Paul Zaharias
email: paul.zaharias@edu.mnhn.fr

Yuri I Kantor

Alexander E Fedosov

Francesco Criscione

Anders Hallan

Yasunori Kano

Jérémie Bardin

Nicolas Puillandre

MADEEP Kavieng

Just the once will not hurt: DNA suggests species lumping over two oceans in deep-sea snails (Cryptogemma)

Keywords: species delineation < Taxonomy, species redescription < Taxonomy, Gastropoda < Taxa

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

The advent of integrative taxonomy [START_REF] Dayrat | Towards integrative taxonomy[END_REF][START_REF] Will | The Perils of DNA Barcoding and the Need 1071 for Integrative Taxonomy[END_REF], mainly driven by 3 the molecular revolution, has led to a great remodeling of the practice of species 4 delimitation, extending the use of standardized approaches in the field of taxonomy (e.g.

Species delimitation

147 COI sequences were aligned manually using MEGA X [START_REF] Kumar | MEGA X: Molecular 1003 Evolutionary Genetics Analysis across Computing Platforms[END_REF]; 12S and 148 28S sequences were aligned using Muscle [START_REF] Edgar | MUSCLE: multiple sequence alignment with high accuracy and high 951 throughput[END_REF] as implemented in MEGA X 149 and then checked by eye. Phylogenetic reconstruction methods were applied to the COI 150 alignment, plus a concatenation of the COI, 12S and 28S alignments with one specimen 151 per species (see Results section).ABGD, GMYC and mPTP methods were applied to 152 the CO1 dataset. All three methods are exploratory and aim at finding clusters of 153 specimens corresponding to species but differ strongly in several aspects. ABGD is 154 based on genetic distances only and automatically detects the gap (the so-called 155 "barcode gap") between intra and interspecific distances, then used to delimit species 156 hypotheses. On the contrary, GMYC and mPTP rely on phylogenetic trees. GMYC uses 157 an ultrametric tree and looks for a transition between speciation and coalescent nodes.

158 mPTP also delimit species by identifying the transition between the inter and 159 intraspecific parts of the tree, but requires only a phylogeny with branch length being 160 proportional to the number of substitutions, and relies on the mutation rate instead of the 161 branching rate.

162

For ABGD, the web version (http://wwwabi.snv.jussieu.fr/public/abgd/) and the default 163 parameters were used, with a Kimura (K80) TS/TV model implemented. Maximum 164 likelihoods trees were constructed for COI, and the concatenated (COI + 12S + 28S) 165 alignments using IQ-TREE v1.6.3 (Nguyen et al. 2014), with 100 bootstraps. For all 166 alignments, COI was consistently divided into three partitions, corresponding to the [START_REF] Kalyaanamoorthy | 989 ModelFinder: fast model selection for accurate phylogenetic estimates[END_REF]. The mPTP approach of species delimitation (Kapli et 170 al., 2017) was performed on the COI maximum likelihood tree, using both ML 171 delimitation with default parameters and MCMC method with a Markov chain of 172 200,000,000 generations, a sampling frequency each 2,000 generations and a burnin set 173 at 25%. Bayesian trees were reconstructed using BEAST v2.5.0 (Bouckaert et al.,174 2019), running 200,000,000 generations on the COI dataset with a sampling frequency 175 every 4,000 generations and a burnin set at 10%. The COI dataset was partitioned by 176 codon position for model evaluation, and prior values were set as in Puillandre et al. 177 (2017). The resulting trees were combined using TreeAnnotator as implemented in 178 BEAST2, with node heights corresponding to mean heights of all trees. The "single 179 threshold" method of GMYC was applied using the resulting tree. Finally, a non-180 ultrametric Bayesian tree was reconstructed for the COI, and concatenated datasets 181 using MrBayes v3.2.6 [START_REF] Ronquist | MrBayes 3: Bayesian phylogenetic inference under 1039 mixed models[END_REF], each of the two runs consisting 182 of 8 Markov chains and 20,000,000 generations and a sampling frequency each 2,000.

183

The analysis was performed on the Cipres Science Gateway 184 (http://www.phylo.org/portal2). The consensus tree was calculated with burnin set at 185 20%.

186 Four species of Turridae were used as outgroups: Turris babylonia (Linnaeus, 1758), 187 Gemmula kieneri (Doumet, 1840), Unedogemmula unedo (Kiener, 1839) and 188 Turridrupa sp. The MolD program [START_REF] Fedosov | Revisiting use of DNA characters in 955 taxonomy with MolD-a tree independent algorithm to retrieve diagnostic nucleotide 956 characters from monolocus datasets[END_REF] was used to identify unique 189 combinations of diagnostic sites for each delimited species. protoconch whorls counted according to Bouchet & Kantor (2004).

196

Variation of shell shapes was analyzed through their outlines. Initially, we made a 197 selection of shells of sequenced specimens that were not broken, truncated or aberrant.

198

To complete this dataset, we obtained photographs of 20 name-bearing types 199 (corresponding to 21 species names) that were deemed high-quality, of which 18 were 200 suitable for morphometric analysis (nearly complete shells). In total, shape acquisition

229

All the material examined for this study is summarized in Table S1. on Turridae (e.g. [START_REF] Powell | The family Turridae in the Indo-Pacific. Part 1. The subfamily 1022 Turrinae[END_REF][START_REF] Tucker | Catalog of Recent and fossil turrids (Mollusca: Gastropoda)[END_REF] and by comparison of type specimens or 264 their images to shells of sequenced specimens, we found 23 names (see Taxonomy 265 section) that we applied tentatively to this partition. A total of 18 name-bearing types 266 corresponding to 19 names were of sufficient quality to be included in the 267 morphometric study. The other name-bearing types were discussed and assigned to 268 molecular species using only the shell photographs or drawings and extrinsic parameters 269 such as the bathymetry or geography (see Taxonomy section).

270

Despite considerable intraspecific morphological heterogeneity, the morphometric 271 analysis yielded instructive results: using shape alone, the leave-one-out cross-272 validation procedure successfully predicted correct species attribution of 84% of 273 sequenced specimens and confirmed placement of 14 types out of 18 a priori attributed 274 to our species hypotheses (see Figure S3). By adding shell size, we increased the score 275 of the leave-one-out cross-validation procedure to 90.64% of the sequenced specimens, 276 and 15 out of 18 types (Fig. 1). The three type specimens whose placement was 277 incorrectly a priori attributed were Bathybermudia carynae (only representative of its 278 species), Pleurotoma praesignis and Ptychosyrinx bisinuata japonica (see also 279 taxonomic section). The most discriminant axis (Fig. 1, axis 1: 71.92% of trace) 280 corresponds to the variation range of the siphonal canal length and the width of the 281 shell, while variation along the second axis (15.57% of trace) mainly corresponds to the 282 intensity of the curvature of the outer aperture lip.

283

The phylogenetic trees resulting from the COI (Fig. 2) and concatenated-gene analyses 284 (Fig. 3) showed poorly supported interspecific relationships, except for the Philippines (see Table S1). All species geographic (Fig. 4) and depth (Fig. 5 Included species: Cryptogemma aethiopica (Thiele, 1925); C. periscelida (Dall, 1889) [START_REF] Powell | The family Turridae in the Indo-Pacific. Part 1. The subfamily 1022 Turrinae[END_REF]; C. timorensis (Tesch, 1915); C. unilineata [START_REF] Powell | The family Turridae in the Indo-Pacific. Part 1. The subfamily 1022 Turrinae[END_REF]. Counting starts right next to the 3' end of the LCO1490 primer (from 1 to 658).

403

Distribution: From the Central Indo-Pacific (Banda Sea) to the North Atlantic Ocean 404 (Bermuda Is.) (Fig. 4A), from ~1400 to ~3000 meters depth (Fig. 5). This species is 405 expected to be found in the Indian Ocean, as is the case with other Indo-Pacific species

F o r R e v i e w O n l y 18 406
of Cryptogemma, but it has not been documented there, due presumably to a lack of 407 sampling at bathyal depths. [START_REF] Barnard | Contribution to the knowledge of South African marine Mollusca. 895 Part 1. Gastropoda; Prosobranchiata: Toxoglossa. Annals of the South African Museum[END_REF], contrasting with the large 467 unicuspid rectangular-based central tooth described in P. bisinuata by Thiele (1929, p. 468 359). [START_REF] Powell | The family Turridae in the Indo-Pacific. Part 1. The subfamily 1022 Turrinae[END_REF] suggested that Thiele could have mixed his radular preparations, 469 but in light of the radulae type of Cryptogemma (Fig. 8), all composed of a unicuspid 470 central formation, it is more likely that either it was Barnard, who mixed up the radula Distribution: This species is known to occur from the West Indian Ocean to the East 481 Pacific Ocean (Fig. 4B), from ~300 to ~1400 m of depth (Fig. 5).

483

Cryptogemma timorensis (Tesch, 1915) 484 (Fig. 8L-P uninterrupted along its length (Fig. 7I).

504

This type of this species, Pleurotoma timorensis, is a fossil specimen from Timor island 505 (Indonesia) described by Tesch, and Martin described another very similar specimen 506 from Buton island (Indonesia), Pleurotoma ktolemandoënsis, without reference to the 507 work of Tesch. However, Martin furtherly recognized the affinities between P. 508 timorensis and P. ktolemandoënsis [START_REF] Martin | Oligocaene Gastropoden von Buton[END_REF]. Finally, [START_REF] Robba | Early Pleistocene gastropods from 1037 Timor[END_REF] 509 synonymized P. ktolemandoënsis with P. timorensis. [START_REF] Powell | The family Turridae in the Indo-Pacific. Part 1. The subfamily 1022 Turrinae[END_REF]

555

The former Pinguigemmula genus is easily distinguishable from Gemmula in having a 556 broadly conic spire, a strongly constricted base and a long, straight siphonal canal 557 [START_REF] Powell | The family Turridae in the Indo-Pacific. Part 1. The subfamily 1022 Turrinae[END_REF]. Although expressing a wide variety of forms, the molecular analysis 558 resulted in a single species hypothesis. Several species were described based on the 559 sculptural details such as the number of gemmate cords (1, 2 or none). The sculpture 560 seems to correlate with geography, with the forms from West Indian Ocean having 561 smooth inter-suture sculpture, while the forms from East Indian-West Pacific usually 562 possess two or three gemmate cords. Some large specimens of this species possess a 563 similar 'tertiary notch' to that of C. praesignis, suggesting sexual dimorphism also for 564 this species [START_REF] Kantor | Sexual Dimorphism in the Apertural Notch of a New 997 Species of Gemmula (Gastropoda: Turridae)[END_REF]. outline and a more tuberculate subsutural fold [START_REF] Powell | The family Turridae in the Indo-Pacific. Part 1. The subfamily 1022 Turrinae[END_REF]. Although readily 596 distinguished from the other Cryptogemma species due to its color pattern and small 597 size, LDA analysis indicates that this species more closely resembles small adults of C.

