Pierre Gueriau
email: pierre.gueriau@hotmail.fr

Farid Saleh
email: farid.nassim.saleh@gmail.com

Lukáš Laibl

Francesc Perez Peris

Lorenzo Lustri

Cristian Mocuta

Solenn Réguer

Serge X Cohen

Loïc Bertrand

Allison C Daley
email: allison.daley@unil.ch

Trace elements discriminate between tissues in highly weathered fossils

Keywords: Ordovician, Fezouata Biota, synchrotron X-ray fluorescence, taphonomy, nervous system

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Elucidating the origin and evolutionary history of life relies on the accurate placing of organisms, extant and extinct, on the tree of life. This has historically been based upon anatomical similarities, with clades being identified by shared derived characteristics (synapomorphies) that can be traced to a most recent common ancestor and are not present in more distant groups and ancestors. For extant organisms, such phylogenetic/cladistic classifications were revolutionised by molecular biology, which utilizes differences and similarities in genetic sequences, predominately DNA and RNA, to build trees of evolutionary relationships. However, despite being successfully applied to recently extinct animals such as the giant moas from New Zealand 1,2 or the iconic mammoth [START_REF] Debruyne | Mitochondrial cytochrome b of the Lyakhov mammoth (Proboscidea, Mammalia): new data and phylogenetic analyses of Elephantidae[END_REF][START_REF] Van Der Valk | Million-year-old DNA sheds light on the genomic history of mammoths[END_REF] , these methods are of limited use for most fossil organisms, because observed rates of DNA degradation indicate that in samples older than 1.5 million years, DNA is either severely crosslinked or non-detectable [START_REF] Willerslev | Long-term persistence of bacterial DNA[END_REF] and the oldest ancient DNA ever extracted (from permafrost-preserved mammoth teeth) is 1.6 million years old [START_REF] Van Der Valk | Million-year-old DNA sheds light on the genomic history of mammoths[END_REF] . Palaeontology has undergone its own revolution with the advent of 3D X-ray microtomography and more recently a series of advanced 2D imaging techniques, which reveal previously inaccessible or invisible anatomical information [START_REF] Tafforeau | Applications of X-ray synchrotron microtomography for non-destructive 3D studies of paleontological specimens[END_REF][START_REF] Smith | Virtual taphonomy using synchrotron tomographic microscopy reveals cryptic features and internal structure of modern and fossil plants[END_REF][START_REF] Bergmann | Archaeopteryx feathers and bone chemistry fully revealed via synchrotron imaging[END_REF][START_REF] Bergmann | Chemical mapping of ancient artifacts and fossils with x-ray spectroscopy[END_REF][START_REF] Gueriau | Trace elemental imaging of rare earth elements discriminates tissues at microscale in flat fossils[END_REF][START_REF] Gueriau | Show me your yttrium, and I will tell you who you are: implications for fossil imaging[END_REF][START_REF] Kaye | Laser-Stimulated Fluorescence in Paleontology[END_REF] . These techniques allow fossil anatomy to be described better than ever before, providing a wide range of new characters that have led to better resolution of phylogenetic analyses [START_REF] Giles | Osteichthyan-like cranial conditions in an Early Devonian stem gnathostome[END_REF][START_REF] Zhai | Three-dimensionally preserved appendages in an Early Cambrian stem-group pancrustacean[END_REF][START_REF] Miyashita | Hagfish from the Cretaceous Tethys Sea and a reconciliation of the morphological-molecular conflict in early vertebrate phylogeny[END_REF] . Furthermore, although DNA is not preserved in ancient fossils, remnants or derivatives of ancient biomolecules can survive in the geological record, associated to organically-preserved fossil remains or isolated within sedimentary rocks [START_REF] Brocks | Sedimentary Hydrocarbons[END_REF][START_REF] Brocks | Building the biomarker tree of life[END_REF][START_REF] Lindgren | Molecular preservation of the pigment melanin in fossil melanosomes[END_REF][START_REF] Greenwalt | Hemoglobinderived porphyrins preserved in a Middle Eocene blood-engorged mosquito[END_REF][START_REF] Briggs | Ancient biomolecules: their origins, fossilization, and role in revealing the history of life[END_REF][START_REF] Colleary | Chemical, experimental, and morphological evidence for diagenetically altered melanin in exceptionally preserved fossils[END_REF][START_REF] Pan | Molecular evidence of keratin and melanosomes in feathers of the Early Cretaceous bird Eoconfuciusornis[END_REF][START_REF] Vajda | Molecular signatures of fossil leaves provide unexpected new evidence for extinct plant relationships[END_REF][START_REF] Bobrovskiy | Ancient steroids establish the Ediacaran fossil Dickinsonia as one of the earliest animals[END_REF][START_REF] Pan | The molecular evolution of feathers with direct evidence from fossils[END_REF][START_REF] Kjaer | A 2-million-year-old ecosystem in Greenland uncovered by environmental DNA[END_REF] . Some of these compounds represent biomolecular signatures (or "biomarkers") diagnostic of particular (often broad) clades of organisms, and therefore represent a powerful complement to fossil anatomies in revealing the history of life [START_REF] Briggs | Ancient biomolecules: their origins, fossilization, and role in revealing the history of life[END_REF] . Nonetheless, such approaches provide little new information when applied to the majority of fossils whose morphology and chemistry have substantially been altered during decay, burial, diagenesis and weathering. As a result, recent studies focus on relatively "unaltered" specimens while many other fossils remain enigmatic in their affinities. New tools and approaches accounting for and exploiting information loss/gain during the taphonomic history are needed to help deciphering the affinities of altered fossils [START_REF] Briggs | Ancient biomolecules: their origins, fossilization, and role in revealing the history of life[END_REF][START_REF] Murdock | The impact of taphonomic data on phylogenetic resolution: Helenodora inopinata (Carboniferous, Mazon Creek Lagerstätte) and the onychophoran stem lineage[END_REF][START_REF] Bertrand | Deciphering the Chemistry of Cultural Heritage: Targeting Material Properties by Coupling Spectral Imaging with Image Analysis[END_REF] .

