R Agnello

S Béchu

I Furno

Ph Guittienne

A A Howling

R Jacquier

G Plyushchev

M Barbisan

R Pasqualotto

Morgal

A Simonin

Negative ion characterization in a helicon plasma 1 source for fusion neutral beams by Cavity 2 Ring-Down Spectroscopy and Langmuir Probe laser 3 photodetachment 4

teaching and research institutions in France or abroad, or from public or private research centers.

Negative ion characterization in a helicon plasma source for fusion neutral beams by cavity ring-down spectroscopy and Langmuir probe laser photodetachment

The neutralization efficiency, however, drops to negligible values for energies greater 40 than 250 keV, rendering gas neutralization impracticable for positive ions [1]. The 41 neutralization efficiency of negative H -or D -ions in gas neutralizers, is around 60% 42 at the energy requirements of NBIs, rendering negative ions much more attractive. For 43 example, ITER's negative ion-based NBI will deliver 16.7 MW of D 0 at 1 MeV [2].

44

The negative ions for ITER will be produced by Inductively-Coupled Plasma (ICP) 45 sources requiring several hundreds of kW of RF power and the evaporation of caesium A c c e p t e d M a n u s c r i p t basic plasma diagnostics including Langmuir Probes (LPs), microwave interferometry, 76 magnetic probes, Optical Emission Spectroscopy (OES) and diagnostics based on 77 laser-plasma interaction, such as LP-assisted laser photodetachment and Cavity Ring-78 Down Spectroscopy (CRDS), are routinely employed [12,[START_REF] Jacquier | [END_REF]. Previous experimental 79 campaigns based on OES and CRDS revealed a significant amount of negative ions both 80 in hydrogen and deuterium plasmas [START_REF] Marini | [END_REF]14]. Both OES and CRDS measure negative ion 81 density in the order of a few 10 16 m -3 , and development is needed to reach the negative 82 ion densities required for NBIs, which is 10 17 m -3 [15]. The main advantage of CRDS 83 is that it allows a direct measurement of the absolute value of the negative ion density 84 without requiring any calibration and being non-invasive. However, the line-integrated 85 measurement does not provide the local density. In a previous experimental campaign,

86
we tested the robustness of CRDS and studied the experimental challenge of how to 87 reduce undesired effects that could deteriorate the accuracy of the measurement [14]. In The paper is organized as follows: Section 2 describes the plasma source RAID.

93

In Section 3, we present the experimental setup of CRDS and LP-assisted laser wave has been measured by means of a magnetic probe [START_REF] Jacquier | [END_REF] confirming that the plasma 111 column is sustained by helicon waves. depending only on the gas pressure [START_REF] Lieberman | Principles of plasma discharges and materials processing 487[END_REF] H 2 (v) is the radiative decay from singlet state, the so called E-V process [21]:

154 H 2 (X 1 Σ + g , v) + e → H 2 (B 1 Σ u ; C 1 Π u ) + e → H 2 (X 1 Σ + g , v ) + e + hν.
Previous experiments based on Optical Emission Spectroscopy (OES) and CRDS 155 revealed a significant volume production of H -and D - [START_REF] Marini | [END_REF]. The radial distribution 156 measured by OES suggests that negative ions reach a maximum density in a radial shell 157 located around r ∼ 4 cm. Since CRDS was demonstrated to be a robust diagnostic, it is 158 used here for an experimental campaign to broaden knowledge of negative ion physics 159 in the present experimental device and to form a database for models of negative ion 160 production in the helicon plasma. In order to make measurements comparable with 161 the previous campaign [14], the same experimental setup was used as described in 3, 162 combined with a movable LP to measure the radial profile of negative ion density using 163 laser photodetachment. the measurement position of the laser is fixed at r = 0.04 m (where we expect the 171 largest density of H -according to OES measurements [START_REF] Marini | [END_REF]) and the laser beam performs 172 multiple reflections between the two high reflectivity (HR) mirrors.

173

The CRDS technique was developed in 1988 by A. O'Keefe [22] to perform optical 174 absorption measurements. CRDS is nowadays routinely used in fusion research as a 175 plasma diagnostic to measure H -and D -line densities in negative ion sources [15,[START_REF] Nakano | 17th International Symposium on Laser-Aided Plasma Diagnostics[END_REF].

176

To detect these ions, a pulsed Nd:YAG laser is employed [START_REF] Pasqualotto | [END_REF]. The photon energy 177 (E = 1.2 eV, corresponding to a wavelength of λ = 1064 nm) is sufficient to strip the 178 weakly bound electron of H -and D -(E binding = 0.75 eV) by photodetachment:

179 hν + H -→ H + e. ( 3
)
180

The CRDS diagnostic on RAID relies on an optical cavity made of two HR mirrors 181 (R > 99.998% [START_REF] Pasqualotto | [END_REF]) to multiply the interaction length with the absorbing medium.

182

The light stored in the cavity is lost with an exponential decay time τ depending on 183 the mirrors' reflectivity and the medium's absorption. Further details on the CRDS 184 diagnostic in RAID can be found in Ref. [14]. An example of a CRDS signal in RAID 185 is shown in the inset in Fig. 5. The line-integrated negative ion density n-is:

186 n-= n -(l) dl, (4)
187

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

where n -is the local negative ion density. The line-averaged negative ion density n - 188 is given by: can be varied simultaneosly with the movable LP, which can be vertically displaced.

189 n -= 1 σ - L cd 1 τ - 1 τ 0 , (5

206

The probe is displaced by a stepper motor and the vacuum is maintained by a bellow.

207

The probe tip is aligned co-axially with the direction of the laser beam and biased 208 at V bias = 26 V, above the plasma potential V plasma , which is V plasma = 12 (±1) V for negative ion density n -can be estimated as follows:

216 n - n e = I pd I dc , ( 6
)
217
where n e is the local electron density, I dc the collected current at the V bias , and I pd 218 the amplitude of the variation of the collected current due to the photodetachment.

219

Eq. ( 6) can be applied if the following criteria are satisfied [25]: 1) the laser wavelength of H 2 and Ar gas (see Fig. 5); the Ar gas is added to facilitate plasma ignition [14].

238

The drop of τ just after plasma ignition is due to a combination of the absorption of 239 the laser pulse by negative ions and the thermal stress of the optical cavity. Just after 240 plasma ignition, Ar is removed in a few seconds and RF power is increased meanwhile.

241

In the plateau where τ ∼ = 37 µs, the plasma consists purely of H 2 and it is steady state.

242

The plasma discharge is then turned off at t ∼ = 80 s causing the sudden increase of We verified that the energy of the laser pulse was enough to strip all the electrons of weaker signal is measured in the case of D 2 . As suggested by Eq. ( 6), the shape of the 299 curve is due to a combination of the negative ion density and the electron temperature. 305 Fig. 8(c) shows the ratios n H -/n e and n D -/n e , given by Eq. ( 6). Although the electrons 306 are magnetized, the electron transport coefficient is the same both for the photodetached 307 electrons and for the electrons collected by the probe when biased above V plasma .

308

Therefore, the transport coefficients tend to cancel out in the ratio. We note the strong 309 difference between the two plasmas. At r = 0.06 m, n H -/n e reaches 0. can be found in [31]. The higher density in D 2 is measured by microwave interferometry 332 at the edge, at measurement position r = 0.04 m, revealing a line-integrated density a 333 factor 2 higher in the case of deuterium.

334

The data shown in Fig. 8 For completeness we also show the temperature profiles in Fig. 10. T e decreases 341 weakly along the radial direction down to 1 eV at r = 0.065 m and 5 kW of power

342
for both H 2 and D 2 . Moreover, a general decrease of T e is observed for increasing RF

n -(x, y) = n peak -F (x, y), ( 7
)
370
where n peak is the negative ion peak density. CRDS measures the integral of ( 7)

371
along the line of sight at the measurement position y = y CRDS , which is:

372 nCRDS - = n peak - x 2 x 1 F (x, y CRDS ) dx, (8)

388

The isotopic effect in negative ion and electron density has been investigated by 389 some authors and it is due to a combination of different phenomena [START_REF] Skinner | [END_REF]34,35].

390

Calculations and experiments show that n e is higher in deuterium than hydrogen effect on the negative ion density, the lower negative ion density in deuterium might be 396 due to the higher degree of dissociation in deuterium leading to increased V-t cooling 397 (cooling of D 2 (v) in collision with D) and then less efficient D -production [START_REF] Skinner | [END_REF]. For a 398 better understanding of the processes, modeling is envisaged. pressures, and future studies at those pressures will be required [START_REF] Hemsworth | AIP Conference Procedings[END_REF]. We showed that LP-406 assisted photodetachment is not sufficient alone to obtain reliable measurements. Thus, densities appear to be comparable, the ratio n -/n e is about 6 times lower in deuterium 417 than hydrogen. This suggests that the co-extracted electrons might be a more important 418 issue for deuterium, if the current magnetic geometry is employed.