638

7D), from ~300 to ~800 meters of depth (Fig. 5). Remarks: This species corresponds to PSH 15 in [START_REF] Puillandre | Large-scale species delimitation method for hyperdiverse groups[END_REF]. Cryptogemma 706 powelli sp. nov. exhibits similar intraspecific variation in shell shape to that of its 707 congeners, based on its convex hull area (Fig. 1). Studied specimens vary in shades of 708 shell color, from light yellowish to light orange, occasionally with a patchy pattern. In 709 terms of both size and shape, C. powelli can in some cases be undistinguishable from C. 710 unilineata (Fig. 1). The morphometric analysis shows considerable overlap of the 711 measured shell features between the two species, with the main distinction between 712 them expressed by the second axis. Interpretation of this result suggests that the C. new species (e.g. [START_REF] Puillandre | A quest for 1028 the lost types of Lophiotoma (Gastropoda: Conoidea: Turridae): integrative taxonomy in 1029 a nomenclatural mess[END_REF]Abdelkrim et al., 2018b). All the synonymized 736 species names in this group were established before the 'molecular era of taxonomy', species. However, our study also shows that using only molecular delimitation of 740 species while ignoring some key specimens such as types would limit knowledge of 741 intra-specific morphological variability, thus obscuring also the ability to relate such 742 variability to extrinsic parameters, such as geographic and bathymetric ranges. In this 743 study, morphological study and taxonomic expertise on types resulted in the extension 744 of intra-specific variability (Fig. 1; e.g. C. aethiopica types). This might be explained 745 because, when describing a new species, there can be a strong subjective effect on the 746 choice of the type specimens (usually the largest of a lot). The recent and promising 747 success in sequencing DNA obtained from empty shells [START_REF] Villanea | Reviving Galápagos snails: ancient DNA 1068 extraction and amplification from shells of probably extinct endemic land snails[END_REF] 748 [START_REF] Sarkissian | Ancient DNA analysis identifies 1045 marine mollusc shells as new metagenomic archives of the past[END_REF] should assist in attributing names to molecularly-defined species 749 by sequencing type specimens. However, sequencing shells implies destructive 750 sampling, and will therefore possibly not be accepted by many museum curators.

751

Furthermore, identifying and attributing material to species within a species complex 752 still requires considerable taxonomic investigation.

753

By exploring the intraspecific variability of the shell, in isolation or in relation to 754 interspecific variability, geographical and bathymetric ranges, or ecological data, we 755 here emphasize a few remarks that may provide directions for the future study of this 756 group.

757

Geographical intra-specific variability. In C. aethiopica, for which many species were 758 synonymized, there are significant geographical variations in the spire whorls. Ecological variability. It is shown (e.g. [START_REF] Bierne | Habitat preference and the marine-speciation 901 paradox[END_REF][START_REF] Hauquier | Distinct genetic 970 differentiation and species diversification within two marine nematodes with different 971 habitat preference in Antarctic sediments[END_REF] that 768 some benthic species have clear preferences with regards to the substrate on which they 769 thrive (e.g. sandy/muddy vs. rocky bottom). However, other species are generalists with 770 respect to the substratum, as seems to be the case for the Cryptogemma species. All 771 species were found both on hard and soft substrate, with or without organic matter (e.g. 772 leaves or sunken wood). A remarkable case is C. tessellata, for which the three 773 available trawl images showed three very different environments (Fig. 12), and, even 774 more remarkably, correlated with shell morphology (Fig. 10C-D); specimens found in 775 sandy or spongy environments exhibited a light pale color ground while the individual 776 found in a volcanic rock bottom has a reddish color ground. However, the holotype of 777 this species shows an intermediate pattern and was collected "in mud and sand" 778 [START_REF] Powell | The family Turridae in the Indo-Pacific. Part 1. The subfamily 1022 Turrinae[END_REF]. More material is needed to properly study the potential effects of 779 substrate on shell color.

780

Sexual dimorphism. C. praesignis is the first Turridae turrid species for which apparent 781 sexual dimorphism of the shell has been documented [START_REF] Kantor | Sexual Dimorphism in the Apertural Notch of a New 997 Species of Gemmula (Gastropoda: Turridae)[END_REF]. The Bathymetric zonation. Bathymetric segregation between sister species has been shown 788 in molluscan species (e.g. [START_REF] Clague | Phylogeography 935 of Acesta clams from submarine seamounts and escarpments along the western margin 936 of North America[END_REF]. In the Turridae, it has been found that a 789 pair of sister species, Lophiotoma abbreviata and L. brevicaudata, show distinct 790 bathymetric preferences, with the former observed at an average depth of 3 m and the 791 latter at an average depth of 15 m [START_REF] Puillandre | A quest for 1028 the lost types of Lophiotoma (Gastropoda: Conoidea: Turridae): integrative taxonomy in 1029 a nomenclatural mess[END_REF]. The present study 792 demonstrates that, at a much wider scale, a similar case was found in the C. 793 praesignis/C. phymatias species pair, with the former species occurring between ~300 794 to ~1400 m and the latter between ~1400 to ~3000m depth (Fig. 5). This example could 795 be added to the list of models used for studying speciation processes in benthic species 796 with high dispersal capabilities and large geographic distributions, for which a 797 topographic barrier would not satisfactorily explain genetic isolation (see Etter et al., 798 2005;Zardus et al., 2006).

799

Geographical ranges. While an Indo-Pacific distribution is common in benthic species, 800 but so far no -molecularly supported -cases of Pacific-Atlantic distribution have been 801 found in benthic gastropods. There are cases of cosmopolitan pelagic gastropods: 802 Glaucus atlanticus [START_REF] Churchill | Molecular and morphological systematics 932 of neustonic nudibranchs (Mollusca : Gastropoda : Glaucidae : Glaucus), with 933 descriptions of three new cryptic species[END_REF], Clione limacina [START_REF] Jennings | Species diversity of 980 planktonic gastropods (Pteropoda and Heteropoda) from six ocean regions based on 981 DNA barcode analysis[END_REF] and 803 Fiona pinnata [START_REF] Trickey | Transoceanic dispersal and cryptic diversity in a 1064 cosmopolitan rafting nudibranch[END_REF]. To this extent, C. phymatias could be the first 804 documented, molecularly supported, case of a benthic gastropod with a Pacific-Atlantic 805 distribution (Fig. 4A). Cases of such molecularly confirmed cosmopolitan benthic 806 species have been found in other phyla such as Annelida [START_REF] Meyer | Morphological and molecular data 1014 suggest a cosmopolitan distribution of the polychaete Proscoloplos cygnochaetus Day, 1015 1954 (Annelida, Orbiniidae)[END_REF]Sun et al., 807 2016;[START_REF] Tomioka | 1061 Cosmopolitan or Cryptic Species? A Case Study of Capitella teleta (Annelida: 1062 Capitellidae)[END_REF]), Arthropoda (Casanova et al., 1998;Havermans et al., 808 2013) and Algae [START_REF] Macaya | Dna Barcoding and Genetic Divergence in the Giant 1010 Kelp Macrocystis (laminariales)1[END_REF]. However, human-mediated introduction 809 was strongly suggested for some of these cases, e.g. annelids. Nothing is known of the 810 biological features of C. phymatias that would enable such a wide dispersal of larvae.

815

However, no correlation was found between the number of whorls of protoconch in a 816 specimen and its associated depth, implying that, from a certain threshold (e.g. 4), the 817 number of whorls is not the only factor determining the ability to disperse. Furthermore, 818 in other neogastropod species complex for which there is a comparable number of 819 protoconch whorls (e.g. [START_REF] Sanders | 1041 One for each ocean: revision of the Bursa granularis species complex (Gastropoda: 1042 Tonnoidea: Bursidae)[END_REF] [START_REF] Kantor | New and unusual deep-water Conoidea 992 revised with shell, radula and DNA characters[END_REF]. Large-scale morphometrics correlated with

837

Radular morphology. The radula is an important morphological character in conoidean 838 taxonomy. In Turridae, the radula is seemingly quite similar between congeneric species 839 with few exceptions, such as for Iotyrris (Abdelkrim et al, 2018b). Cryptogemma

  s, followed by extension at 72 °C for 1 min. The final extension was at 72 144 °C for 5 min. 145 146

  , and 12S and 28S considered each as a single partition. The best 168 model for each partition was evaluated using ModelFinder as implemented in IQ-TREE 169

  190

  by standard methods[START_REF] Kantor | Evolution of the Radular Apparatus in Conoidea 994 (Gastropoda: Neogastropoda) as Inferred from a Molecular Phylogeny[END_REF] and examined 193 by scanning electron microscopes JEOL JSM 840A and Tescan VEGA II LSU at the 194 MNHN. Protoconchs and shells were measured in standard position and the number of

  195

201

  was performed for 197 sequenced specimens, 18 types and 8 unsequenced specimens of 202 C. periscelida. 203 Shell shape was acquired from photographs using a Photoshop CC 2019 script. Outlines 204 were reconstructed with the package Momocs (Bonhomme et al. 2014) from the R 205software. We used Elliptical Fourier analysis[START_REF] Giardina | Accuracy of curve approximation by harmonically related 965 vectors with elliptical loci[END_REF], which presents 206 many advantages over alternative Fourier analyses(Bonhomme et al. 2014), to 207 decompose the closed outlines of the shells into periodic functions. The number of 208 harmonics necessary to describe the shape was selected to reach 99.9% of the 209 cumulative harmonic power, ensuring that the majority of shell shape outlines were 210 considered. Shapes were also checked by eye to ensure that enough complexity was 211 captured. The obtained coefficients were normalized to allow the superposition of the 212 first ellipse and make the shapes comparable. We checked that all the reconstructed 213 outlines were appropriately aligned. A principal component analysis (PCA) was 214 performed on the harmonics coefficients in order to capture an optimal shape variation number of principal components. Finally, a linear discriminant analysis 216 (LDA) was performed to find the linear combination of principal components that 217 maximizes difference between specimens from species delimited with DNA. Fourier 218 analysis does not take size into account; only shapes are compared. As the size of the 219 shells may provide some discriminatory information, we added the length of each 220 specimen as a variable for the LDA. The appropriate number of principal components to 221 use in the LDA model was carefully chosen to find optimal equilibrium between 222 maximizing the differences and overfitting. A leave-one-out cross-validation procedure 223 was used to find the number of PC axes leading to the best percentage of species 224 predictions (see Figure S1). In order to test if our species attribution to each molecular 225 species partition was correct, type specimens were not included in the LDA training set 226 but subsequently added in the LDA morphospace. Another reason not to include the 227 types in the training set was to extract at best the morphological differences between 228 molecular species without the influences of type specimens morphology.