Here we report the major-to-trace elemental composition of 14 strongly weathered fossil animals, including various arthropods, annelids, sponges and an echinoderm, from the Early Ordovician (Tremadocian) Fezouata Shale of Morocco [START_REF] Van Roy | Ordovician faunas of Burgess Shale type[END_REF][START_REF] Van Roy | The Fezouata fossils of Morocco; an extraordinary record of marine life in the Early Ordovician[END_REF] using synchrotron-based X-ray fluorescence (XRF) mapping. In this Burgess Shale-type deposit, fossils were originally preserved as carbonaceous compressions and/or in authigenic minerals including pyrite [START_REF] Saleh | Taphonomic pathway of exceptionally preserved fossils in the Lower Ordovician of Morocco[END_REF][START_REF] Saleh | Insights into soft-part preservation from the Early Ordovician Fezouata Biota[END_REF] , before being extensively weathered by modern water circulations that led to the leaching of carbon from carbonaceous compressions, the oxidation of pyrite crystals into yellow, red to purple iron oxide pseudomorphs, the deposition of new, poorly crystallised Fe-oxides in previously non-pyritised areas, and the dissolution of carbonates from the matrix and from the skeletal elements of animals such as echinoderms [START_REF] Saleh | Taphonomic pathway of exceptionally preserved fossils in the Lower Ordovician of Morocco[END_REF][START_REF] Saleh | Insights into soft-part preservation from the Early Ordovician Fezouata Biota[END_REF][START_REF] Lefebvre | Exceptionally preserved soft parts in fossils from the Lower Ordovician of Morocco clarify stylophoran affinities within basal deuterostomes[END_REF][START_REF] Saleh | Fossil weathering and preparation mimic soft tissues in eocrinoid and somasteroid echinoderms from the Lower Ordovician of Morocco[END_REF] . We consistently extracted mean XRF spectra from the tissues preserved in our fossils, as well as from their surrounding sedimentary matrix (see Figs. 1A-M, Table S1 and Figs. S1-S14 for location and details on the specimens and the selected areas, and methods for the XRF spectra extraction strategy). Linear discriminant analysis (LDA) was then applied to assess systematic variation in the major-to-trace elemental chemistry of different structure types (defined according to Saleh et al. [START_REF] Saleh | Taphonomic bias in exceptionally preserved biotas[END_REF] ), i.e. between biomineralised, sclerotised, cuticularised, and internal tissues, and the sedimentary matrix. We further show the potential of this framework to interpret the anatomy of a highly weathered enigmatic organism from the Fezouata Biota.

Results and Discussion

Highly weathered fossils preserve tissue-specific trace elemental chemistries.

Our results show that although the fossils have all been extensively weathered to Fe-oxides (usually goethite, more rarely hematite, as determined by Raman spectroscopy), their biomineralised, sclerotised, cuticularised, and internal tissues can still be distinguished based on their trace element composition (Fig. 1N). Iron is naturally the main element composing the fossils (as Feoxyhydroxides), but it is also the main element probed by our analyses for the surrounding shale (as Fe-bearing clay minerals) because light elements and particularly silicon cannot be detected with the used setup (see Methods). All fossil tissues are, nevertheless, richer in Fe than the shale, and are also considerably enriched in As and Pb (Fig. 1O). The proximity of biomineralised and sclerotised tissues in the LDA plot indicates they have a similar elemental composition, only discriminated by a slight enrichment in Ti and Zn in biomineralised tissues, and in Fe, Cu, As and Pb in sclerotised tissues. Cuticularised tissues differ by strong depletions in Cu and As, as well as in Rb, Sr and Y.

The only internal tissue available for study contains little to no Ni, Cu, Zn and Ga, and is the most depleted in Rb, Sr and Y. Note that no chemical inventory was collected for soft cellular outer tissues in direct contact with seawater (e.g. tentacles) as they were never found preserved in the Fezouata Biota [START_REF] Saleh | Insights into soft-part preservation from the Early Ordovician Fezouata Biota[END_REF][START_REF] Saleh | Taphonomic bias in exceptionally preserved biotas[END_REF] .

The specific chemical inventory for each type of tissues can be further confirmed considering that analysed fossils were found in numerous and distant (up to >30 km; Fig. S15) contemporaneous outcrops with different modern-weathering history [START_REF] Saleh | Taphonomic pathway of exceptionally preserved fossils in the Lower Ordovician of Morocco[END_REF] . This means that the chemical discrimination between different tissue types is not specific to a particular outcrop, and tissue types can be chemically differentiated within the entire Fezouata Shale, because it was affected by broadly similar water circulation processes in the Draa valley [START_REF] Saleh | Insights into soft-part preservation from the Early Ordovician Fezouata Biota[END_REF] . The high concentrations and chemical speciation of the accessory metals imply that they have accumulated mainly through reactive transport during diagenesis -before the material reaches a stage of depercolation where the formation of specific fluid pathways and remineralisation blocks further uptake-rather than being originally present in the living organism (and persisting through geological times). Indeed, Cu, As, Pb, Ti and Zn concentrations in the 0.02-0.86 wt% range (see Table S2, and Methods for XRF quantification) are exceeding by one to several orders of magnitude those encountered in modern relatives of the Fezouata organisms, even when inhabiting environments heavily contaminated by terrestrial pollutants [START_REF] Burger | Metal levels in horseshoe crabs (Limulus polyphemus) from Maine to Florida[END_REF][START_REF] Liu | Heavy metal concentrations in commercial marine organisms from Xiangshan Bay, China, and the potential health risks[END_REF] . Furthermore, the most contrasting metal Cu is present in all investigated fossils as a Cu-carbonate compound (Fig. S16), which ubiquitous occurrence in such a wide range of organisms and tissues cannot be original, particularly considering that weathering led to the dissolution of carbonates from skeletal elements of animals such as echinoderms [START_REF] Saleh | Taphonomic pathway of exceptionally preserved fossils in the Lower Ordovician of Morocco[END_REF][START_REF] Saleh | Insights into soft-part preservation from the Early Ordovician Fezouata Biota[END_REF][START_REF] Lefebvre | Exceptionally preserved soft parts in fossils from the Lower Ordovician of Morocco clarify stylophoran affinities within basal deuterostomes[END_REF][START_REF] Saleh | Fossil weathering and preparation mimic soft tissues in eocrinoid and somasteroid echinoderms from the Lower Ordovician of Morocco[END_REF] . A description of the precise mechanisms responsible for such a diagenetic uptake (i.e. percolation, deposition, adsorption or substitution) is beyond the scope of the present work, but it clearly indicates that tissue-specific physico-chemical differences persisted long enough after decay and early mineralization to distinctly influence later elemental uptake, leading to elemental signatures that were retained (at least locally) after extensive subsequent weathering.