419

We believe that these experimental investigations add relevant data on the volume

R

  Agnello, Stéphane Béchu, I Furno, P H Guittienne, A A Howling, R Jacquier, G Plyushchev, M Barbisan, R Pasqualotto, I Morgal, et al. To cite this version: R Agnello, Stéphane Béchu, I Furno, P H Guittienne, A A Howling, et al.. Negative ion characterization in a helicon plasma source for fusion neutral beams by cavity ring-down spectroscopy and Langmuir probe laser photodetachment. Nuclear Fusion, 2020, 60, pp.026007. 10.1088/1741-4326/ab5e64. hal-03049113 1. Introduction 35 Negative ion based Neutral Beam Injectors (NBIs) for fusion applications require high 36 accelerated negative ion currents, and current density, with the ion energy of the 37 order of 1 MeV. In conventional NBI systems, positive ions, usually H + or D + , are 38 accelerated by electric fields and neutralized by charge exchange with gas molecules.

  39

88

  this paper, we extensively exploit CRDS by varying plasma parameters in combination 89 with LP-assisted laser photodetachment to deduce absolute negative ion density local 90 profiles. The knowledge of these profiles is important since they are used as input 91 parameters for negative ion extraction and modeling.

  92

94H 2

 2 photodetachment on the RAID device. Section 4 shows the experimental results in 95

97 2 .

 2 The Resonant Antenna Ion Device (RAID) 98 RAID, shown in Fig. 1, produces a magnetized plasma column by means of a resonant 99 antenna in a birdcage geometry [8]. The cylindrical vacuum vessel is surrounded by six 100 coils producing an axial magnetic field up to 800 G on axis. The first coil on the left is 101 usually operated in inverse polarity with respect to the other five to create a gradient of 102 magnetic field along the axis in the antenna region. This enhances the electron confine-103 ment in the antenna probably because slower electrons produced in the antenna region 104 are confined by the bottleneck of the magnetic field. This was experimentally seen to 105 facilitate the ignition of the plasma discharge and to stabilize the helicon regime. A va-106 riety of axial boundary conditions can be implemented. In the present experiments, the 107 plasma column is bounded behind the antenna by a ceramic foam, and on the far end 108 by a water-cooled grounded copper end-plate. A stable helicon regime can be attained 109 with a minimum power of 1000 W for hydrogen and deuterium. A traveling helicon 110

Figure 1 .

 1 Figure 1. A cross section of the RAID plasma device along the axis. The cylindrical vacuum vessel of 1.4 m length and 0.4 m diameter is surrounded by six coils producing an axial magnetic field up to 800 G. The resonant birdcage antenna, placed on the left, produces a plasma column up to the target on the right.

  132

Fig. 3 (Figure 2 .Figure 3 .

 323 Fig. 3(a) shows the electron density radial profile, measured at the axial position

164 3 .

 3 photodetachement are respectively shown in Fig.4(a) and in Fig.4(b). In Fig.4(a), 170

) 190 where τ and τ 0 Figure 4 .

 04 Figure 4. A schematic of the two different experimental setups employed: in (a) the CRDS and, in (b) the LP-laser photodetachment scheme. In (a) the measurements position of the laser is fixed at 4 cm. In (b) the laser beam is vertically displaced simultaneously with the LP.

  209 r < 0.06 m, for both H 2 and D 2 plasmas, and independent of gas pressure. The H -or 210 D -ions in the laser path are photoneutralized and the stripped electrons are collected 211 by the probe. This causes a sudden increase of the signal detected by the LP, in the 212 A c c e p t e d M a n u s c r i p t order of a fraction of microsecond, proportional to the local negative ion density. An 213 example of a photodetachment signal in RAID in shown in Fig. 7. The fast transient 214 is measured by a current probe consisting of a toroidal current transformer. The local 215

220

  is suitable to photo-detach the electrons of the negative ions (i.e. the photon energy and 221 the detachment cross section are sufficiently high), 2) the laser pulse energy is enough 222 to photodetach all negative ions in the target volume, 3) the laser beam must have a 223 minimum diameter from which the amplitude of the signal does not increase with beam 224 diameter and, 4) the probe bias must be above the plasma potential. These criteria are 225 satisfied as described above and in section 4.2.

226 4 .

 4 Experimental Results227The CRDS and the LP-assisted photodetachment were used in two distinct experimental 228 campaigns in H 2 and D 2 plasmas with 200 G magnetic field on the axis of RAID, and 229 variable gas pressures.

230 4 . 1 .

 41 CRDS characterization in RF power and gas pressure at a fixed Line-Of-Sight231 (LOS)232CRDS measurements are performed employing the technique described below, which 233 was already introduced in[14] and reviewed here for clarity. Fig.5shows τ , the ring-234 down time, as a function of time, before, during, and after a H 2 plasma discharge with 235 5 kW RF power. The laser is pulsed at 10 Hz so that the temporal resolution is of the 236 order of 0.1 s. The plasma discharge starts at t ∼ = 30 s in the presence of a mixture

  237

243τ.Figure 5 .

 5 Figure 5. Ring-down time τ before, during, and after a plasma discharge for a H 2 plasma and 5 kW RF power. The plasma discharge starts at t ∼ = 30 s and it is stabilized after a few seconds. Plasma is turned off at t = 80 s causing the jump of τ due to the disappearance of H -ions. The linear fit of tau before and after the plasma off is used to accurately determine τ at plasma off. The jump is used to evaluate the line-averaged H -density. The inset shows a fit as used to calculate the ring-down time for each data point τ .

266 4 . 2 .AFigure 6 .Figure 7 .

 4267 Figure 6. Line-integrated negative ion density as a function of RF power at different pressures for H 2 and D 2 plasmas, demonstrating the steady-state production of negative ions up to 8 kW of RF power.

289A c c e p t e d M a n u s c r i p t H

 t -to satisfy[START_REF] Marini | [END_REF]. This was performed by increasing the laser pulse energy and observing 290 the I pd saturation from a pulse energy of 35 mJ. Consequently, all results were obtained 291 using a pulse energy of 50 mJ.292To radially characterize the plasma, the LP is moved in the vertical direction. For 293 each position, a new alignment laser beam-probe is needed. Fig. 8 summarizes the 294 results obtained with laser photodetachment while varying the RF power. The left and 295 the right column show the results for H 2 and D 2 plasmas respectively. The gas filling 296 pressure was 0.3 Pa. 297 In Fig. 8(a) the amplitudes of the photodetachment signals are shown. On average, a 298

300Fig. 8 (

 8 Fig. 8(b) shows the electron current I dc when the probe is biased at V bias = 26 V, 301

317Fig. 8 (attains 1 . 3 ×

 813 Fig. 8(d). At 5 kW power and r = 0.04 m (where the fixed LOS of CRDS is located) n e 322

Figure 8 .

 8 Figure 8. Results of the laser photodetachment measurements for H 2 (left column) and D 2 (right column) plasmas at 0.3 Pa: (a) Photodetachment amplitudes I pd ; (b) Electron current I dc collected when the probe is biased at V bias = 26 V, above the plasma potential; (c) Ratio n -/n e ; and (d) Electron density n e . On the edge of the plasma column, hydrogen plasma has lower electron density and a higher ratio n -/n e .

H -is 7 . 7 ×Figure 9 .Figure 10 .

 77910 Figure 9. Radial profiles of negative ion density obtained with laser photodetachment and measuring the electron density via IV curves. These data are not calibrated with CRDS.

  343power. The T e values in the edge are in the range where the DA cross section peaks, 344 depending on the rovibrational state of H 2 (v) and D 2 (v). Thus, the T e profiles show 345 that the electron cooling on the edge, caused by the magnetic field needed to sustain 346 the helicon discharge, is also effective to reach a favorable electron temperature for DA 347 process.

348 4 . 3 .Figure 11 .

 4311 Figure 11.Gaussian fits of the relative n -profiles measured with laser photodetachment. The position of CRDS line-of-sight to calibrate n -absolute profiles and the vessel wall position is indicated.

  369

373 where x 1 AFigure 12 .Figure 13 .

 11213 Fig.12shows the absolute n H -and n D -profiles only for 2, 3 and 5 kW RF powers for 376

391AFigure 14 .

 14 Figure 14. Negative ion density vs. electron density in hydrogen and deuterium plasmas.

399 5 .

