Carys E Bennett

Mark Williams

Melanie J Leng

Martin Lee

Magali Bonifacie

Damien Calmels

Richard A Fortey

Alan W Owen

Alex A Page

Thijs R A Vandenbroucke

Oxygen isotope analysis of the eyes of pelagic trilobites: testing the application of sea temperature proxies for the Ordovician

Keywords: Stable isotope geochemistry, marine geochemistry, instruments and techniques, biomineralization, petrography, microstructures, and textures Trilobite, oxygen isotopes, Ordovician, paleotemperature, microstructure

Highlights 1. Preservation assessment shows some well-preserved Ordovician trilobite eyes 2. Ordovician trilobite eyes yield  18 O values similar to Ordovician brachiopods 3. SIMS and clumped isotope results indicate diagenetic alteration of trilobites 4. Classic protocols to assess preservation may be inapt for most ancient carbonates

Introduction

Telephinid trilobites are amongst the few unequivocally pelagic organisms preserved in Ordovician rocks that have a carbonate biomineralized skeleton. The most common telephinid, Carolinites genacinaca, had a global paleoequatorial distribution during the Early Ordovician (Floian) and the morphology of the Carolinites body plan, eye shape and eye position suggest that species were epipelagic, living near the sea surface in the mixed layer (McCormick andFortey, 1998, 1999). The holochroal eyes of telephinid trilobites are composed of hundreds of interlocking lenses of calcite and are sufficiently large to provide sufficient material for stable isotope analysis, which in turn might conceivably yield an estimate of Ordovician sea temperature.

Trilobite cuticle has been used in Ordovician isotope paleoclimate studies in the same way as brachiopod calcite [START_REF] Brand | Carbon, oxygen and strontium isotopes in Paleozoic carbonate components: an evaluation of original seawater-chemistry proxies[END_REF][START_REF] Finnegan | The magnitude and duration of Late Ordovician-Early Silurian glaciation[END_REF]. Studies on the ultrastructure of the biomineralized trilobite exoskeleton (the cuticle) show that original features such as horizontal lamination, relict organic material, and pore canals can be preserved [START_REF] Dalingwater | Trilobite cuticle microstructure and composition[END_REF][START_REF] Dalingwater | Cuticular ultrastructure of the trilobite Ellipsocephalus Polytomus from the Middle Cambrian of Öland, Sweden[END_REF], although evidence for diagenetic alteration is also common [START_REF] Wilmot | Primary and diagenetic microstructures in trilobite exoskeletons[END_REF][START_REF] Budil | Exoskeletal structures and ultrastructures in Lower Devonian dalmanitid trilobites of the Prague Basin (Czech Republic)[END_REF]. Investigations into the original chemical and isotope compositions of trilobite cuticles indicate that they were formed from low-Mg calcite [START_REF] Wilmot | Original mineralogy of trilobite exoskeletons[END_REF][START_REF] Lee | Biomineralisation in the Paleozoic oceans: Evidence for simultaneous crystallisation of high and low magnesium calcite by phacopine trilobites[END_REF][START_REF] Mcroberts | Original spotted patterns on Middle Devonian phacopid trilobites from western and central New York[END_REF][START_REF] Teigler | Microstructure and composition of trilobite cuticles[END_REF]. Some specimens have been recorded with an intermediate-Mg calcite composition [START_REF] Brand | Carbon, oxygen and strontium isotopes in Paleozoic carbonate components: an evaluation of original seawater-chemistry proxies[END_REF][START_REF] Mcalister | Geochemistry of some Ordovician and Devonian trilobite cuticles from North America[END_REF]. However, as the preservation of the cuticle ultrastructure in these latter specimens was not examined in detail, the possibility remains that the chemical composition of these cuticles may reflect diagenetic recrystallization in Mg-rich fluid. The lenses in the schizochroal eyes of phacopine trilobites have been identified as being originally composed of high-Mg calcite of ~7.5 mol% MgCO3, while the cuticle comprises low-Mg calcite of ~1.4 to 2.4 mol% MgCO3 [START_REF] Lee | Biomineralisation in the Paleozoic oceans: Evidence for simultaneous crystallisation of high and low magnesium calcite by phacopine trilobites[END_REF].

As the physical properties of a functional calcite lens are well understood, even if trilobite eyes do not have modern analogues, it is possible to assess their degree of preservation [START_REF] Torney | Microscructure and growth of the lenses of schizochroal trilobite eyes[END_REF]. Previous work on the ultrastructure of schizochroal trilobite eyes using EBSD has revealed that the original structure of the eye lenses can be identified [START_REF] Lee | Magnesium-rich intralensar structures in schizochroal trilobite eyes[END_REF][START_REF] Lee | Biomineralisation in the Paleozoic oceans: Evidence for simultaneous crystallisation of high and low magnesium calcite by phacopine trilobites[END_REF][START_REF] Torney | Microscructure and growth of the lenses of schizochroal trilobite eyes[END_REF]. This finding suggests the possibility that the original chemical and isotope composition of eye calcite may also be retained. Highresolution SEM analysis of the lenses of phacopine trilobite eyes show that the intralensar bowl (present only in schizochroal eyes) was composed of high-Mg calcite with micro-crystals of dolomite indicating diagenetic alteration [START_REF] Lee | Magnesium-rich intralensar structures in schizochroal trilobite eyes[END_REF]2012). SIMS analysis can be used to target small (less than 20 m diameter) areas of a fossil in situ, such as trilobite eye lenses, and SIMS analysis of  18 O has been successfully applied to the analysis of conodont microfossils [START_REF] Wheeley | Oxygen isotope variability in conodonts: implications for reconstructing Paleozoic palaeoclimates and paleoceanography[END_REF]. Biogenic proxies that have been hitherto used to provide Early and Middle Ordovician marine paleotemperatures are the oxygen isotopic compositions of brachiopod calcite and conodont apatite [START_REF] Shields | Sr, C, and O isotope geochemistry of Ordovician brachiopods: A major isotopic event around the Middle-Late Ordovician transition[END_REF][START_REF] Trotter | Did cooling oceans trigger Ordovician biodiversification? Evidence from conodont thermometry[END_REF][START_REF] Wadleigh | 18 O 16 O and 13 C 12 C in lower Paleozoic articulate brachiopods: Implications for the isotopic composition of seawater[END_REF], along with carbonate clumped isotope compositions (47) from trilobite, brachiopod and coral calcite for the Late Ordovician [START_REF] Finnegan | The magnitude and duration of Late Ordovician-Early Silurian glaciation[END_REF]. However, some of these results are controversial. For instance, brachiopod isotope data from the Lower Ordovician can have very low  18 O (down to -10‰). This result implies very high (up to + 60 o C) seawater temperatures [if it is assumed that seawater  18 O was similar to today], or diagenetic alteration at higher temperatures than that of the ocean, diagenetic alteration by a different fluid, or that seawater  18 O was substantially different from present [START_REF] Veizer | 87 Sr/ 86 Sr,  13 C and  18 O evolution of Phanerozoic seawater[END_REF][START_REF] Shields | Sr, C, and O isotope geochemistry of Ordovician brachiopods: A major isotopic event around the Middle-Late Ordovician transition[END_REF], or a combination of those alternatives. In contrast,  18 O from conodont apatite from the Ordovician suggests that seawater temperatures were between 30-40 o C if a value similar to modern  18 Owater of -1‰VSMOW is used [START_REF] Trotter | Did cooling oceans trigger Ordovician biodiversification? Evidence from conodont thermometry[END_REF]. Oxygen isotope data can also reflect other environmental parameters; for example, [START_REF] Bickert | Paleoenvironmental changes in the Silurian indicated by stable isotopes in brachiopod shells from Gotland, Sweden[END_REF] showed that the  18 O values of well-preserved Silurian brachiopods reflect salinity differences due to varying fresh-water input rather than temperatures. Nevertheless, the development of proxies that can provide robust and reasonable estimates of seawater temperature for the early Paleozoic is important for many reasons. For example, such information can help constrain the environmental feedbacks or triggers of the Great Ordovician Biodiversification Event [START_REF] Trotter | Did cooling oceans trigger Ordovician biodiversification? Evidence from conodont thermometry[END_REF]Amberg et al., 2016), and it also represents one of the few available proxies for ground-truthing General Circulation Models of early Paleozoic climate [START_REF] Vandenbroucke | Groundtruthing Late Ordovician climate models using the paleobiogeography of graptolites[END_REF][START_REF] Pohl | Effect of the Ordovician paleogeography on the (in)stability of the climate[END_REF]2016). Changes in seawater temperatures may have had significant effects on the ability of organisms to biomineralize [START_REF] Pruss | Carbonates in skeleton-poor seas: new insights from Cambrian and Ordovician strata of Laurentia[END_REF]. The 47 paleothermometer has great potential for more accurate paleotemperature reconstruction because it is independent of the  18 O of mineralising fluids (Ghosh et al., 2006). Usefully, the recrystallization temperature of diagenetic calcite recorded by 47 is thought to remain stable over hundreds of millions of years, at temperature histories below ~250 o C (Ghosh et al., 2006). However, in deeply buried sedimentary deposits or carbonates having a moderate-temperature burial history (~100°C on timescales of hundreds of million years) the closed-system solid-state diffusive reordering of atoms can modify 47 towards lower values and higher apparent equilibrium temperatures [START_REF] Passey | Carbonate clumped isotope bond reordering and geospeedometry[END_REF][START_REF] Henkes | Temperature limits for preservation of primary calcite clumped isotope paleotemperatures[END_REF][START_REF] Stolper | The kinetics of solid-state isotope-exchange reactions for clumped isotopes: A study of inorganic calcites and apatites from natural and experimental samples[END_REF].

