that the novel species is probably associated with plants, as also suggested by the origin of the studied isolates and closely related culture Exobasidiomycetidae sp. AUMC 10262 (KX011608) from the palm weevil Rhynchophorus ferrugineus that infests palms, which is a primary host of Graphiola.

Although the novel species is known from its asexual state, it is most likely that it has a host like all other Graphiola species. Widespread transcontinental transfer of plants results in a global dissemination of plant pathogens and crop pests [START_REF] Bebber | Range-expanding pests and pathogens in a warming world[END_REF][START_REF] Hurley | Increasing numbers and intercontinental spread of invasive insects on eucalypts[END_REF]). Among them, fungal pathogens currently lead the global invasion of agriculture, despite their more restricted host range [START_REF] Bebber | The global spread of crop pests and pathogens[END_REF][START_REF] Wingfield | The unified framework for biological invasions: a forest fungal pathogen perspective[END_REF]). In our study, a new species of the plant pathogenic genus Graphiola was identified over a broad geographic range that includes the Old World (Iran and Egypt), tropical Asia and the Americas (French Guiana and USA). It is very likely that the ongoing transport of plants and pests promoted dissemination of Graphiola fimbriata between continents, localities, and habitats.

It cannot be precluded that Graphiola fimbriata represents the anamorphic stage of a palm pathogen of which no sequence data are available. Of the 12 Graphiola species described [START_REF] Piepenbring | Revision of the genus Graphiola (Exobasidiales, Basidiomycota)[END_REF]) only three are represented by sequence data in GenBank. Moreover species diversity seems underestimated in the genus Graphiola. Assuming that its host specificity developed to the same degree shown for other plant pathogens [START_REF] Piątek | Cryptic diversity in the Antherospora vaillantii complex on Muscari species[END_REF][START_REF] Savchenko | Revision of Entyloma (Entylomatales, Exobasidiomycetes) on Eryngium[END_REF][START_REF] Vasighzadeh | Discovery of Thecaphora schwarzmaniana on Rheum ribes in Iran and Turkey: implications for the diversity and phylogeny of leaf smuts on rhubarbs[END_REF][START_REF] Choi | Host jumps and radiation, not co-divergence drives diversification of obligate pathogens. A case study in downy mildews and Asteraceae[END_REF][START_REF] Scholler | A taxonomic and phylogenetic study of the Golovinomyces biocellatus complex (Erysiphales, Ascomycota) using asexual state morphology and rDNA sequence data[END_REF], the number of 12 Graphiola species on 38 palm species from 21 plant genera (Farr & Rossman n.d., Piepenbring et al. 2012) is lower than in other known plant-parasite systems. Intensive sampling and molecular analyses were used successfully in other groups to link anamorph and teleomorph stages [START_REF] Sampaio | Diversity, phylogeny and classification of basidiomycetous yeasts[END_REF][START_REF] Boekhout | Extensive colonization of apples by smut anamorphs causes a new postharvest disorder[END_REF][START_REF] Inácio | Farysizyma gen. nov., an anamorphic genus in the Ustilaginales to accommodate three novel epiphytic basidiomycetous yeast species from America, Europe and Asia[END_REF], Wang et al. 2015a[START_REF] Kruse | Asexual and sexual morphs of Moesziomyces revisited[END_REF][START_REF] Piątek | Pattersoniomyces tillandsiae gen. et comb. nov.: linking sexual and asexual morphs of the only known smut fungus associated with Bromeliaceae[END_REF]. However, both sexual and asexual species are yet strongly under sampled in many lineages (Liu et al. 2015a, Wang et al. 2015a, Wang et al. 2015b). Although these studies unified systematics of plant parasites and yeast taxa, the results strongly suggest that the available genetic data is insufficient to resolve anamorph-teleomorph relationships in sister lineages (e.g., [START_REF] Boekhout | Extensive colonization of apples by smut anamorphs causes a new postharvest disorder[END_REF][START_REF] Kruse | Asexual and sexual morphs of Moesziomyces revisited[END_REF].

The lack of sequence data of sexual species also complicates biodiversity assessments and new species discovery. With this study we provide the first overview on Graphiola diversity from sequence data available in public databases. We propose new species Graphiola fimbriata to accommodate yeasts from several localities and habitats. Our results show that sequences related to Graphiola species are rapidly accumulating in public databases as taxonomically unassigned isolates and clones. In our opinion, it is important to describe Graphiola fimbriata to provide a proper name for these isolates to communicate it in the future. We agree that taxonomic redundancy cannot be ruled out in the genus Graphiola considering a very few sequenced species. Rediscovery and description of already known species as new may happen when stable and informative morphological characters for species differentiation are limited, both in plant material and in culture. Thus, future studies should be addressed on resampling and sequencing of new and already existing material in order to better understand genetic diversity and taxonomy in this group of fungi.