 5 Discussion and Conclusions 400 The measurements performed with CRDS and LP-assisted photodetachment show that 401 H -and D -are produced both in hydrogen and deuterium helicon plasmas in steady 402 state regime with significant densities in volume production mode, without using 403 caesium and with a few kWs RF power. The measurements were performed at a 404 fill pressure of 0.3 Pa, relevant to NBIs of ITER. Future NBIs may require lower fill 405

407

  to extract the absolute negative ion density profile, LP-assisted laser photodetachment is 408 employed in combination with line-integrated measurements of CRDS. The LP-assisted 409 laser photodetachment is used to measure the relative negative ion density profile 410 showing that it is peaked off-axis. The line-integrated measurement performed by CRDS 411 is then used to obtain the absolute negative ion density profile. Taking into account the 412 peak negative ion density at 5 kW RF power, H -density reaches 1.9 (±0.3) × 10 16 m -3 , 413 close to 3.0 (±0.3) × 10 16 m -3 measured with OES. However, D -reaches 1.8 × 10 16 m -3 414 quite lower if compared to 4.5 (±2.0) × 10 16 m -3 measured with OES. The reason for 415 this disagreement in the D 2 case is under investigation. Despite the absolute H -and D -416

420

  production of H -and D -in hydrogen and deuterium helicon plasmas. However, the 421 design of a source based on a helicon plasma requires the investigation of different 422 A c c e p t e d M a n u s c r i p t magnetic field geometries and the compatibility with an extractor and an acceleration 423 system. Thus, a next step with the current system is the characterization of negative 424 ions along the plasma column and the study of different magnetic geometries, such as 425 the expanding magnetic field, with the diagnostics mentioned in this paper. In parallel 426 with this, 3D numerical simulations could be an important tool to predict the negative 427 ion production as a function of source parameters.428If used as part of a future ion source for an NBI system, direct extraction of the 429 negative ions from the plasma column in the radial or axial direction should be possible.430If radial extraction is used, because of the inherent cylindrical nature of the helicon 431 driven plasma, the beam would have to be long and narrow, such as that suggested 432 in reference[36]. However significant development may be needed if the extractor 433 perturbs significantly the plasma and because the extraction system would need to 434 be segmented, with each segment fitting between the magnetic coils. Axial extraction 435 seems unattractive as the annular region from which ions could be extracted is short, and 436 hence the extracted current would be low, and a transverse filter field would probably 437 be needed to reduce the co-extracted electron current. It is also to be noted that the 438 resulting annular beam shape has no particular advantage from the point of view of 439 neutralisation or beamline design.440Using the helicon device to provide a plasma that expands towards a conventional 441 multi-aperture system with a caesiated plasma grid should be considered because of 442 the ease of operation at fill pressures < 0.3 Pa and the high efficiency of the helicon 443 device. In this case, it needs to be established that the helicon driven plasma produces 444 sufficient dissociation of the gas in order to have sufficient negative ion production via 445 the impingement of the atoms on the low work function of the plasma grid surface. 446 A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t

. Similar behaviours are observed in other helicon

	140 141 142 143 144 145 146 147 148 149 150 152 A c c e p t e d M sources [17]. The n e and T e profiles are measured with a L-shaped Langmuir probe (0.2 mm diameter and 10 mm long) located at axial position 0.830 m and vertically movable. The peaked T e profile is favorable for the volume production of H -since it provides a central hot region to rovibrationally excite the H 2 molecules and a cold external region where low energy electrons can produce the H -by dissociative attachment (DA) [18, 19]: H 2 (v ≥ 5) + e (≤ 1 eV) → H + H -(DA). (1) There are two possible mechanisms to produce the rovibrationally excited H 2 (v) in volume. The first is the stepwise vibrational excitation of molecules in collision with electrons (e-V process): e + H 2 (v) → H 2 -→ e + H 2 (v + ∆v) (e -V), (2) this reaction, however, requires many collisions to increase the vibrational state to high a n u s c r i p t of ions sources for NBIs, another important contribution to the production of the excited 153

151 values since the most probable change in v is ∆v = ±1 [20]: At the low pressure typical

Acknowledgments 447

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


Mickaël J Mourlam

Maeva J Orliac

Early evolution of the ossicular chain in Cetacea: into the middle ear gears of a semi-aquatic protocetid whale

Modifications of the morphology and acoustic properties of the ossicular chain are among the major changes that accompanied the adaptation of Cetacea to the aquatic environment. Thus, data on the middle ear ossicles of early whales are crucial clues to understand the first steps of the emblematic terrestrial/aquatic transition that occurred in that group. Yet, the delicate nature and very small size of these bones make their preservation in the fossil record extremely rare. Due to the scarcity of available data, major questions remain concerning the sound transmission pathways in early non-fully aquatic whales. Virtual reconstruction of a partially complete ossicular chain of an Eocene protocetid whale documents for the first time the three ossicles of a semi-aquatic archaeocete. Contrary to previous hypotheses, these ossicles present different evolutionary patterns, showing that the ossicular chain does not act as a single morphological module. Functional analyses of the different middle ear units highlight a mosaic pattern of terrestrial and aquatic signatures. This integrative anatomical and functional study brings strong evidence that protocetids were adapted to their dual acoustic environment with efficient hearing in both air and water.

Introduction

The adaptation to the aquatic environment by cetaceans implied drastic modifications of their sensory organs from their terrestrial ancestral condition. Among these, the sense of hearing was particularly reshaped in conjunction with the physical constraints imposed by water, implying modifications of the whole sound transmission pathway. The modality of the adaptation of the earliest cetaceans to underwater hearing is only partly known and major questions remain concerning hearing mechanisms in non-fully aquatic whales. The delicate nature and very small size of the middle ear ossicles make their preservation in the fossil record rather unlikely; when preserved, all three ossicles are rarely found together and almost never in their anatomical position. Yet, few middle ear ossicles are documented for non-ully aquatic early whales. The incus of the earliest archaeocete Pakicetus (early Eocene, Pakistan) [START_REF] Thewissen | Origin of underwater hearing in whales[END_REF] documents the oldest cetacean ossicle and shows an intermediate morphology between terrestrial artiodactyls and cetaceans (e.g. incomplete rotation of the ossicular chain [START_REF] Thewissen | Origin of underwater hearing in whales[END_REF]). In addition, the ossicular chain is nearly fully known for remingtonocetids (one in situ ossicular chain and one incudomallear complex of Remingtonocetus; middle Eocene, India [START_REF] Nummela | Eocene evolution of whale hearing[END_REF][START_REF] Nummela | Sound transmission in archaic and modern whales: anatomical adaptations for underwater hearing[END_REF]) and only partially documented for protocetids (two partially preserved mallei; middle Eocene, India [START_REF] Nummela | Eocene evolution of whale hearing[END_REF][START_REF] Nummela | Sound transmission in archaic and modern whales: anatomical adaptations for underwater hearing[END_REF]). Based on this incomplete record, some authors concluded that underwater hearing in these early whales was almost fully functional, while aerial hearing was strongly altered [START_REF] Nummela | Eocene evolution of whale hearing[END_REF][START_REF] Nummela | Hearing[END_REF]. Middle ear ossicles of extinct, strictly aquatic early whales, such as basilosaurids, are well documented [START_REF] Pompeckj | Das Ohrskelett von Zeuglodon[END_REF][START_REF] Lancaster | The middle ear of the Archaeoceti[END_REF][START_REF] Gingerich | Ancalecetus simonsi, a new Dorudontine Archaeocete (Mammalia, Cetacea) from the Early Late Eocene of Wadi Hitan, Egypt[END_REF][START_REF] Uhen | Form, function, and anatomy of Dorudon atrox (Mammalia, Cetacea): an archaeocete from the Middle to Late Eocene of Egypt[END_REF]. They display the derived morphology observed in modern cetaceans [START_REF] Pompeckj | Das Ohrskelett von Zeuglodon[END_REF][START_REF] Lancaster | The middle ear of the Archaeoceti[END_REF][START_REF] Kellogg | A review of the Archaeoceti[END_REF][START_REF] Oelschläger | Evolutionary morphology and acoustics in the dolphin skull[END_REF], strongly suggesting fully functional underwater hearing abilities Micro-computed tomography (CT) scan investigation of the middle ear cavity of a protocetid whale partial cranium (Lutetian, Togo [START_REF] Gingerich | A new archaeocete and other marine mammals (Cetacea and Sirenia) from lower middle Eocene phosphate deposits of Togo[END_REF][START_REF] Mourlam | Protocetid (Cetacea, Artiodactyla) bullae and petrosals from the middle Eocene locality of Kpogamé, Togo: new insights into the early history of cetacean hearing[END_REF]) provides unprecedented access to the partially complete ossicular chain (composed of malleus, incus and stapes) of this amphibious archaeocete whale. This discovery provides essential clues for understanding the functioning of the ear of a semiaquatic cetacean and to discuss the early evolutionary history of the sound transmission pathway in Cetacea.

Institutional abbreviations

H-GSP, Howard University, Geological Survey of Pakistan; LSUMG, Louisiana State University, Museum of Geoscience, Baton Rouge, Louisiana, USA; MRAC, Musée Royal d'Afrique Centrale, Tervuren, Belgium; NOAA, National Oceanic and Atmospheric Administration, Southwest Fisheries Science Center, Washington, DC, USA; UBC, University of British Columbia, Vancouver, Canada; UM, Université de Montpellier, France.