Trace elemental composition can identify tissue types in enigmatic fossils.

Building on this tissue-specific chemical inventory, we used XRF mapping and the obtained LDA classification to interpret the anatomy of an enigmatic, highly weathered organism (AA-FETB-OI-22; Figs. 2A,B). While it is particularly challenging to optically investigate this specimen owing to its poorly preserved state, the distributions of Fe (enriched in fossil tissues) (Fig. 2C) and to a larger extent Rb (depleted in most fossil tissues) allow us to distinguish the outline of the organism (Fig. 2D), which does not include the entire brownish-purplish weathered surface. Chemically, we can distinguish two different types of tissues in this fossil: one depleted in Rb (Fig. 2D), and a central, 3-mm wide tubular structure strongly enriched in Fe (Fig. 2C), optically yellowish and burgundy in colour, which extends anteriorly into a 28-mm long, yellowish and unsegmented slender appendage that curves along its length and tapers to a pointed end (Figs. 2B,C). We extracted mean XRF spectra from these two tissues and the sedimentary matrix (see Fig. 2C, Table S1 and Fig. S17 for location and details on the selected areas) and added them to our LDA classification (Fig. 2E). The matrix spectrum perfectly clusters with the matrix spectra from the other Fezouata fossils. The composition of the main body part clusters with that of cuticularised tissues. The anterior part of the Fe-rich tubular structure, however, does not cluster with biomineralised, sclerotised or cuticularised tissues. Its central position is in proximity to the one data point from known internal tissue, making this the most likely tissue type for this tubular structure. The posterior part of this tubular structure, which is more burgundy in colour (Fig. 2B) and of different trace elemental composition (richer in Cu and Pb; Fig. 2F), plots distinctively from the anterior part, closer to the internal tissue point (Fig. S17), and may therefore represent the remains of another internal system. Note that the richer in iron the XRF spectra extracted from weathered matrix areas are the closer they plot to cuticularised tissues, following the fact that the shale matrix (clay and silica) is way less concentrated in iron than the fossil tissues (Fe-oxyhydroxides) (Fig. S17).

Using the elemental composition as a guide, a description of the overall anatomy of this animal can be made (Fig. 2G). The organism has a cuticularised, 11-mm wide, elongated and parallel-side trunk region, through which runs a central 3-mm wide band of internal tissues that extends into a 28-mm long slender, tapering and unsegmented appendage. The trunk region shows several faint discontinuous parallel lines that suggest the body was segmented and divided into a minimum of 6 body units. Laterally to the trunk outline, the cuticularised tissues extend outwards into poorly-defined wide lateral flaps that are rounded or roughly triangular in outline. This combination of anatomical features is very reminiscent of Kerygmachela kierkegaardi, a stemgroup euarthropod from the Cambrian Sirius Passet Biota of Greenland [START_REF] Budd | A Cambrian gilled lobopod from Greenland[END_REF][START_REF] Park | Brain and eyes of Kerygmachela reveal protocerebral ancestry of the panarthropod head[END_REF] . An affinity for the Fezouata Shale organism similar to K. kierkegaardi would suggest that one of the pair of frontal appendages has not been preserved in the Fezouata organism, explaining the asymmetrical position of the one that is present. Regardless, due to the 40 Myr gap between the Sirius Passet and the Fezouata Shale, it is unlikely that the Fezouata organism belongs to the same taxon. In any case, stem euarthropod affinities for this organism increase the species richness of the Fezouata Biota.

