H Maury

Philippe Jonnard

K Le Guen

J.-M André

Z Wang

J Zhu

Jia Dong

Zhen Zhang

Françoise Bridou

Franck Delmotte

C Hecquet

N Mahne

A Giglia

S Nannarone

Thermal cycles

Keywords: . 68.65.Ac Multilayers -61.05.cm X-ray reflectometry (surfaces, interfaces, films) -78.70.En X-ray emission spectra and fluorescence -73.90.+f Other topics in electronic structure and electrical properties of surfaces, interfaces, thin films, and low-dimensional structures -66.30.Ny Chemical interdiffusion, diffusion barriers -68.35.Fx Diffusion, interface formation

published or not. The documents may come   L'archive ouverte pluridisciplinaire

Introduction

Periodic multilayers consisting of an alternating and periodic succession of optically dense and less dense films are widely used nowadays as diffractive optical elements in the X-UV range [START_REF] Yanagihara | Multilayers for Soft and Hard X-rays[END_REF]. For the 30 nm region, the Mo/Si system has been commonly employed because of its high time and thermal stability [START_REF] Yoshikawa | [END_REF]3]. Its reflectivity ranges from 15 to 20% in the 25-35 nm spectral range. However for applications like solar imaging or photoemission microscopy using the He II line (30.4 nm, 40.7 eV) multilayers with high reflecting power are needed. Thus Si/Mo 2 C, Si/B 4 C, Si/C and Si/SiC multilayers have been developed for this spectral range [4]. The Si/B 4 C system features a good reflectivity but poor temperature and time stabilities, while Si/SiC was found inadequate due to a measured reflectivity of only 16%. It has been proposed to replace silicon by magnesium in multilayers because of the low absorption coefficient of the Mg atoms after the L absorption edge at 25 nm (50 eV). Ejima et al. have proposed Mg/SiC multilayers and measured a reflectivity of 20% at 30.4 nm [5].

More recently, Takenaka et al. have obtained a reflectivity as high as 40% with the Mg/SiC multilayer at the same wavelength [6].

Because of its recent development, the Mg/SiC system has been little characterized. For example, with respect to Mo or Si, Mg is highly reactive and has a relatively low melting point (649 • C) [START_REF] Caldarelli | Materials Handbook[END_REF][START_REF] Veena Kumari | [END_REF][9][10]. These properties could lead to a difficulty in controling the time and thermal stability a On leave to CEA.

Correspondence to: Dr. P. Jonnard; jonnard@ccr.jussieu.fr of the Mg/SiC multilayers, despite their high reflectivity.

It is thus important to investigate the phenomena that take place at the interfaces and limit the performances of the system, in order to understand how interdiffusion and roughness develop as a function of the annealing. It

Experimental details

The Mg/SiC multilayers, consisting of 30 bilayers, were deposited onto silicon substrates by an ultrahigh vacuum direct current magnetron sputtering system with four 100mm-diameter sputtering sources (JGP560C, made in China).

The base pressure of this sputtering system reaches ∼ 1 × 10 -4 Pa before deposition. The working gas is argon (99.999% purity) at the constant pressure of 0.1 Pa.

The sputtering sources work at the constant power mode, regulated at 10 W and 60 W for Mg and SiC targets, re-spectively. Substrates in size of 20 mm × 30 mm were cut from a polished single-crystal Si (100) wafer. During the deposition of each layer, the substrate was kept stationary over the sputtering source for the time required to deposit the desired thickness. The multilayer was fabricated by alternate facing the substrate in front of each target.

The deposited multilayer was then measured, for quality control and depositing rate control, by using a small angle x-ray diffractometer (D1 system, Bede Ltd., UK). The measurements show that the layer thicknesses of Mg and SiC are 12.1 nm and 4.7 nm, respectively, see table 1. Some samples are annealed during 1.25 h (this does not include the times for increasing and decreasing the temperature) under argon atmosphere or in vacuum in the preparation chamber of the XES apparatus. The annealing temperatures range from 100 to 500 • C with a 50 • C step. For the samples dedicated to the XRR analysis at 0.154 nm the temperature step is 100 • C. The samples are analysed by specular optical reflectivity at three different wavelengths ranging from hard x-rays to ultra-violet. XRR at 0.154 nm (Cu Kα emission at 8048 eV) is performed with a home-made apparatus [15] for the annealed samples. All the multilayers are analysed by XRR at 1.33 nm (Cu Lα emission at 932 eV) with a home-made spectrogoniometer [16].

For the as-prepared samples, UVR measurements at ∼30.4 nm were taken in θ -2θ mode in the 5 • -35 • range in s-incidence at BEAR beamline [START_REF] Nannarone | AIP Conf. Proc[END_REF] at Elettra. The pho- with a high-resolution wavelength dispersive spectrometer [START_REF] Bonnelle | [END_REF]. The x-rays are produced by electron irradiation.

In the case of the analysis of the Si emission band, the electron energy (4 keV) is carefully chosen so that no signal can come from the silicon substrate. No evolution of the intensity and lineshape of the emission bands is detected with the number of accumulations, indicating that the electron irradiation conditions have no significant effect on the sample. SiC samples present differences: for the crystalline powder the main peak is narrower and the structure around 1827 eV is better resolved. The high energy broadening could be ascribed to the presence of occupied defect states in the forbidden gap of the amorphous sample, the slight low energy narrowing to the difference between the crystalline and amorphous states and the poor resolution of the 1827 eV structure for the amorphous samples to the fact that the stoichiometry of the film is not exactly that of SiC [21].

Results and discussion

The spectra of the as-prepared multilayers are shown in figure 2 in comparison with Mg metal for the Mg Kβ emission (Fig. 2a) and with crystalline SiC for the Si Kβ emission (Fig. 2b). For the sake of clarity, only a limited number of references is displayed. It is clear, within the sensitivity of the method, that the magnesium atoms in the multilayer are in the metallic state and the silicon ones are on the carbide state, i.e. there is no interaction or diffusion between the Mg and SiC layers.

The XRR obtained at 0.154 nm (8048 eV) with the asprepared sample is shown on figure 3a. The Bragg peaks are narrow, intense and well-defined up to the fifth diffraction order. Some secondary peaks are also observed between the Bragg peaks. We suggest that their existence is due to a small defect or super-structure in the SiC layers, every two periods. Based on indications from XES, a fit of the experimental XRR curves is made using a two units period. Fitted parameters, i.e. the thickness and density of the Mg and SiC layers and interface roughness, deduced using the IMD code [22] are given in table 1. It can be seen that the rms interfacial roughness is quite large, specially for the Mg layers and that the density of the SiC layers is that of the bulk SiC whereas the Mg layers have a density slightly smaller than the one of the Mg metal.

In figure 4 the UV reflectivity at 30.4 nm (40.7 eV) obtained in a θ -2θ scan for an as-prepared sample is shown. The simulated reflectivity for the corresponding ideal structure with thickness parameters of table 1 is reported. The comparison with the experimental curve shows a higher reflectivity of the order of 20%. A much better agreement with the experiment is obtained when the interface roughnesses reported in table 1, as obtained from XRR measurements at hard x-ray energy, are intro-duced into the simulation. It is important to stress that, in agreement with the indication coming from XES (i.e. absence of important interdiffusion or chemical reaction at the interfaces), the optical performance of as-prepared multilayers is substantially limited by interface roughness.

Annealed samples

We present the Mg Kβ and Si Kβ x-ray analysis of the annealed Mg/SiC multilayers in figure 5 where the lineshape of Mg 2 Si presents its maximum (see Fig. 1). At 400 • C this shoulder is well developed and its maximum is slightly shifted toward the low photon energies, consistent with the shift between the maxima of pure Mg and Mg 2 Si spectra. Then, there is no more evolution up to 500 • C. The silicon emission band, figure 5b, shows that at this highest temperature the silicon atoms are in a chemical environment like the one of Mg 2 Si. This behavior of the magnesium and silicon atoms evidences the formation of the magnesium silicide at the various interfaces of the stack. This is confirmed by fitting the Mg Kβ lineshape by a weighted sum (linear combination) of the reference spectra as shown in the figure 6. An equal contribution of pure Mg and Mg 2 Si is necessary to account for the shape of the spectrum of the sample after an annealing at 500 • C. This means that a part of the magnesium atoms has reacted with the silicon atoms from the SiC layers to form the silicide. This is consistent with the observation that the silicon atoms have reacted to form Mg 2 Si with the magnesium atoms of the Mg layers (Fig. 5b). It could be presumed that the shoulder appearing on the Mg spectrum of the highly annealed multilayers is due to an oxidation of the Mg atoms because the maximum of the MgO emission is at the same emission energy as the shoulder of the Mg 2 Si emission (see Fig. 1a). However, we can rule out this possibility because even when introducing some oxide contribution in the fit, it is not possible to account for the energy shift of the maximum and to improve the fit in the range of the shoulder.

The evolution of the reflectivity obtained at 1.33 nm The simulation of these reflectivity curves shows that the contrast of the refractive index decreases between the Mg and SiC layers when going from 200 to 300 • C. This can be explained by a development of the roughness or of the interdiffusion between the layers [23]. However, the latter can be ruled out because no chemical diffusion has been evidenced by XES. The absence of diffusion is also confirmed by looking at the value of the critical angle for the total reflection (Fig. 8a). It is almost the same (0.211 • ) for both annealing temperatures, meaning that the mean optical index and threfore the composition of the sample does not change. Thus, the evolution of the reflectivity at 0.154 nm between 200 and 300 • C can be explained by an increase of the interfacial roughness. This is probably due to the crystallization of the Mg layers and should be confirmed by diffraction measurements. When annealed at 400 • C, see figure 8b, it is observed that the Bragg peaks are less intense and become broader. The structure of the stack has almost disappeared due to the formation of the Mg 2 Si compound as evidenced by XES. This is also confirmed by the shift of the critical angle down to 0.174 • at 400 • C, showing that the mean index and thus the composition of the multilayer has changed.