Here we test whether: (i) the unique functional morphology and microstructure of trilobite eyes allows for the accurate assessment of the preservation of their calcite lenses; and (ii) the subsequent  18 O analyses of well-preserved eyes of epipelagic trilobites could serve as a sea temperature proxy. Trilobite eye microstructure and composition were compared to those of the trilobite cuticle. Two isotope methods were used on trilobite material from three sedimentary formations considered to have experienced relatively limited diagenesis: (i) conventional determination of oxygen isotope compositions, and (ii) carbonate clumped isotope 47 analysis.

Materials Utilised

Trilobite eyes were analyzed from three broadly coeval Ordovician formations from separate, equatorial basins: 1) Valhallfonna Formation, Spitsbergen; 2) Emanuel Formation, Canning Basin, Australia; and 3) Horn Valley Siltstone, Amadeus Basin, Australia (Figure 1). For all formations, the following materials were analyzed for  18 O,  13 C and clumped isotope 47 compositions, where possible: pelagic and benthic trilobite eyes and cuticle, sedimentary carbonate (host rock) and coarsely crystalline diagenetic calcite cement (spar). For comparison, additional trilobite cuticle was analyzed from the Upper Member of the Dalby Limestone of Västergötland, Sweden, as this basin has experienced significantly higher burial temperatures. The Supplementary Material (extended version) details further the type and number of analyses from each formation.

Valhallfonna Formation

Forty-three trilobite eyes were examined from eleven samples collected from the Floian (Arenigian) Valhallfonna Formation of Ny Friesland, Spitsbergen [START_REF] Fortey | The Ordovician trilobites of Spitsbergen. 11. Asaphidae, Nileidae, Raphiophoridae and Telephinidae of the Valhallfonna Formation[END_REF][START_REF] Fortey | Cambrian-Ordovician rocks adjacent to Hinlopenstretet, North Ny Friesland, Spitsbergen[END_REF]. Four species of the telephinid Carolinites were studied (C. angustagena, C. genacinaca, C. nevadensis and C. sibiricus), along with cuticle (but no eyes) from benthic olenids (species indeterminate). The depositional environment is interpreted to be a marine shelf setting [START_REF] Fortey | Early Ordovician conodont and trilobite communities of Spitsbergen: influence on biogeography[END_REF]. The Ny Friesland succession is thought to have undergone shallow burial, with Conodont Alteration Index (CAI) values of 1 suggesting maximum temperatures of 90 o C [START_REF] Bergström | Conodonts as paleotemperature tools in Ordovician rocks of the Caledonides and adjacent areas in Scandinavia and the British Isles[END_REF]. Calcite veins and calcite spar cements within the cavities of ostracod fossils and in pore spaces in the limestone indicate evidence for some diagenetic alteration.

Emanuel Formation

Trilobites were studied from the Floian (Bendigonian) Emanuel Formation of the Canning Basin, Western Australia [START_REF] Laurie | Early Ordovician trilobite taxonomy and biostratigraphy of the Emanuel Formation, Canning Basin, Western Australia, Parts 1 and 2[END_REF]. Fifty-one trilobite eyes were examined from the telephinid Opipeuterella sp. and benthic asaphids (species indeterminate), occurring in five samples from the type section of the Emanuel Formation. The burial history of the Ordovician Canning Basin indicates low thermal maturation, with a CAI index of 1 [START_REF] Nicoll | Revised stratigraphy of the Ordovician (Late Tremadoc-Arenig) Prices Creek Group and Devonian Poulton Formation, Lennard Shelf, Canning Basin, Western Australia[END_REF] and Apatite Fission Track Analysis (AFTA) indicating temperatures of ~100 o C during the Late Devonian/Early Carboniferous [START_REF] Arne | Regional thermal history of the Lennard shelf, Canning Basin, from apatite fission track analysis: Implications for the formation of Pb-Zn ore deposits[END_REF].

Horn Valley Siltstone

Trilobites were studied from the Floian to Dapignian (Bendigonian to Yapeenian) Horn Valley Siltstone of the Amadeus Basin, Northern Territory, Australia, which can be partly correlated to the upper part of the Emanuel Formation from the Canning Basin [START_REF] Laurie | Ordovician trilobites from the Horn Valley Siltstone and basal Stairway Sandstone, Amadeus Basin, Northern Territory[END_REF]. Over 100 trilobite eyes from Carolinites genacinaca and benthic asaphids (species indeterminate) were examined from three samples collected from a field section at Mt Olifent (fig. 6 in Laurie, 2006). The burial history for the Amadeus Basin has been determined by AFTA and organic maturity data that indicate maximum burial during the Late Carboniferous, with Ordovician strata subjected to maximum temperatures of 140 o C [START_REF] Gibson | Regional perspectives on new and reviewed thermal history data from central Australian basins[END_REF].

Västergötland

Two test samples of Telephina cuticle and its host rock were analyzed for 47 from the Upper Member of the Dalby Limestone, Sandbian (Late Ordovician), in Västergötland, Sweden, introducing contrasting data from the opposite end of the preservation spectrum. The Västergötland rocks have been heated by local Permian intrusions, giving CAI values of 6 to 7 and temperatures of over 300 o C [START_REF] Bergström | Conodonts as paleotemperature tools in Ordovician rocks of the Caledonides and adjacent areas in Scandinavia and the British Isles[END_REF].

Methods

Preservation Assessment Protocol

In total, 198 trilobite eyes were examined under reflected light using a binocular microscope and by SEM, which allowed an initial assessment of eye lens integrity and preservation. Of these, 34 specimens representing a range of preservation states were selected for thin section analysis, based on the eye size (greater than 1.8 mm length), to enable a detailed preservation assessment. Polished thin sections were examined by cathodoluminescence (CL) microscopy and the geochemical variation in eye specimens was quantified using electron microprobe facilities at the universities of Lille (France) and Leicester (UK).

Trilobite eye specimens were imaged by SEM under high vacuum using Secondary Electron and Back Scattered Electron detectors. Electron Backscatter Diffraction (EBSD) analysis of polished thin sections was undertaken at the University of Glasgow to examine intra-lens variations in crystallographic orientations [START_REF] Torney | Microscructure and growth of the lenses of schizochroal trilobite eyes[END_REF].