There is little known about physiological properties of dimorphic plant parasites, whereas fungi traditionally recognized as "yeasts" were intensively studied in this respect. Undersampling and the lack of genetic (including housekeeping genes) and physiological data do not allow delimitation of many genera (e.g., Wang et al. 2015a). In this study, we decided not to propose a new genus for the novel fungus, but to accommodate it in the genus Graphiola in order to reduce the taxonomic complexity in this group. Neither phylogenetic analyses nor the analysis of physiological data revealed a basis to distinguish the new species from other Graphiola species.

Therefore, we modified the description of the genus Graphiola to include information about its asexual form considering own results and previous reports by Tubaki & Yokohama (1971) and [START_REF] Oberwinkler | Graphiolales: Basidiomycetes parasitic on palms[END_REF]. Despite limited genetic data, our study identified several fungal isolates and metabarcoding clones as Graphiola fimbriata and G. phoenicis (Figs. 1 &2). These fungi were found in Indomalayan and Neotropical realms (e.g., [START_REF] Takashima | Taxonomic richness of yeasts in Japan within subtropical and cool temperate areas[END_REF][START_REF] Urbina | DNA-metabarcoding uncovers the diversity of soil-inhabiting fungi in the tropical island of Puerto Rico[END_REF], which are the regions where palms are widely distributed [START_REF] Kissling | Cenozoic imprints on the phylogenetic structure of palm species assemblages worldwide[END_REF].

White TJ, Bruns TD, Lee S, Taylor JW (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White

  are given in Figs 1 & 2.

'

  Minimum Number of Sequences for a Conserved Position': 20, 'Minimum Number of Sequences for a Flank Position': 20, 'Maximum Number of Contiguous Non-conserved Positions': 8, 'Minimum Length of a Block': 5, and 'Allowed Gap Positions' to 'With half', for the ITS part of the ITS+LSU dataset: 11/11/8/5/'With half' and for the LSU part of the ITS+LSU dataset: 12/12/8/5/'With half'. After alignment the ITS and LSU part of the ITS+LSU dataset were concatenated. The resulting alignments [LSU dataset: new number of positions: 591 (32% of the original 1801 positions), number of variable sites: 279; ITS+LSU dataset: new number of positions: 1121 (34% of the original 3264 positions), number of variable sites: 468] were used for phylogenetic analyses using a Maximum Likelihood (ML) and a Bayesian Approach (BA) following Vasighzadeh et al. (2014).

Graphiola

  Poit., Ann. Sci. Nat. (Paris): 473 (1824) emend. "S. Nasr, M. Lutz, D. Stien & A.Yurkov" According to the current genus concept of Graphiola[START_REF] Piepenbring | Revision of the genus Graphiola (Exobasidiales, Basidiomycota)[END_REF], the genus comprises plant pathogens on palms (Arecaceae).[START_REF] Tubaki | Cultural aspects of Graphiola phoenicis[END_REF] Yokohama (1971) and[START_REF] Oberwinkler | Graphiolales: Basidiomycetes parasitic on palms[END_REF] obtained and studied Graphiola phoenicis in culture. They provided also the diagnosis of this species, including morphological and physiological properties observed in culture. Emendation of the diagnosis of the genus Graphiola is proposed to allow classification of asexual, known only from a saprobic state yeast species in the genus.Sexual reproduction is observed in some species. Fungi are dimorphic with a filamentous sexual form parasitizing on plants and free-living saprobic yeast states. On malt extract, colonies are white, pale orange to pink in color. Budding cells are present in culture. Colonies are white, pale orange to pink in color. Ballistoconidia are not produced. Pseudohyphae may be present in cultures. Fermentation is absent. DBB reaction is positive. Urease activity is positive.Graphiola fimbriataS. Nasr, M. Lutz, D. Stien & A. Yurkov, sp. nov. (Fig. 3) MycoBank MB 825077 Etymology: Referring to the margin of the colonies.

  Molecular characteristics: nucleotide sequences of ITS and LSU (D1/D2 domains) rDNA sequences are deposited in GenBank (Table 1.) Deposits: holotype, PC1 T (= IBRC-M 30158 T ) isolated as a contaminant of an unidentified plant culture, preserved in a metabolically inactive state at the Iranian Biological Resource Centre, Teheran (holotype), Iran. Ex-type cultures are deposited in the CBS yeast collection of the Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands (= CBS 13945 T ) and in the German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany (DSM 104832 T ). Strain examined: PC1 T , SNB-CN72 (= DSM 104833), IBL 03150 DISCUSSION Morphological and physiological characteristics of the three yeast isolates examined in this study are uniform, differences in ITS and LSU sequences are low all that suggesting assignment to a single species. Molecular phylogenetic analyses reveal the three yeast isolates within the Graphiolaceae as a sister lineage of Graphiola cylindrica, G. geonomae, and G. phoenicis. Thus, we propose Graphiola fimbriata sp. nov. to accommodate the three yeast strains. On the basis of the pair-wise sequence similarities (ITS and D1/D2 regions) and subsequence phylogenetic analyses (Figs 1 & 2), the novel species differs from all other Graphiola species.

Fig. 1 .

 1 Fig. 1. Bayesian inference of phylogenetic relationships within the sampled Exobasidiales:

Fig. 2 .