A closer look at the regions of the specimen interpreted as internal tissues reveals anatomical detail that allows us to identify the organ systems that are present. Microscopic examination of the anterior yellowish region of the internal tissues reveals the presence, besides numerous rock fractures, of lateral extensions (Fig. 3A). A higher-resolution XRF map (25-µm pixel size) further unveils four Fe-rich spherical structures organised antero-posteriorly, as well as another pair of two spherical structures, not as rich in Fe, positioned laterally to the first spherical structure and connected to it by an elbow duct (Figs. 3B,C). The only interpretation for such an arrangement of structures is that they represent four ganglia of the nervous system and a pair of lateral eyes, respectively (Fig. 3C,D). Although rare, the fossilisation of nervous tissues is being documented in a growing number of fossils [START_REF] Park | Brain and eyes of Kerygmachela reveal protocerebral ancestry of the panarthropod head[END_REF][START_REF] Ma | Complex brain and optic lobes in an early Cambrian arthropod[END_REF][START_REF] Tanaka | Chelicerate neural ground pattern in a Cambrian great appendage arthropod[END_REF][START_REF] Cong | Brain structure resolves the segmental affinity of anomalocaridid appendages[END_REF][START_REF] Yang | Fuxianhuiid ventral nerve cord and early nervous system evolution in Panarthropoda[END_REF][START_REF] Parry | Canadia spinosa and the early evolution of the annelid nervous system[END_REF][START_REF] Lan | Leanchoiliidae reveals the ancestral organization of the stem euarthropod brain[END_REF][START_REF] Ortega-Hernández | Neuroanatomy in a middle Cambrian mollisoniid and the ancestral nervous system organization of chelicerates[END_REF][START_REF] Strausfeld | The lower Cambrian lobopodian Cardiodictyon resolves the origin of euarthropod brains[END_REF][START_REF] Figueroa | Exceptional fossil preservation and evolution of the ray-finned fish brain[END_REF] . It is explained by the presence of highly reactive biogenic Fe in the nervous system, which has been proposed to initiate selective pyritisation very rapidly during early diagenesis while other tissues are still decaying [START_REF] Saleh | Biogenic iron preserves structures during fossilization: a hypothesis: iron from decaying tissues may stabilize their morphology in the fossil record[END_REF] . Nonetheless, the undisputable identification of fossilised internal anatomical features as remains of nervous tissues may be challenging. Aria et al. proposed a set of criteria for their recognition, namely the consideration of the specimen topology, the morphoanatomical consistency, the taphonomic context and redundancy [START_REF] Aria | Interpreting fossilized nervous tissues[END_REF] . The remains observed in AA-FETB-OI-22 meet most of the proposed criteria: surface relief has been carefully considered, as shown in our interpretative drawing (Fig. 3C), especially as the Fe distribution probed by XRF is not limited to the surface (see Methods) allowing us to confidently distinguish between relief and fossilised remains; features are consistent with those described from closely-related fossils [START_REF] Park | Brain and eyes of Kerygmachela reveal protocerebral ancestry of the panarthropod head[END_REF] ; detailed knowledge of the taphonomy at the site [START_REF] Saleh | Taphonomic pathway of exceptionally preserved fossils in the Lower Ordovician of Morocco[END_REF][START_REF] Saleh | Insights into soft-part preservation from the Early Ordovician Fezouata Biota[END_REF][START_REF] Lefebvre | Exceptionally preserved soft parts in fossils from the Lower Ordovician of Morocco clarify stylophoran affinities within basal deuterostomes[END_REF][START_REF] Saleh | Fossil weathering and preparation mimic soft tissues in eocrinoid and somasteroid echinoderms from the Lower Ordovician of Morocco[END_REF][START_REF] Saleh | Taphonomic bias in exceptionally preserved biotas[END_REF] and the dorso-ventral plane of preservation associated with some 3D preservation uncovered by XRF ensure that taphonomic variations have been considered. We, however, acknowledge that the redundancy criterion is not met, but we argue that if our method could uncover such new features in a random specimen then more are expected to be discovered. The internal anatomical features preserved in the analysed specimen therefore represent the first remains of a nervous system ever reported from the Ordovician period. The other regions of the internal tubular structure that have a different chemistry (Fig. 2F) likely represent remains of other internal tissues or systems (Fig. 2G).

Towards a more exhaustive documentation of the history of life

The identification, based upon the tissue-specific incorporation of inorganic minor and trace elements, of a stem euarthropod yielding remains of the nervous system from Fezouata extends both its biodiversity and the range of tissues that could be preserved there. It also illustrates the potential of novel uses of advanced spectro-imaging techniques to identify pivotal yet poorly preserved and/or highly weathered fossils such as lower stem euarthropods, which are neither biomineralised nor sclerotised and as such have a low fossilisation potential. Tissue-specific chemistry is known to be a powerful tool for interpreting the soft tissue anatomy of exceptionally preserved fossils. This approach has been developed with XRF mapping of a wide range of vertebrates retaining organometallic compounds derived from the original melanin pigments [START_REF] Wogelius | Trace metals as biomarkers for eumelanin pigment in the fossil record[END_REF][START_REF] Egerton | The mapping and differentiation of biological and environmental elemental signatures in the fossil remains of a 50 million year old bird[END_REF][START_REF] Manning | Pheomelanin pigment remnants mapped in fossils of an extinct mammal[END_REF] . Recently, the discovery that tissue-specific chemical signatures can persist in fossilised internal melanosomes (quite common in the fossil record) has even suggested that it could potentially be used to constrain the affinities of enigmatic fossil vertebrates [START_REF] Rossi | Tissue-specific geometry and chemistry of modern and fossilized melanosomes reveal internal anatomy of extinct vertebrates[END_REF] . Yet, such an approach is limited to literally exceptionally preserved fossils, i.e. retaining molecular and/or organelles remains, which have only been affected by limited diagenetic [START_REF] Rossi | Tissue-specific geometry and chemistry of modern and fossilized melanosomes reveal internal anatomy of extinct vertebrates[END_REF][START_REF] Rossi | Hierarchical biota-level and taxonomic controls on the chemistry of fossil melanosomes revealed using synchrotron X-ray fluorescence[END_REF][START_REF] Rossi | Maturation experiments reveal bias in the chemistry of fossil melanosomes[END_REF] . In the case of most other fossils, which only preserve hard parts bones, shells, plates, ossicles, biomineralised cuticle), and occasionally soft parts replicated in minerals during fossilisation and/or, diagenesis, the use of XRF mapping has been limited to acquiring chemical information and visualising anatomical features superficially hidden beneath the sample surface [START_REF] Gueriau | Trace elemental imaging of rare earth elements discriminates tissues at microscale in flat fossils[END_REF][START_REF] Gueriau | Show me your yttrium, and I will tell you who you are: implications for fossil imaging[END_REF][START_REF] Brayard | the Paris Biota team, Glow in the dark: Use of synchrotron μXRF trace elemental mapping and multispectral macro-imaging on fossils from the Paris Biota (Bear Lake County, Idaho, USA)[END_REF][START_REF] Saucède | the Paris Biota team, A new holocrinid (Articulata) from the Paris Biota (Bear Lake County, Idaho, USA) highlights the high diversity of Early Triassic crinoids[END_REF][START_REF] Gueriau | A new Devonian euthycarcinoid evidences the use of different respiratory strategies during the marine-to-terrestrial transition in the myriapod lineage[END_REF] . Our study unexpectedly shows that the combination of both the spatial and chemical information provided by elemental mapping with appropriate sampling and data processing can represent, for a given locality, a powerful toolkit to decipher the anatomy and affinities of poorly preserved and/or highly weathered soft-bodied fossils.