Material and methods

Material

The specimen UM-KPG-M 73, investigated in this study, was collected in Lutetian phosphate deposits (ca 46-43 Ma) at Kpogamé, Togo [START_REF] Gingerich | A new archaeocete and other marine mammals (Cetacea and Sirenia) from lower middle Eocene phosphate deposits of Togo[END_REF][START_REF] Mourlam | Protocetid (Cetacea, Artiodactyla) bullae and petrosals from the middle Eocene locality of Kpogamé, Togo: new insights into the early history of cetacean hearing[END_REF]. It consists of a skull fragment of an adult protocetid belonging to an indeterminate species, referred to as 'morphotype γ' by Mourlam & Orliac [START_REF] Mourlam | Protocetid (Cetacea, Artiodactyla) bullae and petrosals from the middle Eocene locality of Kpogamé, Togo: new insights into the early history of cetacean hearing[END_REF]. Micro-CT scan investigation revealed the presence of the almost complete ossicular chain: the stapes and the incus are finely preserved, whereas the malleus was damaged during the physical preparation of the specimen [START_REF] Gingerich | A new archaeocete and other marine mammals (Cetacea and Sirenia) from lower middle Eocene phosphate deposits of Togo[END_REF]. In order to facilitate anatomical comparisons, we provide a simplified 'incudomalleo-centred' orientation system considering the two articular facets as referential (see electronic supplementary material, text S1 and figure S1). Detailed descriptions of the petrotympanic complex and bony labyrinth of Protocetidae indet γ were provided by Mourlam & Orliac [START_REF] Mourlam | Protocetid (Cetacea, Artiodactyla) bullae and petrosals from the middle Eocene locality of Kpogamé, Togo: new insights into the early history of cetacean hearing[END_REF][START_REF] Mourlam | Infrasonic and ultrasonic hearing evolved after the emergence of modern whales[END_REF].

Micro-computed tomography scan investigation

UM-KPG-M 73 was scanned with a resolution of 70 μm using a General Electric Phoenix Nanotom S at the AniRA-ImmOs (SFR Biosciences Gerland-Lyon) microtomography facility. The three ossicleswere virtuallyextractedmanuallyslicebysliceusing the segmentation tools of AVIZO 9.0 [START_REF]Avizo: 3D analysis software for scientific and industrial data[END_REF].No smoothingwas performed in the rendering process for the surfaces used in the following analyses (see [START_REF] Orliac | Fallen in a dead ear: intralabyrinthine preservation of stapes in fossil artiodactyls[END_REF]). Three-dimensional models are available at MorphoMuseuM.com (tympanic bulla, M3#134_UM KPG-M 73; stapes, M3#407_UM KPG-M 73; incus, M3#408_UM KPG-M 73; malleus, M3#409_UM KPG-M 73).

Hearing parameters measurement

Linear measurements follow Uhen ([8], fig. 48) and Hemilä et al. ( [START_REF] Hemilä | What middle ear parameters tell about impedance matching and high frequency hearing[END_REF], pp. [START_REF] Farina | Avian soundscapes and cognitive landscapes: theory, application and ecological perspectives[END_REF][START_REF] Nummela | The anatomy of the killer whale middle ear (Orcinus orca)[END_REF] for the lever arm lengths. The volume of the incus and stapes were measured with AVIZO 9.0 [START_REF]Avizo: 3D analysis software for scientific and industrial data[END_REF] and their estimated mass ranges follow Nummela et al. [START_REF] Nummela | Eocene evolution of whale hearing[END_REF][START_REF] Nummela | Sound transmission in archaic and modern whales: anatomical adaptations for underwater hearing[END_REF] considering a minimum ossicular bone density of 2.0 g cm-3, based on terrestrial mammals, and a maximum of 2.7 g cm-3, based on odontocetes. Bone thickness quantification of the tympanic bulla was computed as the minimum Euclidean distance between a mesh node of the outer tympanic surface and a mesh triangle of the inner tympanic surface [START_REF] Guy | Prospective in (Primate) dental analysis through tooth 3D topographical quantification[END_REF][START_REF] Lazzari | Quantitative threedimensional topography in taxonomy applied to the dental morphology of catarrhines[END_REF][START_REF] Thiery | Beyond the map: enamel distribution characterized from 3D dental topography[END_REF] using the 'Surface Distance' tools of AVIZO 9.0 [START_REF]Avizo: 3D analysis software for scientific and industrial data[END_REF]. The resultant matrix was used to generate a 3D chromatic map with its associated isopleth bone thickness information [START_REF] Lazzari | Topographic maps applied to comparative molar morphology: the case of murine and cricetine dental plans (Rodentia, Muroidea)[END_REF]. The tympanic plate area has been estimated following Nummela et al. [START_REF] Nummela | Scaling of the cetacean middle ear[END_REF] and the isopleth of 2 mm. Above this value, thickness of the tympanic bone increases markedly (see electronic supplementary material, figure S2). The estimation of the tympanic membrane area follows Nummela [START_REF] Nummela | Scaling of the mammalian middle ear[END_REF] and includes the pars flaccida (electronic supplementary material, figure S3). All surface areas were taken using the measurement tools of Fiji [START_REF] Schindelin | Fiji: an open-source platform for biological-image analysis[END_REF]. Measurement of sound input areas (sensu [START_REF] Nummela | Eocene evolution of whale hearing[END_REF][START_REF] Nummela | Sound transmission in archaic and modern whales: anatomical adaptations for underwater hearing[END_REF]) along with the incudal and stapedial masses of other mammals (see electronic supplementary material, table S1) have been compiled from Nummela [START_REF] Nummela | Scaling of the mammalian middle ear[END_REF] and Nummela et al. [START_REF] Nummela | Eocene evolution of whale hearing[END_REF][START_REF] Nummela | Sound transmission in archaic and modern whales: anatomical adaptations for underwater hearing[END_REF][START_REF] Nummela | Scaling of the cetacean middle ear[END_REF], to which we added data for Choeropsis liberiensis (MRAC RG 35715) and Delphinus capensis (NOAA-cet 436B-KXD0307). Mallear and incudal lever arm lengths data come from Hemilä et al. [START_REF] Hemilä | What middle ear parameters tell about impedance matching and high frequency hearing[END_REF] and the geometric transformer ratio of the middle ear has been computed following these authors (i.e. product between the area ratio ((tympanic membrane area × 2/3)/fenestra vestibuli area) and the lever arm ratio (length of mallear lever arm/length of incudal lever arm)).

Results

Description and comparisons

The partially complete ossicular chain of UM-KPG-M 73 is preserved in the middle ear cavity (figure 1a). The stapes is close to its original anatomical position and the natural articulation between the incus and malleus appears to be roughly maintained. However, the incudomallear complex moved post-mortem and it is not possible to provide a robust reconstruction of the original position of the ossicular chain due to the partial preservation of the auditory region (electronic supplementary material, text S1).

(i) Stapes

The stapes, partially sunk (post-mortem) within the bony labyrinth, has kept its original anteroposterior orientation (figure 1b-e). Compared with available data on early whales, the UM-KPG-M 73 stapes is lightly built, with very thin crura of the same width, and it does not show the pachyostosis observed in basilosaurids and extant cetaceans [START_REF] Pompeckj | Das Ohrskelett von Zeuglodon[END_REF][START_REF] Lancaster | The middle ear of the Archaeoceti[END_REF][START_REF] Uhen | Form, function, and anatomy of Dorudon atrox (Mammalia, Cetacea): an archaeocete from the Middle to Late Eocene of Egypt[END_REF] (electronic supplementary material, figure S4). It is high and narrow, and bears a wide foramen intercrurale of the same diameter on both faces. The capitulum is broad and presents a large articular facet for the contact with the crus longum of the incus [START_REF] Kellogg | Kentriodon pernix, a Miocene porpoise from Maryland[END_REF][START_REF] Fleischer | Evolutionary Principles of the Mammalian Middle Ear[END_REF][START_REF] Mead | The therian skull: a lexicon with emphasis on the odontocetes[END_REF]. Its long axis does not have the same orientation as that of the stapedial footplate (figure 1e). The distinction between the capitulum and the crura is clearly marked by a neck, contrary to the artiodactyl sample described by Orliac & Billet ([15], fig. 2) and to the fully aquatic cetaceans [START_REF] Lancaster | The middle ear of the Archaeoceti[END_REF][START_REF] Uhen | Form, function, and anatomy of Dorudon atrox (Mammalia, Cetacea): an archaeocete from the Middle to Late Eocene of Egypt[END_REF][START_REF] Mead | The therian skull: a lexicon with emphasis on the odontocetes[END_REF][START_REF] Fraser | Hearing in cetaceans[END_REF]. The muscular process, serving as attachment site for the stapedial muscle and located on the posterior crus, is wide and blunt. The footplate, articulating with the petrosal at the fenestra vestibuli, is oblong with a stapedial ratio of 1.42. It is fully concave on the vestibular face, like basilosaurids and modern cetaceans [START_REF] Kellogg | Kentriodon pernix, a Miocene porpoise from Maryland[END_REF][START_REF] Doran | XVIII. Morphology of the mammalian Ossicula auditûs[END_REF], and unlike non-cetacean artiodactyls. The concave vestibular face of the stapedial footplate is concurrent with an increase in the footplate thickness near the base of the two crura.