 2 Fig. 2. Bayesian inference of phylogenetic relationships within the sampled Exobasidiales:

Fig. 3 .

 3 Fig. 3. Scanning electron micrographs of the strain IBRC-M 30158 T showing budding cells and

Table 1 .

 1 TJ (eds) PCR Protocols: a guide to methods and applications. Academic Press, San Yamagishi D, Akamatsu H, Izawa H,Kodama M, Otani H (2006). Meira nashicola sp. nov., a novel basidiomycetous, anamorphic yeastlike fungus isolated from Japanese pear fruit with reddish stain. Mycoscience 47: 36-40 Yuan ZL, Rao LB, Chen YC, Zhang CL, Wu YG (2011). From pattern to process: species and functional diversity in fungal endophytes of Abies beshanzuensis. Fungal Biol 115: 197-Designations and GenBank accession numbers of the yeast strains used in this study.

	Diego, pp 315-322
	Yasuda F, Yamagishi D, Akamatsu H, Izawa H, Kodama M, Otani H (2005). Fruit stain of
	Japanese pear caused by basidiomycetous, yeast-like fungi, Acaromyces ingoldii and
	Meira sp. Jpn J Phytopathol 71: 156-165
	Yasuda F, 213
	TABLES
		Strain designation	GenBank accession numbers
	Yeast strains
		IBRC-M	CBS	LSU	ITS
	PC1	30158 T	13945 T	KM403453	KM403454
	SNB-CN72	30163	-	KP229360	KJ023736
	IBL 03150	-	14052	KP308195	DQ682574

ACKNOWLEDGEMENTS

This work has benefited from an "Investissement d'Avenir" grant managed by the Agence Nationale de la Recherche (CEBA, ref ANR-10-LABX-0025). The authors are grateful to C. Nirma for isolating SNB-CN72 and to R. Constantino for identification of the host termite. The authors gratefully acknowledge financial support from the Iranian Biological Resource Centre (IBRC), ACECR. Authors are grateful to Prof. M. Catherine Aime (Purdue University, USA) and Dr. Marizeth Groenewald (Westerdijk Fungal Biodiversity Institute, The Netherlands) for granting access to the strain IBL 03150.

CONFLICTS OF INTEREST

The authors declare that there are no conflicts of interest.


Claudia S Bianchini

Léa Chèvrefils
email: leachevrefils@gmail.com

Claire Danet
email: claire.danet@gmail.com

Patrick Doan

Morgane Rébulard
email: morgane.rebulard@gmail.com

Adrien Contesse
email: adriencontesse@gmail.com

Dominique Boutet
email: dominique_jean.boutet@orange.fr

Coding Movement in Sign Languages: the Typannot Approach

Keywords: Sign Language, Transcription System, Handshape, Movement, Initial Localization, Intrinsic Frame of Reference, Motion Capture, User Interface ACM Reference format:

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

INTRODUCTION

The analysis of any language requires the implementation of a system of graphical representation to set the characteristics that make them analyzable and comparable. Linguistics started its development through written materials [START_REF] Goody | La Raison graphique: la domestication de la pensée sauvage. Collection "Le sens commun[END_REF][START_REF] Auroux | La révolution technologique de la grammatisation: introduction à l'histoire des sciences du langage[END_REF], and only in the last few decades it has been involved with spoken languages. Even the development of new multimedia technologies has not changed the situation: even if we can record sound and gestures associated with any language without problems, organizing and storing data on the basis of the elements deemed relevant requires a transcription system (TranSys) to pass from a simple collection to a usable corpus of data: i.e., to record is not to categorize.

To make possible accurate analyses and their reading, TranSys must meet 5 main criteria [START_REF] Boutet | Systèmes graphématiques et écritures des langues signées[END_REF]: readability, writability, searchability, genericity, modularity. A TranSys must be able to be written and read by human transcribers but also by the machines that manage the information (readability and writability). On one hand, the system shall allow a detailed and structured representation of languages using a systematic graphematic formula (genericity) and, on the other hand, and shall propose an integration of these minimal components within larger formal structures, allowing synthetic representations (modularity). The data thus transcribed should allow computer queries for fine and distinct characteristics (searchability) while displaying different levels of visual synthesis in order to facilitate their understanding (readability).

For the vocal languages (VLs), these criteria are satisfied by more or less detailed adaptations of existing writing systems, in particular by the Latin alphabet [START_REF] Bigi | Influence de la transcription sur la phonétisation automatique de corpus oraux[END_REF] and by its derivation, the International Phonetic Alphabet (IPA) [START_REF]Handbook of the International Phonetic Association: a guide to the use of the International Phonetic Alphabet[END_REF]. But what about Sign Languages (SLs)?

SLs are languages used by most deaf 1 people to communicate within their community. SLs are produced through the simultaneous movement of several articulators located on the upper limbs (hands, forearms and arms) and the face (eyes and mouth, mainly) of the signer.