  303 COI, 32 28S and 18 12S sequences of Cryptogemma were used for this 234 study, of which respectively 255, 8 and 16 were newly obtained. The USNM and 235 ZMMU samples yielded poor DNA quality, making them difficult to sequence with a 236 classical Sanger sequencing approach. Nevertheless, the 12S was obtained for the 237 USNM96485 and USNM123087 paralectotypes of Gemmula benthima collected 128 238 years ago. For the ZMMU paratype, the USNM123092 paralectotype and the

  According to WoRMS (checked July 2019), Cryptogemma comprises 11 344 species, and has never been revised. A consultation of available type photos and 345 published images on Cryptogemma species indicated that all of 11 species except the 346 type species C. benthima and C. phymatias, the senior synonym of C. benthima, should 347 be excluded from the genus, as the majority of them lack key characters such as the 348 narrow fusiform shell and the well-marked peripheral anal sinus. Moreover, studied 349 samples from Japan most certainly corresponding to the Cryptogemma corneus as 350 pictured in Hasegawa (2009: figs 335-338), showed closer affinity to other conoidean 351 families (e.g. Horaiclavidae) than to the Turridae, based on both, the sequence of the 352 barcode fragment of COI, and radular morphology (results not shown). The eight 353 species of Cryptogemma listed herein have wide distributions, most of them covering 354 the whole Indo-Pacific tropical region, and are not found at depths shallower than 355 200m. 356 Protoconch consisting of 4 to 5.25 whorls. Shell shape of the teleoconch combined with 357 size is an important factor to distinguish species (see Results section), despite high 358 intra-specific variability. Widest variability range observed in shell proportions, with last whorl and long siphonal canal being at one extreme of the range, 360 and more elongated shells with short siphonal canal on the other.

  R. B. Watson, 1886. 1920 m, Philippine Islands, 16°42'N, 366 119°22'E (Expedition H.M.S. Challenger on November 13, 1874, st. 205).367Gemmula benthimaDall, 1908Dall, . 2323 m, Gulf of Panama. 368 Gemmula benthina -Dall, 1918: 318 (lapsus calami); Powell, 1964: 279 (lapsus 369 calami). 370 Pleurotoma truncata Schepman, 1913. 2798 m, Banda Sea, Indonesia, 6°24'S, 371 124°39'E. 372 Bathybermudia carynae Haas, 1949. 3109 m, off Bermuda, 32°08.2'N, 64°33'W.373 374 Remarks: This species was not present in the sampling of Puillandre et al. (2012) but 375 was included in the phylogeny of Puillandre et al. (2011) under the name Ptychosyrinx 376 carynae. 377 Due to the frequent loss of the shell apex, there are very few available protoconchs for 378 this species. Only the eroded protoconch of the type specimen of Bathybermudia 379 carynae could be measured, with PD= 1.3 mm and PL > 1.6 mm, consisting of more 380 than 3 whorls. 381 Radula medium long, about 2.6 mm in length (0.44 of AL), composed of 74 transverse 382 rows of teeth. Marginal teeth 118-128 µm long (mean 120 µm, n = 5), duplex. Anterior

  tooth length solid, very narrow in dorsal view, pointed, in posterior 2/3 384 major and accessory limbs broadly bifurcating, accessory limb with clear constriction at 385 about half tooth length, slightly shorter than major limb. Central formation with distinct 386 narrow carinated cusp and lateral inconspicuous flaps with indistinct lateral and anterior 387 margins (Fig. 7B-C). 388 Although this species can be distinguished from its congeners by the frequent loss of the 389 first whorls, and from its sister species Cryptogemma praesignis by the slightly larger 390 diameter of the shell, the bathymetry is its most distinct characteristic. C. praesignis is 391 found between ~300 and ~1400 m depth, while Cryptogemma phymatias is the only 392 Turridae to our knowledge to be exclusively found below ~1400 m deep. Moreover, it is 393 the only Turridae so far, and possibly the first reported benthic gastropod, to have a 394 Pacific-Atlantic distribution (Fig. 4; see Discussion). Most studied type specimens have 395 very eroded shells, but the variability of the last whorls, siphonal twist and aperture 396 among them is in agreement with the variability in the sequenced specimens 397 Noteworthily, the etymology of this species combines "Cryptogemma" (hidden gems) 398 and "phymatias" (one who has tubercules), resulting in a confusing combination.399 400 List of COI diagnostic sites (position: character state): [379: T, 493: C, 622: C].

  401

?

  Smith, 1895. 1234 m, off Colombo, Ceylon. 413 Pleurotoma microscelidaDall, 1895. 642 m, South of Oahu Island Albatross st. 3475 414 Pleurotoma (Subulata) bisinuata Martens, 1901. 1134 m, off East Africa, 1°49'N, 415 45°29'E. 416 Pleurotoma rotatilis Martens, 1902. 1134 m, off Mogadishu, Somalia, East Africa, 417 1°49'N, 45°29'E. 418 Pleurotoma (Surcula) lobata G. B. Sowerby III, 1903. 805 m, off Cape Natal, Durban419 and 567 m, off Buffalo River, East London, S. Africa. E. A. Smith, 1906. 420 Ptychosyrinx bisinuata japonica Okutani, 1964. 620 m, Sea of Enshu-nada, Japan, 421 34°25.7'N, 137°58.5'E. 422 Ptychosyrinx lordhoweensis Kantor & Sysoev, 1991:205, figs. 1, 2. 1210 m, Lord Howe 423 Rise, off eastern Australia, 30°24'S, 161°51'E. 424 425 Remarks: This species corresponds to PSH 11 in Puillandre et al. (2012). 426 The protoconch exhibits a wide size range, with PD = 1.05-1.38 mm and PL = 1.4-1.93 427 mm, and from 4 to 5.2 whorls. 428 Radula long, about 3.4 mm in length (0.44 of AL), formed of 72 transverse rows of 429 teeth. Marginal teeth 142-151 µm long (mean 149 µm, n = 5, or 1.9% of AL), duplex.

  ) 1/3 of tooth length solid, narrow in dorsal view, pointed, in posterior 431 2/3 major and accessory limbs broadly bifurcating, accessory limb with clear 432 constriction and bent at about half tooth length, shorter than major limb. Central 433 formation with distinct narrow carinated cusp and lateral inconspicuous flaps with 434 indistinct lateral and anterior margins (Fig.7A).435This species is morphologically close to Cryptogemma timorensis, although it differs 436 from that species in its generally smaller size (~15-35mm SL), in having a narrower last 437 whorl, and the presence of a tertiary apertural notch in mature females. Cryptogemma. 438 praesignis also differs from C. timorensis in having a much longer radula (0.44 vs 0.2 of 439 AL) and relatively longer marginal teeth (1.9% of AL vs 1.35%). Anatomical 440 examination of Australian material (C571714 and C571704) shows that mature males 441 (SL 29.4 and 34.9 mm respectively) can obtain an extremely large and muscular penis, 442 with a width almost equal to the width of the animal itself, and with a very large, long 443 lateral appendage situated distally. The penial tip is rather blunt, with the opening 444 situated distally. A third, smaller Australian specimen (C571757; SL 25.7 mm), did not 445 possess a well-developed papilla, suggesting that such a feature may develop with 446 increasing maturity and therefore not be present in subadults. Furthermore, the shells of 447 C571714 and C571704 did not possess a tertiary apertural notch. The observation of 448 C571714 and C571704 supplements the findings of Kantor & Sysoev (1991) that the 449 mature females of this species develop a tertiary apertural notch. The authors 450 hypothesized that this structure was possibly connected with the process of fertilization. 451 Such a feature has been observed in C. aethiopica specimens as well (Fig. 9G, I), but 452 not in other Cryptogemma species.

  morphology, and more specifically the presence of a tertiary apertural notch 454 in Pleurotoma bisinuata and Ptychosryinx bisinuata japonica, supports the 455 synonymization of these names with C. praesignis. Because the holotype of P. bisinuata 456 japonica is a comparatively large specimen (39 mm), the result of the LDA and the 457 leave-one-out cross-validation procedure attributed it to C. timorensis. Possibly for this 458 same reason, the holotype of P. praesignis (42 mm) also fell into the C. timorensis 459 range. The result of the LDA analysis and the leave-one-out cross-validation procedure 460 when considering only shape predicted the two holotypes as C. praesignis, (see Figure 461 S3) thus justifying the attribution of P. praesignis to this molecular species. The 462 sequencing of a paratype of P. lordhoweensis confirmed its conspecificity with C. 463 praesignis. The name Pleurotoma lobata, although conchologically very similar to P. 464 bisinuata, has never been synonymized based on the reportedly different morphology of 465 the radula: the absence of a central tooth (= central formation as understood herein) in 466 the specimens of P. lobata examined by

  471 preparation, or that the specimens examined by Barnard are not Cryptogemma 472 specimens. The holotype of Pleurotoma rotatilis is almost identical conchologically to a 473 sequenced juvenile from the East African coast (MNHN-IM-2013-62900; Fig. 8J).

474

  Some doubts remain as to the status of Pleurotoma microscelida, but the size and 475 overall last whorl morphology of the type specimen show a stronger resemblance to C. 476 praesignis than to any other Cryptogemma species.

  diagnostic sites (position: character state): [232: A, 319: T, 407: C, 409: T] 479 480

  Pliocene of Timor Island.

  This species corresponds to PSH 14 of[START_REF] Puillandre | Large-scale species delimitation method for hyperdiverse groups[END_REF]. 494 Protoconch commonly eroded, with PD = 1-1.2 mm and PL = 1.3-1.675 mm and 495 number of whorls varying from 4 to 4.5. 496 Radula medium short, about 2.5 mm in length (0.2 of AL), formed of 45 transverse 497 rows of teeth. Marginal teeth 162-172 µm long (mean 168 µm, n = 5, or 1.35% of AL), 498 duplex. Anterior (inner) 1/3 of tooth length solid, medium broad in dorsal view, 499 lanceolate, in posterior 2/3 major and accessory limbs broadly bifurcating, accessory 500 limb with clear constriction and bent at about half tooth length, nearly the same length . Central formation with short and obtuse cusp and lateral inconspicuous 502 flaps with indistinct lateral and anterior margins. Posterior margin thickened and 503

  described the 510 subspecies Ptychosyrinx timorensis teschi based on an extant specimen, which Sysoev 511 (1996) elevated to species rank. Both authors' arguments for separating the fossil 512 species from the extant was the 'much broader' shell of the extant Ptychosyrinx 513 timorensis teschi specimen. Examination of the morphological variability of the 514 sequenced specimens showed that dimensions and shape of fossil specimens is close to 515 the inferred average breadth of the species; some specimens show broader aperture and 516 a taller last whorl than others, despite having the same number of teleochonch whorls. 517 The results of the LDA showed that P. timorensis and P. timorensis teschi fall into the 518 variability of the sequenced specimens. The broader last whorl and the generally larger 519 size (~30-55mm) are characteristic for C. timorensis when compared to C. praesignis. 520 521 List of COI diagnostic sites (position: character state): [73: G, 214: G, 334: G, 511: G] about 3.4 mm in length (0.37 of AL), composed of 77 transverse rows of 548 teeth. Marginal teeth 150-159 µm long (mean 155 µm, n = 5, or 1.66% of AL), duplex. 549 Anterior (inner) 0.4 of tooth length solid, narrow in dorsal view, awl-shaped, in 550 posterior part major and accessory limbs broadly bifurcating, accessory limb with clear 551 constriction and bent at about half tooth length, thin, nearly same length as major limb. 552 Central formation with long, very narrow, sharp, carinated cusp and lateral flaps with 553 distinct posterior and antero-lateral margins. Flaps not completely fused with cusp (Fig.5547E).

  diagnostic sites (position: character state): [115: C, 307: A, 418: G] 567 568Distribution: Found in East Africa to Central Indo-Pacific (Fig.4E), from ~400 to ~850 569 meters of depth (Fig.5).

  diagnostic sites (position: character state): [46: C, 61: A, 316: T] 601 602 Distribution: The sequenced specimens were found in New Caledonia only and the 603 holotype is from the Hawaiian Islands (Fig. 4C), from ~200 to ~500 meters depth (Fig. 604 5). The protoconch characteristics, similar in shape, size and number of whorls to C. 605 praesignis, imply that the species may have a much broader range than what is currently 606 documented, possibly covering the entire Central Indo-Pacific.