(ii) Incus

In ventral view, the incus is stocky and conical in shape, slightly compressed anteroposteriorly (figure 1f-i). The incus of UM-KPG-M 73 is of 'cetacean type' and presents several characters retrieved in cetaceans (electronic supplementary material, figure S5): (i) the size and orientation of the crura are of cetacean type with a posteriorly oriented larger crus longum extending from the inferior articular facet and a more lateral, thinner, crus breve [START_REF] Mead | The therian skull: a lexicon with emphasis on the odontocetes[END_REF][START_REF] Lambert | Macroraptorial sperm whales (Cetacea, Odontoceti, Physeteroidea) from the Miocene of Peru[END_REF]; (ii) the strong size difference between the facets of the articular surface for the malleus is typical of what is observed in cetaceans [START_REF] Thewissen | Origin of underwater hearing in whales[END_REF][START_REF] Lancaster | The middle ear of the Archaeoceti[END_REF][START_REF] Fleischer | Evolutionary Principles of the Mammalian Middle Ear[END_REF]; and (iii) the angulation of the two articular facets is positioned at a right angle [START_REF] Pompeckj | Das Ohrskelett von Zeuglodon[END_REF][START_REF] Uhen | Form, function, and anatomy of Dorudon atrox (Mammalia, Cetacea): an archaeocete from the Middle to Late Eocene of Egypt[END_REF][START_REF] Fraser | Hearing in cetaceans[END_REF][START_REF] Kinkel | Rotation of middle ear ossicles during cetacean development[END_REF]. Among Cetacea, the incus of UM-KPG-M 73 is morphologically closer to that of Pakicetus than that of remingtonocetids and pelagicetes (basilosaurids and neocetes sensu [START_REF] Uhen | New protocetid whales from Alabama and Mississippi, and a new cetacean clade[END_REF]), with: (i) no inflation of the incudal body; (ii) slight inflation of the crus longum; and (iii) a posterior location of the base of the crus breve.

(iii) Malleus

The malleus was damaged during the preparation of the specimen (mechanical abrasion of body and head; figure 1j-k). Nevertheless, the convex inferior articular facet and part of the superior articular facet are intact. The former facet matches its counterpart on the incus (figure 1j ), which allows assessment of the whole shape of the articular surface (electronic supplementary material, figure S6). The malleus was broad and pachyostotic. The lateral part of the mallear head is perforated by a wide canal that we interpret as part of the lodge of the lateral extension of the chorda tympani (electronic supplementary material, text S2 and figure S7). On the mallear body, the gonial was completely abraded by mechanical preparation. In dorsal view, medial to the main broken area, a tuberosity surmounted by a small circular pit (dotted line in figure 1j ) corresponds to the muscular process, which is the site of insertion for the tendon of the tensor tympani muscle [START_REF] Lancaster | The middle ear of the Archaeoceti[END_REF][START_REF] Mead | The therian skull: a lexicon with emphasis on the odontocetes[END_REF][START_REF] De Muizon | Nouvelles données sur le diphylétisme des Dauphins de rivière (Odontoceti, Cetacea, Mammalia)[END_REF]. The mallear body bears a short hook-like manubrium. The malleus of UM-KPG-M 73 is closer to Pelagiceti than to its terrestrial relatives (electronic supplementary material, figures S6 and S8): the muscular process is small and blunt instead of spike-like, the manubrium is short, and the chorda tympani probably passes through the mallear head. Based on the preserved structures, the general shape and dimensions of UM-KPG-M 73 malleus are also close to the protocetid Indocetus ramani and the remingtonocetid Remingtonocetus sp. ( [START_REF] Nummela | Sound transmission in archaic and modern whales: anatomical adaptations for underwater hearing[END_REF], fig. 2E-G).

Ecoacoustic niche investigation

Various isometric correlations have been found between the different components of the sound transmission chain, from the outer/middle ear interface (sensu [START_REF] Nummela | Eocene evolution of whale hearing[END_REF][START_REF] Nummela | Sound transmission in archaic and modern whales: anatomical adaptations for underwater hearing[END_REF]) to the middle/inner ear interface [START_REF] Nummela | Scaling of the cetacean middle ear[END_REF][START_REF] Nummela | Scaling of the mammalian middle ear[END_REF]. Since the middle ear is acting as an impedance matching device, these correlations are dependent on the soundtope (i.e. living environment of uniform acoustic conditions; see also [START_REF] Farina | Avian soundscapes and cognitive landscapes: theory, application and ecological perspectives[END_REF]) of a taxon and are therefore informative ecological proxies [START_REF] Nummela | Eocene evolution of whale hearing[END_REF][START_REF] Nummela | Sound transmission in archaic and modern whales: anatomical adaptations for underwater hearing[END_REF]. Figure 2 presents a mechanical dissection of the middle ear of UM-KPG-M 73 through a set of correlations between different links in the sound transmission chain, from the sound input area (sound wave entrance to the middle ear; see [START_REF] Nummela | Eocene evolution of whale hearing[END_REF][START_REF] Nummela | Sound transmission in archaic and modern whales: anatomical adaptations for underwater hearing[END_REF]) to the fenestra vestibuli (sound wave entrance to the inner ear). Figure 2a,b represents the distribution of incudal mass (figure 2a) values and fenestra vestibuli area (figure 2b), respectively, relative to sound input area for fully terrestrial mammals and phocids (sound input area = tympanic membrane surface) and for cetaceans (sound input area = tympanic plate surface, following [START_REF] Nummela | Eocene evolution of whale hearing[END_REF][START_REF] Nummela | Sound transmission in archaic and modern whales: anatomical adaptations for underwater hearing[END_REF][START_REF] Nummela | Scaling of the cetacean middle ear[END_REF]). According to both plots, based on its tympanic membrane surface, UM-KPG-M 73 lies slightly above the terrestrial mammals' morphospace, whereas it lies within the odontocetes' morphospace according to its tympanic plate area, suggesting optimal hearing abilities both in air and underwater. Interestingly, based on the measurements provided by Nummela et al. [START_REF] Nummela | Sound transmission in archaic and modern whales: anatomical adaptations for underwater hearing[END_REF], Pakicetus follows the same pattern (figure 2a) and lies on the edge of the terrestrial's morphospace (tympanic membrane area), close to UM-KPG-M 73, and within the odontocetes' morphospace (tympanic plate area).This suggests that Pakicetus also had middle ear proportions suited to hear in air and underwater. As already shown in previous studies [START_REF] Nummela | Eocene evolution of whale hearing[END_REF][START_REF] Nummela | Sound transmission in archaic and modern whales: anatomical adaptations for underwater hearing[END_REF], based on their tympanic plate surface, the basilosaurids Zygorhiza and Basilosaurus are found within the cetaceans' morphospace, between odontocetes and mysticetes. Yet, no data are available for the tympanic membrane surface of basilosaurid and their aerial hearing abilities could not be investigated here. Within the ossicular chain and at the end of the sound transmission pathway (figure 2c-e), UM-KPG-M 73 shows a rather terrestrial signature. According to the relative mass of the incus and stapes (figure 2c), it lies within the terrestrial's morphospace, halfway between artiodactyls and cetaceans. When the mass of the stapes relative to the fenestra vestibuli area is considered, UM-KPG-M 73 is found between the terrestrial and modern cetaceans' morphospaces (figure 2d). In addition, according to the relative proportion of the length of the two lever arms of the incudomallear complex [START_REF] Hemilä | What middle ear parameters tell about impedance matching and high frequency hearing[END_REF], the protocetid lies within the terrestrial morphospace, where it is located halfway between artiodactyls and phocids (figure 2e). Modern cetaceans possess a different lever mechanism [START_REF] Nummela | The anatomy of the killer whale middle ear (Orcinus orca)[END_REF][START_REF] Hemilä | A model of the odontocete middle ear[END_REF][START_REF] Hemilä | Anatomy and physics of the exceptional sensitivity of dolphin hearing (Odontoceti: Cetacea)[END_REF] and there are, to our knowledge, no available data in the literature to directly compare this parameter with that of terrestrial mammals. Nevertheless, according to our measurements of the two lever arms on the incudomallear complex of D. capensis, this odontocete is found out of the terrestrial mammals' morphospace (figure 2e). Finally, for comparison purposes, the geometric transformer ratio (sensu [START_REF] Hemilä | What middle ear parameters tell about impedance matching and high frequency hearing[END_REF]; see 'Material and methods' and also [START_REF] Mason | Structure and function of the mammalian middle ear. II: Inferring function from structure[END_REF][START_REF] Koyabu | Into the dark: patterns of middle ear adaptations in subterranean eulipotyphlan mammals[END_REF]) of UM-KPG-M 73 is 18.7, which is lower than most terrestrial mammals (see electronic supplementary material, table S1) and close to that of phocids (transformer ratio between 19.20 and 22.35) and human (transformer ratio = 21.38). S1. The grey dotted lines outline the morphospace of terrestrial mammals and that of neocetes. Blue silhouettes, pelagicetes; dark blue dots, mysticetes; light blue dots, odontocetes; navy blue dots or lines, basilosaurids (including at least Basilosaurus cetoides (LSUMG V1)); dark green, non-cetaceans artiodactyls; light green, terrestrial mammals excluding artiodactyls; orange, Pakicetus (H-GSP 91035); purple, phocids; red, protocetid indeterminate γ (UM-KPG-M 73). Illustrations by R. Mourlam. The results of these five analyses are summed up in the functional profile of the middle ear of protocetid indeterminate γ (UM-KPG-M 73) at the centre of this figure. Each pair of links is symbolized by a circle. The asterisk next to the circle between the malleus and the incus refers to the corresponding graph (e). The double circles stand for the double sound input area (tympanic membrane and tympanic plate). In green, functional aerial hearing; in cyan, functional underwater hearing. Damaged malleus is symbolized in grey (see also electronic supplementary material, figure S9). (Online version in colour.)