Despite various attempts over the centuries [START_REF] Bébian | Mimographie ou essai d'écriture mimique, propre à régulariser le langage des sourds muets[END_REF][START_REF] Déléage | Le geste et l'écriture: langue des signes, amérindiens, logographies[END_REF], SLs have not yet developed a notation system of their own, so there is a lack of a graphic base that can be adapted to create a TranSys. The absence of a TranSys was felt as a problem since the early researches on SLs done by W. Stokoe [START_REF] William | Sign Language structure: an outline of the visual communication systems of the American deaf[END_REF], who for this reason invented a rudimentary TranSys focused on the shapes and movements of the hands, known as the "Stokoe notation".

Given the difficulty of taking into account the formal characteristics of SLs, some researchers have opted for TransSys that encode only the signified of VLs (for example Id-Gloss [START_REF] Johnston | From archive to corpus: transcription and anoatation in the creation of signed language corpora[END_REF]). In contrast, a small number of researchers tried in the '80s to find solutions to this problem, generating various systems, e.g. SignFont [START_REF] Newkirk | SignFont handbook[END_REF], D'Sign [START_REF] Jouison | Ecrits sur la Langue des Signes Française[END_REF], SignWriting (SW) [START_REF] Sutton | Lessons in SignWriting: textbook & workbook. Deaf Action Committee for Sign Writing[END_REF][START_REF] Bianchini | Analyse métalinguistique de l'émergence d'un système d'écriture des Langues des Signes: SignWriting et son application à la Langue des Signes Italienne (LIS)[END_REF], Hamburg Notation System (HamNoSys) [START_REF] Prillwitz | Hamburg notation system for sign languages: an introductory guide[END_REF]: today, only the last two are still in use, SW being aimed more to deaf people and educators, HamNoSys to linguists. SW (Figure 1) is a system based on a set of characters detailing all the components of SLs (manual and non-manual). The characters are arranged in a two-dimensional space that analogically represents the signing space. SW is easily readable by humans, but this does not go together with its very laborious writability, by both hand and computer. Moreover, born outside of the concerns of the world of linguistic research, SW is not easy to search. Finally, its characters are a global representation of a sign parameter, and therefore they are neither generic nor modular.

HamNoSys (Figure 1) is a more modern and detailed version of the Stokoe Notation. It is a linear graphic system that accurately represents the manual components of the SLs. It has been conceived to promote computer writability and searchability, perhaps to the detriment of human readability. The system is partially modular but not generic. This article starts with the description of the HandShapes (HS) parameter to explain the concept of Typannot and its user interface (UI), followed by a presentation of initial localization (LOCini) and movement (MOV) parameters, which obey multiple, intrinsic Frames of Reference (iFoR), allowing to understand the relationship between the position of the limb articulators and the movement. Finally, the article shows how data transcribed using Typannot and its iFoR make it possible to give an answer to these two questions: -how is it possible that MOV in SLs is standardized and simple, while its very principle brings it to continual changes? -how may we determine MOV from its preparation phase?

PRESENTATION OF TYPANNOT

Typannot is a typographic system that offers a description of the SLs forms using both symbolic and explicit visual analogies, thus enabling the advancement of linguistic knowledge [START_REF] Boutet | Systèmes graphématiques et écritures des langues signées[END_REF]. It allows coding different parameters, both manual (HS, LOCini and MOV) and non-manual (mouth action and eye expressions).

It is designed to meet the 5 criteria of a TranSys: readability, writability, searchability, genericity and modularity. The analysis of the HS parameter allows understanding the general framework of the approach, since for all the other components the creation process follows the same steps and generates the same levels of representation (Figure 2).

2.1 Typannot levels of representation: the HandShape example

Graphematic formula

In the first place, it has been necessary to establish a graphematic formula [START_REF] Bianchini | La notation des corpus de Langue des Signes: un défi encore ouvert mais parfois ignoré[END_REF], i.e. an ordered list of features relevant for the description (Figure 2.1).

The HS parameter is the most studied by the SLs linguists [START_REF] William | Sign Language structure: an outline of the visual communication systems of the American deaf[END_REF][START_REF] Boyes-Braem | Preliminaries to a distinctive feature analysis of handshapes in American Sign Language[END_REF][START_REF] Scott | Structures for representing handshape and local movement at the phonemic level[END_REF][START_REF] Ann | On the relation between ease of articulation and frequency of occurrence of handshapes in two sign languages[END_REF]. After evaluating various studies on this subject, it has been chosen to refer to the phonological study of 9 SLs by Eccarius and Brentari [START_REF] Eccarius | Handshape coding made easier: a theoretically based notation for phonological transcription[END_REF], which seems to be quite complete to serve as a descriptive basis for all the HS of the 142 SLs of the world [START_REF] Lewis | Ethnologue: languages of the world[END_REF]. For HS, the graphematic formula is composed by 33 features (Figure 3). It was not the same for the other parameters, where the GestualScript team had to establish the list of the relevant traits by itself.

Generic characters

On a second step, the 33 features of the graphematic formula are translated into 33 generic characters called "primary generics" (G1) (Figure 2.2), that are written in a linear way following strict syntactic rules.

For better readability and faster understanding, those 33 G1 characters can be combined to form generic compound characters ("secondary generics", G2) (Figure 2.2, 3 rd character). Combinations are performed automatically, thanks to the OpenType typographic "ligature" functionality.