  Powell, 1967:437(22-716a), pl. 313. 366 m, off Waikiki, 612 Oahu Island, Hawaii (Expedition Pele, June 13 1964).

  This species corresponds to PSH 13 in[START_REF] Puillandre | Large-scale species delimitation method for hyperdiverse groups[END_REF]. 615 Protoconch commonly retained, with PD = 1.075-1.275, PL = 1.375-1.925 and number 616 of whorls ranging from 4.75 to 5.5. 617 Radula long, about 3 mm in length (0.31 of AL), composed of 99 transverse rows of 618 teeth. Marginal teeth 121-127 µm long (mean 125 µm, n = 5, or 1.27% of AL), duplex.

  ) 0.4 of tooth length solid, very narrow in dorsal view, awl-shaped, in 620 posterior part major and accessory limbs broadly bifurcating, accessory limb with clear 621 constriction and bent at about half tooth length, slightly shorter than major limb. Central 622 formation with long sharp carinated cusp and lateral flaps with distinct posterior and 623 antero-lateral margins. Flaps not completely fused with cusp (Fig. 7G). 624 This species has been named based on the characteristic carinated brown-orange 625 subsutural band. The brown-orange subsutural band has been found in C. powelli n. sp. 626 and in some specimens of C. praesignis, as well as in other Turridae such as Gemmula 627 cosmoi (Sykes, 1930), but it is generally smoother and thinner to that of C. unilineata. 628 The LDA analysis indicated that C. unilineata has generally a more angulated shape on 629 the outer aperture lip of the last whorl, indicating a more concave subsutural ramp in 630 comparison to other Cryptogemma. However, we note that the convex hulls (Fig. 1) of 631 C. unilineata and C. powelli sp. nov. are greatly overlapping, so the strong carinated 632 brown-orange subsutural band remains the best suited character for distinguishing C. 633 unlineata from C. powelli. 634 635 List of COI diagnostic sites (position: character state): [172: G, 307: T, 361: G, 370: C] 636 637 Distribution: From East Africa to Central Pacific (Hawaiian and Society islands) (Fig.

  unknown, as it is consistently eroded in all examined material, even 649 in younger specimens. 650 Radula medium long (part of youngest section lost), more than 1.9 mm in length (>0.22 651 of AL), composed of more than 50 transverse rows of teeth. Marginal teeth 107-116 µm 652 long (mean 110 µm, n = 5, or 1.25% of AL), duplex. Anterior (inner) 0.4 of tooth length 653 solid, narrow lanceolate in dorsal view, in posterior part major and accessory limbs 654 medium broadly bifurcating, accessory limb with clear constriction and bent at less than 655 half tooth length, thin, nearly same length and width as major limb. Central formation 656 with long, narrow, sharp carinated cusp and lateral flaps with distinct posterior and 657 postero-lateral margins. Flaps completely fused with cusp (Fig. 7D). 658 This species has been described from the Gulf of Mexico, although specimens from 659 Suriname and from the MNHN recent expedition GUYANE 2014 (French Guyana) 660 were also collected. This is the only 'Gemmula'-like species to be found exclusively in 661 the Atlantic Ocean, and it occurs in a 200-800 m depth range.662 663 List of COI diagnostic sites (position: character state): [352: C, 412: G, 655: C] 664 665 Distribution: Found in the Gulf of Mexico and further south to French Guyana (Fig.6664B), from ~200 to ~500m of depth (Fig.5).

  Holotype MNHN-IM-2013-68787; Paratype 1, MNHN IM-2007-40795, 672 paratype 2, MNHN-IM-2007-40765, all live collected and processed for DNA 673 extraction. 674 675 Type locality: New-Caledonia, South-West of Ile des Pins, 22°48'S, 167°15'E, 449-465 676 meters depth, sand and debris (Expedition KANACONO, st. DW4697).

  Named after the New Zealand malacologist A.W.B.Powell (1901Powell ( -1987)), 679 who contributed enormously to the systematics of Conoidea, and in particular, who 680 revised the family Turridae in 1964 and described several species of Cryptogemma. 681 682 Description (holotype): Shell narrowly fusiform, with high spire, and medium-long 683 siphonal canal. Protoconch conical, eroded, with a diameter of 1.125 mm, of about 4 684 convex whorls. Teleoconch of 9 whorls; suture shallow, impressed. Shell height 38 mm, 685 aperture height 10.9 mm and shell diameter 12.5 mm. Whorls strongly shouldered, with 686 slightly concave shoulder slope, very weakly convex, nearly cylindrical periphery. 687 Spiral sculpture of fine, broadly spaced, wavy subequal spiral cords on subsutural ramp, 688 10 on last whorl, uppermost being much more pronounced than others, colored light 689 orange-brown. Sinus cord strongly gemmate, with gemmules clearly bisected (39 on the 690 body whorl). Spiral cords becoming thicker on whorl periphery, sometimes nodulose, than others. Axial sculpture of fine growth lines. Last whorl shortly 692 constricted to long siphonal canal, with 32 cords below sinus, of which about 20 on 693 canal. Aperture irregular oval, outer lip thin, simple. Anal sinus moderately deep, U-694 shaped. Shell color straw-yellow, subsutural cord orange-brown, gemmae slightly 695 lighter than background. 696 Radula medium long, around 3.1 mm in length (0.28 of AL), composed of 75 transverse 697 rows of teeth. Marginal teeth 129-135 µm long (mean 133 µm, n = 5, or 1.22% of AL), 698 duplex. Anterior (inner) 1/3 of tooth length solid, lanceolate in dorsal view, in posterior 699 part major and accessory limbs broadly bifurcating, accessory limb with clear 700 constriction at half tooth length, thin, about half length and width of major limb. Central 701 formation with medium long and medium broad sharp carinated cusp and lateral flaps 702 with distinct posterior and postero-lateral margins. Flaps not completely fused with cusp 703 (Fig. 7H).

713

  powelli on average possess a more concave curvature of the outer aperture lip, 714 compared to C. unilineata. Besides, distinction of C. powelli and C. unilineata mostly degree of pronunciation of the subsutural cord -it is typically not 716 significantly stronger than the succeeding cords in the former species, but is always 717 thick and gemmate in the latter. The subsutural cord is, however, thicker in younger 718 specimens, rendering the distinction of juvenile C. powelli and C. unilineata specimens taxonomy approach, by combining DNA-based species 729 delimitation methods with classical morphological taxonomic methods, we reevaluated 730 the Cryptogemma group studied in Puillandre et al. (2012) with new sequences -in 731 some cases from type material -resulting in eight delimited species. Among the eight 732 delimited species, C. phymatias, C. praesignis, C. timorensis and C. aethiopica 733 comprise 20 of the available species names, with C. powelli the only one described as 734 new. Our results contrast with the usual tendency in this family toward the discovery of 735

737

  reinforcing the recommendation -yet not always followed (Bouchet & Strong, 2010) -738 of systematically relying on molecular analysis, combined with other data, to delimit

759

  Specimens from the West Indian Ocean were found to have very smooth spire whorls, 760 while specimens from the central Indo-Pacific were found with either two or three well-761 marked gemmate cords. Considering the few available discrete shell features, these 762 distinguishable characteristics were used by authors to justify the creation of several

  No such intraspecific variation in shell sculpture was found in the other 764 species of Cryptogemma, although other geographically-structured morphological 765 variability has been observed in the Turridae genus Lophiotoma (Puillandre et al. 2017), 766 with shell color varying from one side of the Indo-Pacific to the other.

  767

782(

  mature) females possess one or several tertiary notches that is probably linked to 783 fertilization according to the authors, since mature males possess an extremely large and 784 muscular penis. The authors proposed the hypothesis that these notches appear 785 periodically during the breeding period, mainly based on the observation of several 786 successive notches on the shells.

  As reported inBouchet & Warren (1979;[START_REF] Folmer | DNA primers for 960 amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan 961 invertebrates[END_REF], many deep-812 water gastropods' larval shells indicate a planktotrophic larval development, which is 813 generally associated with good dispersal abilities and gene flow. The Cryptogemma 814 species are no exception to this, with protoconch varying from 4 to 5.25 whorls.

  the study showed a strict ocean-delimited 820 (e.g. Indian only, Pacific only) distribution. As for C. phymatias, the (eroded) 821 protoconch of the holotype of B. carynae is to our knowledge the only one preserved 822 (but slightly eroded) in this species, and it shows no major difference 823 (diameter/height/number of whorls) from other Cryptogemma species with regards to 824 this character that could explain its very large Pacific-Atlantic distribution. 825 Morphological stasis in teleoconch. C. praesignis and C. timorensis, previously 826 assigned to Ptychosyrinx in Puillandre et al. (2012), show considerable similarity in 827 shell sculpture despite not being sister species, and the two taxa would be difficult to 828 distinguish without their significant difference in size. These two species represent a 829 clear case of morphological homogeneity in Conoidea, for which other examples have 830 been reported

831

  phylogeny may serve to better characterize and understand the conserved 'Gemmula-832 like' form across geological time. Some specimens from the Mio-Pliocene have been 833 attributed to species of Cryptogemma (see 'Taxonomy'), however, an incommensurable 834 number of 'Ptychosyrinx-like' or 'Gemmula-like' Turridae from the Paleogene will to a clade until a more comprehensive understanding, 836 characterization and formalization of the family is achieved.

840Figure 1 :Figure 2 :Figure 3 :

 123 Figure 1: Projection of specimens along the two first discriminant axes. Convex hulls

Figure 4 :

 4 Figure 4: Map showing the species distributions. Filled circles represent localities of

Figure 5 :

 5 Figure 5: Candlestick chart of depth distributions of the eight species. The high and low

Figure 6 :Figure 12 :

 612 Figure 6: Cryptogemma phymatias (Watson, 1886). (A) Lectotype of Gemmula

Figure S1 :

 S1 Figure S1: Result of the leave-one-out cross-validation procedure that was used to find

Figure S3 :

 S3 Figure S2: Results of the ABGD, mPTP and GMYC analysis

) ranges 302 were represented using only sequenced specimens, except for C. periscelida. For the 303 latter, the COI sequencing did not work for the 13 MNHN samples available but 304 information on geography and bathymetry was included nonetheless.