Discussion

Early evolutionary history of the ossicular chain in Cetacea

The preservation of a partially complete protocetid ossicular chain represents a unique opportunity to document the sound transmission pathway in a non-fully aquatic cetacean and to refine the current knowledge on their early evolutionary history. The stapes is morphologically close to terrestrial artiodactyls and does not show the pachyostosis observed in fully aquatic cetaceans (basilosaurids and neocetes). Likewise, the incus body is only slightly inflated and shows proportions similar to Pakicetus or modern hippos. On the other hand, the preserved portions of the malleus indicate that it was composed of a massive pachyostotic head, morphologically close to the basilosaurid condition. Hence, the stapes and the incus do not show the same bulkiness as the malleus, which goes against the hypothesis postulated by Fleischer [START_REF] Fleischer | Evolutionary Principles of the Mammalian Middle Ear[END_REF] and Lancaster [START_REF] Lancaster | The middle ear of the Archaeoceti[END_REF] that the three ossicles acquired cetacean characters (among which pachyostosis) all together, as a single evolutionary unit. The decoupling of acquisition of derived functional characters between the different links of the ossicular chain is illustrated in figure 3, which shows, in a phylogenetic context, the repartition of morphological characters for the three ossicles, at the Cetancodonta [START_REF] Árnason | The mitochondrial genome of the sperm whale and a new molecular reference for estimating Eutherian divergence dates[END_REF][START_REF] Árnason | Whole-genome sequencing of the blue whale and other rorquals finds signatures for introgressive gene flow[END_REF][START_REF] Zurano | Cetartiodactyla: updating a time-calibrated molecular phylogeny[END_REF] scale. The ossicular chain of UM-KPG-M 73 indeed shows a mosaic of plesiomorphic and derived characters, with a pachyostotic malleus (first link of the chain) and no to little pachyostosis of the incus and stapes (second and last link of the chain). The fully pachyostotic condition of the three ossicles is observed in basilosaurids [START_REF] Pompeckj | Das Ohrskelett von Zeuglodon[END_REF][START_REF] Lancaster | The middle ear of the Archaeoceti[END_REF][START_REF] Uhen | Form, function, and anatomy of Dorudon atrox (Mammalia, Cetacea): an archaeocete from the Middle to Late Eocene of Egypt[END_REF] and neocetes [START_REF] Kellogg | Kentriodon pernix, a Miocene porpoise from Maryland[END_REF][START_REF] Kellogg | Two additional Miocene porpoises from the Calvert Cliffs, Maryland[END_REF][START_REF] Kellogg | Fossil marine mammals from the Miocene calvert formation of Maryland and Virginia, parts 5-8[END_REF][START_REF] Kellogg | Cetothere skeletons from the Miocene choptank formation of Maryland and Virginia[END_REF][START_REF] Westgate | Balaena ricei, a new species of bowhead whale from the Yorktown formation (Pliocene) of Hampton, Virginia. In Cenozoic mammals of land and Sea: tributes to the career of clayton[END_REF][START_REF] Bianucci | Arimidelphis sorbinii a new small killer whale-like dolphin from the Pliocene of Marecchia river (Central Eastern Italy) and a phylogenetic analysis of the Orcininae (Cetacea: Odontoceti)[END_REF][START_REF] Bianucci | A new delphinid record (Odontoceti, Cetacea) from the Early Pliocene of Tuscany (Central Italy): systematics and biostratigraphic considerations[END_REF][START_REF] Bouetel | The anatomy and relationships of Piscobalaena nana (Cetacea, Mysticeti), a Cetotheriidae s.s. from the early Pliocene of Peru[END_REF][START_REF] Bosselaers | A new fossil rorqual (Mammalia, Cetacea, Balaenopteridae) from theEarly Pliocene of the North Sea, with a review of the rorqual species described by Owen and Van Beneden[END_REF][START_REF] Marx | A new Early Oligocene toothed 'baleen' whale (Mysticeti: Aetiocetidae) from western North America: one of the oldest and the smallest[END_REF][START_REF] Boessenecker | A new eomysticetid from the Oligocene Kokoamu Greensand of New Zealand and a review of the Eomysticetidae (Mammalia, Cetacea)[END_REF][START_REF] Lambert | Yaquinacetus meadi, a new latest Oligocene-early Miocene dolphin (Cetacea, Odontoceti, Squaloziphiidae, fam. nov.) from the Nye Mudstone (Oregon, USA)[END_REF] at the Pelagiceti node, and could be related to strictly underwater hearing in cetes. The early evolutionary history of the cetacean's auditory region is also marked by the rotation of the ossicular chain [START_REF] Thewissen | Origin of underwater hearing in whales[END_REF][START_REF] Lancaster | The middle ear of the Archaeoceti[END_REF][START_REF] Fleischer | Evolutionary Principles of the Mammalian Middle Ear[END_REF][START_REF] Fleischer | Structural analysis of the Tympanicum complex in the bottlenosed dolphin (Tursiops truncatus)[END_REF]. Despite damage on the tympanic bulla, petrosal and malleus of UM-KPG-M 73 (see 'Description and comparisons' and electronic supplementary material, text S1), some anatomical elements of the incus provide evidence that the rotation of the incudomallear complex was similar to that of Pakicetus [START_REF] Thewissen | Origin of underwater hearing in whales[END_REF] and intermediate between fully terrestrial artiodactyls and cetaceans. Indeed, compared to terrestrial artiodactyls, the two crura of the incus of UMKPG-M 73 show a rotated position relative to the articular surface (figure 3, character I3, turquoise state) and a change of their relative proportions (figure 3, character I2, turquoise state). The rotation of the incudomallear complex is also accompanied by drastic changes of body proportions of the malleus (figure 3, character M1) and of the shape of its manubrium (figure 3, character M2). The malleus of UM-KPG-M 73 is intermediate in terms of body proportions and similar to some modern cetaceans in terms of manubrium size (e.g. Balaenoptera musculus ( [START_REF] Lillie | Observations on the anatomy and general biology of some members of the larger Cetacea[END_REF], pl. 74) and Eubalaena australis ( [START_REF] Doran | XVIII. Morphology of the mammalian Ossicula auditûs[END_REF], pl. 63). In addition to these characters (which states vary within Cetacea), three others are common to all cetaceans of our sample: a large difference in size between the superior and inferior articular facets of the malleus (figure 3, character M3, blue state), a right angle between these two facets and a concave stapedial footplate (figure 3, character S2, blue state). It is noteworthy that among Artiodactyla, C. liberiensis (figure 3; electronic supplementary material, figures S5G and S8C) and probably Hippopotamus amphibius ( [START_REF] Doran | XVIII. Morphology of the mammalian Ossicula auditûs[END_REF], pl. 61) present the same 'cetacean' structural organization of their incudomallear articular surface, indicating either an amphibious hearing signal (functional convergence) or a potential osteological synapomorphy for Cetancodonta [START_REF] Árnason | The mitochondrial genome of the sperm whale and a new molecular reference for estimating Eutherian divergence dates[END_REF][START_REF] Zurano | Cetartiodactyla: updating a time-calibrated molecular phylogeny[END_REF][START_REF] Boisserie | Evolving between land and water: key questions on the emergence and history of the Hippopotamidae (Hippopotamoidea, Cetancodonta, Cetartiodactyla)[END_REF]. Contrary to previous hypotheses, we show here that the ossicular chain of UM-KPG-M 73 does not act as a single morphological module [START_REF] Wagner | The biological role of homologues: a building block hypothesis[END_REF][START_REF] Wagner | Homologues, natural kinds and the evolution of modularity[END_REF][START_REF] Eble | Modularity: understanding the development and evolution of natural complex systems[END_REF] within the middle ear. The mosaic pattern of character states (figure 3) shows a relative independence of evolutionary tempo and mode (sensu [START_REF] Simpson | Tempo and mode in evolution[END_REF]) between the different ossicles, with a rather plesiomorphic stapes and a more derived malleus. This morphological modularity can be explained by the fact that the stapes derives from the hyoid arch (second pharyngeal arch), whereas the incudomallear complex originates from the mandibular arch (first pharyngeal arch) [START_REF] Sienknecht | Developmental origin and fate of middle ear structures[END_REF][START_REF] Burford | Early development of the malleus and incus in humans[END_REF]. In addition, the incus and malleus derive from two different b one units, the quadrate and articular, respectively [START_REF] Fleischer | Evolutionary Principles of the Mammalian Middle Ear[END_REF][START_REF] Reichert | Ueber die Visceralbogen der Wirbelthiere im Allgemeinen und deren Metamorphosen bei den Vögeln und Säugethieren[END_REF][START_REF] Luo | Transformation and diversification in early mammal evolution[END_REF][START_REF] Luo | Developmental patterns in Mesozoic evolution of mammal ears[END_REF][START_REF] Maier | Evolution of the mammalian middle ear: a historical review[END_REF]. Moreover, even within a single ossicle, a combination of derived and plesiomorphic character states is always observed (i.e. different modules can be identified within the same ossicle; figure 3). At that level of integration (i.e. intra-bone), data on the development of the mammalian middle ear are rather limited and mainly rely on mice [START_REF] Anthwal | The development of the mammalian outer and middle ear[END_REF]. However, two modules have been identified within the stapes of these rodents: the outer edge of the footplate derives from the otic capsule (mesoderm origin), whereas the rest of this ossicle comes from the neural crest [START_REF] Anthwal | The development of the mammalian outer and middle ear[END_REF][START_REF] Thompson | The origin of the stapes and relationship to the otic capsule and oval window[END_REF]. These different developmental origins could explain the mosaic pattern of characters of the protocetid stapes.