For example (Figure 4), the G1 "shape = curved" and the G1 "angle = open" can be combined to form a G2 compound "curved open"; of course, it will then be possible to request both one of the two G1 or the entire G2 (Figure 4). In order to ensure data queries with every operating system and software, the GestualScript team aims to have Typannot recognized by the Unicode Consortium. The set of possible HS being of several millions (although those actually recorded in 9 SL [START_REF] Eccarius | Handshape coding made easier: a theoretically based notation for phonological transcription[END_REF] do not exceed half a thousand), only the 33 G1 characters will be registered in Unicode.

Composed forms

Although using just G1s is sufficient to identify a HS, Typannot allows a composed typographic form for each HS generic transcription. This final level of representation aims at providing a better readability and writability. It integrates a set of typographic components into a single unit (oppose to the linear vector of the generic forms) to build a logographic form. Similarly to the generic characters, composed forms are displayed automatically using the OpenType ligature functionality, replacing an entire line of G1 transcription by a single composed form (Figure 2.3).

Thus, like in the Latin alphabet "a+e→ae", typing several G1 may create alternative level of representations (G2 or composed forms) that are back-decomposable (and therefore searchable).

For the HS, the three levels of representation have been fully realized. A font allowing to visualize both generic and composed characters of each HS is at advanced stage. Moreover, Typannot allows to represent mouth actions, eye expressions and LOCini: for these components, development reached the second level. Conversely, MOV representation is still at the early level, i.e. the availability of a sketch of the graphematic formulas without its development into character sets.

Typannot User Interface (UI)

To enable the use of Typannot in every platform and software, a UI is under development, allowing to write it according to different modalities, according to the users' requirements and skills, thanks to the presence of three complementary sub-interfaces (sUI): -generic sUI (selection of generic characters -G1, G2); -module sUI (selection of "morphological" modules of the specific typographic formula); -gestural sUI (selection through the physical reproduction of the component to be described, thanks to the use of capture devices, i.e. LeapMotion for HS [Figure 5] and Inertial Measurement Unit [IMU] for LOCini).

It is possible to switch from one sUI to another to refine or correct the description, and a signing avatar should allow, at any time, to verify the accuracy of the transcript.

Whatever the sUI used, the UI restores both the generic formula and the specific "morphological" character. Using the Typannot font allows viewing the result in all OpenType supporting programs. Along with the HS, Typannot proposes a new system to transcribe three interdependent parameters: the position and the orientation of the hand, and the posture of the arm. These data are translated through a single notion, i.e. LOCini, a principle to characterize MOV in a novel way.

INITIAL LOCALIZATION (LOCini) AND MOVEMENT (MOV)

The movements of the upper limbs (MOV) are the result of a concatenation of actions of different articulators that act simultaneously: for this reason, MOV is considered the most difficult parameter to describe and, therefore, to represent [START_REF] Goldin | Gesture, sign and language: the coming of age of sign language and gesture studies[END_REF].

In order to successfully overcome the drawbacks of MOV representation, most SLs TranSys focus on describing the trajectory of the hands, annotating trajectory and/or position of the other segments only if it cannot be easily deduced by the information about the hand itself [START_REF] Johnson | A segmental framework for representing signs phonetically[END_REF][START_REF] Wilbur | Contributions of sign language research to gesture understanding: what can multimodal computational systems learn from sign language research[END_REF]. Conversely, the GestualScript team starts on a completely different basis.

The LOCini is classically seen according to an extrinsic, egocentric Frame of Reference (eFoR) in which only the position of the hand is considered, according to a frame centered the speaker (front, back, left, right, top and bottom).

The approach chosen by GestualScript considers that LOCini and MOV are both accountable for the relative positions and movements of each segment (SEG: hand, forearm and arm) and therefore novel iFoR, intrinsic to each SEG and multiplied by all the SEGs, shall constitute the structuring level of the signs [START_REF] Boutet | Structuration physiologique de la gestuelle: modèle et tests[END_REF].

Novel approach for the movement (MOV)

3.1.1 Degrees of Freedom (DoF) and intrinsic Frames of Reference (iFoR) Typannot therefore proposes a novel approach based on taking into account the three SEGs of the upper limbs: arm, forearm and hand. For each of them, the different Degrees of Freedom (DoF) [START_REF] Adalbert | Anatomie fonctionnelle -Tome 1: membre supérieur[END_REF] (Figure 6) are underlined, together with their grades, going from one pole extremity to the other: -arm: flexion/extension (with grades Flex2, Flex1, Flex0, Ext1, Ext2); abduction/adduction (Abd1, Abd2, Abd0, Add1, Add2); internal/external rotation (RInt2, RInt1, RInt0, RExt1, RExt2); -forearm: flexion/extension (Flex2, Flex1, Flex0, Ext1, Ext2); pronation/supination (Pron2, Pron1, Pron0, Supi1, Supi2); -hand: flexion/extension (Flex2, Flex1, Flex0, Ext1, Ext2); abduction/adduction (Abd1, Abd0, Add1, with just 3 grades). To allow the description of DoF, the usual eFoR is not suitable. Therefore, Typannot is based on an iFoR for each SEG: -the shoulder iFoR allows describing MOV and LOCini of the arm; -the arm iFoR allows describing those of the forearm; -the forearm iFoR allows describing those of the hand.