Documenting new anatomical features completes our knowledge of past biodiversity and ecology, while uncovering hidden evolutionary patterns. As such, this approach holds great promise in allowing a detailed understanding of overlooked aspects of the history of life. S1. Precise locality information is curated with the specimens, and available upon request from the authors. Specimens were selected to represent a variety of taxa and tissue types, for which remains are thicker than 300 µm to make sure that the elemental signal recorded using µXRF only comes from the fossil itself and not also from the underlying sedimentary matrix. Details about the scanning steps and dwell times used for each map, as well as about the areas from which spectra have been extracted are also given in Table S1.

Materials and Methods

Fossil

Elemental mapping. Synchrotron micro X-ray fluorescence (µXRF) major-to-trace elemental mapping was performed at the DiffAbs beamline of the SOLEIL synchrotron source (France), using a monochromatic beam of 18 keV (∆E/E ~1-2×10 -4 ), selected for excitation of K-lines from phosphorus to yttrium and L-lines from cadmium to uranium. In order to map the specimen with a high lateral resolution, the beam was reduced down to a diameter of 50 µm using a molybdenum pinhole. The sample was mounted on a scanner stage allowing 90 mm movements (in both horizontal and vertical directions) with micrometre accuracy, and orientated at 45° with respect to the incident beam. XRF was collected using a 4-element silicon drift detector (SDD, Vortex ME4, Hitachi High-Technologies Science America, Inc., total active area: 170 mm 2 ) oriented at 90° with respect to the incident beam, in the horizontal plane [START_REF] Gueriau | Show me your yttrium, and I will tell you who you are: implications for fossil imaging[END_REF] in order to minimize the elastic (Thompson) scattering signal. Two-dimensional spectral images, i.e. images for which each pixel is characterised by a full XRF spectrum, were collected on the fly [START_REF] Leclercq | Flyscan: a fast and multi-technique data acquisition platform for the SOLEIL beamlines[END_REF] over the specimens at a 20-200 µm lateral resolution with a 20-70 ms dwell time (effective counting time was 90% of the dwell time) depending on the samples (see Table S1 for the precise scanning steps and dwell times used for each map). Spectra were then reduced by summing intensities from the four elements of the XRF detector, and integrating intensities every 100 eV. All elemental distributions presented herein correspond to integrated intensities from the main XRF peaks, produced using ImageJ and represented using grey, RGB composite or Green Fire Blue colour scales.

XRF spectra extraction. Based on the obtained elemental distributions, we consistently extracted mean XRF spectra from the different tissues preserved in our fossils, as well as from their surrounding sedimentary matrix, carefully selecting homogeneous areas (see Figs. 1A-M, Table S1 and Figs. S1-S14 for location and details on the specimens and the selected areas). We particularly avoided Mn-rich areas that resulted from recent weathering and covered parts of some fossils (Figs.

1D, G and L) as they would obscure other chemical signals; weathering in the Fezouata Shale was also responsible for the leaching of carbon, oxidation of pyrite, deposition of Fe-oxides and dissolution of carbonates [START_REF] Saleh | Taphonomic pathway of exceptionally preserved fossils in the Lower Ordovician of Morocco[END_REF][START_REF] Saleh | Insights into soft-part preservation from the Early Ordovician Fezouata Biota[END_REF][START_REF] Lefebvre | Exceptionally preserved soft parts in fossils from the Lower Ordovician of Morocco clarify stylophoran affinities within basal deuterostomes[END_REF][START_REF] Saleh | Fossil weathering and preparation mimic soft tissues in eocrinoid and somasteroid echinoderms from the Lower Ordovician of Morocco[END_REF] . To compensate for the use of different exposure times between datasets, the number of pixels for each area was adapted such that the collection time (and consequently the signal-to-noise ratio) was similar between all extracted spectra (see Table S1).

Another important parameter to consider is the thickness of the fossil material. At 18 keV, the attenuation length for goethite (the main iron oxyhydroxide in the Fezouata fossils) is about 80 µm: it is therefore essential that extracted spectra are collected from fossil tissues thicker than this length, so that the extracted signal is not the sum of contributions from the fossil and the underlying sediment.

Linear discriminant analyses. Differences in elemental abundances between the tissue types of the 14 identified fossils were assessed using a linear discriminant analysis (LDA), a method that explains (and predicts) the affiliation of an individual to a predefined class (group) based on the measured predictive variable characteristics. The analyses were performed in the R statistical environment using the MASS package 62 , and plotted using the ggplot2 package [START_REF] Wickham | ggplot2: Elegant Graphics for Data Analysis[END_REF] . The statistical analysis is based on the integrated intensity of the XRF signals of the different elements, with partial reabsorption by the matrix. To discriminate between tissues, we directly rely on the fact that the signal results from the statistical realisation of the photon-matter interaction (rather than quantitative estimates). Discrimination therefore takes into account both the average content in each element and its local distribution within each probed voxel (containing material of varying composition). Prior to the LDA, bending followed by a rubberband baseline corrections were applied to the log-normalised spectra using the wl.eval and spc.rubberband function of the hyperSpec package [START_REF] Beleites | hyperSpec: a package to handle hyperspectral data sets in R, R package version 0[END_REF] . Spectra from the enigmatic organism were then added to the LDA plot using the predict function. Spectra and the R script used in this work are available via the following Dryad Digital Repository: https://datadryad.org/stash/share/LGEGhXAwq2FZjiDl2k9MPFfZ1D0Vt0Z_M1wLWXSKzSw (private, randomized URL for Peer Review until the related manuscript has been accepted).