Figure 3. Evolution of some characters of the ossicular chain components, malleus (M), incus (I) and stapes (S), character states of terrestrial artiodactyls are in pink, character states found in fully aquatic cetaceans are in blue and intermediate states are in turquoise. Character list: S1, crura pachyostosis ( pink, absent; blue present); S2, stapedial footplate ( pink, convex or flat; blue, concave); I1, inflation of the body ( pink, no inflation; turquoise, moderate; blue, important); I2, relative size of crus longum versus crus breve ( pink, crus breve bigger; turquoise, crus breve smaller; blue, crus breve much smaller); I3, reorientation of crura relative to the articular surface ( pink, no rotation; turquoise, intermediate rotation of the crura; blue, full rotation of the crura); M1, proportions ( pink, body longer; turquoise, body and head of relative similar length; blue, head longer); M2, manubrium shape ( pink, long process; blue, from a hook-like process to a vestigial site of insertion for the tympanic ligament); M3, relative size of articular facets ( pink, subequal; blue, inferior articular facet smaller than the superior). CB, crus breve; CL, crus longum. Question marks represent missing data and white boxes represent character state ambiguity related to missing data for Pakicetus. Ossicles not to scale. Megaptera novaeangliae malleus, stapes (UBC-cet 416B 16-4358) and incus after Bosselaers & Post ([49], fig. 12). Information related to other specimens illustrated is provided in the 'Material and methods' section and in the anatomical plates of the electronic supplementary material. (Online version in colour.)

Functional profile of the protocetid amphibious middle ear

Every medium has its own resistance to sound transmission. This resistance is called the acoustic impedance (Z), and can be quantified as the ratio between sound pressure ( p) and particle velocity (v) (i.e. Z = p/v) [START_REF] Nummela | Sound transmission in archaic and modern whales: anatomical adaptations for underwater hearing[END_REF][START_REF] Hemilä | What middle ear parameters tell about impedance matching and high frequency hearing[END_REF][START_REF] Nummela | The physics of sound in air and water[END_REF]. At the interface between two different media, the less their acoustic impedance differs, the better the sound is transmitted. The hearing mechanism consists in sound transmission between the external environment, where sound waves are born, and the internal environment of the inner ear, where sound waves are transduced into electrical signals transmitted to the brain. The acoustic impedance of the fluid-filled cochlea of the inner ear is about 10 times lower than that of water and 375 times higher than that of air [START_REF] Nummela | Hearing[END_REF][START_REF] Hemilä | Anatomy and physics of the exceptional sensitivity of dolphin hearing (Odontoceti: Cetacea)[END_REF][START_REF] Nummela | Hearing in aquatic mammals[END_REF]. These large differences of acoustic impedance between the soundtope and the inner ear prevent a direct transmission of sound (most of the acoustic energy would be lost at their interface through sound reflection; see [START_REF] Hemilä | Anatomy and physics of the exceptional sensitivity of dolphin hearing (Odontoceti: Cetacea)[END_REF][START_REF] Nummela | Hearing in aquatic mammals[END_REF]). In tetrapods, the middle ear plays an essential role in hearing by minimizing the acoustic impedance mismatch between the soundtope and the inner ear [START_REF] Hemilä | What middle ear parameters tell about impedance matching and high frequency hearing[END_REF][START_REF] Koyabu | Into the dark: patterns of middle ear adaptations in subterranean eulipotyphlan mammals[END_REF][START_REF] Burda | The ear in subterranean insectivora and rodentia in comparison with ground-dwelling representatives. I. Sound conducting system of the middle ear[END_REF][START_REF] Hetherington | Comparative anatomy and function of hearing in aquatic amphibians, reptiles, and birds[END_REF]. The sound pressure depends mainly on the ratio between the sound input area (tympanic membrane or tympanic plate) and the surface of the fenestra vestibuli, while the particle velocity relies on different lever arms [START_REF] Hemilä | What middle ear parameters tell about impedance matching and high frequency hearing[END_REF][START_REF] Hemilä | Anatomy and physics of the exceptional sensitivity of dolphin hearing (Odontoceti: Cetacea)[END_REF][START_REF] Koyabu | Into the dark: patterns of middle ear adaptations in subterranean eulipotyphlan mammals[END_REF][START_REF] Burda | The ear in subterranean insectivora and rodentia in comparison with ground-dwelling representatives. I. Sound conducting system of the middle ear[END_REF]. Thus, to match a low acoustic impedance soundtope (air) with the fluid-filled cochlea, the role of the middle ear will be to increase sound pressure and decrease particle velocity. The contrary goes for matching a high acoustic impedance soundtope (water) with the inner ear [START_REF] Nummela | Hearing[END_REF][START_REF] Nummela | The physics of sound in air and water[END_REF]. Consequently, at first glance, it seems counterintuitive that a single sound transmission pathway could be tuned for both airborne and waterborne sounds (i.e. being able to increase and decrease simultaneously sound pressure). The assessment, for the first time, of a partially complete middle ear of a non-fully aquatic archaeocete enhances our understanding of amphibious hearing in early whales. The anatomical survey of the tympanic bulla of UM-KPG-M 73 highlights the presence of two potential sound input areas [START_REF] Mourlam | Protocetid (Cetacea, Artiodactyla) bullae and petrosals from the middle Eocene locality of Kpogamé, Togo: new insights into the early history of cetacean hearing[END_REF]: (i) the tympanic membrane, collecting sound from air; (ii) the tympanic plate, for underwater sound. This duality of potential sound input area provides two independent possibilities to regulate sound pressure through the sound transmission pathway within the middle ear. In air, the ear of UM-KPG-M 73 functioned the same way as terrestrial mammals. The tympanic membrane area is broader than that of the fenestra vestibuli, which implies an increase in sound pressure within the sound transmission chain. In parallel, the lever ratio between the mallear and incudal lever arms of UM-KPG-M 73 is typical of that of land mammals (figure 2e) [START_REF] Hemilä | What middle ear parameters tell about impedance matching and high frequency hearing[END_REF], indicating a decrease in particle velocity. By contrast, the geometric transformer ratio (sensu [START_REF] Hemilä | What middle ear parameters tell about impedance matching and high frequency hearing[END_REF]) of UMKPG-M 73 is lower than that of most land mammals and similar to that of phocids (see electronic supplementary material, table S1), probably in relation with their amphibious lifestyle. Underwater, the ear of UM-KPG-M 73 functioned in a way close to modern cetaceans. The surface of the tympanic plate is broader than that of the fenestra vestibuli, which increases sound pressure within the middle ear. However, as explained above, sound pressure should be decreased to avoid impedance mismatch between water and the cochlea. Thus, to compensate the sound pressure increase induced by the tympanic plate surface, particle velocity should be even more increased. Hemilä et al. [START_REF] Hemilä | Anatomy and physics of the exceptional sensitivity of dolphin hearing (Odontoceti: Cetacea)[END_REF] propose two lever mechanisms involving the tympanic bulla and the ossicular chain that would be able to increase particle velocity within modern cetacean's middle ear. Unfortunately, in UM-KPGM 73, the gonial and part of the tympanic plate are broken, and it is not possible to quantify these parameters. Nevertheless, based on strong morphological similarities and equivalent sound pressure ratio (figure 2b), the middle ear of UM-KPG-M 73 probably performed an efficient acoustic impedance matching between the aquatic soundtope and the inner ear, such as that of modern odontocetes. This mosaic functional profile of the middle ear is also observed in phocids (figure 2; electronic supplementary material, figure S9) that are able to hear efficiently in both aquatic and aerial soundtopes [START_REF] Kastak | Low-frequency amphibious hearing in pinnipeds: methods, measurements, noise, and ecology[END_REF][START_REF] Kastak | In-air and underwater hearing sensitivity of a northern elephant seal (Mirounga angustirostris)[END_REF][START_REF] Wartzok | Marine mammal sensory systems[END_REF][START_REF] Hemilä | High-frequency hearing in phocid and otariid pinnipeds: an interpretation based on inertial and cochlear constraints[END_REF][START_REF] Sills | Amphibious hearing in spotted seals (Phoca largha): underwater audiograms, aerial audiograms and critical ratio measurements[END_REF][START_REF] Sills | Amphibious hearing in ringed seals (Pusa hispida): underwater audiograms, aerial audiograms and critical ratio measurements[END_REF][START_REF] Loza | Adaptations to a semiaquatic lifestyle in the external ear of southern pinnipeds (Otariidae and Phocidae, Carnivora): morphological evidences[END_REF]. Although phocids and UM-KPG-M 73 present different morphological adaptations towards amphibious hearing (such as a strong increase in incudal mass for the former [START_REF] Nummela | Hearing in aquatic mammals[END_REF] and a potential double sound input area for the latter), their middle ears' functional profiles are very similar. The patchwork of functional signals observed in these two phylogenetically distant groups appears to be a hallmark of the mammalian amphibious ear.