The three iFoR make it possible to precisely locate the position of all the SEGs participating in the MOV. If at first this approach seems more complicated than the eFoR, the iFoR allow many savings on the transcription of the MOV, as shown below.

Initial Location (LOCini)

The second basic premise of Typannot for MOV is that the LOCini, i.e. the stable position assumed by the SEGs of the signer's upper limb(s) before the deployment of MOV, is the key towards a simple description of MOV itself.

In SLs, MOV is a nearly continuous and complex flow that oscillates between a preparation moment (which culminates in a LOCini) and a deployment phase (that is, the MOV itself). However, these displacements are not the result of chance and one can find simple and standardized patterns. This paradox leads to two questions: -how can MOV be standardized and simple, as it changes continuously? -can we, and how, predict MOV from the observation of LOCini?

Before MOV takes place, the signer his/her action for a while: he/she blocks the LOCini, and his/her limbs are positioned to allow deploying MOV whilst minimizing the motor control. It is therefore possible to say that the preparatory phase for LOCini also consists in setting up the SEGs in a balanced and sufficiently stable LOCini to serve as anchor point and as basis for a simple MOV flow (from the point of view of the motion scheme). In other words, in order to reach the LOCini, the signer performs a series of potentially complex actions implementing each SEG, so that the sign MOV can be deployed through a simple motor scheme.

Movement (MOV)

The representation of LOCini renders superfluous the description of the preparation phase, thus strongly reducing the elements to be described in MOV. After registering the LOCini, it is sufficient to consider the DoF on which MOV has repercussions and the type of impulse. Although it often seems visually that several SEGs are in action, it is possible to deduce without much difficulty which is the SEG that "carries" the MOV impulse and how MOV propagates.

An example allows to better understand these last affirmations. The two signs on Table 1

means [THANK YOU] in French SL (LSF).

Table 1. Two signs to say [THANK YOU] in French Sign Language with transcription of initial location (LOCini), movement (MOV) and impulse (differences shown in bold).

LOC ini

Arm Add0 Flex0 Add0 Flex0 Forearm Flex2 RInt2

Flex2 RInt0 Hand Supi1 Flex0 Add1 Supi1 Flex1 Add1 MOV However, looking at their transcript it is possible to note that: their LOCini differs slightly in terms of grades and not in terms of poles (-2 grades for the forearm, which remains in RInt, and +1 grade for the hand which stays in Flex); the SEGs affected by MOV are not the same, and if the pulses are different, they affect in any case two aligned poles, i.e. the extension of the arm and the extension of the forearm.

They are thus two realizations of the same sign, which go visually in different directions but which are physiologically quite similar: the difference of LOCini being only a question of grades. Only an approach, such as that of Typannot, based on iFoR and a description of the SEGs makes it possible to note this difference: the egocentric observation of the only trajectory of the hand would probably not have made it possible to reconcile these two realizations of which one sees a MOV directed inward and downward, while for the other MOV is directed forward and downward.

How Typannot generates new knowledge

As stated in the presentation of Typannot ( §2), the goal of the GestualScript team is not only to create a TranSys, but also to provide an instrument capable of advancing knowledge on SLs and gestures: the following section shows how Typannot makes it possible to investigate SLs.

Hypotheses

The first assumption looks at the complexity of the motor control. By observing the sign [NEVER]2 in LSF, we see that it can be done with the palm in a horizontal plane and downwards or in a vertical plane, but in both cases MOV will be a simple abduction of the hand. The motion scheme is simple and identical for this sign, regardless of the achievements. This example, and many others, allows us to formulate a first hypothesis: -Hypothesis A: MOV has a simple motor scheme aimed at minimizing motor control during MOV.

The second supposition concerns the MOV propagation flow (FLOW) along the upper limb. If we compare the signs [LOCATION] and [TO GRASP] in LSF (Figure 7), we notice that MOV follows a proximal-distal flow (hand→fingers, FLOW Prox→Dist ) in the first case, and a distal-proximal flow (hand→arm; FLOW Dist→Prox ) in the second case. The FLOW Prox→Dist only affects the hand and the fingers, and only appears in the few realizations showing a change of HS during signing. Generally, the great fixity of the HS during signing seems to show that the flow is mainly a FLOW Dist→Prox . However, working on MOV in general (and not on the SLs), Dumas [START_REF] Dumas | Joint and segment coordinate systems revisited[END_REF] states that the inertia of the hand compared to the forearm is only 24% and that of the forearm to the arm is 65%: there is an inertial tendency towards a FLOW Prox→Dist which should also affect the fingers (change of HS). The difference between what Dumas asserts and the observations on HS changes leads to a second hypothesis: -Hypothesis B: the inertial tendency towards a FLOW Prox→Dist of MOV is predominant also in SLs.