Conclusion

We studied the thermal stabilty of periodic Mg/SiC multilayers up to 500 • C by the combination of XRR, UVR and XES techniques to show the possible thermally activated evolution of the system both through morphology and interface processes. We have shown that the as-prepared samples do not present interfacial compounds but have quite large interfacial rms roughness.

Upon annealing, a critical temperature for a strong decrease of the reflectivity has been reported in the literature, but the various authors do not agree on its value, This paper shows that the knowledge and control of the microscopic processes governing the interface chemistry and morphology are the key aspects to be improved in order to optimize the optical performances and the overall stability of Mg/SiC multilayers.

  may then be possible to obtain knowledge-based Mg/SiC structures both optimized for thermal and time stabilities. In this paper, we present the study of periodic Mg/SiC multilayers prepared by magnetron sputtering and designed for application at 30.4 nm (40.7 eV). They are analysed by a combination of non-destructive techniques [11-14]: xray reflectivity (XRR) in the hard and soft x-ray ranges, UV reflectivity (UVR) and x-ray emission spectroscopy (XES), in order to obtain structural and chemical informations about the Mg/SiC stack together with the monitoring of the optical performances in the working range. The study is performed for the sample obtained by magnetron sputtering and for samples annealed at temperatures up to 500 • C.

  ton energy was calibrated by a He gas absorption cell; the calibrated value resulted 40.70 eV (30.4 nm). The θ go-niometer angular resolution was 1 100 • . Energy resolution E ∆ , beam divergence and spot dimension of the incident beam at the sample were respectively: ∼1000, 20 mρ× 20 mρ (horizontal × vertical), 400µm × 200 µm (horizontal × vertical). Impinging and reflected intensities were measured by an IRD SXUV100 solid state diode within two separate runs; incident intensities were monitored by a Au mesh inserted in the beam path whose drain current was used for normalisation. The reflectometer features an overall accuracy on the absolute reflectivity of ∼1%. The x-ray spectra are measured with the IRIS (Instrument for Research on Interfaces and Surfaces) equipped

3. 1

 1 As-prepared samplesThe Mg 3p and Si 3p valence states are studied by XES through the Mg Kβ and Si Kβ emissions originating from the 3p -1s transition. In fact, the Mg and Si 3p valence states are very sensitive to the chemical state[19,20] of the Mg and Si atoms and give information about the com-pounds formed at the interfaces of the Mg/SiC multilayers. We compare the spectra from the multilayers to those from reference compounds: a Mg polished plate, a Mg 2 Si powder (from Alfa AEsar, 99,5% purity), a SiC crystalline powder (from Alfa AEsar, 99,8% purity), a 250 nm thick SiC film deposited in the same conditions as the SiC layers of the Mg/SiC stacks and a MgO single crystal. If no interaction takes place within the multilayer, one should observe the Mg lineshape of Mg in pure metal and that of Si in SiC. We present in figure 1 the spectra from the reference compounds that demonstrate the chemical dependence of the x-ray valence band emissions. The Mg Kβ spectra (figure 1a) of Mg and Mg 2 Si can be easily distinguished by their shape and the position of their maximum. In the same way, the Si Kβ spectrum (figure 1b) of Mg 2 Si is clearly distinct from those of the SiC samples. The two

  . Because it is not possible to acquire both emissions at the same time, the Si emission band is obtained only at 500 • C. It is observed in figure 5a that the Mg valence states are those of the metal up to a temperature of 300 • C. At 350 • C a slight shoulder begins to grow at a photon energy of 1296 eV

(

  figure 3a-f up to 500 • C. Five Bragg peaks can be observed.There is no significant evolution of the intensity and width

  200• C[5] or 300 • C[6]. In this work, we have found two critical temperatures for which we have observed an important change of the optical and chemical properties. The reflectivity is quite constant up to 200 • C after which it drops drastically, very probably due to the development of the interfacial roughness. Thus, this kind of multilayer is thermally stable only for application requiring no heating higher than 200 • C.A second critical temperature has been observed between 350 and 400 • C that corresponds to the destruction of the multilayer due to the formation of magnesium silicide resulting from the chemical reaction between the magnesium and silicon atoms. This leads to another very strong loss of reflectivity.

Fig. 1 .Fig. 2 .

 12 Photon energy (eV)

Fig. 3 .

 3 Fig. 3. Reflectivity curves at 0.154 nm (8048 eV) of the Mg/SiC multilayers: as-prepared (a), annealed at 100 • C (b), 200 • C (c), 300 • C (d), 400 • C (e) and 500 • C (f).

Fig. 4 .Fig. 5 .Fig. 6 .

 456 Fig.4. Reflectivity curve at 30.4 nm (40.7 eV) of the as-prepared Mg/SiC multilayer (dots) compared to simulated curves for a perfect structure (solid line) and for a structure with the parameters listed in the table 1 (dotted line).

Fig. 7 .

 7 Fig. 7. Evolution of the reflectivity at 1.33 nm (932 eV) of the Mg/SiC multilayer as a function of the annealing temperature.

Fig. 8 .

 8 Fig. 8. Reflectivity curves at 0.154 nm (8048 eV) of the Mg/SiC multilayer: (a) annealed at 200 (solid line) and 300 • C (dotted line); (b) annealed at 300 (solid line) and 400 • C (dotted line). The vertical bars indicate the critical angles for total reflection.

Table 1 .

 1 Parameters of the as-prepared Mg/SiC multilayer deduced from XRR at 0.154 nm. The relative density is the density of the layer divided by the density of the bulk corresponding material.

	layer	thickness (nm) relative density roughness (nm)
	Mg	12.1	0.94	1.7
	SiC	4.7	1.00	0.9

Acknowledgments : Pr. R. Gauvin from McGill University is thanked for providing us with the polished Mg sample.


Hsin Lee

Wei-Jen Chen

Nicolas Puillandre

Laetitia Aznar-Cormano

Mong- Hsun Tsai

Sarah Samadi

Incorporation of deep-sea and small-sized species provides new insights into gastropods phylogeny

Keywords: Deep sea, Gastropoda, Phylogeny, Mitogenome, Next Generation Sequencing, Chemosynthetic environment

The use of phylogeny with uneven or limited taxon sampling may bias our interpretation of organismal evolution, for instance, the origin(s) of the deep-sea animals. The Mollusca is the second most speciose phylum, in which the Gastropoda forms the largest group. However, the currently proposed hypotheses of gastropod phylogeny are mainly based on part of their taxonomic diversity, notable based on the large-sized and shallow-water species. In this study, we aimed at correcting this bias by reconstructing the phylogeny with new mitogenomes of deep-sea gastropods including Anatoma sp., Bathysciadiidae sp., Bayerotrochus teramachii, Calliotropis micraulax, Coccocrater sp., Cocculina subcompressa, Lepetodrilus guaymasensis, Peltospira smaragdina, Perotrochus caledonicus, Pseudococculinidae sp., and Shinkailepas briandi. This dataset provided the first reports of the mitogenomes for the Cocculiniformia, three vetigastropod superfamilies: Pleurotomarioidea, Lepetelloidea, and Scissurelloidea, and the neritimorph family Phenacolepadidae. The addition of deep-sea representatives also allowed us to evaluate the evolution of habitat use in gastropods. Our results showed a strongly supported sister-group relationship between the deep-sea lineages Cocculiniformia and Neomphalina. Within the Vetigastropoda, the Pleurotomarioidea was revealed as the sister-group of the remaining vetigastropods. Although this clade was presently restricted to the deep sea, fossil records showed that it has only recently invaded this habitat, thus suggesting that shallow waters was the ancestral habitat for the Vetigastropoda. The deep-sea Lepetelloidea and Lepetodriloidea formed a well-supported clade, with the Scissurelloidea sister to it, suggesting an early transition from shallow water to deep sea in this lineage. In addition, the switch between different chemosynthetic habitats was also observed in deep-sea gastropod lineages, notably in Neomphalina and Lepetelloidea. In both cases, the biogenic substrates appeared as the putative ancestral habitat, confirming the previously proposed hypothesis of a wooden-step to deep-sea vents scenario of evolution of habitat use for these taxa.

Introduction

The deep sea is the most extensive ecosystem on earth, but it is yet poorly studied [START_REF] Ramirez-Llodra | Deep, diverse and definitely different: unique attributes of the world's largest ecosystem[END_REF]. This vast area includes the water depths below the light zone (> 200 meters). The deep-sea floor has long been thought to be infertile [START_REF] Anderson | Deserts on the sea floor: Edward Forbes and his azoic hypothesis for a lifeless deep ocean[END_REF] and it is only with the great historical expeditions at the end of the 19 th century, notably the circumnavigations of the Challenger, that life was shown to be present and diverse at every depths. In the late seventies, the unexpected discovery of the hydrothermal vents revealed moreover a dense community of organisms living in very unusual environmental conditions and displaying original morphological and physiological traits [START_REF] Tunnicliffe | A biogeographical perspective of the deep-sea hydrothermal vent fauna[END_REF][START_REF] Van Dover | The Ecology of Deep-Sea Hydrothermal Vents[END_REF].