Summary and conclusions

The anatomical and functional studies of the first partially complete protocetid middle ear support the amphibious hearing signal found with the cochlea of its inner ear [START_REF] Mourlam | Infrasonic and ultrasonic hearing evolved after the emergence of modern whales[END_REF]. Functional analyses of the different links of the sound transmission pathway within the middle ear highlight a combination of aerial and aquatic signals (figure 2; electronic supplementary material, figure S9). This indicates that the ossicular chain is able to process the sound coming from the two acoustic portals of the bulla (tympanic membrane and tympanic plate) and to transmit sound waves efficiently to the inner ear. This functional duality is tightly linked with the morphology of the three ossicles that present a mosaic of plesiomorphic (terrestrial artiodactyl) and derived (fully aquatic cetacean) character states (figures 1 and 3). Indeed, the morphological modularity of the ossicular chain increases the adaptability of the protocetid's hearing organ [START_REF] Koyabu | Into the dark: patterns of middle ear adaptations in subterranean eulipotyphlan mammals[END_REF][START_REF] Wagner | The biological role of homologues: a building block hypothesis[END_REF][START_REF] Wagner | Homologues, natural kinds and the evolution of modularity[END_REF][START_REF] Eble | Modularity: understanding the development and evolution of natural complex systems[END_REF][START_REF] Sienknecht | Developmental origin and fate of middle ear structures[END_REF], and thus makes it possible for a unique middle ear to be in tune with two soundtopes with drastically different physico-acoustic properties.

Science Center) for collecting and preparing the middle ear of Delphinus and to M. Morell (Institute for Neurosciences of Montpellier, France-Inserm Unit 1051) for providing us with the scan of this specimen along with that of Megaptera. We also acknowledge S. Raverty (Animal Health Center, Canada), who helped M. Morell extracting the ear of Megaptera. We are grateful to M. Bouchet, C. Charles and J. Martin for access to the AniRA-ImmOs (SFR Biosciences Gerland-Lyon) microtomography facility. We acknowledge the MRI platform member of the national infrastructure France-BioImaging supported by the French National Research Agency (ANR-10-INBS-04, 'Investments for the future'), the labex CEMEB (ANR-10-LABX-0004) and NUMEV (ANR-10-LABX-0020). We are also very grateful to P.-O. Antoine for his comments and to R. Mourlam for her help in improving the English of the manuscript, along with her illustrations of archaeocetes and other mammals. We also thank the three anonymous reviewers, who greatly helped to improve this article. This is ISE-M publication no. ISEM 2019-137.

Figure 1 .

 1 Figure 1. Three-dimensional models of the partially complete left ossicular chain of protocetid indeterminate γ (UM-KPG-M 73). (a) Location of the ossicular chain preserved within the middle ear cavity in the anterior view (red, stapes; blue, incus; orange, malleus; bony transparent elements, petrosal on top and tympanic bulla underneath). (b-e) Stapes in, from left to right, medial, posterior, dorsal and ventral views. ( f-i) Incus in, from left to right, ventral, dorsal, anterior and lateral views. ( j-k) Malleus in ( j ) dorsal and (k) anterior views. The orientation of incus and malleus based on a spatial 'incudomallear-centred' referential (see electronic supplementary material, text S1 and figure S1). ac, anterior crus; br, broken or incomplete structure; cb, crus breve; cl, crus longum; fic, foramen intercrurale; iaf, inferior articular facet; ib, incudal body; in, incus; lct, lodge of the lateral extension of the chorda tympani; mn, manubrium; mp, muscular process of malleus; pc, posterior crus; pm, processus muscularis stapedis; saf, superior articular facet; sf, stapedial footplate; sh, head of stapes. Scale bar in (a), 1 cm; other scale bars, 1 mm. (Online version in colour.).

Figure 2 .

 2 Figure 2. Mechanical dissection of the middle ear of protocetid indeterminate γ (UM-KPG-M 73). Set of bivariate plots: (a) incudal mass versus sound input area; (b) fenestra vestibuli area versus sound input area; (c) stapedial mass versus incudal mass; (d ) stapedial mass versus fenestra vestibuli area; (e) incudal lever arm length versus mallear lever arm length. Measurements are available in electronic supplementary material, tableS1. The grey dotted lines outline the morphospace of terrestrial mammals and that of neocetes. Blue silhouettes, pelagicetes; dark blue dots, mysticetes; light blue dots, odontocetes; navy blue dots or lines, basilosaurids (including at least Basilosaurus cetoides (LSUMG V1)); dark green, non-cetaceans artiodactyls; light green, terrestrial mammals excluding artiodactyls; orange, Pakicetus (H-GSP 91035); purple, phocids; red, protocetid indeterminate γ (UM-KPG-M 73). Illustrations by R. Mourlam. The results of these five analyses are summed up in the functional profile of the middle ear of protocetid indeterminate γ (UM-KPG-M 73) at the centre of this figure. Each pair of links is symbolized by a circle. The asterisk next to the circle between the malleus and the incus refers to the corresponding graph (e). The double circles stand for the double sound input area (tympanic membrane and tympanic plate). In green, functional aerial hearing; in cyan, functional underwater hearing. Damaged malleus is symbolized in grey (see also electronic supplementary material, figureS9). (Online version in colour.)

Acknowledgements. We are especially grateful to J. G. M. Thewissen (NEOMED) for access to 3D models of the ossicles of Pakicetus and Basilosaurus, to S. Usip and D. A. Waugh from NEOMED for the scans of these specimens, to K. Danil (NOAA-Southwest Fisheries

Funding. This study was supported by the ANR/ERC funding project PALASIAFRICA, headed by L. Marivaux; the ANR programme SPLASH (no. ANR-15-CE32-0010-01), headed by J.-R. Boisserie and F. Lihoreau; and the CeMEB project PygmHippoCom (coPi M. J. Orliac).

Data accessibility.

Additional data relating to this study are available from the Dryad Digital Repository: https://dx.doi.org/10.5061/ dryad.0k3fv2j [START_REF] Mourlam | Data from: Early evolution of the ossicular chain in Cetacea: into the middle ear gears of a semi-aquatic protocetid whale[END_REF]. Three-dimensional models of the three ossicles and the tympanic bulla of UM-KPG-M 73 are available at: https:// morphomuseum.com/ (tympanic bulla, M3#134_UM KPG-M 73; stapes, M3#407_UM KPG-M 73; incus, M3#408_UM KPG-M 73; malleus, M3#409_UM KPG-M 73).

Authors' contributions. M.J.M. and M.J.O. conceived and designed the study. M.J.O. performed the data segmentation and M.J.M. performed the functional analyses. Both authors wrote the manuscript.

Competing interests. We declare we have no competing interests.