Materials and methods

To test these hypotheses, Typannot was used to transcribe three short extracts (about 1 min each) of narrations, each told in a different SL. These are "L'histoire du cheval" [START_REF] Huillier | L'histoire du cheval, extract from Corpus LS-Colin. Collection[END_REF] in French SL (LSF) (duration: 60 sec), "Pinocchio in LIS" [START_REF] Bertone | Pinocchio in LIS (chapter 1). DVD[END_REF] (duration: 113 sec) in Italian SL (LIS) and "Holiday in Lanzarote (BF21n)" [START_REF] Cormier | Holidays in Lanzarote (extract #BF21n) from the British Sign Language (BSL) Corpus. Economic and Social Research Council[END_REF] (duration: 50 sec) in English SL (BSL).

The extracts have been transcribed with the ELAN software [START_REF] Lausberg | Coding gestural behavior with the NEUROGES-ELAN system[END_REF], using the Typannot G1 characters to encode MOV.

It must be remembered that only the graphematic formula has been developed for LOCini and MOV (see §1) and that the UI is still under realization. The transcription work could therefore only be based on the graphical aspect of Typannot: in fact, the corpus has been annotated with an alphanumeric keyboard, without resorting to the generic and composed characters and without recourse to the UI. In these conditions, the transcription of a single minute may require up to 5 hours of annotation (ratio: 1/300), which more or less corresponds to the time required using other TranSys [START_REF] Onno | Research methods in sign language studies: a practical guide[END_REF]. This length depends not only on the complexity of the parameters but also on the ergonomics of the tools used, both in terms of graphic signs and input interfaces.

A collateral hypothesis emerges from these considerations: -Collateral hypothesis: using iFoR common to the IMU MoCap device and to Typannot, together with a suitable interface (such as the Typannot UI), drastically reduces MOV transcription times.

Tests and preliminary results

In the corpus, 333 MOVs have been counted: 92 in LSF, 153 in LIS and 88 in BSL. These MOVs were then categorized differently to test the hypotheses: Test 1 pertains to Hypothesis A, Test 2 to Hypothesis A and the collateral hypothesis, and Test 3 to Hypothesis B.

Test 1. MOVs were ranked on the basis of the number of MOV-deploying SEGs and within each SEG the number of DoF at the MOV origin, if this is the case (Table 2). The predominance of MOVs concerning only one DoF (initiator of the rest of the MOV) on a single SEG is highly significant (p<0.001): whatever the SLs considered, nearly 80% of MOVs are directed by a single DoF and the other SEGs reposition accordingly with the MOV-initiating DoF.

Therefore, the preliminary results seem to validate Hypothesis A: despite the apparent complexity of MOVs, these are, for the most part, governed by a simple motor scheme.

Test 2. MOVs were also classified on their potentiality for being tracked by a MoCap system, i.e. MOVs being initiated by a single DoF and those whose form matches an algorithm like the circle; Table 3 reports these numbers and their sum. It appears that for more than 90% of the signs it is possible to determine the MOV origin, irrespective of MOV performed.

These data make it possible to show the interest of a UI integrating a system of IMU-like MoCap. This is based on an iFoR system, so the relative data that such equipment provides are easily convertible into Typannot categorical data, based on an iFoR system too. If the IMU and the development implemented in the UI allow to recognize 90% of MOVs, it will be sufficient for the transcriber to redo the movements that he/she visualizes in the video to automatically transcribe them in Typannot, thus minimizing the transcription duration.

Test 3. MOVs were classified based on their flow direction (FLOW Prox→Dist or FLOW Dist→Prox ) (Table 4).

Results are less marked here, but it appears that FLOW Dist→Prox is dominant in more than 60% of MOVs (p<0.001), while the remaining 40% is subdivided between FLOW Prox→Dist (less than 10%), absence of a well-defined flow, MOV on a single DoF (without transfer to another SEG), and total absence of MOV (static). These preliminary results do not corroborate Hypothesis B: in spite of the inertial tendency towards a FLOW Prox→Dist established by Dumas [START_REF] Dumas | Joint and segment coordinate systems revisited[END_REF] for the movements in general, it is the FLOW Dist→Prox to be predominant in SLs. As for Test 1, a classical approach, based on the trajectories of the hands and an eFoR, would not have made it possible to unfold this phenomenon, which on the contrary appears when using Typannot.

CONCLUSIONS

Typannot is a SLs representation system that wants to satisfy 5 basic criteria for transcription: readability; writability; searchability; genericity and modularity. To meet these criteria, it is based on a rigorous protocol for creating characters which makes it possible to obtain a system having at the same time a robust graphematic and a coherent typographic formulas.

The use of the novel TranSys Typannot makes it possible to understand the SLs signifier form in a new light that emphasizes features of the SLs which remained hidden with the previous TranSys. This is shown by the MOV analysis, based not on the trajectory of the hands in an eFoR, but on the observation of the movement propagation flow on the different SEGs and DoF, in an iFoR and from a steadied LOCini.

Two hypotheses have been tested with Typannot on a short corpus allowing to obtain some preliminary but promising results.