Isotope Analyses

A total of 182 isotope analyses were undertaken on trilobite eyes, cuticle and host rock from all three formations (Appendix C).  18 O,  13 C and 47 analyses were performed using three methods: 1. Conventional isotope analysis of carbonate powder, with lens/cuticle extraction using both a hand-held dental drill and automated micro-mill, at the British Geological Survey and at the University of Erlangen, Germany; 2. Secondary Ion Mass Spectrometry (SIMS) analysis of 15 m diameter areas of thin sections (Figure 2) at the Centre de Recherches Pétrographiques et Géochimiques (CRPG-CNRS) facility in Nancy, France; 3.

Clumped isotope analysis (47) and  18 O and  13 C analysis of carbonate powder at the Institut de Physique du Globe de Paris (IPGP, Stable Isotope team). The extraction of material by dental drill is subject to human error and samples likely contain some underlying sediment matrix material. Micro-mill extraction is accurate to an error of ~ 5 m, however it is possible that underlying sediment is sometimes sampled. SIMS analysis is the preferred technique in this context because it can accurately sample intra-lens material from thin sections. For a more detailed account of the methods, refer to the Supplementary Methods (extended version) section. Arrows indicate examples of lenses that were analyzed for oxygen isotopes. Scale bars 500 m (A-D) and 100 m (E-F).

Results

Preservation

The majority of the trilobite eyes examined had intact lenses that look superficially well-preserved. The hexagonal lenses are arranged in a single layer, which is on average 70 m in thickness. Individual eye lenses are on average 50 m in diameter, with no mineral partitions between them. Lens size varies between species, with telephinids having significantly larger lenses than asaphids, and moreover, a different eye morphology. Here we describe the results of the preservation assessment protocol from 34 specimens examined in thin section. Specimens are classified as 'well-preserved' or 'poorly preserved' based on their microstructural and geochemical properties (Table 1). Trilobite eye preservation assessment data table, for specimens examined in polished thin section. Some specimens were not observed under EBSD due to their lenses being heavily dolomitised, silicified, or fractured/broken. Note that only specimens from the Valhallfonna Formation were analyzed on the electron microprobe (see Appendix 1). Abbreviations: CL = cathodoluminescence; L = luminescent; NL = non-luminescent, SL = slightly luminescent; SEM = Scanning Electron Microscope; EBSD = Electron back scatter diffraction. In those specimens classified as poorly preserved, the eye lenses exhibit major alteration and lack pristine microstructures such as trabeculae. The microstructural evidence for extensive alteration of calcite lenses is as follows: 1. indistinct lens boundaries where the calcite crystals cross the lens-sediment boundary, form sub-crystals, or exhibit calcite twinning; 2. extensive pitting, resulting from the loss of small crystals and the presence of micropores, indicating that the lenses are recrystallized (Figure 3F); 3. a wide range of crystallographic orientations within a lens, indicating large-scale recrystallization (Fig. 3G); 4. The lens calcite is luminescent (Figure 3H). Minor alteration of lens calcite is evident by variable or partial luminescence due to trace amount variations in Mn and Fe, which can be detected by SEM and electron microprobe analysis. Under CL, recrystallization can be recognised by zoning in the lens calcite crystals and a luminescence intensity that is high, and similar to calcite spar cements in the host rock. Lenses that are more significantly altered can contain diagenetic quartz, dolomite and pyrite crystals.

Micro-dolomite crystals are present within the lenses of nine eye specimens (some well-preserved and others poorly preserved), and are associated with a pervasive microporosity/micro-pitting (Figure 3C). In some specimens, celestine can be recognised in BSE images as small white (i.e., high mean atomic number) crystals. Micro-crystalline dolomite is absent from the eyes of the Horn Valley Siltstone Formation trilobites, which are the most highly altered, and also does not occur in the cuticle. Micro-crystalline dolomite of a similar crystal size, celestine and a microporous texture are also features of the eyes of phacopine trilobites [START_REF] Lee | Magnesium-rich intralensar structures in schizochroal trilobite eyes[END_REF][START_REF] Lee | Biomineralisation in the Paleozoic oceans: Evidence for simultaneous crystallisation of high and low magnesium calcite by phacopine trilobites[END_REF].

Trilobite eyes from the Valhallfonna Formation were analyzed using the electron microprobe to test for geochemical variation. The range in trace elements for calcite eyes is 0.6 -1.9 wt% MgCO3, 0 -0.9 wt% FeCO3 and 0 -0.5 wt% MnCO3. Trilobite eyes that are luminescent (and classified as poorly preserved)

have slightly elevated Mn concentrations relative to non-luminescent lenses. There is no significant difference in the composition of eye calcite compared to that of the associated trilobite cuticle, or carbonate in the host sedimentary deposit (micrite and calcite spar) (Appendix B). Eyes containing micro-crystalline dolomite do not exhibit higher levels of Mg compared to those without micro-crystalline dolomite.

Specimens of trilobite cuticle from the Valhallfonna Formation were examined (Appendix A and B). Eight of the 44 studied had an internal structure comprising two layers of aligned crystals (Figure 4A), although most specimens lacked any internal structure. The cuticle is composed of prismatic, interlocking calcite micro-spar, with a consistent crystal size (Figure 4B), and no evidence of an outer prismatic layer of coarser crystals. EBSD of cuticle associated with trilobite eyes showed that in all specimens the cuticle crystals are randomly oriented. Some specimens have small euhedral pyrite crystals on the margins of the cuticle fragment, or in the interior. Most specimens are non-luminescent, but others exhibit partial or bright orange luminescence. Cuticle from six samples was analyzed by electron microprobe, with a maximum concentration of 2.3 wt% MgCO3, 0.2 wt% FeCO3, 0.4 wt% MnCO3 and 0.6 wt% SrCO3. In comparison to the trilobite eyes, the cuticles contain relatively pure calcite with low trace element concentrations. The size of the calcite crystals within the cuticle was measured from SEM images (Figure 4C), and average size does not correlate with the size of the cuticle fragment examined or its chemical composition (Appendix A and B).

Stable Isotope Data

In total, 50 trilobite eyes were analyzed for isotope composition from the three formations, and where possible, the preservation state of each specimen was assessed prior to isotope analyses using the protocols described above (Appendix C). There is no difference in  13 C and  18 O between trilobite eye calcite extracted by a hand-held dental drill and that extracted by micro-mill (conventional isotope analysis methods), or from clumped isotope analysis. For  18 O, there is a significant difference between conventional isotope analysis (Figure 5) and SIMS isotope results (Figure 6).

Conventional Isotope Results

The total ranges in isotope composition for trilobite eyes (independent of preservation state), cuticle, and host rock (sediment and spar) analyzed are  18 O -6.3‰ to -10.5‰ and  13 C +0.3‰ to -3.9‰. Each formation plots in a different, but overlapping,  13 C and  18 O field (Figure 5A), and within each formation the trilobite eye isotope values are within the range of the results from the trilobite cuticle and host rock (Figure 5B-D).

With regards to the Valhallfonna Formation, the combined isotope results from all material (trilobite eyes, cuticle and host rock) show a low covariance between  13 C and  18 O (R 2 = 0.28) with values ranging from  13 C +0.3‰ to -2.7‰ and  18 O -6.3‰ to -10.5‰. This result is different from the other formations, which lack any covariance between  13 C and  18 O (R 2 = 0.13 Emanuel Formation; R 2 = 0.01 Horn Valley Siltstone). Well-preserved trilobite eyes yield  18 O that are on average higher than those of poorly preserved eyes, ranging from  18 O -7.2‰ to -7.7‰, although there is some overlap (Figure 5B). There is no significant difference in isotope composition between Carolinites species, stratigraphic units, or between the cuticle of Carolinites and olenid trilobites (Appendix C). The  13 C or  18 O variation between cuticle specimens that were identified as having relatively small or large calcite crystals is also negligible.