The complete exploration of deep-sea habitats is far from being achieved [START_REF] Higgs | Biases in biodiversity: wide-ranging species are discovered first in the deep sea[END_REF] and other habitats such as seamounts, cold seeps or biogenic substrates have been recently explored and revealed diversified communities [START_REF] Distel | Marine ecology -Do mussels take wooden steps to deep-sea vents?[END_REF][START_REF] Richer De Forges | Diversity and endemism of the benthic seamount fauna in the southwest Pacific[END_REF][START_REF] Smith | Ecology of whale falls at the deep-sea floor[END_REF]. Some animal lineages are restricted -or more diverse -in the deep sea but this diversity is poorly represented in the phylogenetic reconstruction of entire phyla, classes, orders or even families. The question of the evolutionary origin(s) of deep-sea organisms is thus very poorly documented.

The Mollusca is the second largest phylum in the animal kingdom, next to the Arthropoda, and the largest marine phylum [START_REF] Aktipis | Gastropoda: an overview and analysis[END_REF]. Molluscan species are found in all the marine environments, from shallow water to deep sea. The bestknown species assemblages are relatively large in size and from shallow waters.

However, these species represent only a small part of the diversity of marine mollusks [START_REF] Bouchet | Assessing the magnitude of species richness in tropical marine environments : exceptionally high numbers of molluscs at a New Caledonia site[END_REF][START_REF] Bouchet | How many species of molluscs are there in the world's oceans, and who is going to described them?[END_REF]. Some molluscan families and genera are less known because their members are tiny or they are difficult to be collected, with some groups known exclusively from the bathyal or the hadal zones [START_REF] Warén | New and little known skeneimorph gastropods from the Mediterranean Sea and the adjacent Atlantic Ocean[END_REF][START_REF] Warén | Description of Bathysciadium xylophagum Warén and Carrozza, sp.n. and comments on Addisonia excentrica (Tiberi) two Mediterranean cocculiniform gastropods[END_REF][START_REF] Warén | New records, species, genera, and a new family of gastropods from hydrothermal vents and hydrocarbon seeps[END_REF][START_REF] Warén | Gastropoda and Monoplacophora from hydrothermal vents and seeps; New taxa and records[END_REF], 2009). Massive collecting efforts, especially from the deep sea, are required to assess the magnitude of species richness, the biogeography and the phylogenetic relationships.

The Gastropoda is the largest class in the phylum Mollusca. This class comprises seven main groups: Caenogastropoda, Cocculiniformia, Heterobranchia, Neomphalina, Neritimorpha, Patellogastropoda, and Vetigastropoda. While each group contains deep-sea lineages, the members of Cocculiniformia and Neomphalina are mostly found in deep-sea habitats [START_REF] Marshall | Recent and Tertiary Cocculinidae and Pseudococculinidae (Mollusca, Gastropoda) from New-zealand and New-south-wales[END_REF][START_REF] Mclean | Cocculiniform limpets (Cocculinidae and Pyropeltidae) living on whale bone in the deep-sea off California[END_REF][START_REF] Warén | New records, species, genera, and a new family of gastropods from hydrothermal vents and hydrocarbon seeps[END_REF][START_REF] Mclean | Review of western Atlantic species of cocculinid and pseudococculinid limpets, with descriptions of new species (Gastropoda: Cocculiniformia)[END_REF][START_REF] Warén | Description of Bathysciadium xylophagum Warén and Carrozza, sp.n. and comments on Addisonia excentrica (Tiberi) two Mediterranean cocculiniform gastropods[END_REF][START_REF] Leal | Deepest Atlantic molluscs: hadal limpets (Mollusca, Gastropoda, Cocculiniformia) from the northern boundary of the Caribbean Plate[END_REF][START_REF] Warén | Gastropoda and Monoplacophora from hydrothermal vents and seeps; New taxa and records[END_REF][START_REF] Kiel | Deep-sea food bonanzas: early Cenozoic whale-fall communities resemble wood-fall rather than seep comminities[END_REF][START_REF] Heß | Microanatomy, shell structre and molecular phylogeny of Leptogyra, Xyleptogyra and Leptogyropsis (Gastropoda: Neomphalida: Melanodrymiidae) from sunken wood[END_REF][START_REF] Sasaki | Gastropods from Recent hot vents and cold seeps: systematics, diversity and life strategies[END_REF][START_REF] Warén | Molluscs on biogenic substrates[END_REF]. The Vetigastropoda (including abalones, slit snails, true limpets, keyhole limpets, seguenzid snails and turban snails) is the most diverse group with approximately 3,700 described living species occurring in a wide range of marine habitats from all oceans [START_REF] Aktipis | Gastropoda: an overview and analysis[END_REF]), yet it has not been exclusively studied. Among the eight superfamilies (Fissurelloidea, Haliotoidea, Lepetelloidea, Lepetodriloidea, Pleurotomarioidea, Scissurelloidea, Seguenzioidea, and Trochoidea) and the currently recognized 37 families [START_REF] Bouchet | Revised classification, nomenclator and typification of gastropod and monophacophoran families[END_REF], several are exclusively found in the deep sea (e.g., Lepetodriloidea and Seguenziidae). The Neritimorpha is supposedly a relic lineage of an early radiation with a morphological and ecological diversity equivalent to that achieved by all the other gastropods [START_REF] Lindberg | Patellogastropoda, Neritimorpha, and Cocculinoidea[END_REF] During the last decade many studies exploring the phylogenetic relationships within the Gastropoda were based on Next Generation Sequencing (NGS) techniques to improve the accuracy for the inference by increasing the character sampling [START_REF] Williams | The complete mitochondrial genome of a turbinid vetigastropod from MiSeq Illumina sequencing of genomic DNA and steps towards a resolved gastropod phylogeny[END_REF][START_REF] Zapata | Phylogenomic analyses of deep gastropod relationships reject Orthogastropoda[END_REF][START_REF] Osca | Caenogastropod mitogenomics[END_REF][START_REF] Lee | Eight new mitogenomes for exploring the phylogeny and classification of Vetigastropoda[END_REF]Uribe et al., 2016aUribe et al., , 2016b[START_REF] Uribe | Denser mitogenomic sampling improves resolution of the phylogeny of the superfamily Trochoidea (Gastropoda: Vetigastropoda)[END_REF][START_REF] Wort | Testing the contribution of individual genes in mitochondrial genomes for assessing phylogenetic relationships in Vetigastropoda[END_REF]. Noteworthily, [START_REF] Zapata | Phylogenomic analyses of deep gastropod relationships reject Orthogastropoda[END_REF] reevaluated the phylogenetic relationships among the five main gastropod clades (Vetigastropoda, Caenogastropoda, Heterobranchia, Neritimorpha, and Patellogastropoda) by analyzing a 56-taxon matrix comprising forty new transcriptomes, completed by publicly available data. This study confirmed the Apogastropoda clade (Caenogastropoda + Heterobranchia) and rejected the monophyletic Orthogastropoda (including all major main groups except Patellogastropoda). In this latter study, Cocculiniformia and Neomphalina were lacking and the rooting of the reconstructed tree remained under debate. In [START_REF] Aktipis | Testing relationships among the Vetigastropod taxa: a molecular approach[END_REF], the authors re-examined the phylogenetic relationships within the Vetigastropoda based on a multi-gene dataset (five genes) and a dense taxonomic sampling, which included 69 terminal taxa of the Vetigastropoda, covering all the vetigastropod superfamilies and 22 out of the 37 vetigastropod families [START_REF] Bouchet | Revised classification, nomenclator and typification of gastropod and monophacophoran families[END_REF]. The authors of this study suggested excluding the Pleurotomarioidea and the deep-sea vetigastropod lineage Lepetelloidea from the Vetigastropoda sensu stricto. However, the relationships were not robustly resolved in this and other previous studies; further investigations are required. Similarly, Uribe et al (2016b) examined the relationships within the Neritimorpha based on mitogenomic information, but here again deep-sea lineages were lacking.

Overall, recent molecular studies investigating gastropod phylogeny and/or specific lineages such those within the Vetigastropoda focused more on the shallow water species (e.g. [START_REF] Zapata | Phylogenomic analyses of deep gastropod relationships reject Orthogastropoda[END_REF][START_REF] Lee | Eight new mitogenomes for exploring the phylogeny and classification of Vetigastropoda[END_REF]Uribe et al., 2016a;Uribe et al., 2016b) and used often only a few number of genetic markers (e.g., in [START_REF] Aktipis | Testing relationships among the Vetigastropod taxa: a molecular approach[END_REF]. Deep-sea representatives were indeed only occasionally included and generally restricted to a few species or specimens [START_REF] Colgan | Gastropod phylogeny based on six segments from four genes representing coding or noncoding and mitochondrial or nuclear DNA[END_REF][START_REF] Geiger | Molecular phylogeny of Vetigastropoda reveals non-monophyletic Scissurellidae, Trochoidea, and Fissurelloidea[END_REF][START_REF] Lee | Eight new mitogenomes for exploring the phylogeny and classification of Vetigastropoda[END_REF][START_REF] Osca | Caenogastropod mitogenomics[END_REF][START_REF] Smith | Resolving the evolutionary relationships of molluscs with phylogenomic tools[END_REF]Uribe et al., 2016a[START_REF] Uribe | Denser mitogenomic sampling improves resolution of the phylogeny of the superfamily Trochoidea (Gastropoda: Vetigastropoda)[END_REF][START_REF] Zapata | Phylogenomic analyses of deep gastropod relationships reject Orthogastropoda[END_REF]. Such a bias in the taxonomic or character sampling may strongly distort our understanding of the causes of the observed pattern of diversity and thus of the evolution of the Gastropoda. For instance, although some phylogenetic studies included many (>20) deep-sea taxa, they generally suffered from a lack of resolution on deep nodes (e.g. [START_REF] Kano | Vetigastropod phylogeny and a new concept of Seguenzioidea: independent evolution of copulatory organs in the deep-sea habitats[END_REF][START_REF] Aktipis | Testing relationships among the Vetigastropod taxa: a molecular approach[END_REF][START_REF] Geiger | Santa Barbara Museum of Natural History[END_REF].