XRF quantification. Full quantification of trace elements from XRF maps of heterogeneous materials such as fossils is generally hampered by local heterogeneity (both laterally and in depth), which limits the precision of corrections for matrix X-ray reabsorption, with strong impact on the calculated concentrations depending on the hypothesis on the matrix composition. Nonetheless, by carefully selecting homogeneous areas (based on elemental distributions) and defining the matrix, statistics are sufficient to estimate semi-quantitative contents of trace elements in fossils [START_REF] Gueriau | Cerium anomaly at microscale in fossils[END_REF][START_REF] Gueriau | Oxidative conditions can lead to exceptional preservation through phosphatization[END_REF] . We estimated elemental concentrations in our fossils from a full spectral decomposition performed with the PyMCA data analysis software 67 using an Hypermet peak shape, a polynomial approximation of the baseline and experimental parameters, applying reabsorption corrections considering a goethite matrix (as determined by Raman spectroscopy). The photon flux was estimated to 3.4×10 9 photons•s -1 taking Fe as internal standard (62.85wt% in pure goethite). Resulting concentrations expressed as wt% are presented in Table S2. S1 for specimen details and acquisition parameters). (N) Linear discriminant analysis of the mean XRF spectra extracted from the white box areas in A-M; white arrows locate spectra for the shale matrix that were taken outside of the displayed field of view, their length being proportional to the distance from the sampled area (see Figs. S1-S14 for precise locations). (O) Mean XRF spectrum for each tissue type and the matrix, vertically shifted for readability. The 'Fe ×2' peak in the fossil spectra corresponds to a Fe pile-up peak, an artefactual peak [START_REF] Brouwer | Theory of XRF[END_REF] that increases with concentration and further indicate that fossil tissues are richer in Fe than the shale. Classification of the all 7 extracted XRF spectra within our linear discriminant analysis. Note that weathered matrix spectra plot closer to cuticularized tissues the richer they are in iron, following the fact that the shale matrix (clay and silica) is way less concentrated in iron than the fossil tissues (iron oxides). Of note is also the classification of the green XRF spectrum, corresponding to the remains of an internal system, which plots close to the trilobite internal, most likely digestive, tissue, suggesting that this area may preserve remains of the digestive system. S2. XRF minor to trace element quantification of the mean spectra for the sclerotized, biomineralized and cuticularized tissues (wt%)

[planned for 2-column width]

  specimens. The studied material is housed in the collections of the Musée cantonal de Géologie de Lausanne (MGL), and of the Cadi-Ayyad University (Faculté des Sciences et Techniques, Guéliz), Marrakesh, Morocco. The MGL material was collected by authorised and academically recognised avocational Moroccan collector Mohamed Ben Moula and his family over the period of 2015 to 2016; A.C.D. worked in collaboration with them to collect the metadata associated with the collected fossils. Mohamed Ben Moula has a long-standing working relationship with several academics, has received the Mary Anning Award from the Palaeontological Association, and has a radiodont fossil named after him in honour of his great contribution to the field of palaeontology. The collection was purchased with funds from the University of Lausanne and the Swiss National Science Foundation, following all regulations for purchases. The material was transported to Casablanca and subjected to export approval by the Ministry of Energy, Mines and the Environment of the federal government of the Kingdom of Morocco and approved for shipment to Switzerland on 11.05.2017 (export permits curated with the collection). The material from the Marrakesh collection studied herein was loaned to the University Lyon 1 for study and will be returned to the Cadi-Ayyad University after study. Details about the fossil specimens investigated herein (identification, inventory numbers, and localities) are given in Table

Figure 1 .

 1 Figure 1. Synchrotron-based XRF major-to-trace elemental mapping of 14 fossil animals from the Early Ordovician Fezouata Shale of Morocco. (A-M) Optical photograph and false-colour overlays of iron (red), manganese (green), and rubidium (blue) distributions of the analysed fossils (see TableS1for specimen details and acquisition parameters). (N) Linear discriminant analysis of the mean XRF spectra extracted from the white box areas in A-M; white arrows locate spectra for the shale matrix that were taken outside of the displayed field of view, their length being proportional to the distance from the sampled area (see Figs. S1-S14 for precise locations). (O) Mean XRF spectrum for each tissue type and the matrix, vertically shifted for readability. The 'Fe ×2' peak in the fossil spectra corresponds to a Fe pile-up peak, an artefactual peak[START_REF] Brouwer | Theory of XRF[END_REF] that increases with concentration and further indicate that fossil tissues are richer in Fe than the shale.[planned for 2-column width]

Figure 2 .Figure 3 .Figure S9 .

 23S9 Figure 2. Synchrotron-based XRF major-to-trace elemental mapping of a highly weathered organism (AA-FETB-OI-22) from the Early Ordovician Fezouata Shale of Morocco. (A) Optical photograph of the entire slab. (B) Close-up on the specimen, from the dotted white box area in A. (C) False-colour overlay of iron (red), manganese (green), and rubidium (blue) distributions from the area in B. Acquisition parameters: 100×100 μm 2 scan step, 20 ms dwell time, 215 280 pixels. Numbered white box areas indicate location of XRF spectra in E. (D) Rubidium distribution only. (E) Classification of the mean XRF spectra extracted from the white box areas in C within our LDA. (F) False-colour overlay of arsenic (red), lead (green), and copper (blue) distributions. (G) Interpretative line drawing of the anatomy of the specimen. Abbreviations: al, anterior lobe; fa, frontal appendage; fl, lateral flaps; ns, nervous system; ?oris, other remains of internal system(s); str, segmented trunk. [planned for 2-column width]

Figure S10 .

 S10 Figure S10. Synchrotron-based XRF major-to-trace elemental mapping of the marrellomorph euarthropod Enosiaspis hrungnir Legg, 2016, specimen MGL 102321, from the Early Ordovician Fezouata Shale of Morocco. Optical photograph (left), false-colour overlays of iron (red), manganese (green), and rubidium (blue) distributions (centre), and the extracted mean XRF spectra from box areas of corresponding colours in the elemental overlay (right). Refer to Fig. 1O for peak identification.

Figure S11 .

 S11 Figure S11. Synchrotron-based XRF major-to-trace elemental mapping of the demosponge Choia sp., specimen MGL 107663, from the Early Ordovician Fezouata Shale of Morocco. Optical photograph (left), false-colour overlays of iron (red), manganese (green), and rubidium (blue) distributions (centre), and the extracted mean XRF spectra from box areas of corresponding colours in the elemental overlay (right). Refer to Fig. 1O for peak identification.