	311	FMNH: Field Museum of Natural History, Chicago, USA.
	312	MNHN: Muséum national d'Histoire naturelle, Paris, France.
	313	NHMUK: Natural History Museum of United Kingdom, London, UK.
	314	USNM: National Museum of Natural History, Smithsonian Institution, Washington,
	315	DC, USA.
	316	ZMB: Museum für Naturkunde, Humboldt-Universität, Berlin, Germany.
	317	ZMMU: Zoological museum of Moscow University, Moscow, Russian Federation.
	318	F ZSIC: Zoological Survey of India, Calcutta, India.
	319 320	Others	o r
	321 322 323 324	AL: Shell aperture length. PD: Protoconch diameter. PL: Protoconch length. R/V: research vessel.	R e v i e w
	325	St.: station.		O
				n l
				y
	305
		14

326 327 Taxonomy 328 329 Superfamily Conoidea Fleming, 1822 330 Family Turridae H. & A. Adams, 1853 (1838) 331 Genus Cryptogemma Dall, 1918

	335	Bathybermudia Haas, 1949 (type species Bathybermudia carynae Haas, 1949 -OD)
	336	†Pinguigemmula McNeil, 1960 (type species †Pinguigemmula okinavensis McNeil,
	337	1960 -OD)
	338
	339
		F
		o r
		R
		e v i e w
		O
		n l
		y
	332
	333	Type species: Gemmula benthima Dall, 1908 (OD)
	334	Ptychosyrinx Thiele, 1925 (type species Pleurotoma bisinuata Martens, 1901 -OD)
		15

  ;

	340	C. phymatias (Watson, 1886); C. powelli sp. nov.; C. praesignis (Smith, 1895); C.

341

tessellata

)

  485 486 †Pleurotoma timorensisTesch, 1915. Timor. Pliocene.

487 †Pleurotoma ktolemandoënsis K.

Martin, 1933. Ktolemando. Miocene. 488

Ptychosyrinx timorensis teschi

Powell, 1964: 291 (22-853), pl. 223, figs. 5, 6. 759 m, 489

NW off Sipadan Island, Borneo.

Zoological Journal of the Linnean Society

constituting the Lophogastrida (Crustacea: Mysidacea) inferred from morphological and

Acknowledgments

This work was supported by the Service de Systématique Moléculaire (UMS 2700 CNRS MNHN), the CONOTAX project funded by the French National Research Agency (grant number ANR-13-JSV7-0013-01), the bilateral cooperation research funding from the Ministry of Science and Technology, Taiwan (grant number MOST 102-2923-B-002-001-MY3) and the French National Research Agency (grant number ANR 12-ISV7-0005-01). The contribution of Y. Kantor and A. Fedosov was supported by the grant from Russian Science Foundation no. 16-14-10118-П (PI Y. Kantor), F. Criscione and A. Hallan supported by a grant from the Australian Biological Resources Study (ABRS grant RF217-57 to PI F. Criscione), and Y. Kano by the Japan Society for the Promotion of Science (KAKENHI grant nos 15H04412 and 18H02494). The material in this paper originates from numerous shore-based expeditions and deep sea cruises, conducted respectively by MNHN and Pro-Natura International (PNI) as part of the Our Planet Reviewed programme (ATIMO VATAE, MAINBAZA, GUYANE 2014, PAPUA NIUGINI, KAVIENG 2014; PI P. Bouchet)) and/or by MNHN and Institut de Recherche pour le Développement (IRD) as part of the Tropical Deep-Sea Benthos programme (BIOMAGLO, TARASOC, MIRIKY, AURORA 2007, EBISCO, TAIWAN 2013, DongSha 2014, NANHAI 2014, BIOPAPUA, SALOMON 2, SALOMONBOA 3, CONCALIS, EXBODI, NORFOLK 2, TERRASSES, ZHONGSHA 2015, KANACONO, KANADEEP, MADEEP; PIs Bertrand Richer de Forges, Philippe Bouchet, Sarah Samadi, Nicolas Puillandre, Wei-Jen Chen, Tin-Yam Chan). Scientific partners included the University of Papua New Guinea (UPNG); National Fisheries College, Kavieng, Papua New Guinea; the National Museum of the Philippines; Institut d'Halieutique et Sciences Marines (IH.SM), Université de Tuléar, Madagascar; University of Taipei and University of Keelung, Taiwan; Universidade Eduardo Mondlane, Maputo; the Madagascar bureau of the Wildlife Conservation Society (WCS); and Instituto Español de Oceanografia (IOE). Funders and sponsors included the Total Foundation, Prince Albert II of Monaco Foundation, Stavros Niarchos Foundation, Richard Lounsbery Foundation, Vinci Entrepose Contracting, Fondation EDF, European Regional Development Fund (ERDF), the Philippines Bureau of Fisheries and Aquatic Research (BFAR), the French Ministry of Foreign Affairs, Fonds Pacifique and the Government of New Caledonia. Additional field work included PANGLAO 2004, a joint project of MNHN and University of San Carlos, Cebu City. All expeditions operated under the regulations then in force in the countries in question and satisfy the conditions set by the Nagoya Protocol for access to genetic resources. The authors would like to thank Virginie Héros, Barbara Buge, and Julien Brisset (MNHN) for their help in curating the vouchers; Alexander Sysoev (ZMMU) for photographs and tissue loan; Christine Zorn (ZMB) for photographs and type specimens loan; Ellen Strong, Mark. S. Florence and Kathy Hollis (USNM) for photographs and specimens loan; Jochen Gerber (FMNH) for photographs; Philippe Maestrati (MNHN) for photographs; Regina Kawamoto & Norine Yeung (BPBM) for photographs; Severine Hannam (AWMM) for photographs; Andreia Salvador (NHMUK) for photographs;Jun Hashimoto (Nagasaki University) for organizing the Nagasaki-maru cruise N275; the Plateau Technique de Microscopie Électronique (PtME) of MNHN for SEM pictures. Posthumous thank goes to Richard Kilburn for photograph of P. praesignis.

completed this sampling with specimens from the Australian museum (AMS), 99 Zoological Museum of Moscow State University


Wadii Abid

Imen Ben Mahmoud

Saoussan Masmoudi

Mohamed Ali Triki

Stéphane Mounier

Emna Ammar

Physico-chemical and spectroscopic quality assessment of compost issued from date palm waste valorization

Keywords: palm waste, goat manure, compost, FTIR, fluorescence spectroscopy, PARAFAC analysis

In order to increase organic matter availability, date palm waste was co-composted with goat manure according to an industrial ecological approach for a sustainable development. During the composting process, the organic matter biodegradation and compost quality were controlled using physico-chemical and biological parameters associated to spectroscopic techniques. To promote the composting process, date palm waste was previously water soaked in basin for 5 days and the resulted wastewater was used to humidify the windrow.

The results showed a rapid temperature increase during the first 6 days and a relatively long compost maturity phase. Organic matter content was reduced by 36% and C/N ratio shifted from 60 to 20 at the process end that held 6 months. The microbial activity and the germination index exceeding 80% indicated respectively the compost stability and its phytotoxicity lack. UV-visible spectra confirmed the organic matter biodegradation by the SUVA 254 , SUVA 269 and SUVA 280 values increase. During the process, the fourier transform infrared spectroscopy (FTIR) analysis of the different collected samples showed qualitatively similar spectra differing in the absorbance bands intensity, proving the organic matter 1 1 2 3 4 5 6 7 8 9 1 2

biodegradation and mineralization. A total of 12 fluorescence excitation emission matrixces of composts were successfully decomposed into a four-factor model using PARAFAC analysis. For the 4 main evidenced components, the excitation/emission (Ex/Em) wavelengths were of 350/450 nm, 400/500 nm 450/530 nm and 325/400 nm attributable respectively to humic-like and fulvic-like substances.

Introduction

At present, organic waste valorization is an important strategy for human and environment safeguard, reducing agricultural costs and chemical products use. Composting was considered as an effective biological, economical and sustainable process to reuse the organic matter wastes (Sellami et al., 2008;Hachicha et al., 2009a;[START_REF] Masmoudi | Characterization of olive mill wastes composts and their humic acids: stability assessment within different particle size fractions[END_REF][START_REF] El Fels | Artemia salina as a new index for assessment of acute cytotoxicity during co-composting of sewage sludge and lignocellulose waste[END_REF]. Indeed, the composting process stabilizes the organic waste by its conversion into humic substances and inactivate pathogens, allowing compost use for soil amendment [START_REF] Hachicha | Compost of poultry manure and olive mill wastes as an alternative fertilizer[END_REF]Zhang and Sun, 2015). Several studies had developed the organic agricultural wastes composting such as olive mill waste [START_REF] Hachicha | Compost of poultry manure and olive mill wastes as an alternative fertilizer[END_REF][START_REF] Hachicha | Quality assessment of composts prepared with olive mill wastewater and agricultural wastes[END_REF][START_REF] Masmoudi | Characterization of olive mill wastes composts and their humic acids: stability assessment within different particle size fractions[END_REF], sludge issued from the olive mill waste evaporation ponds and poultry manure (Hachicha et al., 2009b), horticultural wastes [START_REF] Gavilanes-Terán | Windrow composting as horticultural waste management strategy -A case study in Ecuador Irene[END_REF], green waste [START_REF] Abid | Gestion et valorisation des margines par co compostage avec les déchets verts et amendements des sols agricoles pour l'amélioration des rendements[END_REF]Zhang and Sun, 2015) and Acacia residues [START_REF] Brito | Co-composting of invasive Acacia longifolia with pine bark for horticultural use[END_REF]. Consequently, the composting process and the final product quality depend on many factors such as the raw materials nature and their formulation, the windrow and the ambient temperatures, the windrow materials water content and returning frequency and the microbial activity [START_REF] Masmoudi | Characterization of olive mill wastes composts and their humic acids: stability assessment within different particle size fractions[END_REF][START_REF] Cesaro | Compost from organic solid waste: Quality assessment and European regulations for its sustainable use[END_REF]. Previous studies established that compost quality was especially related to its stability and maturity (Hachicha et al., 2009a;[START_REF] Cesaro | Compost from organic solid waste: Quality assessment and European regulations for its sustainable use[END_REF][START_REF] Nikaeen | Respiration and enzymatic activities as indicators of stabilization of sewage sludge composting[END_REF]. Further, compost quality was assessed with different physico-chemical (pH, electrical conductivity (CE), carbon measure, C/N, temperature and moisture) and biological (microbial population, respiration, germination index, solvitan test) parameters, associated to spectroscopic techniques (FTIR, UV-visible and fluorescence spectroscopy) (Sellami et al., 2008;Yu et al., 2011;[START_REF] Masmoudi | Characterization of olive mill wastes composts and their humic acids: stability assessment within different particle size fractions[END_REF][START_REF] El Fels | Assessment of biotransformation of organic matter during co-composting of sewage sludge-lignocelullosic waste by chemical, FTIR analyses, and phytotoxicity tests[END_REF][START_REF] Mehta | Compost: its role, mechanism and impact on reducing soil-borne plant diseases[END_REF]Song et al., 2015;[START_REF] Kulikowska | Kinetics of organic matter removal and humification progress during sewage sludge composting[END_REF].