In this study we reexamined the evolutionary relationships of the Gastropoda based on a dataset enriched with deep-sea gastropods from the Vetigastropoda (notably from Lepetelloidea, Scissurelloidea and Pleurotomarioidea), Cocculiniformia, Neomphalina and Neritimorpha. These taxa are rare and difficult to collect, making them the least-studied groups of gastropods. We focused on mitogenomic data that have been successfully used for resolving deep phylogenies of several gastropod groups [START_REF] Grande | Evolution of gastropod mitochondrial genome arrangements[END_REF][START_REF] Cunha | Neogastropod phylogenetic relationships based on entire mitochondrial genomes[END_REF][START_REF] Allcock | What can the mitochondrial genome reveal about higher-level phylogeny of the molluscan class Cephalopoda?[END_REF][START_REF] Medina | Crawling through time: Transition of snails to slugs dating back to the Paleozoic, based on mitochondrial phylogenomics[END_REF][START_REF] White | Ten new complete mitochondrial genomes of pulmonates (Mollusca: Gastropoda) and their impact on phylogenetic relationships[END_REF][START_REF] Kocot | Phylogenomics supports Panpulmonata: Opisthobranch paraphyly and key evolutionary steps in a major radiation of gastropod molluscs[END_REF][START_REF] Williams | The complete mitochondrial genome of a turbinid vetigastropod from MiSeq Illumina sequencing of genomic DNA and steps towards a resolved gastropod phylogeny[END_REF][START_REF] Osca | Caenogastropod mitogenomics[END_REF][START_REF] Lee | Eight new mitogenomes for exploring the phylogeny and classification of Vetigastropoda[END_REF]Uribe et al., 2016aUribe et al., , 2016b[START_REF] Uribe | Denser mitogenomic sampling improves resolution of the phylogeny of the superfamily Trochoidea (Gastropoda: Vetigastropoda)[END_REF][START_REF] Wort | Testing the contribution of individual genes in mitochondrial genomes for assessing phylogenetic relationships in Vetigastropoda[END_REF] to investigate the phylogenetic relationships among deep-and shallow-water gastropods. In addition, the data from three independent nuclear gene markers, 18S rRNA (18S), 28S rRNA (28S), and Histone 3 (H3), were also used as additional data for the phylogenetic hypothesis evaluation.

Material and methods

The eleven specimens used to reconstruct mitogenomes in this study were listed in Table 1. The specimens were mainly collected during the biodiversity expeditions under the Tropical Deep-Sea Benthos program and the cooperation project between Taiwan and France, TFDeepEvo (Table 1).

For most of the specimens, the genomic DNA was extracted using DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) or NucleoSpin 96 Tissue kit microkit with the Eppendorf automated pipetting systems epMotion 5075 (Macherey-Nagel, France) following the manufacturer's protocols. For the smaller specimens (Anatoma sp. and Bathysciadiidae sp.), the genomic DNA was extracted using QIAamp DNA Micro-Kit (Qiagen, Hilden, Germany) to ensure sufficient quantity for subsequent molecular work. For Bayerotrochus teramachii, genomic DNA was extracted using a phenol-chloroform extraction method as described in [START_REF] Lee | Eight new mitogenomes for exploring the phylogeny and classification of Vetigastropoda[END_REF].

Mitogenome reconstruction

To reconstruct the mitogenome of Bayerotrochus teramachii, the long-range PCR and primer-walking Sanger sequencing were used to obtain its whole mitogenomic sequence by following the protocol described in [START_REF] Lee | Eight new mitogenomes for exploring the phylogeny and classification of Vetigastropoda[END_REF].

Seven gastropod mitogenomes were reconstructed by using whole genome shotgun sequencing approach with NGS on Illumina platform (Table 1). The library construction and the procedure for shotgun sequencing on Illumina platform followed those described in [START_REF] Lee | Eight new mitogenomes for exploring the phylogeny and classification of Vetigastropoda[END_REF]. The quality of the reads was checked using the program FastQC v. 0.10.1 [START_REF] Andrews | FastQC: a quality control tool for high throughput sequence data[END_REF]. Low quality reads were trimmed according to the Phred quality score (< 20) using the program Trimmomatic v.0.33 [START_REF] Bolger | Trimmomatic: a flexible tremmer for Illumina sequence data[END_REF]. The raw data were deposited in the NCBI SRA (Sequence Read

Table

Archive) database (accession number: PRJNA516567). The methods of the postsequencing assemblages of the mitogenomes followed also those described in [START_REF] Lee | Eight new mitogenomes for exploring the phylogeny and classification of Vetigastropoda[END_REF]. Three other gastropod mitogenomes were reconstructed by using whole genome shotgun sequencing approach with NGS on Ion Torrent platform (Table 1). Ion Torrent libraries were constructed for three other specimens by using NEB next Library preparation kit (New England Biolabs, MA, USA) for Ion Torrent. Template preparation of these libraries was performed by emulsion PCR on an Ion OneTouch robotic system, and the subsequent sequencing was performed by an Ion Torrent PGM sequencer using Hi-Q chemistry (Life technologies, France). The quality of the reads was checked by default program implemented in the sequencing analyzer and the passed reads were exported for post-sequencing assemblages.

The assemblage of mitochondrial sequence reads exported from Ion Torrent analyzer was performed using Geneious R9 assembler, in de novo mode with default settings (Biomatters Ltd., Auckland, New Zealand). Contigs with matching ends where circularized. To map their mitochondrial genes, individual gene sequences from the reference genomes of Nerita melanotragus [START_REF] Castro | The phylogenetic position of Neritimorpha based on the mitochondrial genome of Nerita melanotragus (Mollusca: Gastropoda)[END_REF],

Bayerotrochus teramachii (this study), and Cocculina subcompressa (this study) were used. Bait mapping was performed as follows: twenty-five iterations with assembly sensitivity set to 'med-low' (and max mismatch percentage ranging 15-20%), without any postulate on the gene order. Individual contigs were compared and aligned to generate larger contigs.

Mitochondrial gene annotation

The DNA sequences of each reconstructed mitogenome were edited with sequence assembly and alignment software CodonCode Aligner v. 6.0.2 (Codoncode Corporation, Dedham, MA, USA) and Se-Al v. 2.0 [START_REF] Rambaut | Se-al: Sequence alignment editor version 1.0 a1[END_REF]. Gene annotation was carried out using MITOS Webserver [START_REF] Bernt | MITOS: Improved de novo metazoan mitochondrial genome annotation[END_REF] with the invertebrate genetic codes for protein coding genes and default settings. Gene boundaries were further determined by eye using the mitogenome of Lunella granulata as the reference [START_REF] Lee | Eight new mitogenomes for exploring the phylogeny and classification of Vetigastropoda[END_REF].

Nuclear gene assemblages

Three nuclear genes, 18S, 28S, and H3, were also mapped and assembled from the read-pools of each NGS sample using Geneious R9 (Biomatters Ltd., Auckland, New Zealand). The reference sequences for baiting and mapping were published sequences from species of the same family or superfamily as the target sample.

Reference sequences were selected from the dataset of [START_REF] Aktipis | Testing relationships among the Vetigastropod taxa: a molecular approach[END_REF] Octopoda, and Bivalvia. The polyplacophoran Katharina tunicata was used as the distant out-group to root the inferred tree.

From this master dataset, we further compiled another mitogenomic dataset (called hereafter 'small mt dataset'). The data included all gastropod mitogenomes except some long-branch taxa that have been identified in the analysis with the 'large mt dataset' and in several previous studies [START_REF] Grande | Evolution of gastropod mitochondrial genome arrangements[END_REF][START_REF] Williams | The complete mitochondrial genome of a turbinid vetigastropod from MiSeq Illumina sequencing of genomic DNA and steps towards a resolved gastropod phylogeny[END_REF]Uribe et al., 2016a). This 46 taxa data matrix was herein used to infer the phylogeny.

Octopus vulgaris (cephalopod) and Graptacme eborea (scaphopod) were chosen as distant out-groups to root the inferred tree.

A nuclear dataset was also compiled for the three targeted nuclear gene markers (18S, 28S, and H3). Using both the sequences from this study and previously published sequences, we compiled data for 45 species [START_REF] Aktipis | Testing relationships among the Vetigastropod taxa: a molecular approach[END_REF] (Supplementary table S2).

Finally, a combined dataset of the mitochondrial sequences (from the small mt dataset) and the nuclear sequences was compiled by selecting the representative taxa from each superfamily or family which were from the same genus or family in both mitogenomic and nuclear gene datasets. The resulting dataset included 32 taxa (Supplementary table S3).