In the first place, it has been shown that the motor control necessary for the realization of the signs can be reduced to very simple motor schemes (1 DoF on 1 SEG) for the parameter which appears as the most unstable, i.e. MOV. This observation is only detectable if we adopt iFoR.

Subsequently, it was highlighted how the LOCini participates in the conditions of realization of the sign standardization. This standardization goes through a simple motor scheme, requiring only minimal motor control. This is the case here for more than 90% of signs.

These data, obtained thanks to Typannot, open the way to other hypotheses, for example that the preparation phase of LOCini is probably more unstable, more complex and with a greater occurrence of FLOW Prox→Dist compared to the MOV deployment phase. These hypotheses need to be verified through recordings made with MoCap.

Figure 1 .

 1 Figure 1. [WHAT?] in American Sign Language, written with (from left to right): SignWriting, Stokoe Notation, HamNoSys. Source: www.signwriting.org Since 2013, a third TranSys of SLs is emerging: it is Typannot [15], realized within the l'École supérieure d'art et design (ESAD, Advanced School for Art and Design) of Amiens (France) by the GestualScript team, a multidisciplinary group of linguists, graphic designers, calligraphers, designers and computer scientists.This article starts with the description of the HandShapes (HS) parameter to explain the concept of Typannot and its user interface (UI), followed by a presentation of initial localization (LOCini) and movement (MOV) parameters, which obey multiple, intrinsic Frames of Reference (iFoR), allowing to understand the relationship between the position of the limb articulators and the movement. Finally, the article shows how data transcribed using Typannot and its iFoR make it possible to give an answer to these two questions: -how is it possible that MOV in SLs is standardized and simple, while its very principle brings it to continual changes? -how may we determine MOV from its preparation phase?

Figure 2 .

 2 Figure 2. The three levels of representation of Typannot: example of a HandShape.

Figure 3 .

 3 Figure 3. Typannot primary generics characters (G1) for HandShapes.

Figure 4 .

 4 Figure 4. Generic compound: G1 "Curved" + G1 "Open" = G2 "Curved Open".

Figure 5 .

 5 Figure 5. Typannot User Interface: gestural sub-interface for handshapes (back-end version).

Figure 6 .

 6 Figure 6. The degrees of freedom (DoF) of the upper limbs.

Figure 7 .

 7 Figure 7. The flows in French Sign Language signs [LOCATION] and [TO GRASP].

Table 2 .

 2 Distribution of movement complexity. ⊖ ←---Movement complexity --→ ⊕

	Ø SEG Ø DoF		1 SEG 1 DoF	1 SEG 2 + DoF	2 + SEG 2 + DoF	Proba-bility
	LSF 7	% 7.6%	72	% 78.3% 5		% 5.4%	8	% 8.7% 10 -7
	LIS 15	9.8%	119 77.8% 11	7.2%	8	5.2% 10 -12
	BSL 9	10.2% 70	79.5% 2		2.3%	7	6.9% 10 -8
	TOT 31	9.3%	261 78.4% 18	5.4%	23	6.9% 10 -26

Table 3 .

 3 Frequency of trackable movements (MOV).

	1 SEG 1 DoF		Circles		Trackable MOV	Non-trackable MOV
		%		%		%		%
	LSF 72	78.3%	11	12.0%	83	90.2%	9	9.8%
	LIS 119	77.8%	27	17.6%	146	95.4%	7	4.6%
	BSL 70	79.5%	14	15.9%	84	95.5%	4	4.5%
	TOT 261	78.4%	52	15.6%	313	94.0%	20	6.0%

Table 4 .

 4 Distribution of flow.

		FLOW Dist→Prox	FLOW Prox→Dist	Static	Un-defined	1 DoF	Proba -bility
		%	%	%	%	%
	LSF	54 58.7% 8 8.7% 7 7.6% 0 0.0% 23 25.0% 0.1174
	LIS	98 64.1%. 15 9.8% 15 9.8% 2 1.3% 23 15.0% 0.0006
	BSL	61 69.3% 0 0.0% 9 10.2% 2 2.3% 16 18.2% 0.0004
	TOT 213 63.7% 23 6.9% 31 9.3% 4 1.2% 62 18.6%	10 -8

We decided to use the term "deaf" because of a cultural issue: even if, for hearing people, this term might seems "politically incorrect", most deaf people, especially those who use SLs, do prefer to be called "deaf" (which underlines a difference) than "hearing impaired" (that underlines a handicap).

[NEVER] in LSF, with vertical palm: see Elix-LSF.fr https://goo.gl/aPGC7u[NEVER] in LSF, with horizontal palm: see Sematos.eu https://goo.gl/svStQH

ACKNOWLEDGMENTS

This work was supported by: École supérieure d'art et de design (ESAD) d'Amiens; Amiens Métropole; Conseil regional des Hauts-de-France; Délégation générale à la langue française et aux langues de France (DGLFLF); Ministère de la Culture; Robofont; Crédit Agricole; Dynamique du langage in situ (DyLIS) de l'Université de Rouen-Normandie.