In the Mediterranean basin, date palm is largely cultivated. Indeed, in Tunisia, there are more than 4.5 million trees producing an important waste quantities essentially palm leaf waste (16 kg/tree yearly) [START_REF] Ben Salah | Le recyclage des sous-produits des oasis : acquis et perspective[END_REF].

In the present study, an industrial ecological interest was considered in the date palm waste valorization by its co-composting with goat manure and compost quality assessment, using physico-chemical and biological parameters associated to spectroscopic techniques (FTIR, UV-visible and fluorescence spectroscopy) to evaluate the organic matter biodegradation. The final product availability would contribute to a sustainable development.

Materials and methods

Composting procedure

In this study, goat manure and crushed date palm waste composed essentially of palm leaflets both collected from the Oasis of Cheneni-Gabes (South of Tunisia) were used for the composting procedure. The physico-chemical characteristics of the mentioned materials are presented in Table 1.

The composting procedure was carried out at the Chenini Oasis Protection Association composting site, as described in [START_REF] Abid | Date palm wastes co-composted product: an efficient substrate for tomato (Solanum lycopercicum L.) seedling production[END_REF]. The windrow was watered with the previous date palm waste soaking-water, characterized by acidiclow pH (5.83) and its important EC (5.61 mS cm -1 ) and mesophilic flora (4.1 x 10 7 ufc mLl -1 ) to keep a moisture range of around 50%.

During the composting procedure, homogeneous and representative samples were collected from the windrow and refrigerated until analysis. Each sample consists of a mixture of 9 subsamples taken at different points of the windrow. Before analysis, the samples were dried at 105 °C, and then mechanically crushed to obtain homogeneous powder.

Physico-chemical analysis

The windrow temperature was measured using digital thermometer (type Alla, France)

weekly during the mesophilic and the thermophilic composting phases and monthly until its maturity. The presented temperature value consists of a mean of 12 measurements at different points and depths of the windrow.

All the dried and crushed samples were submitted to physico-chemical analysis using standard methods. The pH and EC values were measured in a solution consisting of 20 g of sample in 100 ml of distilled water. Organic matter (OM) was determined by dry matter and weight loss on ignition at 550 °C for 4 h in a muffle furnace (Hachicha et al., 2009b). The total Kjeldahl nitrogen (TKN) was determined according to [START_REF] Afnor) | Determination of nitrogen Kjeldahl -Titrimetric determination method after mineralization and distillation[END_REF]. Phosphorus content, macro and micro elements were determined using standard methods as mentioned in our previous study [START_REF] Abid | Date palm wastes co-composted product: an efficient substrate for tomato (Solanum lycopercicum L.) seedling production[END_REF].

2.3.

Dissolved organic matter extraction (DOM) The organic matter extraction was achieved according to Song et al. (2015) with a slight modification. All the dried compost samples were dissolved in distilled water (solid to water ratio of 1:10, w/v) for 24 h in a rotary shaker (Heidolph reax 20) at room temperature. The suspensions were then centrifuged at 5000 rpm for 15 min, filtered through a 0.22 µm membrane filter and refrigerated until spectroscopy analysis. The Ddissolved Oorganic Ccarbon (DOC) was measured using multi TOC/TN analyzer type (Analytic Jena multi N/C 2100S).

Biological analysis

Microbial respiration

Microbial counts

The microbial count was achieved by number of colony forming units count (cfu). A 10 g mass of matured compost was suspended in 90 mLl of a sterile peptone water solution and stirred at 150 rpm for 10 min at 28 °C. The suspension was used for serial successive decimal dilutions (10 -1 to 10 -7 ) and 1 mLl was used to inoculate Plate Count Agar medium (PCA). The cell enumeration was for 72 h ± 4 h at 37 °C ± 1 °C [START_REF] Hachicha | Compost of poultry manure and olive mill wastes as an alternative fertilizer[END_REF].

Phytotoxicity test

The compost phytotoxicity was evaluated using the germination index (GI) according to the method described by [START_REF] Cesaro | Compost from organic solid waste: Quality assessment and European regulations for its sustainable use[END_REF]. This was determined as follows: 20 g of dry weight compost were extracted with 200 mLl of distilledated water, stirred for 2 h and then centrifuged at 9000 rpm. Ten Lipidium sativum seeds were evenly distributed on filters papers in Petri dishes (10 cm diameter) and moistened with 10 mLl of the compost extract. For each sample, three replicate dishes were incubated at 25 °C for three days in the dark. The numbers of germinating seeds per dishes were counted and the length of each seed root was measured.

For the control, 10 mLl of distilledated water were used to moister the filter. The GI was calculated using the following formula: GI (%) = [(Seed germination x Treated root length)/(Seed germination x Control root length)]

x 100.

Spectral analysis 2.5.1. UV-visible

The Dissolved Organic Matter (DOM) UV absorbance measurements from 200 to 800 nm were recorded on the Shimadzu UV-1800 spectrophotometer. 2015). The ratio of the absorbance at 250 nm to that at 365 nm (E 2 /E 3 ) was determined to describe the humification process intensity (reference). The ratio of the absorbance at 280 nm to that at 472 nm (E 2 /E 4 ) was used as an indicator of the relative lignin amounts at the beginning of the humification (reference). Finally the ratio between the absorbance at 472 and that at 664 nm (E 4 /E 6 ) was the most indicative parameter related to the humification degree (Sellami et al., 2008).

Fourier transform infrared spectroscopy

The dried compost samples were analyzed using an infrared spectrometer type FTIR Nicolet iS50 (Thermo Scientific, USA). The spectra were scanned over a wavenumber range of 4000-400 cm -1 with a resolution of 4 cm -1 .

Excitation-emission matrix (EEM) fluorescence spectra

The EEM spectra were achieved on the DOM of the different samples collected during the composting process using a spectrofluorimeter HITACHI F4500 with scanning emission (Em)

and excitation (Ex) wavelengths spectra from 200 to 800 nm at 10 nm increments and the scan speed was set at 2,400 nm. Min -1 . Excitation and emission slit were fixed at 10 nm and the detector time response in automatic mode. Fluorescence spectroscopy was carried out on et al., 2011;[START_REF] Mouloubou | An adapted sequential chemical fractionation coupled with UV and fluorescence spectroscopy for calcareous soil organic matter study after compost amendment[END_REF].

Statistical analysis

The data were submitted to variance analysis using Portable IBM SPSS statistics version 19. All analyses were performed in triplicate. Principal component analysis (PCA) was used to examine multivariate relationship between the physico-chemical and biological parameters progress during composting. Pearson's correlation coefficient (R) was used to evaluate the linear correlation between two parameters at a confidence level of 95% (p < 0.05).

Results and discussion

Composting performance

Temperature profiles 8

Temperature is one of the main parameters used to monitor the composting process. As shown in Figure 1, the temperature profile of the date palm waste and goat manure cocomposting was divided into 4 phases: mesophilic, thermophilic, cooling and maturity.

During the first 6 days of the composting process, windrow temperature showed a rapid increase and exceeded the 40 °C (Fig. 1). This period, represented mesophilic phase, characterized by the easily degradation of the organic matter by mesophilic microorganisms.

After this period, the temperature rose to above 53 °C, corresponding to the thermophilic phase and remained approximately at 50 °C for 7 days. This high temperature enhanced the windrow sanitation (Zhang et al., 2014;[START_REF] He | Insight into the composition and evolution of compost-derived dissolved organic matter using high-performance liquid chromatography combined with Fourier transform infrared and nuclear magnetic resonance spectra[END_REF]. This temperature increase was due to the intense microbial activity.

By the 20 th day of composting, the temperature started to decrease announcing the cooling phase, characterized by the mesophilic microorganism colonization which contributed to the cellulose and lignin degradations and the humus formation [START_REF] El Fels | Assessment of biotransformation of organic matter during co-composting of sewage sludge-lignocelullosic waste by chemical, FTIR analyses, and phytotoxicity tests[END_REF]. According to Zhang et al. (2014), the traditional composting requires between 90 and 270 days to produce mature compost. In this study, the maturity was reached after 190 days of composting; this fact may be attributed to the effect of the initial date palm waste soaking step. However, in another study date palm waste composting was held in 14 months (El Ouaquoudi et al., 2015).

At the maturity, the temperature was 35 °C. At the 62 and 133 days, the two observed temperature increases were the result of the windrow return reinforcing the aeration, which stimulate the microorganism activity [START_REF] Gavilanes-Terán | Windrow composting as horticultural waste management strategy -A case study in Ecuador Irene[END_REF]. Also, at the maturity phase, the windrow temperature was highly related to the ambient temperature which increased by the summer beginning from the day 62 until the process end.

pH and EC progress

During the date palm waste and goat manure co-composting process, the pH value showed a high correlation with the composting time (R 2 = 0.91) and varied slightly but remained globally neutral while an increase in EC value was noticed with a linear model and also high correlation with the composting time (R 2 = 0.89). However, a pH decrease by 0.43 units was registered at day 84 (Table 2). According to the literature, this decrease was explained by the raw material degradation essentially lignicellulose, and the organic acids release [START_REF] Hachicha | Quality assessment of composts prepared with olive mill wastewater and agricultural wastes[END_REF]Tripetchkul et al., 2012;[START_REF] El Fels | Assessment of biotransformation of organic matter during co-composting of sewage sludge-lignocelullosic waste by chemical, FTIR analyses, and phytotoxicity tests[END_REF]. After this fluctuation, the pH value was stabilized with the humic substances biosynthesis, these acted as pH buffer [START_REF] El Fels | Assessment of biotransformation of organic matter during co-composting of sewage sludge-lignocelullosic waste by chemical, FTIR analyses, and phytotoxicity tests[END_REF]. It could be noticed that the pH profile was different from that found by El Fels et al.

(2014) who mixed palm waste with sewage sludge characterized by an acidic pH. The observed difference would be attributed to the composted raw materials typology and pretreatment. The mature compost pH value was in the range of the compost standard value (between 6 and 7.5), confirming its suitability for agricultural use as specified by [START_REF] Gavilanes-Terán | Windrow composting as horticultural waste management strategy -A case study in Ecuador Irene[END_REF].

The EC was also measured during the date palm waste co-composting and reflected the compost salinityion content and its importance for plant growth [START_REF] Gavilanes-Terán | Windrow composting as horticultural waste management strategy -A case study in Ecuador Irene[END_REF]Singh and Kalamdhad, 2015). The EC values increased during composting process from 2.71 to 4.33 mS cm -1 (Table 2) reflecting the OM mineralization and the ammonium ions and mineral salts release [START_REF] El Fels | Assessment of biotransformation of organic matter during co-composting of sewage sludge-lignocelullosic waste by chemical, FTIR analyses, and phytotoxicity tests[END_REF][START_REF] Gavilanes-Terán | Windrow composting as horticultural waste management strategy -A case study in Ecuador Irene[END_REF].