Results for all materials from the Emanuel Formation range from  18 O of -7.3‰ to -9.1‰ and  13 C of -1.3‰ to -3.9‰. Well-preserved trilobite eyes have a similar range in  13 C and  18 O to that of poorly preserved eyes (Figure 5C). There is no significant difference in isotope composition between the planktonic Opipeuterella sp. and benthic asaphid trilobites, or between different samples (Appendix C). It is possible that the outlier well-preserved eye result of  18 O -8.8‰ is lower than the other well-preserved eye results due to the presence of diagenetically altered matrix material underlying the calcite lenses. Post-sampling photographs after the micro-mill had powdered the lenses do not indicate that this is the case, however the imprecision of the micro-mill technique compared to intra-lens sampling by SIMS means that all micro-mill samples must be considered to be subject to some sediment contamination.

Results for all materials from the Horn Valley Siltstone have a fairly narrow range of  18 O -6.4‰ to -7.4‰

and a broader range of  13 C -1.1‰ to -3.3‰. All eyes examined were poorly preserved and there is no difference in isotope composition between the planktonic Carolinites genacinaca and benthic asaphid trilobites, or between different samples (Appendix C).

The isotope composition of lenses containing micro-crystalline dolomite does not differ from those composed purely of calcite. All samples were analyzed as calcite and it is uncertain whether dolomite crystals even contributed to the signal due to the small quantity of dolomite present (<1%). The presence of dolomite does not correspond to the preservation state of the eyes (Table 1).

Four larger well-preserved specimens were analyzed to test the average isotope variation across the eye. For each specimen, two sub-samples were taken for isotope analysis using the micro-mill or dental drill. Two of the specimens had a consistent composition in both sub-samples, while the other two showed a different composition, with a variability of up to circa 1‰ in  18 O and  13 C (Appendix C).

Twenty-one specimens with cuticle material attached to the eye (such as the librigena or glabella) were analyzed to compare the composition of the cuticle and adjacent eye lenses. In well-preserved specimens from the Valhallfonna Formation, the cuticle is similar in composition to that of the eyes (with a maximum difference in  18 O of 0.6‰) while in poorly preserved specimens the difference is greater (increasing to

 18 O 2.4‰) (Appendix C).
In the Emanuel Formation, the composition of the cuticle is similar to that of the eyes (with a difference in  18 O of 0.8‰) for asaphids and Opipeuterella sp., with no significant difference between poorly preserved and well-preserved specimens (Appendix C). However, the difference in  18 O between eyes and cuticle from the Horn Valley Siltstone, which contains only poorly preserved specimens, is less than 0.2‰.

SIMS Oxygen Isotope Results

SIMS analysis of individual lenses across trilobite eyes in thin section reveals a wide range in  18 O, from -2.4‰ to -10.4‰ (Table 2). The range can dramatically vary even within specimens identified as wellpreserved, and is often different from the conventional isotope analysis results of the same specimen extracted using the dental drill or micro-mill (Fig. 6). For most of the 12 specimens analyzed, the lens composition is within the general range of  18 O as that of cuticle and host rock. However, many of the SIMS results for trilobite eyes, cuticle and host rock yield  18 O values that are substantially more positive than the conventional isotope analysis results.

Specimens identified as well-preserved and poorly preserved have SIMS  18 O results within the same range, and there is no correlation with specimens that have micro-crystalline dolomite within the lenses. The average  18 O, and the range of  18 O within a single eye, does not correlate with the state of preservation of the eyes. There is no difference between the results from telephinids and asaphid specimens from the same formation, even in well-preserved eyes.

Multiple lenses of individual specimens were analyzed using SIMS, giving an intra-eye  18 O range, with the greatest range seen within a single eye specimen of 2.7‰ and the lowest range of 0.3‰. Eight specimens have a relatively high  18 O range (1‰ or greater), while four have a low range in values (less than 1‰)

(Figure 6).

The SIMS and conventional isotope analysis results for individual specimens show some variations. All trilobite cuticle analyses of specimens from the two methods show a high disparity (with  18 O difference greater than 1‰) (Figure 6). Eleven trilobite eye specimens were assessed by SIMS and conventional isotope analysis, and of these, six specimens have a high disparity between the SIMS and conventional methods, while five specimens had similar results ( 18 O <1‰ difference). There is no apparent link between the analysis conditions (beam intensity, chamber pressure, measurement error, run order) and those trilobite eye samples with a large range in  18 O (see Supplementary Information). In general,  18 O results obtained by SIMS are more positive (by as much as 6‰) in comparison to those obtained by conventional isotope analysis.

Clumped Isotope Results

Of the 12 samples examined for their 47 compositions, the cuticle and limestone analyzed from Västergötland had markedly low 47 values of 47CDES25 = +0.383 and +0.396‰, respectively [reported against the absolute Carbon Dioxide Equilibrated Scale -hereafter CDES -reference frame described by [START_REF] Dennis | Defining an absolute reference frame for 'clumped' isotope studies of CO2[END_REF] and referring to CO2 extracted by phosphoric acid digestion at 25°C]. These values correspond to very high "apparent equilibrium temperature" (over 200 o C, see Appendix D) representing the "blocking temperature" with respect to diffusional resetting of the calcite clumped isotope thermometer (e.g., [START_REF] Passey | Carbonate clumped isotope bond reordering and geospeedometry[END_REF][START_REF] Bonifacie | Clumped isotope thermometry of marbles as an idicator of the closure temperatures of calcite and dolomite with respect to solid-state reordering of C-O bonds[END_REF].

In contrast, the range in 47CDES25 values for the three other basins investigated in this study is much higher (with 47CDES25 from 0.581 to 0.610‰ for Spitsbergen, 0.608 to 0.623‰ for the Canning Basin and 0.606 to 0.608‰ for the Amadeus Basin; Appendix D). These 47 values correspond to temperature ranges of 55- The  18 Owater from which the calcite in these three formations was precipitated was calculated using the temperature estimated from 47 and the  18 O of the carbonate, and referenced to published Ordovician  18 OVSMOW estimates. They correspond to  18 Owater of -0.4 to +1.0‰ (average 0.2‰) for the Valhallfonna Formation, -1.6 to -0.2‰ (average -0.9‰) for the Emanuel Formation and 0.0 to +0.6‰ (average +0.3‰)

for the Horn Valley Siltstone (Appendix D). Because the 47 signatures found here do not represent those acquired over original carbonate crystallisation from seawater, the calculated  18 Owater of the mineralizing fluid cannot be interpreted in terms of the original seawater  18 O, but rather likely reflects the properties of diagenetic fluids. However because the  18 Owater results are fairly comparable to that estimated for Ordovician seawater (-1‰  18 OVSMOW: [START_REF] Trotter | Did cooling oceans trigger Ordovician biodiversification? Evidence from conodont thermometry[END_REF] it is probable that diagenesis may have occurred due to the flow of seawater through the rocks during burial, down to a maximum depth of two kilometres.

Discussion

The eye preservation assessment using a combined methodology (SEM, CL, EBSD and electron microprobe) indicates a range in degrees of alteration. The preservation of the trabeculae has been described

as an indicator of good eye preservation [START_REF] Schoenemann | Light guide lenses in trilobites?[END_REF], but it has also been noted that trabeculae can be preserved in diagenetically altered specimens [START_REF] Torney | Microscructure and growth of the lenses of schizochroal trilobite eyes[END_REF]. The cornea (outermost layer of the trilobite eye) is likely to have been a transparent sheet in vivo [START_REF] Clarkson | The eyes of trilobites: The oldest preserved visual system[END_REF], and its present micro-crystalline state indicates that it was recrystallized during diagenesis. The loss of in vivo microstructures from trilobite cuticle indicates that the majority have been recrystallized: the outer prismatic layer [START_REF] Dalingwater | Trilobite cuticle microstructure and composition[END_REF][START_REF] Dalingwater | Cuticular ultrastructure of the trilobite Ellipsocephalus Polytomus from the Middle Cambrian of Öland, Sweden[END_REF] is absent, the presence of pyrite within the cuticle is consistent with diagenetic alteration [START_REF] Wilmot | Primary and diagenetic microstructures in trilobite exoskeletons[END_REF], as is the homogeneous microstructure of the calcite [START_REF] Budil | Exoskeletal structures and ultrastructures in Lower Devonian dalmanitid trilobites of the Prague Basin (Czech Republic)[END_REF]. As the cuticle is thought to mineralize in an organic mesh-like framework, similar to that of an ostracod carapace [START_REF] Teigler | Microstructure and composition of trilobite cuticles[END_REF], the lack of correspondence between crystal size and size of the exoskeleton may indicate diagenesis. Large cuticle crystals may be expected to have incorporated trace-elements or have a different isotope composition to microcrystalline cuticle calcite.