Sequence alignment and phylogenetic analysis

Sequences for each gene in the four compiled datasets were first aligned using the automatic multiple-alignment program MUSCLE [START_REF] Edgar | MUSCLE: a multiple sequence alignment method with reduced time and space complexity[END_REF] and then adjusted by eye. For protein-coding genes we translated the nucleotide sequences to amino acids before aligning the sequences in order to avoid erroneous creation of gaps that would lead to shifts in the open reading frame. Each gene block was modified using Gblocks v. 0.91b [START_REF] Castresana | Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis[END_REF] to remove the ambiguous alignments gene-bygene, with default settings except that gap positions within the final blocks were allowed. To reduce the impact of homoplasy at the third codon position sites on phylogenetic inferences, RY-coding strategy was used by recoding "A" and "G" into "R", and "C" and "T" into "Y" at the third codon positions [START_REF] Chen | Molecular systematics of the Cyprinoidea (Teleostei: Cypriniformes), the world's largest clade of freshwater fishes: further evidence from six nuclear genes[END_REF] for each protein-coding gene. The final alignments respectively consisted of 10,393 bp for the large mt dataset, 11,129 bp nucleotides for the small mt dataset, 3,313 bp nucleotides for the nuclear dataset, and 14,442 bp nucleotides for the combined dataset. Four partitions (three partitions for each codon position for mitochondrial protein-coding genes and one partition for the 12S plus 16S rRNA genes) were set for large mt dataset and small mt datasets. Five partitions were set for nuclear dataset (by gene and by codon position). Finally, nine partitions were set for combined dataset (Supplementary table S4). Phylogenetic analyses were performed using both partitioned maximum likelihood method (ML) and Bayesian inference (BI). ML was conducted by using the RAxML v. 8.0 [START_REF] Stamatakis | RAxML version 8: a tool for phylogenetic analysis and postanalysis of large phylogenies[END_REF] with the GTR+G+I model. Nodal support was assessed by bootstrapping [START_REF] Felsenstein | Confidence-limits on phylogenies -an approach using the bootstrap[END_REF] with 1,000 pseudo-replicates. BI was performed with MrBayes V. 3.2.6 [START_REF] Ronquist | MrBayes 3.2: Efficient Bayesian Phylogenetic Inference and Model Choice Across a Large Model Space[END_REF] on the CIPRES Science Gateway [START_REF] Miller | Creating the CIPRES Science Gateway fir inference of large phylogenetic trees[END_REF] with best-fit nucleotide substitution models suggested by model test implemented in PartitionFinder [START_REF] Lanfear | PartitionFinder: combined selection of partitioning schemes and substitution models for phylogenetic analyses[END_REF]) (Supplementary table S4). Four Markov chains were performed in each of two parallel runs for 30,000,000 generations for the large mt dataset, small mt dataset, and nuclear dataset, and 60,000,000 generations for the combined dataset, with a sampling frequency of one tree every one thousand generations. The default setting from MrBayes of the burn-in value (0.25) was used. The convergence of the likelihood scores for parameters was evaluated using Tracer v. 1.6 [START_REF] Rambaut | Tracer v1.6[END_REF] to make sure that all the ESS values were over 200.

Results

Reconstructed mitogenomes and nuclear genes

The size of the nearly complete mitogenome of Bayerotrochus teramachii, reconstructed by the primer walking and Sanger Sequencing was 13,474 bp (Table 2).

It contained 12 protein-coding genes, 2 rRNAs, and 11 tRNAs. Although we assembled a final contig showing overlapping sequences from the two ends of the circular genome, several genes were missing (trnY,trnC,trnW,trnQ,trnG,trnE,cox3,trnK,trnA,trnR,trnN,trnI and part of 12S rRNA). These genes are commonly found next to each together (e.g. in [START_REF] Grande | Evolution of gastropod mitochondrial genome arrangements[END_REF][START_REF] Cunha | Neogastropod phylogenetic relationships based on entire mitochondrial genomes[END_REF][START_REF] Stöger | Mitogenomics does not resolve deep molluscan relationships (yet?)[END_REF][START_REF] Schrödl | A review on deep molluscan phylogeny: old markers, integrative approaches, persistent problems[END_REF][START_REF] Williams | The complete mitochondrial genome of a turbinid vetigastropod from MiSeq Illumina sequencing of genomic DNA and steps towards a resolved gastropod phylogeny[END_REF][START_REF] Lee | Eight new mitogenomes for exploring the phylogeny and classification of Vetigastropoda[END_REF]Uribe et al., 2016aUribe et al., , 2016b[START_REF] Uribe | Denser mitogenomic sampling improves resolution of the phylogeny of the superfamily Trochoidea (Gastropoda: Vetigastropoda)[END_REF] and we thus suspected that a hairpin of mitochondrial fragment occurred during the PCR leading to skip the twisted part during the elongation.

Ten other new mitogenomes, yet not fully complete, were reconstructed using the shotgun sequencing approach described in [START_REF] Lee | Eight new mitogenomes for exploring the phylogeny and classification of Vetigastropoda[END_REF]. After the assembly, the depths of the contigs were mostly over 25x. The amino acid sequences of each contig were further examined by comparing with the reference sequences to avoid the possibility of shifts in reading frames, especially for those having lower coverage. Finally, for the undetermined regions, Ns were used to replace the uncertain positions. We assembled each read pool into a single contig for Anatoma sp., Cocculina subcompressa, Peltospira smaragdina, Bathysciadiidae sp., Shinkailepas briandi, and Calliotropis micraulax. For Perotrochus caledonicus we assembled four non-Table overlapped contigs containing the mitochondrial sequences from two long segments, and from two short fragments with nd2 and nd3, respectively. For Coccocrater sp. we were able to assemble three contigs. From both read pools of Lepetodrilus guaymasensis and Pseudococculinidae sp., we assembled two non-overlapping final contigs. Finally, for Peltospira smaragdina, the final contig we assembled showed overlapping sequences from the two ends of the circular genome and a full set of animal mitochondrial genes.

The evolution of animal mitogenomes in terms of gene content is generally conservative. A typical animal mitogenome contains 13 protein-coding genes, 2 ribosomal RNAs, and 22 transfer RNAs; this gene content was observed in most of our reconstructed mitogenomes with a few exceptions. In the mitogenome of Cocculina subcompressa, a duplicate trnD was found. The trnM and trnY could not be detected in the reconstructed mitogenome of Bathysciadiidae sp. In the reconstructed mitogenome of Pseudococculinidae sp., the trnQ, trnG, and trnE could not be found.

Besides, a large segment could not be assembled from the read pool of Anatoma sp., containing partial 16S rRNA, trnV, 12S rRNA, and the tRNA series MYCWQGE. In the reconstructed mitogenome of Perotrochus caledonicus, the segment containing cox3, the tRNA series YCWQGE and KARNI, and one trnS could not be found. In the mitogenome of Shinkailepas briandi, the segment of the tRNA series MYCWQGE, trnV, and 12S rRNA could not be detected. For Coccocrater sp., the segment of partial nd5, trnH, nd4, nd4l, trnT, and trnS could not be found. The main structures of the reconstructed mitogenomes were shown in Table 2 and the gene orders of the newly reconstructed mitogenomes were shown in Supplementary figure S1.

For the three nuclear genes targeted in this study, sequences were assembled from only four read pools. In the read pool of Bathysciadiidae sp., we assembled the 18S. In the read pool of Pseudococculinidae sp., H3 was assembled. For Perotrochus caledonicus, we assembled the 18S and the H3. Lastly, the 18S and 28S were assembled from the read pool of Shinkailepas briandi (Supplementary table S1).

Inferred phylogenetic trees

The topologies of the inferred ML and BI trees based on the big mt dataset were almost identical except for the position of Stomatella planulata (Supplementary figure S2), however, both positions were not supported. In these trees, Gastropoda was not monophyletic. The cephalopod (Octopus vulgaris), scaphopod (Graptacme eborea), bivalves (Pyganodon grandis, Mytilus edulis, Chlamys farreri), heterobranchs (Pupa strigosa, Peronia peronii, Siphonaria pectinata), patellogastropod (Lottia digitalis), and one neritimorph (Pleuropoma jana) were grouped together, but this clade was not meaningfully supported (bootstrap value [BP] below 50%; posterior probability [PP] = 0.9). This clade was placed as the sister-group of the rest (except Polyplacophora) and included the taxa of patellogastropod and bivalves with extreme long branches. We thus removed these taxa from the subsequent analyses.

The phylogenetic trees inferred with respectively the small mt, the nuclear, and the combined datasets are shown in figures 1-3 and supplementary figure S3. From all of the analyses, the Gastropoda and all the main groups within gastropods, including the Neritimorpha that was observed to be non-monophyletic in the inferred tree based on the big mt dataset (Supplementary figure S2), were resolved as monophyletic groups with mediocre or strong nodal supports. As to their inter-relationships, none were well resolved with an exception for the sister-group relationship between Cocculiniformia and Neomphalina (small mt dataset: BP = 100%, PP = 1.00; nuclear dataset: BP = 70%, PP = 0.64; combined dataset: BP = 100%, PP = 1.00).

Within the Vetigastropoda, the Pleurotomarioidea was inferred to be the sistergroup of the clade containing the rest of the vetigastropod superfamilies in all the analyses, and this relationship was highly supported in almost all the analyses (Figs 123). Within the latter clade, two major groups could be delimited based on the results from the analyses conducted with the small mt and combined datasets. The first group included Fissurelloidea, Seguenzioidea, Haliotoidea, Scissurelloidea, Lepetelloidea, and Lepetodriloidea, whereas the second group included all the trochoidean families. However, the first group was not well supported (Fig. 1 and3). Within these two major groups, some well-supported clades were resolved: the Fissurelloidea (full support in all analyses), the clade containing the Haliotoidea and the Seguenzioidea (small mt dataset: BP = 87%, PP = 0.98; combined dataset: BP = 71%, PP = 1.00), the clade containing the Scissurelloidea, the Lepetelloidea, and the Lepetodriloidea (small mt dataset: BP = 92%, PP = 1.00; combined dataset: BP = 85%, PP = 1.00), the clade grouping the Lepetelloidea and the Lepetodriloidea (full support in all analyses), and the clade containing Tegulidae and Turbinidae (full support in all analyses). In the analyses based on the nuclear dataset, most of the clades mentioned above were found, but support values were generally weak.