Organic matter degradation and C/N progress

During the composting process, the OM and C/N progress were the important key parameter reflecting raw materials biodegradation and transformation. At the process end, the OM was reduced by 63% exhibiting the raw materials deterioration and their oxidation to CO 2 and H 2 O (Table 2). Indeed, the OM was decomposed and transformed by microorganisms to stable humic substances; the change was highlighted by a mass loss (Singh and Kalamdhad, 2015). According to Kulikowska et al. (2015), the OM mineralization amount and rate depend on the raw materials chemical composition, and especially the fibers content like lignin. The date palm waste and goat manure co-composting showed an OM loss of 36.58% although in another study El [START_REF] El Ouaqoudi | Evaluation of lignocelullose compost stability and maturity using spectroscopic (FTIR) and thermal (TGA/TDA) analysis[END_REF] observed an OM loss of 29% and 62% respectively for date palm waste and date palm/couch-grass clipping wastes mixture subjected to composting during 14 months. These results proved the important effects of date palm waste soaking step and goat manure use investigated in this study.

Simultaneously, the OM oxidation led to carbon loss and CO 2 release, increasing the total nitrogen content in the medium. In fact, palm waste and goat manure co-composting exhibited a C/N ratio decrease by 3 folds at the process end confirming its maturity (Table 2) (Tian et al., 2012).

Biological parameters evolution

Respiration

Respiration was usually used in compost stability and quality assessment as it refers to the microorganisms activity and OM degradation (Varma and Kalamdhad, 2014;[START_REF] Nikaeen | Respiration and enzymatic activities as indicators of stabilization of sewage sludge composting[END_REF]. In this experiment, the initial respiration value was low, expressing microorganism adaptation to the environment. After 6 days, the highest respiration value was observed corresponding to thermophilic phase (Fig. 2) with intense microorganisms activity. According to [START_REF] Nikaeen | Respiration and enzymatic activities as indicators of stabilization of sewage sludge composting[END_REF], this activity would be attributed to labile OM availability which stimulates microbial activity. After this stage, the respiration decreased then a second pic was observed at day 33 just after the windrow return that stimulated the microbial activity. From the day 34 and until the day 190, the respiration decreased slowly to reach 5.53 mg/50 g reflecting compost stability [START_REF] Gómez | The use of respiration indices in the composting process: a review[END_REF].

Germination index (GI)

During the composting process, the compost quality of the different collected samples was assessed by phytotoxicity control expressed as GI (Fig. 3). For the first 45 days, the GI showed fluctuations related to the OM degradation, the organic acids and the ammonia releases at relatively high concentrations considered as phytotoxic (Zhang et al., 2014). Also, phenols, alkaloids and ketones as well as others flavonic compounds are considered as seed germination inhibitory agents (Hachicha et al., 2009a;[START_REF] El Fels | Artemia salina as a new index for assessment of acute cytotoxicity during co-composting of sewage sludge and lignocellulose waste[END_REF]. From the 60 th day, the germination index increased gradually, indicating the toxic compounds reduction and the presence of stable humic substances, rich in organic matter synthesized and accumulated during the cooling phase [START_REF] El Fels | Assessment of biotransformation of organic matter during co-composting of sewage sludge-lignocelullosic waste by chemical, FTIR analyses, and phytotoxicity tests[END_REF][START_REF] El Fels | Artemia salina as a new index for assessment of acute cytotoxicity during co-composting of sewage sludge and lignocellulose waste[END_REF]. According to Sellami et al. characterized with a GI exceeding 50%, could be used in agriculture without any phytotoxic effect. The GI followed a linear model with a relatively correlation with time during the composting process (R 2 = 0.89).

Spectral analysis

UV-visible spectroscopy

The UV-visible spectroscopy was used to characterize the DOM, reflecting the organic matter humification during the co-composting of date palm waste and goat manure, as well as the final compost quality. The UV-visible spectra of the initial and final composted materials showed an intensity decrease from the wavelength of 267 to 295 nm (Fig 4). According to [START_REF] Lim | Determination of maturity in the vermicompost produced from palm oil mill effluent using spectroscopy, structural characterization and thermogravimetric analysis[END_REF], this decrease is related to lignin biodegradation, since the absorbance region around 280 nm is assigned to lignin and aromatic compounds.

Different specific ultraviolet absorbance SUVA 254, SUVA 269 and SUVA 280 were calculated (Table 3) and showed respectively an important increase by 2 times, while comparing the initial state (raw materials) to the mature compost. Both of the SUVA 254 and the SUVA 280 were relative to the aromatic constituent contents in the DOM (Wang et al., 2013), while the SUVA 269 was related to lignin derived aromatic carbon [START_REF] He | Influence of chemical and structural evolution of dissolved organic matter on electron transfer capacity during composting[END_REF]. The SUVAs values increase, confirmed the organic matter degradation and lignin oxidation into aromatic compounds during composting process. In this study, the absorbance ratio E 2 /E 3, E 2 /E 4 and E 4 / E 6 , used to control the organic matter biodegradation and humic substances synthesis, varied during the composting process (Table 3); this may be attributed to the organic matter rich in lignin, difficult to be biodegraded. However, in the first 6 composting days, E 4 /E 6 decreased because of the mesophilic microorganism activity and increased after the windrow returns to reach 7.40 by the day 21, then decreased successively as a result of raw organic materials humification and humic substances synthesis (Zbytniewski and Buszewski, 2005). By the day 84 of composting, the ratio E 4 /E 6 increased again after windrow return and then decreased at the end of the process. This ratio intensity would inform on the lignin and other materials at the beginning of humification (Zbytniewski and Buszewski, 2005). Furthermore, the observed decreases in E 2 / E 4 index reflected an intense depolymerisation and microbial monomeric compounds degradation (Sellami et al., 2008;[START_REF] Lim | Determination of maturity in the vermicompost produced from palm oil mill effluent using spectroscopy, structural characterization and thermogravimetric analysis[END_REF].

FTIR spectroscopy

The infrared spectroscopy was usually used as a qualitative analysis to identify and monitor the chemical groups and bands present in the composted materials, describing the organic matter degradation. Figure 5 represents the materials FTIR spectra at various composting stages. The major compost FTIR bands are listed in Table 4, where the bands between 4,000 and 2,000 cm -1 were attributed to the humic substances extracted from compost [START_REF] El Fels | Assessment of biotransformation of organic matter during co-composting of sewage sludge-lignocelullosic waste by chemical, FTIR analyses, and phytotoxicity tests[END_REF]. At 3,290 cm -1 , the pic was more important at the initial composting process than at the end, and was assigned to phenol O-H group vibration; this group was degraded during the composting process until the compost maturity [START_REF] Masmoudi | Characterization of olive mill wastes composts and their humic acids: stability assessment within different particle size fractions[END_REF]. This result is in accordance with the phytotoxicity test results. The bands at 2,920 and 2,850 cm -1 , characteristic of the aliphatic C-H groups, were more intense at the initial composting samples. Indeed, during the composting process, the organic matter was biodegraded and the carbon content decreased [START_REF] El Fels | Assessment of biotransformation of organic matter during co-composting of sewage sludge-lignocelullosic waste by chemical, FTIR analyses, and phytotoxicity tests[END_REF]. Also, the bands at 1,690 cm -1 attributed to C=O group in the organic matter, decreased during composting process by the microbial activity. The bands between 1,600 and 900 cm -1 were attributed to cellulose and early decomposition product [START_REF] Grube | Evaluation of sewage sludge-based compost by FTIR spectroscopy[END_REF]. El Ouaquoudi et al. ( 2015) had assigned the peak at 1,525 cm -1 to lignin considering the date palm waste lignocellulosic material.

In the region between 1,300 and 1,400 cm -1 , the bands were more intense in the final compost sample. According to [START_REF] Grube | Evaluation of sewage sludge-based compost by FTIR spectroscopy[END_REF], this region is characteristic of nitrate bands appearance, usually detected at the latter composting stage, when raw materials were well composted. However, the peak at 1,170 cm -1 was more intense at the initial mixture and was attributed to C-O-C groups of cellulose and hemicellulose as reported by Xu et al. ( 2013) at 1,160 cm -1 . At 1,560 cm -1 , the band decreased with the composting time, revealing peptide degradation in manure (Hachicha et al., 2009b).

The changes in the organic matter content and in aromaticity degree were also assessed by the calculation of the 3 absorption ratios 1,650/2,845, 1,525/2,925 and 2,920/1,640 (Table 5).

During the composting process, the 1,650/2,845 and 1,525/2,925 ratios showed an increase while the specific ratio of aliphatic C/aromatic C (2,920/1,640) decreased. These results could be explained by an increase in aromaticity and the biosynthesis of aromatic structures, such as humic-like and fulvic-like substances, with the decomposition/transformation of aliphatic components, polysaccharides, and alcohols (Wu et al., 2011;El Ouaquoudi et al., 2015).

These infrared spectroscopy findings were in accordance with those of the physico-chemical parameters, both of the data confirmed the date palm waste and goat manure co-composting product maturity and quality.

Fluorescence spectroscopy

The three dimensional EEM fluorescence spectra of the DOM composted material progress are presented in Figure 6 (A). These spectra were characterized by the presence of a fluorophore picpeak having different intensities depending on the composting progress and it highest intensity was registered in the final compost spectra after 190 days of composting.

The variable fluorophore picpeak had an excitation/emission wavelength pairs between 350-400 (mean value = 375)/400-500 (mean value = 470) nm, characteristic of humic-like substances and high molecular weight (Albretch et al., 2015;Song et al., 2015;[START_REF] Mouloubou | An adapted sequential chemical fractionation coupled with UV and fluorescence spectroscopy for calcareous soil organic matter study after compost amendment[END_REF]. According to [START_REF] He | Insight into the composition and evolution of compost-derived dissolved organic matter using high-performance liquid chromatography combined with Fourier transform infrared and nuclear magnetic resonance spectra[END_REF], the fluorophores with emission wavelengths exceeding 380 nm are ascribed to humic-like species, while those less than 380 nm represent protein-like species, such as tyrosine and tryptophan-like species. Based on these data, no proteins like fluorophore were detected during the process. Consequently, the total luminescence would be issued from the humic-like compounds. However, the humic like signal could be generated by a mixture of fluorescent compounds. The ratio of the red zone to the total EEM fluorescent area had increased by 76% at the end process revealing the formation of humic substances (Figure 6 B). The ratio fluctuation during the first 60 days of composting was due to the microbial activity and the degradation of organic matter.