Moreover, the crystallographic orientation of the cuticle crystals (observed to be random under EBSD) has changed, with crystal c-axis perpendicular to the cuticle surface [START_REF] Teigler | Microstructure and composition of trilobite cuticles[END_REF].

In terms of the visual protocols used to assess preservation, the most useful method was EBSD analysis, due to the crystallographic orientation information it shows. SEM microstructural observations were also valuable and identified porosity and micro-crystals (Table 1). While CL generally corresponded to other indicators, with luminescent lenses showing an elevated trace-element profile and evidence for recrystallization, lenses that were recrystallized were sometimes non-luminescent. Thus, CL is the least reliable technique, however it does generate visual data that can be used as a starting point for more detailed preservation analysis via other techniques.

The SIMS results differ significantly from the corresponding conventional isotope analysis results in over half the trilobite eye specimens analyzed. This discrepancy may be due to presence of a fine-scale mixture of primary and secondary calcite within the lenses. In addition, little is known about the composition of trilobite cuticle, or vital effects, in comparison to brachiopods where studies on modern specimens can be used to determine  18 O paleotemperature equations [START_REF] Brand | Oxygen isotopes and MgCO3 in brachiopod calcite and a new paleotemperature equation[END_REF]. However, it must also be considered that the inconsistency between analytical datasets could reflect a systematic analytical error in the SIMS data in this study. Previous work has found differences in the trace element content (Mg/Ca, Sr/Ca and Mn/Ca ratios) of marine bivalves analyzed using conventional bulk dissolution and SIMS. These differences have been interpreted as reflecting variable shell organic content, crystal structures and small inter-crystalline heterogeneities in trace element concentrations [START_REF] Freitas | Ion microprobe assessment of the heterogeneity of Mg/Ca, Sr/Ca and Mn/Ca ratios in Pecten maximus and Mytilus edulis (bivalvia) shell calcite precipitated at constant temperature[END_REF]. It is therefore possible that the presence of organic matter within trilobite eyes, sub-crystal boundaries, or pitted surfaces could have affected our SIMS data. However, the cross-comparison of the SIMS results with the micro-crystalline characteristics of each specimen (Table 1) does not reveal a correspondence between the presence of microcrystalline features (porosity, crystal zoning, micro-dolomite) and wide ranging or positive  18 O values. The size of the ion beam used for the SIMS analysis (15 m) may have resulted in the crossing of crystal boundaries, although most of the spots sampled the centre region of the eye lens and were verified by photographs. It is more likely that the results represent genuine intra-eye variation rather than contamination from the host rock. Further detailed work is needed to attempt to correct the SIMS data for the variables of inter-crystalline heterogeneities. The SIMS analysis has revealed large intra-eye variations of lens geochemistry that would otherwise remain undetected. Despite the limitations of the method, for example micro-scale pitting and sub-crystal boundaries that may cause analytical error, SIMS is still a useful tool for assessing diagenesis.

The range of isotope compositions within a single eye specimen (as revealed by both SIMS and conventional isotope analysis) indicates cryptic recrystallization. This means that recrystallization has likely occurred in specimens that were identified as well-preserved using the SEM, CL, EBSD and electron microprobe protocols outlined above. Ten out of the sixteen specimens examined had ranges in  18 O of greater than 1‰ within the same eye. If, as seems reasonable, all the lenses in an eye were biomineralized (or indeed recrystallized) at the same time, in the same conditions, then they should have the same isotope composition. For each formation, there is no significant difference in  18 O or  13 C between trilobite eye calcite or cuticle and the host rock. In addition: 1. all trilobite cuticle examined is interpreted as diagenetically altered, with a complete loss of crystallographic structure, indicating recrystallization was pervasive; 2. calcitic spar within the host rock, typically resulting from diagenetic growth, is within the same isotope range as the trilobite eye and cuticle results (Fig. 5B); and 3. the counterintuitive finding that pelagic trilobite species, which lived in relatively warm waters near the sea surface, have the same  18 O as benthic species, indicates that all  18 O has been reset. Therefore, most, if not all, of the material analyzed is interpreted to have been diagenetically altered, despite our original assessment of good preservation using microstructural criteria. In terms of eye preservation, there is little geochemical difference between eyes identified as well-preserved or poorly preserved, both in terms of conventional isotope analysis and SIMS results. In addition, the degree of recrystallization (complete or partial) has no significant correspondence with the isotope results.

Detailed SIMS analysis shows a large range in  18 O values within a single eye specimen, even those that on microstructural criteria are well-preserved. Our preservation assessment can therefore be criticised for not identifying subtle preservation features. For example, the presence of trabeculae and lens crystallographic continuity as identified by EBSD mapping was interpreted to indicate well-preserved eyes, but the boundaries of the trabeculae were not examined in detail using TEM as they were by [START_REF] Torney | Microscructure and growth of the lenses of schizochroal trilobite eyes[END_REF].

Micro-crystalline dolomite was observed in well-preserved and poorly preserved specimens, and further analysis using TEM is needed to further understand the origin of this mineral. Despite these limitations, the present study has made the most extensive preservation assessment of any trilobite material prior to isotope analysis.

The presence of micro-crystalline dolomite within the holochroal eyes examined here may indicate an original high-Mg calcite composition (as interpreted for the eyes of phacopine trilobites by [START_REF] Lee | Biomineralisation in the Paleozoic oceans: Evidence for simultaneous crystallisation of high and low magnesium calcite by phacopine trilobites[END_REF], with high-Mg calcite being diagenetically altered to low-Mg calcite. The presence of micro-crystalline dolomite in both poorly-and well-preserved eyes, supports this interpretation. The absence of microcrystalline dolomite in the Horn Valley Siltstone samples may be due to their more extensive diagenetic alteration. However, the lenses containing micro-crystalline dolomite do not have an elevated Mg content, as recorded in phacopine eyes, of up to 6 mol% MgCO3 [START_REF] Lee | Magnesium-rich intralensar structures in schizochroal trilobite eyes[END_REF][START_REF] Lee | Biomineralisation in the Paleozoic oceans: Evidence for simultaneous crystallisation of high and low magnesium calcite by phacopine trilobites[END_REF]. This could be due to the diagenetic loss of magnesium over time. However, the sporadic occurrence of microcrystalline-dolomite -it is not present in all eyes identified as well-preserved -means that at present we cannot conclude that the holochroal lenses were originally composed of high-Mg calcite. An alternative explanation might be that the dolomite grew during diagenetic alteration and micro-recrystallisation of low-Mg calcite. Further detailed investigation of the micro-crystalline dolomite at the nanometre scale is needed to determine an original high-Mg calcite composition of the lenses.