Discussion

Mitogenomic phylogeny of the Gastropoda

The mitogenome is widely used for reconstructing the molluscan phylogeny with highly conserved sizes [START_REF] Stöger | Mitogenomics does not resolve deep molluscan relationships (yet?)[END_REF]. However, mitochondrial genes have generally faster evolutionary rates than nuclear genes, leaing to ambiguous results due to saturation when considering deep phylogenetic relationships [START_REF] Burton | A disproportionate role for mtDNA in Dobzhansky-Muller incompatibilities ?[END_REF][START_REF] Stöger | Mitogenomics does not resolve deep molluscan relationships (yet?)[END_REF][START_REF] Wort | Testing the contribution of individual genes in mitochondrial genomes for assessing phylogenetic relationships in Vetigastropoda[END_REF]. [START_REF] Stöger | Mitogenomics does not resolve deep molluscan relationships (yet?)[END_REF] have suggested that long-branch attraction might provide misleading relationships in deep molluscan phylogeny. We detected such phenomenon when using the large mt dataset with some gastropod branches clustering with the longbranches of the bivalves. With this dataset, the non-monophyly observed for the Gastropoda and Neritimorpha should result from a long-branch attraction [START_REF] Grande | Evolution of gastropod mitochondrial genome arrangements[END_REF][START_REF] Williams | The complete mitochondrial genome of a turbinid vetigastropod from MiSeq Illumina sequencing of genomic DNA and steps towards a resolved gastropod phylogeny[END_REF]Uribe et al., 2016a). Actually, the monophyly of the Gastropoda as well as the Neritimorpha, supported by nuclear data set [START_REF] Zapata | Phylogenomic analyses of deep gastropod relationships reject Orthogastropoda[END_REF], was recovered when the long-branch taxa were removed (Figs. 123).

Another characteristic of mitogenomic datasets that should be taken into account in phylogenetic reconstructions is the absence of recombination. In fact, mitochondrial genes as a whole should be regarded as a single locus that experienced the same evolutionary history. Consequently, a particular regime of selection, different rate of evolution or introgression in a given lineage may distort the reconstructed tree. In this context, nuclear genes that are not linked to mitochondrial genes and thanks to recombination not linked among them, may be used as independent evidence to evaluate the reliability of the phylogenetic reconstruction, especially when mitochondrial introgression occurred [START_REF] Chen | A phylogenomic perspective on the new era of ichthyology[END_REF]. In our dataset, for the well supported branches, we found congruent results between the nuclear and mitochondrial datasets (Figs 123). Finally, the most critical issue that may affect phylogenetic reconstructions on gastropods is the taxonomic sampling bias. Many recent mitogenomic studies investigating the gastropod phylogeny were conducted with only shallow-water lineages [START_REF] Grande | Evolution of gastropod mitochondrial genome arrangements[END_REF][START_REF] Cunha | Neogastropod phylogenetic relationships based on entire mitochondrial genomes[END_REF][START_REF] Williams | The complete mitochondrial genome of a turbinid vetigastropod from MiSeq Illumina sequencing of genomic DNA and steps towards a resolved gastropod phylogeny[END_REF][START_REF] Osca | Caenogastropod mitogenomics[END_REF]Uribe et al., 2016b;[START_REF] Wort | Testing the contribution of individual genes in mitochondrial genomes for assessing phylogenetic relationships in Vetigastropoda[END_REF] or a few deep-sea taxa [START_REF] Lee | Eight new mitogenomes for exploring the phylogeny and classification of Vetigastropoda[END_REF]Uribe et al., 2016a). In Uribe et al. (2016a), the authors presented that the deep-sea Neomphalina was the sister-group of the clade containing all other gastropods sampled in the study, but this topology was considered as an artefact. In the present study, we reconstructed 11 nearly complete mitogenomes from deep-sea gastropods that included novel reports of the mitogenomes from taxa difficult to collect, such as the Cocculiniformia, Pleurotomarioidea, Scissurelloidea, and Lepetelloidea. Our results demonstrated that the two major deep-sea gastropod groups Cocculiniformia (not sampled in Uribe et al., 2016a) and Neomphalina are each monophyletic and together form a strongly supported clade (Figs 1-3), supporting the results of [START_REF] Aktipis | Testing relationships among the Vetigastropod taxa: a molecular approach[END_REF]. This implies that during the early diversifications of gastropods colonization of the deep sea occurred only once. The topology does not exclude the hypothesis of deep-sea origin of the entire Gastropoda or Vetigastropoda. However, this hypothesis is just unrealistic notably from a paleontological point of view. This topology is better interpreted as a transition to the deep sea occurring at the latest for the common ancestor of the Cocculiniformia and Neomphalina. Our results revealed that multiple transitions between shallow water and deep-sea habitats occurred later during the evolution of the vetigastropods (Fig. 1-3) (see discussion below). Within the Neritimorpha, deep-sea taxa (Bathynerita, Olgasolaris and Shinkailepas) are placed in the Phenacolepadidae that, together with the Neritidae, belong to the superfamily Neritoidea. In our study Shinkailepas branches with Neritidae species, this result was congruent with the fossil records and the phylogenetic reconstruction based on partial 28S rRNA sequence [START_REF] Kano | Major adaptive radiation in neritopsine gastropods estimated from 28S rRNA sequences and fossil records[END_REF].

The evolutionary significance of the deep-sea taxa among vetigastropods

The definition of the Vetigastropoda was unsettled mainly due to an uncertain placement of a deep-sea lineage, the Pleurotomarioidea. It was placed as the sistergroup of the remaining vetigastropod superfamilies in most molecular studies but the support for this relationship was not robust [START_REF] Colgan | Gastropod phylogeny based on six segments from four genes representing coding or noncoding and mitochondrial or nuclear DNA[END_REF][START_REF] Mcarthur | Molecular systematics of the major lineages of the Gastropoda[END_REF][START_REF] Geiger | Molecular phylogeny of Vetigastropoda reveals non-monophyletic Scissurellidae, Trochoidea, and Fissurelloidea[END_REF][START_REF] Williams | Molecular phylogeny suggests polyphyly of both the turban shells (family Turbinidae) and the superfamily Trochoidea (Mollusca : Vetigastropoda)[END_REF][START_REF] Kano | Vetigastropod phylogeny and a new concept of Seguenzioidea: independent evolution of copulatory organs in the deep-sea habitats[END_REF][START_REF] Williams | Molecular systematics of Vetigastropoda : Trochidae, turbinidae and trochoidea redefined[END_REF]. [START_REF] Aktipis | Testing relationships among the Vetigastropod taxa: a molecular approach[END_REF], using molecular data, even suggested excluding it from the Vetigastropoda. Conversely, several morphological analyses found it to be nested within the Vetigastropoda [START_REF] Ponder | Towards a phylogeny of gastropod molluscs: Analysis using morphological characters[END_REF][START_REF] Sasaki | Comparative anatomy and phylogeny of teh recent Archaeogastropoda (Mollusca: Gastropoda)[END_REF]. With a denser character-and taxon-sampling strategy, the hypothesis of a sister-group relationship between the Pleurotomarioidea and the rest of the vetigastropods is confirmed with high support values in this study.

Our analyses resolve some other conflicting issues about the phylogenetic relationships and classification of the Vetigastropoda. For instance, [START_REF] Aktipis | Testing relationships among the Vetigastropod taxa: a molecular approach[END_REF] suggested removing the deep-sea Lepetelloidea from Vetigastropoda sensu stricto. However, in accordance with [START_REF] Zapata | Phylogenomic analyses of deep gastropod relationships reject Orthogastropoda[END_REF], we found this superfamily to be sister to the monophyletic Lepetodriloidea that also include deepsea taxa. Moreover, we further identified the Scissurelloidea as the closest allies of this deep-sea vetigastropod clade (Figs 123). This particular finding thus resolved the close relationships among the Scissurelloidea, Lepetodriloidea, and Lepetelloidea.

Actually, no Lepetelloidea has been included in the phylogenetic studies based on which a close affinity between Lepetodriloidea and Scissurelloidea was proposed [START_REF] Geiger | Molecular phylogeny of Vetigastropoda reveals non-monophyletic Scissurellidae, Trochoidea, and Fissurelloidea[END_REF][START_REF] Williams | Molecular phylogeny suggests polyphyly of both the turban shells (family Turbinidae) and the superfamily Trochoidea (Mollusca : Vetigastropoda)[END_REF][START_REF] Williams | Molecular systematics of Vetigastropoda : Trochidae, turbinidae and trochoidea redefined[END_REF][START_REF] Geiger | Santa Barbara Museum of Natural History[END_REF]. However, some studies questioned the monophyly of the Scissurelloidea and found that the scissurelloid family, Anatomidae, might be sister to the Lepetodriloidea, and that together these two lineages should be the sister-group of the other scissurelloid family, Scissurelloidea [START_REF] Kano | Vetigastropod phylogeny and a new concept of Seguenzioidea: independent evolution of copulatory organs in the deep-sea habitats[END_REF][START_REF] Geiger | Santa Barbara Museum of Natural History[END_REF]. Our nuclear gene results showed a similar topology (Fig. 2), although the concerned nodes were not supported. The inclusion of more scissurelloid mitogenomes in the data analyses is necessary to test the hypothesis of a scissurelloid monophyly.