  Figure S12. Synchrotron-based XRF major-to-trace elemental mapping of the demosponge Pirania auraeum Botting, 2007, specimen MGL 107764, from the Early Ordovician Fezouata Shale of Morocco. Optical photograph (left), false-colour overlays of iron (red), manganese (green), and rubidium (blue) distributions (centre), and the extracted mean XRF spectra from box areas of corresponding colours in the elemental overlay (right). Refer to Fig. 1O for peak identification.

  Figure S13. Synchrotron-based XRF major-to-trace elemental mapping of the trilobite Bavarilla zemmourensis Destombes, Sougy & Willefert, 1969, specimen MGL 102222, from the Early Ordovician Fezouata Shale of Morocco. Optical photograph (left), false-colour overlays of iron (red), manganese (green), and rubidium (blue) distributions (centre), and the extracted mean XRF spectra from box areas of corresponding colours in the elemental overlay (right). Refer to Fig. 1O for peak identification.

  Figure S14. Synchrotron-based XRF major-to-trace elemental mapping of the stylophoran echinoderm Thoralicystis sp., specimen MGL 107952, from the Early Ordovician Fezouata Shale of Morocco. Optical photograph (left), false-colour overlays of iron (red), manganese (green), and rubidium (blue) distributions (centre), and the extracted mean XRF spectra from box areas of corresponding colours in the elemental overlay (right). Refer to Fig. 1O for peak identification.

Figure S15 .

 S15 Figure S15. Satellite view of the Draa valley locating the different outcrops that yielded the fossils studied herein. The '~14km' measurement refers to the perimeter encompassing all the north localities. Precise locality information is curated with the specimens, and available upon request from the authors.

Figure S16 .

 S16 Figure S16. Synchrotron-based Cu K-edge X-ray absorption spectroscopy (XAS) of a range of fossils from the Early Ordovician Fezouata Shale of Morocco. (A) Optical photograph of the undescribed "xiphosuran" MGL 102701. (B) false-colour overlays of copper (red), iron (green), and manganese (blue) distributions. (C) Cu K-edge XAS spectrum (red line) from the yellow dot in B superimposed upon spectra collected on reference Cu compounds (grey to black dotted lines) for comparison. The spectrum obtained for the fossil is inconsistent with metallic copper (Cu), copper sulfide (CuS) or cupric oxide (CuO) but more consistent with copper hydroxide chloride (Cu(OH)Cl) and to a greater extent copper carbonate (CuCO3), suggesting a similar environment for Cu in the fossil. (D-H) Optical photographs of Enosiaspis hrungnir MGL 102321 (D), the undescribed "xiphosuran" MGL 102705 (E), the undescribed "synziphosurine" MGL 107210 (F), Thoralicystis sp. MGL 107952 (G), and Pirania auraeum MGL 107764 (H). (I) Cu K-edge XAS spectra from the yellow dot in D-H, showing that Cu is present in all investigated fossils in the same chemical environment. XAS was performed at the DiffAbs beamline (SOLEIL synchrotron) with the following acquisition parameters: fluorescence mode, 230×150 μm 2 (H×V) beam spot size, 3 s counting time per energy step; energy step sizes were 0.5 eV between 8970 and 9030 eV, 1 eV between 9031 and 9100 eV, and 2 eV between 9102 and 9200 eV. Spectra have been normalised, and the references corrected from self-absorption, using the Athena software 68 . 68. B. Ravel, M. Newville, ATHENA, ARTEMIS, HEPHAESTUS: data analysis for X-ray absorption spectroscopy using IFEFFIT. Journal of Synchrotron Radiation 12, 537-541 (2005).

Figure S17 .

 S17 Figure S17. Synchrotron-based X-ray fluorescence major-to-trace elemental mapping of AA-FETB-OI-22, an undescribed "enigmatic organism" from the Early Ordovician Fezouata Shale of Morocco. (Top left) Optical photograph. (Top centre) False-colour overlays of iron (red), manganese (green), and rubidium (blue) distributions. (Top right) Mean XRF spectra from the box areas of corresponding colours (numbered 1 to 3) in the elemental overlay, corresponding to the same areas as in Fig. 2C. (Bottom left) Additional mean XRF spectra, extracted from the orange, brown, red (weathered matrix) and green (other internal remains) box areas of corresponding colours in the XRF false-colour overlay. Refer to Fig. 1O for peak identification. (Bottom right)Classification of the all 7 extracted XRF spectra within our linear discriminant analysis. Note that weathered matrix spectra plot closer to cuticularized tissues the richer they are in iron, following the fact that the shale matrix (clay and silica) is way less concentrated in iron than the fossil tissues (iron oxides). Of note is also the classification of the green XRF spectrum, corresponding to the remains of an internal system, which plots close to the trilobite internal, most likely digestive, tissue, suggesting that this area may preserve remains of the digestive system.

  counts; log scale)

Table S1 .

 S1 Details on the specimens. acquisition parameters and selected spectra studied herein. (** Denotes 2 spectra for which it was not possible to obtain a collection time over the selected area close to 22.5 s)