The host rock and fossil material from each of the three basins have a distinctive range in  13 C and  18 O values, which may reflect their different diagenetic histories. The 47 data reveal a minimum temperature of trilobite cuticle (re)crystallisation of 50 o C, which is well in excess of the threshold for most organisms living in the surface layers of the modern oceans [START_REF] Brock | Life at high temperatures[END_REF] and must be interpreted as resulting from the diagenetic alteration of the original cuticle. The Västergötland cuticle clumped isotope results are at the extreme temperature end (200°C), where the original 47 values (i.e. the original 13 C-18 O bonding distribution acquired over crystallisation) have been reset due to closed-system solid-state diffusion alteration. This conclusion is consistent with independent Västergötland CAI values of 6 to 7, indicative of peak temperatures exceeding 300 o C resulting from heating by Permian igneous intrusions [START_REF] Bergström | Conodonts as paleotemperature tools in Ordovician rocks of the Caledonides and adjacent areas in Scandinavia and the British Isles[END_REF]. In contrast, the Australian and Spitsbergen samples have lower temperatures calculated from the 47 (from 50-68 o C; Figure 7), which reflect early recrystallization temperatures.

The thermal history of the sedimentary formations can be linked to the fidelity of eye preservation. The Emanuel Formation was subject to the lowest burial temperatures (maximum temperatures of 70-80 o C: [START_REF] Nicoll | Revised stratigraphy of the Ordovician (Late Tremadoc-Arenig) Prices Creek Group and Devonian Poulton Formation, Lennard Shelf, Canning Basin, Western Australia[END_REF] and its sample set contains the best-preserved trilobite eyes. The Valhallfonna Formation was subject to a slightly higher burial temperature (up to 90 o C: [START_REF] Bergström | Conodonts as paleotemperature tools in Ordovician rocks of the Caledonides and adjacent areas in Scandinavia and the British Isles[END_REF] and its sample set contains just one well-preserved specimen. While the Horn Valley Siltstone experienced a much higher burial temperature (up to 140 o C: [START_REF] Gibson | Regional perspectives on new and reviewed thermal history data from central Australian basins[END_REF] and yielded no well-preserved specimens.

It could be argued that the conventional  18 O and  13 C results for the trilobite cuticle, eyes and host rock reflect original compositions and were not affected by partial recrystallization and only the 47 composition changed. Indeed, solid-state re-ordering can alter the C-O bonds within a shell while retaining original shell microstructures and trace-element concentrations [START_REF] Henkes | Temperature limits for preservation of primary calcite clumped isotope paleotemperatures[END_REF]. Experimental studies on brachiopod shells by [START_REF] Henkes | Temperature limits for preservation of primary calcite clumped isotope paleotemperatures[END_REF] [START_REF] Mcalister | Geochemistry of some Ordovician and Devonian trilobite cuticles from North America[END_REF][START_REF] Brand | Carbon, oxygen and strontium isotopes in Paleozoic carbonate components: an evaluation of original seawater-chemistry proxies[END_REF][START_REF] Finnegan | The magnitude and duration of Late Ordovician-Early Silurian glaciation[END_REF], which is within the range of that recorded in the present study. Ordovician-Silurian studies comparing brachiopods and trilobites found them to have a similar  18 O to each other (McAlister and Brand, 1998;[START_REF] Wilmot | Original mineralogy of trilobite exoskeletons[END_REF]. While Finnegan et al. with trilobite cuticle data from the literature, and brachiopod data [START_REF] Shields | Sr, C, and O isotope geochemistry of Ordovician brachiopods: A major isotopic event around the Middle-Late Ordovician transition[END_REF][START_REF] Rasmussen | Onset of main Phanerozoic marine radiation sparked by emerging Mid Ordovician icehouse[END_REF]. Grey and black bars are isotope ranges, with the mean isotopic value indicated by the symbol in the centre.

The  18 O data from trilobites in the present study are within the range of contemporaneous brachiopods, or more positive (Figure 8). The exceptions are new data from Baltica which have higher  18 O values [START_REF] Rasmussen | Onset of main Phanerozoic marine radiation sparked by emerging Mid Ordovician icehouse[END_REF]. Given our results, this raises questions about the possibility of brachiopod calcite recrystallization from some earlier studies. Brachiopod specimens from the Lower to Middle Ordovician that were analyzed by conventional isotope techniques were assessed for preservation using trace element geochemistry and SEM and CL characterization [START_REF] Veizer | 87 Sr/ 86 Sr,  13 C and  18 O evolution of Phanerozoic seawater[END_REF][START_REF] Shields | Sr, C, and O isotope geochemistry of Ordovician brachiopods: A major isotopic event around the Middle-Late Ordovician transition[END_REF], but not by EBSD or SIMS. Significant cryptic diagenesis may have altered the isotope composition of these specimens: in general it is thought that only the secondary layer of the brachiopod shell is reliably unaltered and the preservation state of this layer can vary between taxonomic groups [START_REF] Garbelli | Micromorphology and differential preservation of Upper Permian brachiopod low-Mg calcite[END_REF]; In addition, SIMS analysis of brachiopods reveals that within certain groups, the secondary layer is only precipitated in isotope equilibrium with seawater towards the innermost part of the shell [START_REF] Cusack | Brachiopods recording seawater temperature -A matter of class or maturation[END_REF]. This fact implies that isotope studies where the entire secondary layer was analyzed (for example [START_REF] Shields | Sr, C, and O isotope geochemistry of Ordovician brachiopods: A major isotopic event around the Middle-Late Ordovician transition[END_REF] may reflect non-equilibrium fractionation. This is variable between brachiopod groups, for example in spiriferid brachiopods the prismatic tertiary layer is more resistant to diagenesis than the secondary layer [START_REF] Grossman | Stable isotopes in Late Pennsylvanian brachiopods from the United States: Implications for Carboniferous paleoceanography[END_REF]. Even in samples that have experienced low burial temperatures, recrystallisation can occur: in a study of Silurian brachiopods, [START_REF] Cummins | Carbonate clumped isotope constraints on Silurian ocean temperature and seawater  18 O[END_REF] argued that elevated clumped isotope temperatures (up to 56 o C) found in samples with CAI = 1 were likely resulting from diagenetic alteration due to recrystallization. This result cautions the use of apparently pristine biogenic specimens to interpret ancient paleotemperatures, without prior carbonate clumped isotope analysis. Due to the effects of sold-state reordering of clumped isotopes, which may not alter  13 C and  18 O values, analysis using the range of methods utilised in this study (SIMS, CL, SEM and EBSD) should be combined to jointly assess fossil preservation and isotope geochemistry.