Habitat use in gastropods

To evaluate the evolutionary significance of the deep-sea habitats in gastropods, we examined the literature to evaluate the relative importance of four distinct habitats:

shallow water, deep-sea floor, biogenic substrates, and hot vents and cold seeps (Fig. 4). Caenogastropoda, Neritimorpha and Vetigastropoda are present in all four habitats whereas the sister-clades Cocculiniformia and Neomphalina are mainly inhabit two deep-sea habitats (biogenic substrates, and vents and seeps), which are recognized as chemosynthetic environments.

Cocculiniform species are all restricted to biogenic substrates [START_REF] Marshall | Recent and Tertiary Cocculinidae and Pseudococculinidae (Mollusca, Gastropoda) from New-zealand and New-south-wales[END_REF][START_REF] Haszprunar | Anatomy and affinities of cocculinid limpets (Mollusca, Archaeogastropoda)[END_REF][START_REF] Mclean | Review of western Atlantic species of cocculinid and pseudococculinid limpets, with descriptions of new species (Gastropoda: Cocculiniformia)[END_REF][START_REF] Lesicki | Checklist of gastropod species refereed to the order Cocculinidormia Haszprunar, 1987 (Gastropoda: Cocculinoidea et Lepetelloidea) with some remarks on their food preferences[END_REF][START_REF] Warén | Molluscs on biogenic substrates[END_REF] whereas the members of Neomphalina can be found in both biogenic substrates (most of the species of Melanodrymiidae except those in Melanodrymia) and vents or seeps (species of Neomphalidae, and Peltospiridae) [START_REF] Heß | Microanatomy, shell structre and molecular phylogeny of Leptogyra, Xyleptogyra and Leptogyropsis (Gastropoda: Neomphalida: Melanodrymiidae) from sunken wood[END_REF]. The sunken wood associated species in Leptogyra and Leptogyropsis (Neomphalina, Melanodrymiidae) display putatively plesiomorphic characters in the morpho-anatomy, which led [START_REF] Heß | Microanatomy, shell structre and molecular phylogeny of Leptogyra, Xyleptogyra and Leptogyropsis (Gastropoda: Neomphalida: Melanodrymiidae) from sunken wood[END_REF] to conclude that the biogenic substrates might be the ancestral habitat of the Neomphalina. From our phylogenetic results, we found that the Neomphalina is sister to the Cocculiniformia, which suggests that the biogenic substrates might serve as an (ancestral) 'step' for the deep-sea gastropod colonization of chemosynthetic Fig. 4 environments (Fig. 4), and thus support the stepping stone hypothesis [START_REF] Distel | Marine ecology -Do mussels take wooden steps to deep-sea vents?[END_REF].

In our analysis, the sister-clades Neomphalina and Cocculiniformia branches with the Vetigastropoda based on the small mt dataset with mediocre supports (Fig. 1). In the Vetigastropoda most lineages are present in all three types of deep-sea habitats, but only the two sister-clades Lepetelloidea and Lepetodriloidea are mainly or exclusively inhabit the deep-sea chemosynthetic environments. Two potential scenarios could be thus proposed. Either the common ancestor of Cocculiniformia, Neomphalina and Vetigastropoda was restricted to deep-sea chemosynthetic environments, and the colonization of other deep-sea habitats and shallow-waters occurred later, or conversely, the colonization toward deep-sea chemosynthetic environments occurred several time independently with two major colonization events (i.e. the common ancestor of Cocculiniformia and Neomphalina, and the common ancestor of Lepetelloidea and Lepetodriloidea). Among Scissurelloidea, Lepetodriloidea, and Lepetelloidea, only one family (Scissurellidae) mainly inhabit shallow waters (< 75m) [START_REF] Geiger | Santa Barbara Museum of Natural History[END_REF]. Additional sampling of mitogenomes within Scissurelloidea is required to determine if as suggested by the nuclear gene analysis (Fig 2 ) and previous studies (e.g. [START_REF] Kano | Vetigastropod phylogeny and a new concept of Seguenzioidea: independent evolution of copulatory organs in the deep-sea habitats[END_REF][START_REF] Geiger | Santa Barbara Museum of Natural History[END_REF], the Scissurellidae is the sister-group of the other deep-sea lineages from this clade. The members of the Lepetodriloidea inhabit mainly deep-sea hydrothermal vents [START_REF] Mclean | New archaeogastropod limpets from hydrothermal vents: superfamily Lepetodrilacea I. Systematic Descriptions[END_REF][START_REF] Sasaki | Gastropods from Recent hot vents and cold seeps: systematics, diversity and life strategies[END_REF] and most of the Lepetelloidea can be found on biogenic substrates from bathyal to hadal zones [START_REF] Marshall | Recent and Tertiary Cocculinidae and Pseudococculinidae (Mollusca, Gastropoda) from New-zealand and New-south-wales[END_REF][START_REF] Haszprunar | Anatomy and affinities of cocculinid limpets (Mollusca, Archaeogastropoda)[END_REF][START_REF] Mclean | Review of western Atlantic species of cocculinid and pseudococculinid limpets, with descriptions of new species (Gastropoda: Cocculiniformia)[END_REF][START_REF] Lesicki | Checklist of gastropod species refereed to the order Cocculinidormia Haszprunar, 1987 (Gastropoda: Cocculinoidea et Lepetelloidea) with some remarks on their food preferences[END_REF][START_REF] Warén | Molluscs on biogenic substrates[END_REF], Kano et al. 2016) and some, namely the Pyropeltidae, inhabit deep-sea vents or seeps [START_REF] Sasaki | Gastropods from Recent hot vents and cold seeps: systematics, diversity and life strategies[END_REF]. In addition, [START_REF] Kano | Phylogenetic position and systematics of the wood-associate limpet genus Caymanabyssia and implications for ecological radiation into deep-sea organic substrates by lepetelloid gastropods[END_REF] suggested that sunken wood habitat might be the ancestral habitat of the Lepetelloidea based on the potentially plesiomorphic radula in this superfamily, which includes species mostly associated with woods. Although additional data are still required, the available results suggest an evolution from shallow waters (Scissurellidae) to deeper habitat (Anatomidae, Lepetelloidea, Lepetodriloidea), with sunken-wood (Lepetelloidea) serving as evolutionary stepping stones to vent and seeps (Lepetodriloidea).

In addition to the main deep-sea vetigastropod groups mentioned above, a few particular deep-sea lineages or species within each other vetigastropod superfamily can be found and some of them inhabit chemosynthetic environments (Fig. 4). There are, for example, some species in Cornisepta (Fissurelloidea) [START_REF] Araya | Cornisepta guzmani new species: first species of genus confirmed from Pacific cold seep environments off central Chile (Gastropoda: Vetigastropoda: Fissurellidae)[END_REF] found in cold seeps [START_REF] Araya | Cornisepta guzmani new species: first species of genus confirmed from Pacific cold seep environments off central Chile (Gastropoda: Vetigastropoda: Fissurellidae)[END_REF] and in hydrothermal vents [START_REF] Sasaki | Gastropods from Recent hot vents and cold seeps: systematics, diversity and life strategies[END_REF], the species in Puncturella found on sunken woods (Fissurelloidea) [START_REF] Warén | Molluscs on biogenic substrates[END_REF][START_REF] Pante | Exploration of the Deep-Sea Fauna of Papua New Guinea[END_REF] and from hydrothermal vents [START_REF] Sasaki | Gastropods from Recent hot vents and cold seeps: systematics, diversity and life strategies[END_REF]; most of the species attributed to Seguenziidae (Seguenzioidea) are from the deep-sea floor, and all the species of Cataegidae (Seguenzioidea) are from chemosynthetic environments (e.g. [START_REF] Warén | New gastropods from deep-sea hydrocarbon seeps off West Africa[END_REF][START_REF] Olu | Biogeography and Potential Exchanges Among the Atlantic Equatorial Belt Cold-Seep Faunas[END_REF][START_REF] Warén | Molluscs on biogenic substrates[END_REF]. The Pleurotomarioidea were first known from the fossil records. It was thought to be one of the most ancient gastropod clades [START_REF] Geiger | Vetigastropoda[END_REF]. They have diversified in shallow water habitats during the Paleozoic and Mesozoic and during these periods they dominated the shallow water gastropod fauna [START_REF] Harasewych | Pleurotomarioidean gastropods[END_REF]. Only a single family (Pleurotomariidae) survived the Cretaceous extinction and the recent species are now restricted to bathyal depth [START_REF] Harasewych | Pleurotomarioidean gastropods[END_REF]. Therefore, together the position of Pleurotomariidae within Vetigastropoda and the fossil data suggest that vetigastropods originated from shallow water ancestors and that the colonization of deep-sea habitats, including chemosynthetic environments happened later.

Our investigation of the taxonomic literature combined with new phylogenetic results offers a first glimpse into the evolution of habitat use among gastropods.

However, in each lineage, more taxa from the different habitats are required to reconstruct the ancestral states of habitat use at the major nodes in the phylogenetic tree of gastropods. Moreover, the phylogenetic relationships among and within gastropod superfamilies/families are far from being complete, especially for those groups containing the deep-sea lineages. For example, the Fissurellidae contains 55 described genera [START_REF] Aktipis | Another step towards understanding the slit-limpets (Fissurellidae, Fissurelloidea, Vetigastropoda, Gastropoda): a combined five-gene molecular phylogeny[END_REF], including 7 genera restricted to the deep-sea habitats (references shown in Fig. 4) but only one was included in the recent phylogenetic study of Fissurellidae [START_REF] Aktipis | Another step towards understanding the slit-limpets (Fissurellidae, Fissurelloidea, Vetigastropoda, Gastropoda): a combined five-gene molecular phylogeny[END_REF]. Conversely, the scissurelloid species inhabit all kinds of marine habitats (Fig. 4) but we included so far only the deep-sea floor representative from the genus Anatoma. Mitogenomic data from other habitats and lineages are still lacking because of their very small size, these gastropods are difficult to collect and to identify, whatever the habitat. The scissureloid case emphasizes that the size of organisms is another important sampling bias in the reconstruction of the evolutionary history of the Gastropoda. Our study provides a new framework for the study of the evolution of the Gastropods by indicating the needed sampling efforts: sampling more thoroughly the deep-water habitats and the smaller organisms.