As shown in Figure 7, the PARAFAC analysis decomposed the EEM fluorescent spectra into 4 fluorophoric components (ConcordiaCORCONDIA > 66%). The first component is located at 350/450 nm (S1) and it is representative of the humic like compounds. The second (S2) is located at 400/500 nm and is less conjugated than the third (S3) one located at 450/530 nm. These entire three components represent the humic-like group. The fourth (S4) represent the fulvic like component with an excitation at 325/400 nm. From S4 to S1 to S2 to S3 we can observe a conjugate trend inducing a red shift evolution for both excitation and emission wavelengths. Observing the ratio of S3 over S2 contribution during the composting process, we may note that the S1 contribution increase more than other components. This is a confirmation of the FTIR trend were C=C is increasing during the same time. Moreover, considering the increase of the SUVAs, the fluorescence intensity and the GI while COD and microbial respiration decreases, we may suggest that at the end of the process (over 120 d), both of conjugated carbon and aromaticity increased.

Compost specification

The compost prepared with date palm waste and goat manure co-composting was characterized by a neutral pH and relatively high EC. It also exhibited a low C/N ratio and a high GI value (88%) reflecting its stability (Table 6). Considering its total nitrogen content inferior to 3% and organic matter inferior to 55%, the produced compost could be classified as vegetal compost (French standards NF U 44-051, 1981).

After six months of palm waste and goat manure co_ composting while comparing to El Fels et al. ( 2014) compost who also studied the palm waste co_ composting with sludge, the produced compost present higher carbon content than that previously described but a lower nitrogen content.

Statistical analysis

The correlation matrix between the physico-chemical and the biological parameters progress during the composting process (Table 7) and the PCA plan representation (Fig 8)

showed a significant positive correlation between pH, C/N ratio and OM content. However, during the composting process, the organic matter biodegradation decreased and as a result, the carbon content was reduced as well as C/N ratio. In addition, temperature and respiration were highly correlated revealing that microorganism activity depends on the temperature and specificity for each composting stage (mesophilic and thermophilic). Moreover, a negative correlation was observed between the germination index and the EC. These are opposed to the The ratio evolution of the red zone specific of humic substances to the total fluorescent area. A la place de la table 4 (pic IR) je mettrais les formules ou les noms sur le spectre, encore une fois si besoin seulement, le texte suffit à lui même je pense.

  Microbial respiration was assayed according toPallo et al. (2009) andYu et al. (2011) with some modifications. A sample of 50 g compost was incubated for 24 h in bottle of 1 Ll where a tube containing 10 mLl of NaOH (0.1 N) was placed. The bottles have the same windrows temperature when the sample was collected. The method involves the CO 2 trapping by sodium hydroxide (0.1 N NaOH), and then titration with hydrochloric acid (0.1 N HCl) with the phenolphthalein as a pH indicator. The amount of CO 2 released (C-CO 2 ) is given by the following formula: CO 2 -C (mg /50 g of compost) = (Control V HCl -Sample V HCl ) x 2.2; Control V HCl (ml): the volume of HCl (0.1 N) used for the titration of NaOH in the control; sample V HCl (ml): the volume of HCl (0.1 N) used for compost sample titration; 2.2 = a coefficient corresponding to 1 mLl of HCl (0.1 N) CO 2 (g) determined. 5 9

4

  two time diluted extract to avoidcorrect the inner filter effect (luciani). The obtained fluorescent EEM were processed by Parallel Factor analysis (PARAFAC) and PROGMEEFprogMEFF interface provided by PROTEE laboratory, Univ. Toulon (France), allowing the extraction of the most representative fluorescent components to characterize the source and nature of the sample organic matter (Yu

(

  2008) and Zhang et al. (2014), the final GI value about 89% may reflect compost maturity and phytotoxicity lack. According to Gavilanes-Terán et al. (2015) the obtained compost

Figure captions Figure 1 .

 captions1 Figure captions

Figure 2 .

 2 Figure 2. Respiration profile reflecting microorganism activity during composting process.

Figure 3 .

 3 Figure 3. The germination index progress during date palm waste and goat manure cocomposting.

Figure 4 .

 4 Figure 4. The UV-visible spectra of the initial and final compost samples.

Figure 5 .

 5 Figure 5. Infrared spectra of various composting stage. T0: initial compost sample; T6: compost sample at 6 d ; T14: compost sample at 14 d ; T21: compost sample at 21 d ; T28: compost sample at 28 d ; T33: compost sample at 33 d ; T42: compost sample at 42 d ; T62: compost sample at 62 d ; T84: compost sample at 84 d ; T119: compost sample at 119 d ; T134: compost sample at 134 d ; T190: final compost sample.

Figure 6 .

 6 Figure 6. (A) Excitation-emission matrix (EEM) fluorescent spectra of the different DOM extracted from compost materials during co composting process expressed in days (d); (B)

Figure 7 .

 7 Figure 7. Excitation-emission matrix spectra of principal fluorescent components identified by the PARAFAC analysis. (S1) component 1; (S2) component 2; (S3) component 3; (S4) component 4.

Figure 8 .

 8 Figure 8. Principal component analysis of the physico-chemical and biological parameters. CE: electrical conductivity, OM: organic matter, GI: germination index.

Figure 2 :

 2 Figure 2

  Figure 3

  Figure 5

  4 /E 6 becomes low the compost is mature and humus particles are complexes.Sellami et al. (2008) associated the large values of E 4 /E 6 ratio to the presence of small size organic molecules or more aliphatic structures with high functional groups content. The E 2 /E 4 index progress presented the same trend as that of the E 4 /E 6 ratio during the composting process.

	Wang et al. (2013) found a reduction in E 4 /E 6 during the active phase
	that rose slowly thereafter. According to Zbytniewski and Buszewski (2005), the final E 4 /E 6
	value 5.7 is an index of mature humic acids. More recently, Haddad et al. (2015) had also
	found an E

4 /E 6 ratio value of 6 at the end process of composting olive pomace and cattle manure. They had admitted this ratio as an indicator of humification intensity. Thus, when the 13 ratio E

Table 1 .

 1 Pallo, F.J.P.,Lemée, L., Ambles, A., 2009. Les lipides libres des sols sous formations naturelles au Burkina Faso: Nature, origine et relation avec quelques facteurs de fertilité. Li, M.,Xi, B., Wei, Z., Zhao, Y., Jia, X., Qi, H., Zhu, C., 2015. Characterisation of dissolved organic matter extracted from the bio-oxidative phase of co-composting of biogas residues and livestock manure using spectroscopic techniques. Int. Biodeter. UV-vis, GPC, FTIR and EEM. Int. Biodeter.Biodegr. 85, 617-623. Wu, H., Zhao, Y., Long, Y., Zhu, Y., Wang, H., Lu, W., 2011. Evaluation of the biological stability of waste during landfill stabilization by thermogravimetric analysis and Fourier transform infrared spectroscopy. Bioresour. Technol. 102, 9403-9408. The main physico-chemicals characteristics of the initial raw materials (dry weight basis).

	Etude et gestion des sols, 16, 101-112.
	Singh, J., Kalamdhad, A.S., 2015. Assessment of compost quality in agitated pile composting
	of water hyacinth collected from different sources. Int. J. Recycl. Org. Waste Agricult. 4,
	175-183.
	Sellami, F., Hachicha, S., Chtourou, M., Medhioub, K., Ammar, E., 2008. Maturity
	assessment of composted olive mill wastes using UV spectra and humification
	parameters. Bioresour. Technol. 99, 6900-6907.
	Song, C., Biodegr. 103, 38-50
	Tian, W., Li, L., Liu, F., Zhang, Z., Yu, G., Shen, Q., Shen, B., 2012. Assessment of the
	maturity and biological parameters of compost produced from dairy manure and rice
	chaff by excitation-emission matrix fluorescence spectroscopy. Bioresour. Technol. 110,
	330-337.
	Tripetchkul, S., Pundee, K., Koonsrisuk, S., Akeprathumchai, S., 2012. Co-composting of
	coir pith and cow manure: initial C/N ratio vs physico-chemical changes. Int. J. Recycl.
	Org. Waste Agricult. 15, 2-8.
	Varma, S.V., Kalamdhad, A.S., 2014. Stability and microbial community analysis during
	rotary drum composting of vegetable waste. Int. J. Recycl. Org. Waste Agricult. 52, 2-9.
	Wang, K., Weiguang, L., Gong, X., Li, Y., Wu, C., Ren, N., 2013. Spectral study of
	dissolved organic matter in biosolid during the composting process using inorganic
	bulking agent:

Table 2 .

 2 The main palm waste and goat manure physico-chemical characteristics during co-

	composting.
	Compostin
	g			MM
	duration	pH	CE	(%)	OM	C	N	C/N
	(Day)		(mS cm -1 )		(%)	(%)	(%)

Table 3 .

 3 The DOM characteristics progress during the composting process.

	Composting			SUVA (x10 -3 )
	duration	DOC
	(Day)	(mg C/l)	254	269	280	E 2 /E 3	E 2 /E 4	E 4 /E 6

Table 4 .

 4 The mean infrared spectral bands related to the compost.

	Wavenumber (cm -1 )	Assignment
	3200-3600	Hydroxyl stretching vibration of phenol, alcohol and organic acid and
		N-H vibrations from amines and amides
	2850-2964	Aliphatic C-H group
	1750-1600	C=O amount in the organic matter
	1716	C=C stretching vibration of acids and ketones carboxyl groups
	1620-1650	Unsaturated aromatic C=C or ketone acids
	1540-1580	Amide II bands and carboxylic group, N-H deformation
	1525	Lignin pic
	1420-1450	Aliphatic C-H deformation
	1380-1400	O-H deformation of phenolic structure, C-H deformation in cellulose
		and hemicellulose
	1220-1270	C-OH stretching of aromatic groups
	1003-1200	C-O stretching of polysaccharides
	1112	C-OH stretching of aliphatic OH
	720-590	Alcohol, OH out of plane bend

Table 5 . Evolution of the humification ratios during the composting process.

 5

	Compostin	Absorbance ratios et définit wavenumber
	g
	progress
	(Day)	1,650/2,845 1,525/2,925 2,920/1,640
	Initial	1.43	0.37	1.60
	6	1.51	0.49	1.31
	14	1.94	0.79	1.00
	21	1.67	0.61	1.22
	28	1.65	0.60	1.12
	33	1.68	0.71	1.07
	42	1.80	0.66	1.02
	62	1.50	0.50	1.32
	84	1.64	0.80	0.96
	119	1.70	0.67	1.01
	134	1.70	0.69	0.98
	190	1.95	1.13	0.71

Table 6 .

 6 The palm waste and goat manure compost characteristics.

	CE: Electrical

previous group including OM, pH and C/N. Indeed, the organic acids and the other phytotoxic components released during composting process affected the seed germination.

Conclusions

Date palm waste and goat manure co-composting is an efficient solution providing organic matter for a sustainable agriculture. During the composting process, physico-chemical and biological parameters were in correlation, proving the organic matter biodegradation and the compost produced quality. The use of spectroscopic techniques approved the lignin degradation and the humic substance components formation. The obtained compost was without phytotoxicity and ready for agriculture application.

Aknowledgements

The authors would like to express their gratitude to the staff at the Oasis Cheninni Safeguard Association for their help in providing the material necessary for this study.