	Taxon Bavarilla zemmourensis Destombes. Sougy & Willefert. 1969 Element K	Collection number MGL 102222 Sclerotised 0.5112	Locality A16 (2015) Biomineralised 1.956	Figure(s) Figs. 1L, S13 Cuticularised Spectrum 1 2 0.4588	Tissue type (Following [35]) biomineralised biomineralised	XRF map scan step (µm) 200 200	XRF map dwell time (ms)	Number of pixels selected 1122 858	Time over slected 22.44 area (s) 17.16 **
	undescribed "xiphosuran" undescribed "synziphosurine" Ca Thoralicystis sp. (Lefebvre et al. Ti 2019) Cr	MGL 102701 MGL 107210 0.5725 MGL 107952 0.1771 0.03413	A5 (2015) A16 (2015) 0.5226 A9 (2015) 0.8605 0.04596	Figs. 1A, S1 Figs. 1B, S2 0.3237 Figs. 1M, S14 0.2255 0.03328	1 2 1 3 1 2	sclerotised matrix sclerotised matrix biomineralised biomineralised	20 20 20 200 70 70		900 900 1122 1122 729 638	22.5 22.5 22.44 21.87 22.44 19.14 **
		Mn	0.2118	0.2076	0.2984	2 3	matrix matrix	20 70		1122 729	22.44 21.87
	undescribed "synziphosurine" undescribed "enigmatic organism" Ni	MGL 102841 AA-FETB-OI-22 0.03054	"Synz. Loc." ZF2(3b) 0.03625	Figs. 1C, S3 Figs. 2, S17 0.08007	1 1	sclerotised ?	20 100		1122 1122	22.44 22.44
		undescribed "marrellid" Cu Zn	0.3035 AA-BIZ31-OI-39 0.2127	0.2087 ZF4(31) 0.4004	0.02415 Figs. 1D, S4 0.2436	2 2 1 3	matrix ? sclerotised matrix	20 100 70 100		1122 1124 324 1122	22.44 22.48 22.68 22.44
		undescribed "xiphosuran" Ga As Br	MGL 102705 0.007206 0.6792 0.006563	A5 (2015) 0.05585 0.522 0.00397	Figs. 1E, S5 0.01741 0.04578 0.0003272	2 1 2 4 5	matrix sclerotised matrix weathered matrix weathered matrix	70 70 70 100 100		324 441 441 1122 1122	22.68 22.05 22.05 22.44 22.44
	Bavarilla zemmourensis Destombes. Sougy & Willefert. 1969 Rb Sr Y	MGL 102177 0.2398 0.1391 0.007973	A17 (2015) 0.4184 0.2448 0.03406	Figs. 1F, S6 0.1613 0.09511 0.01968	1 2 6 7	internal matrix weathered matrix ?	70 70 100 100		1128 1122 1122 1121	22.56 22.44 22.42 22.44
	undescribed "radiodont" appendages Pb	MGL 103593 0.5217	A27 (2016) 0.3441	Figs. 1G, S7 0.3683	1	cuticularised	70		900	22.5
						2	matrix	70		900	22.5
	Table	undescribed "annelid"	MGL 107866	A2 (2015)	Figs. 1H, S8	1	cuticularised	70		1127	22.54
						2	matrix	70		1122	22.44
		undescribed "annelid"	AA-TGR1c-OI-47	ZF51c	Fig. S9	1	cuticularised	70		1124	22.48
						2	matrix	70		1122	22.44
	Enosiaspis hrungnir Legg. 2016	MGL 102321	A6 (2015)	Figs. 1I, S10	1	sclerotised	70		380	22.8
						2	matrix	70		380	22.8
		Choia sp. (Botting 2007)	MGL 107663	A17 (2015)	Figs. 1J, S11	1	biomineralised	70		441	22.05
						2	biomineralised	70		441	22.05
						3	matrix	70		441	22.05
	Pirania auraeum Botting. 2007	MGL 107764	A26 (2016)	Figs. 1K, S12	1	biomineralised	70		552	22.08
						2	matrix	70		552	22.08

Mean intensity (counts; log scale)

Acknowledgments

We are grateful to SOLEIL synchrotron for provision of beamtime, D. Thiaudière, and P. Joly for assistance at the DiffAbs beamline, as well as B. Lefebvre and E. Robert for facilitating access to the material from the Marrakesh collection, and G. Potin for help with the Musée cantonal de Géologie de Lausanne collection. P.G., F.P.P., L.Lu. and this research were funded by the Swiss National Science Foundation, grant number 205321_179084 entitled "Arthropod Evolution during the Ordovician Radiation: Insights from the Fezouata Biota" and awarded to A.C.D. L.La. was supported by the Center for Geosphere Dynamics (UNCE/SCI/006), and by the institutional support RVO 67985831 of the Institute of Geology of the Czech Academy of Sciences. F.S. acknowledges funding from the Faculty of Geosciences and Environment of the University of Lausanne.

Extended Data Figures.


domain: sde sdv

text:
Emeline Vernhes

Nuria Ramos

Donna-Joe Bigot

Virginie Ramillon-Delvolve

Cédric Y Montanier

Gilles Truan

Sébastien Nouaille

Régis Fauré

Enhancing non-canonical amino acid incorporation towards enzyme engineering upgrading Genetic code expansion tool improvement towards biocatalytic reprogramming

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

Enzyme engineering benefits from the > 150 non-canonical amino acids (ncAAs) available for the introduction of non-naturally encountered chemical functions [START_REF] Agostini | Biocatalysis with Unnatural Amino Acids[END_REF]. NcAAs may be incorporated at a target position of a protein of interest by genetic code expansion. This method is based on the reassignment of a nonsense codon to an ncAA by introducing an orthogonal amino-acyl tRNA synthetase (aaRS)/tRNA pair. In E. coli, the pEVOL system is the historical and most widely used [START_REF] Young | An Enhanced System for Unnatural Amino Acid Mutagenesis in E. coli[END_REF]. The pUltra system allows improved incorporation efficiencies in some conditions and can be combined with the pEVOL system for the incorporation of two different ncAAs [START_REF] Chatterjee | A Versatile Platform for Single-and Multiple-Unnatural Amino Acid Mutagenesis in Escherichia coli[END_REF].

Background

Improving the incorporation efficiency emeline.vernhes@insa-toulouse.fr regis.faure@insa-toulouse.fr

•

  TBI -Toulouse Biotechnology Institute, Bio & Chemical Engineering • www.toulouse-biotechnology-institute.fr The incorporation efficiency depends on: -the incorporation position -the nature of the ncAA -the protein Genetic code expansion tool improvement towards biocatalytic reprogramming Emeline Vernhes, Nuria Ramos, Donna-Joe Bigot, Virginie Ramillon-Delvolve, Cédric Y. Montanier, Gilles Truan, Sébastien Nouaille, Régis Fauré Toulouse Biotechnology Institute (TBI), Université de Toulouse, CNRS, INRAE, INSA, Toulouse, France

The incorporation efficiency can be improved by:

-aaRS and tRNA engineering -strain engineering -modulation of the aaRS and tRNA expression levels  New inducible orthogonal expression system: pINS