Acknowledgements

The research was mainly supported by the bilateral cooperation research funding from the Ministry of Science and Technology, Taiwan (MOST 102-2923-B-002-001-MY3 to WJC) and the French National Research Agency (ANR 12-ISV7-0005-01 to SS), and partially supported by the personal research funding from the MOST, Taiwan (MOST 107-2611-M-002-007-to WJC). We thank the Institut Français in Taipei and the National Taiwan University for supporting H. Lee's Ph.D. fellowship. We appreciate the help from S.-P. Wu for collecting samples from Taiwan. We are also grateful to A. Warén for providing the samples of L. guaymasensis and P. smaragdina from the expeditions DIVA 1 and Alvin 2009, and the great help for identifying the samples. We thank all the PIs and the participants of the expeditions EXBODI, BIOPAPUA, PAPUA NIUGINI, AURORA 2007, NORFOLK 2, and CONCALIS conducted by MNHN and IRD as part of the program 'Tropical Deep-Sea Benthos'. We also thank L. Corbari for the sample collected during the BICOSE cruise organized by M-A Cambon-Bonavita. We thank M. Hennion for providing the images for neomphalinid and neritimorph; M. Caballer for the images of the caenogastropod, seguenzioid, haliotoid, and phasianelloid; and A. Le Goff for the image of the pleurotomariid; all these images were obtained under the MNHN collection website (science.mnhn.fr). We thank B. Buge for the curation of specimens; H.-H. Chen from M.-H. Tsai's laboratory and J. Abdelkrim and R. Debruyne from the Service de Systématique Moléculaire (SSM -UMS2700) for helping in managing and analyzing the NGS data; J.-N. Chen and P.-C. Lo from W.-J. Chen's laboratory and P. Bouchet, V. Héros, P. Lozouet and P. Maestrati from MNHN for discussion and collection management; and S. Lavoué, Y. Kano, and an anonymous reviewer for discussion and for providing helpful comments on this article.

Figure captions

Figure 1. Phylogenetic relationships of the Gastropoda inferred using partitioned maximum-likelihood (ML) methods based on 11,129 bp nucleotide sequences combined from 13 mitochondrial protein-coding genes and 2 ribosomal RNA genes (small mt dataset). Nodal supports are shown as bootstrap (BP) values in percentage (above) and posterior probabilities (PP) (below). Values below 50% in BP and 0.8 in PP are not shown. Values higher than 80% in BP and 0.95 in PP are considered significant and shown in bold. Taxa names in bold represent the deep-sea lineages. Figure 2. Phylogenetic tree reconstructed using the partitioned maximum-likelihood (ML) methods based on 3,313 bp nucleotide sequences combined from 18S, 28S, and H3 (nuclear dataset). Numbers above/below the nodes are bootstrap (BP) values in percentage / posterior probabilities (PP). Values within superfamily and values below 50% (BP) and 0.8 (PP) are not shown. Values higher than 80% (BP) and 0.95 (PP) are shown in bold. Asterisk indicates the superfamilies not found monophyletic in the tree. Figure 3. Partitioned maximum-likelihood (ML) tree based on 14,442 bp nucleotide sequences combined from 13 mitochondrial protein-coding genes, 2 ribosomal RNA genes, and nuclear genes including 18S, 28S, and H3 (combined dataset). Nodal supports are shown as bootstrap (BP) values in percentage / posterior probability (PP). Values below 50% in BP and 0.8 in PP are not shown. Values higher than 80% in BP and 0.95 in PP are considered significant and shown in bold. Taxa from Cocculiniformia, Neomphalina, Caenogastropoda, Neritimorpha, and from the same vetigastropod superfamily or family are collapsed. The uncollapsed tree is shown in supplementary figure S3.

Fig. 1

 1 Fig.1

  Fig.

Figure 4 .

 4 Figure 4. On the left: a simplified phylogeny modified from the inferred tree based on

Table 1 .

 1 New gastropod mitogenomes assembled in this study. MNHN: Muséum National d'Histoire Naturelle. NTUM: National Taiwan University Museums. The corresponding field data of the specimens from MNHN are found at https://expeditions.mnhn.fr/

		Species	Voucher number	mitogenome	Sequencing method	Expedition, station, and locality (depth)	Habitat
	Cocculiniformia,
	Cocculinoidea
	Bathysciadiidae	Bathysciadiidae sp.	MNHN-IM-2013-40843 MH837532	NGS, Illumina	EXBOBI, CP3791, Canal de la Havannah, New	cephalopod beak
						Caledonia (806m)
	Cocculinidae	Cocculina subcompressa	MNHN-IM-2013-40775 MH837536	NGS, Illumina	BIOPAPUA, CP3645, Huon Gulf, Papua New Guinea	sunken wood
						(410m)
	Cocculinidae	Coccocrater sp.	MNHN-IM-2013-41044 MH837535	NGS, Iron Torrent	PAPUA NIUGINI, CP3967, Sek Island, Papua New	sunken wood
						Guinea (980m)
	Neomphalina,
	Neomphaloidea
	Peltospiridae	Peltospira smaragdina	NTUM-Inv-00001	MH837538	NGS, Illumina	DIVA 1, DV09, Lucky Strike vent, Mid-Atlantic Ridge	hydrothermal vent
						(1653m)
	Neritimorpha,
	Neritoidea
	Phenacolepadidae	Shinkailepas briandi	MNHN-IM-2013-41914 MH837541	NGS, Iron Torrent	BISCOSE 1, PL568 ASPI2, Snake Pit, Mid-Atlantic	hydrothermal vent
						Ridge (3466m)
	Vetigastropoda,
	Lepetelloidea
	Pseudococculinidae Pseudococculinidae sp.	MNHN-IM-2013-40847 MH837540	NGS, Illumina	AROURA 2007, CP2736, East Philippines (346m)	sunken wood
	Lepetodriloidea
	Lepetodrilidae	Lepetodrilus guaymasensis	NTUM-Inv-00002	MH837537	NGS, Illumina	Alvin 2009, AT 15-59 sta. dive 4590, Off Punta	methane seep
						Arenas, Costa Rica (1800m)
	Pleurotomarioidea
	Pleurotomariidae	Bayerotrochus teramachii	N/A	MH837533	LR-PCR	North-East of Taiwan (unknown)	dee-sea floor
	Pleurotomariidae	Perotrochus caledonicus	MNHN-IM-2009-7483	MH837539	NGS, Iron Torrent	NORFOLK 2, DW2156, Ile des Pins, New Caledonia	dee-sea floor
						(484m)
	Scissurelloidea
	Anatomidae	Anatoma sp.	MNHN-IM-2013-42003 MH837531	NGS, Illumina	CONCALIS, DW3025, Grand Passage, New Caledonia	dee-sea floor
						(398m)
	Seguenzioidea
	Calliotropidae	Calliotropis micraulax	MNHN-IM-2009-31774 MH837534	NGS, Illumina	EXBODI, CP3842, Passe de la Sarcelle, New	dee-sea floor
						Caledonia (763m)

Table 2 .

 2 Summary of features of the new gastropod mitogenomes, including total size of the genome (bp), percentage of A and T (AT %), and the sizes of protein-coding genes (bp).

		Anatoma	Bathysciadiid	Calliotropis	Coccocrater	Cocculina	Lepetodrilus	Bayerotrochu	Peltospira	Perotrochus	Pseudococculinid	Shinkailepas
		sp.	ae sp.	micraulax	sp.	subcompressa	guaymasensis	s teramachii	smaragdina	caledonicus	ae sp.	briandi
	size	13,568	17,138	17,040	13,783	18,067	17,060	13,473	15,112	13,782	15,380	13,618
	AT%	61.0	63.9	63.3	65.8	70.6	63.4.	63.6	69.4	60.0	72.0	61.7
	cox1	1,548	1,539	1,554	1,533	1,542	1,545	1,536	1,542	1,536 b	1,548	1,548
	cox2	699	684	684	690	690	708	693	702	702	711	690
	cox3	780	774	780	780	780	780	-a	780	-a	774	780
	atp6	633	504	309 b	579	717	714	696	678	696	495	696
	atp8	165	339	207	225	249	222	174	162	174	231	165
	cytb	1,137	1,149	1,164	348 b	1,137	1,137	1,140	1,140	1,140	1,137	1,137
	nd1	945	948	1,002	852 b	948	939	921	948	936	951	933
	nd2	1,086	1,038	1,095	1,116	1,119	1,089	1,059	1,095	1,099	1,002	1,131
	nd3	354	357	354	354	372	351	354	381	323 b	354	354
	nd4	1,347	1,344	1,302	-a	1,359	1,386	1,305	1,227	1,324 b	1,383	1,398
	nd4l	300	285	297	-a	300	300	276	300	297	303	297
	nd5	1,737	1,743	1,746	1,351 b	1,725	1,734	1,784	1,701	1,731	1,701	1,716
	nd6	489	525	510	495	516	519	507	498	507	504	507

a: gene could not be sequenced; b: incomplete sequence