Brachiopod data have been used to suggest that the  18 O of Ordovician seawater was lower than at the present-day (-3‰  18 OVSMOW) in order to account for a reasonable paleotemperature calculation for the most negative  18 O results [START_REF] Veizer | 87 Sr/ 86 Sr,  13 C and  18 O evolution of Phanerozoic seawater[END_REF][START_REF] Shields | Sr, C, and O isotope geochemistry of Ordovician brachiopods: A major isotopic event around the Middle-Late Ordovician transition[END_REF]. Modelling of long-term seawater geochemical composition estimates of Ordovician  18 OVSMOW at approximately -6‰ [START_REF] Jaffrés | The oxygen isotopic evolution of seawater: A critical review of a long-standing controversy and an improved geological water cycle model for the past 3.4 billion years[END_REF], and a revised calibration of the brachiopod data gives a value of -5‰  18 OVSMOW for the Early Ordovician at 475 Ma [START_REF] Veizer | Temperatures and oxygen isotopic composition of Phanerozoic oceans[END_REF]. However, [START_REF] Trotter | Did cooling oceans trigger Ordovician biodiversification? Evidence from conodont thermometry[END_REF] used a value of -1‰ to interpret the temperature of formation of Ordovician conodont apatite, giving similar ocean temperatures for the Early Ordovician to that interpreted from the brachiopod data. The Ordovician brachiopod calcite, trilobite calcite and conodont apatite [START_REF] Trotter | Did cooling oceans trigger Ordovician biodiversification? Evidence from conodont thermometry[END_REF][START_REF] Veizer | Temperatures and oxygen isotopic composition of Phanerozoic oceans[END_REF] isotope data show an increase in carbonate or phosphate  18 O values over the Ordovician, which indicates either cooling oceans, changing seawater chemistry, or increasing diagenetic imprints at higher temperature with time. Conodont apatite has been demonstrated to be well-preserved [START_REF] Wheeley | Oxygen isotope variability in conodonts: implications for reconstructing Paleozoic palaeoclimates and paleoceanography[END_REF] and is therefore useful as a paleotemperature proxy in the Paleozoic [START_REF] Joachimski | Conodont apatite δ18O signatures indicate climatic cooling as a trigger of the Late Devonian mass extinction[END_REF]. The results of [START_REF] Trotter | Did cooling oceans trigger Ordovician biodiversification? Evidence from conodont thermometry[END_REF] indicate that conodont apatite may be a much more reliable proxy than brachiopod calcite, as it yields more reasonable ocean paleotemperature calculations. How do the trilobite results influence this debate? The diagenetic alteration of trilobites demonstrated here reveals that the most negative  18 O values in the Early to Middle Ordovician (those less than  18 O -7‰) are probably the result of diagenesis rather than a distinctive seawater composition. Results of the present study therefore bring into question the fidelity of some Early-Middle Ordovician brachiopod records and call for a re-appraisal. The new positive  18 O brachiopod data by [START_REF] Rasmussen | Onset of main Phanerozoic marine radiation sparked by emerging Mid Ordovician icehouse[END_REF] for the Early Ordovician highlight the need for a review of all brachiopod data from this time. As no EBSD or SIMS work has been undertaken on Lower to Middle Ordovician trilobite or brachiopod specimens analyzed for their isotope composition prior to this study, the possibility remains that significant cryptic diagenesis may have altered these specimens. We therefore caution the interpretation of isotope results without rigorous preservation assessment and advise that SIMS analysis should be added to the tool-box of preservation studies.

Conclusions

 The holochroal eyes of species of the pelagic trilobite genera Carolinites and Opipeuterella from the  The  18 O and  13 C results show a different range for each formation. However, intra-eye isotope analyses using SIMS reveals a large range in  18 O in some specimens, of up to 2.7‰. There is also no systematic isotope difference between rock, cuticle, eyes and diagenetic cements, or between benthic and pelagic trilobite eyes or cuticle.

 A sub-set of trilobite cuticles from the three formations was analyzed with carbonate clumped isotope thermometry, generating a temperature range of 50-68 o C and so indicating low-temperature diagenetic alteration of the trilobite calcite.

 Despite rigorous preservation assessment protocols, the SIMS and 47 data both show that all trilobite eye and cuticle specimens analyzed here have, at least in part, been diagenetically altered, including those that were interpreted to be well-preserved using microstructural criteria. The presence of sparse micro-crystalline dolomite within some eye specimens hints at cryptic recrystallization processes, although the impact of the presence of dolomite on the isotope results remains unclear.

 Despite clear evidence for alteration,  18 O obtained in the present study are similar to those that have been previously used to infer Ordovician paleoceanographic conditions.

 We suggest that standard preservation assessment protocols may be insufficient for Paleozoic carbonates because of the difficulty in elucidating cryptic recrystallization that has taken place at low temperatures. Extensive microstructural analysis of Ordovician biogenic carbonates using EBSD in conjunction with SIMS and carbonate clumped isotope analysis is recommended before stable isotope signatures are used for paleoclimate or paleoenvironmental interpretation.

Figure 1 .

 1 Figure 1. Paleogeographical map of trilobite sample sites: 1) Spitsbergen, Norway (Valhallfonna

Figure 2 .

 2 Figure 2. Lens extraction methods for isotope analysis. A-B) Hand drilling of lenses using a dentist drill for

Figure 3 .

 3 Figure 3. Trilobite eye preservation. A: Opipeuterella sp., with intact lenses and libriginal cuticle (specimen

Figure 4 .

 4 Figure 4. Trilobite cuticle preservation. A-B: Polished thin section, BSE images. A: Carolinites sibiricus

Figure 5 .

 5 Figure 5. Dental drill and micro-mill  18 O and  13 C conventional isotope results for the three formations

Figure 6 .

 6 Figure 6.  18 O results from SIMS analysis plotted against conventional isotope results, for individual

  68 o C (average 63 o C) for the Valhallfonna Formation, 50-56 o C (average 52 o C) for the Emanuel Formation and 56-57 o C for the Horn Valley Siltstone Formation, respectively (Figure7). These temperature estimates were calculated from the 47-T universal calibration published by[START_REF] Bonifacie | Calibration of the dolomite clumped isotope thermometer from 25 to 350°C, and implications for a universal calibration for all (Ca, Mg, Fe)CO3 carbonates[END_REF] defined on all (Ca, Mg, Fe)CO3 carbonates. Only small differences in  18 O are observed between cuticle and limestone (up to 0.45 ‰: Appendix D) and there is no difference in temperatures (derived from 47 data) between those of the host rock and cuticle from the same sample. These results indicate that all fossil calcite materials analyzed have experienced (and imprinted a 47 signature characteristic of) higher temperatures than those at which they originally precipitated in Ordovician seawater.

Figure 7 .

 7 Figure 7. Clumped isotope temperature estimates from 47 for the three main formations studied. For further

(

  2011) found Upper Ordovician trilobites to have a similar  18 O composition to that of contemporaneous brachiopods and corals. Of all the published trilobite isotope studies, that by[START_REF] Finnegan | The magnitude and duration of Late Ordovician-Early Silurian glaciation[END_REF] is the only one where the preservation of the trilobite cuticle has been examined via trace element and microstructural studies, prior to isotope analysis.

Figure 8 .

 8 Figure 8. Trilobite eye and cuticle data from this study (conventional isotope analysis and SIMS), plotted

Floian(

  Lower Ordovician) Valhallfonna Formation, Spitsbergen, and the Floian-Dapingian (Lower-Middle Ordovician) Emanuel and Horn Valley Siltstone formations of Australia were examined to assess their preservation state.  The use of trilobite eyes for vision places strict constraints on the in vivo microstructure of their constituent calcite lenses, and as such is a unique means of guiding our preservation assessment.  The microstructure and chemical composition of the eyes was assessed using SEM, CL, EBSD and electron microprobe. The Valhallfonna and Emanuel formations contain the best-preserved specimens, based on these assessment protocols. Well-preserved eyes are composed of low-Mg calcite, are non-luminescent, have clear crystallographic boundaries and retain their original optical structures such as trabeculae.  Trilobite cuticle is composed of low-Mg calcite, but the wide range of crystal size within the cuticle, random crystal orientation and lack of original structures indicates that all specimens have been recrystallized.

Table 1 .

 1 Trilobite eye preservation 1

	Specimen	Trilobite Species	CL	SEM Observations	EBSD Observations	Preservation
				Valhallfonna Formation, Spitsbergen
	1.1A_sp1	Carolinites sibiricus	n/a	lenses fractured, silicified	n/a	poor
	1.1B_sp1	Carolinites sibiricus	L	pervasive porosity, recrystallized, lens boundary overgrowths	lenses undefined, major recrystallization	poor
	1.1B_sp2	Carolinites sibiricus	L	pervasive porosity, recrystallized, fractured lenses	no lens boundaries preserved, major recrystallization	poor
	1.2C_sp1	Carolinites genacinaca	L	pervasive porosity, recrystallized, zoned calcite, micro-dolomite	lenses defined, major recrystallization	poor
	1.2C_sp2	Carolinites genacinaca	L	pervasive porosity, recrystallized, zoned calcite, micro-dolomite	lenses undefined, major recrystallization	poor
	2.1B_sp1	Carolinites genacinaca	NL	lenses defined, no apparent recrystallization	lenses defined, minor recrystallization, trabeculae?	good
	2.1B_sp2	Carolinites genacinaca	SL-L	pervasive porosity, recrystallized, micro-dolomite crystals	lenses defined, recrystallized internally, trabeculae	poor
	2.6_sp1	Carolinites angustagena NL	recrystallized, large crystals of dolomite, euhedral pyrite	n/a	poor
	2.6_sp2	Carolinites angustagena NL-L	recrystallized, zoned calcite-dolomite	n/a	poor
	2.6_sp3	Carolinites angustagena L	recrystallized, zoned calcite-dolomite, euhedral pyrite	n/a	poor
	2.6_sp4	Carolinites angustagena NL-L	recrystallized, calcite-dolomite, euhedral pyrite	n/a	poor
				Emanuel Formation, Canning Basin, Australia
	T_178_sp1	Opipeuterella sp.	NL	lenses defined, no apparent recrystallization	lenses defined, minor recrystallization at base of lenses	good
	T_178_sp3	Opipeuterella sp.	L	lenses defined, crystal zoning in centre of lenses	lenses defined, minor recrystallization at lens boundaries	poor
	T_178_sp4	Opipeuterella sp.	L	lenses defined, crystal zoning in centre of lenses	lenses defined, minor recrystallization	poor
	T_178_sp10 Opipeuterella sp.	NL	lenses defined, pervasive porosity, micro-dolomite crystals	lenses defined, minor recrystallization, trabeculae	good
	T_205_sp3	Opipeuterella sp.	NL	lenses defined, pervasive porosity, micro-dolomite crystals	lenses defined, minor recrystallization at cornea, trabeculae	good
	T_205_sp5	Opipeuterella sp.	L	recrystallized, fractured lenses	n/a	poor
	A_159_sp1	Asaphid indet.	NL	lenses defined, pervasive porosity, micro-dolomite crystals	lenses defined, minor recrystallization, trabeculae	good
	A_159_sp4	Asaphid indet.	SL	lenses defined, no apparent recrystallization	n/a	good
	A_178_sp1	Asaphid indet.	NL	lenses poorly defined, internal porosity	lenses defined, trabeculae, cuticle recrystallized	good
	A_205_sp1	Asaphid indet.	NL	lenses defined, pervasive porosity, micro-dolomite crystals	lenses defined, minor recrystallization, trabeculae	good
	A_205_sp3	Asaphid indet.	NL	recrystallized, lens boundaries poorly defined	lenses undefined, recrystallized	poor
	A_205_sp5	Asaphid indet.	NL	lenses defined, pervasive porosity, micro-dolomite crystals	lenses defined, minor recrystallization, trabeculae	good
	A_205_sp10 Asaphid indet.	NL	lenses defined, pervasive porosity, micro-dolomite crystals	lenses defined, minor recrystallization	good
				Horn Valley Siltstone, Amadeus Basin, Australia
	HV_sp2	Asaphid indet.	L	lenses distinct, recrystallized	lenses defined, calcite twinning across all lenses	poor
	HV_sp4	Carolinites genacinaca	SL-L	lenses undefined, recrystallized, euhedral pyrite within lenses	lenses defined, major recrystallization	poor
	HV_sp5	Carolinites genacinaca	SL	lenses undefined, some recrystallization, pyrite within lenses	lenses defined, major recrystallization	poor
	HV_sp6	Asaphid indet.	L	lenses fractured, partly recrystallized, pyrite within lenses	lenses undefined, recrystallization, calcite twinning	poor
	HV_sp20	Asaphid indet.	L	lenses defined, partly recrystallized, pyrite within lenses	lenses defined, calcite twinning across all lenses	poor
	HV_sp27	Asaphid indet.	L	lenses undefined, recrystallized, internal porosity	no lens boundaries preserved, major recrystallization	poor
	HV_sp37	Carolinites genacinaca	NL	lenses undefined, recrystallization, euhedral pyrite within lenses	lenses defined, recrystallization, calcite twinning	poor

  have shown that solid-state reordering can start to reset the original C-O bonds (and thus 47 compositions) of calcite if it experienced temperatures above 100 o C for more than hundreds of millions of years. Stolper and Eiler (2015) argue that reordering can start to occur at temperatures of 75 o C, and becomes more significant over 120 o C. To explore this possibility further, the thermal history of each basin must be examined in terms of the duration spent at higher temperatures during burial. The detailed burial history of Ny Friesland, Spitsbergen has not yet been reconstructed. The Canning Basin experienced temperatures of 70-80 o C for approximately 200 Myr in the Mesozoic, but only underwent a brief higher temperature interval of ~100 o C (Arne et al., 1989) or <90 o C (Wallace et al., 2002) in the early Carboniferous, making solid state diffusion unlikely. The Stairway Sandstone, which overlies the Horn Valley Siltstone in the Amadeus Basin, experienced burial temperatures of over 100 o C in the Permian-Triassic (for approximately 80 million years), and Cretaceous (for approximately 110 million years)[START_REF] Gibson | Regional perspectives on new and reviewed thermal history data from central Australian basins[END_REF]. It is possible that solid-state diffusion may have affected the specimens from the Horn Valley Siltsone, although the time that the rocks experienced temperatures over 100 o C is fairly short. Overall, lowtemperature recrystallization, over solid-state diffusion, is the most likely mechanism responsible for altering the trilobite eyes and cuticle.Figure8shows trilobite  18 O data from the present study plotted against published geochemical data from trilobite cuticle and brachiopods through the Ordovician. There are no published data available for Lower Ordovician trilobites, but those from the Middle and Upper Ordovician range from  18 O of -3‰ to -7‰

Well-preserved lenses are composed of a single calcite crystal, which is non-luminescent. The outer 1-2 m of the lens surface consists of the cornea, which is micro-crystalline (Figure3B) and luminescent. EBSD maps show that calcite had a uniform crystallographic orientation within each lens, with the c-axis of the calcite parallel to the lens axis (Figure3D). Micro-scale pitting (Figure3C) and sub-crystal boundaries (Figure3D) are observed in some lenses. In some specimens the lenses contain trabeculae, which are microcrystallites oriented perpendicular to the lens surface and are thought to be an original structure[START_REF] Clarkson | The eyes of trilobites: The oldest preserved visual system[END_REF][START_REF] Schoenemann | Light guide lenses in trilobites?[END_REF]. The trabeculae can also be seen in SEM images of broken lens sections, and can be identified in EBSD maps by slight differences in the crystallographic orientations of the constituent sub-crystals (Figure3E). Apparently syntaxial calcite cements occur next to the interior lens surface of some specimens, but EBSD shows these cements have a different crystallographic orientation to the lens calcite.

Acknowledgements

Geoscience Australia is thanked for the loan of trilobite specimens from the Emanuel Formation and Horn Valley Siltstone. Thanks also go to Claire Torney for invaluable discussions on Carolinites eye structure and preservation; Seth Finnegan (UC -Berkeley) for pilot clumped isotope investigations; Diederik Depla and Henk Vrielinck (Ghent University) for use of micro-saw facilities; Philippe Recourt (CNRS, Univ. Lille 1) for SEM photography; Claire Rollion-Bard, Michel Champenois and Denis Mangin (CRPG, CNRS, Nancy) for help with SIMS analyses. The Research Foundation -Flanders (FWO) is acknowledged for funding of isotope analyses (research grant 1.5.079.08); CNRS-INSU is acknowledged for funding of SIMS analyses (action SYSTER). The University of Lille 1 and the Research Foundation -Flanders (FWO) are thanked for funding the postdoctoral fellowships of CEB and TRAV respectively, during which most of this research was carried out. We would like to thank two anonymous reviewers for their thorough and helpful reviews of this manuscript. This is a contribution to IGCP 591: 'The Early to Middle Paleozoic Revolution', and IGCP 653: 'The onset of the Great Ordovician Biodiversification Event'. The data to support the conclusions in this text can be found in Table 1 and in appendices A-D of the Supplementary Information.

Amberg, C., T. Collart, W. Salenbien, L. M. Egger, A. Munnecke, A. T. Nielsen, C. Monnet, Ø. Hammer, and T. R. A. Vandenbroucke (2016), The nature of Ordovician Limestone-mudstone alternations in the