Crypsis in the pelagic realm: evidence from exceptionally preserved fossil fish larvae from the Eocene Stolleklint Clay of Denmark

Marine deposits of earliest Eocene age in northern Jutland, Denmark, are renowned for yielding diverse teleost assemblages that have proved central for enhancing our understanding of the early evolution of many extant actinopterygian clades. In this study, we investigate diminutive larval fish fossils from the Stolleklint Clay, Ølst Formation, that retain multiple soft‐tissue features preserved as distinct dark‐coloured stains. To examine the elemental and molecular composition of these soft parts, we employed a combination of time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS), scanning electron microscopy (SEM) and energy‐dispersive x‐ray spectroscopy (EDS). Our analyses revealed that the preserved structures contain chemically identifiable eumelanin intimately associated with densely aggregated microbodies that are morphologically consistent with melanosome organelles. Thus, we conclude that the carbonaceous structures represent traces of originally melanized body parts, including the eyes and peritoneum. Comparable pigmentation patterns are seen in many extant teleost larvae that use semi‐transparency as a means of camouflage in pelagic environments, to suggest a similar visual appearance of the Stolleklint Clay fish fossils. This in turn suggests that adaptations for concealment and UV‐protection had already evolved by the beginning of the Eocene, notably during a time interval characterized by an extreme greenhouse climate, when the global fish fauna become increasingly modern in composition.

T H E Limfjord region of north-western Denmark is home to marine deposits of earliest Eocene age that have yielded diverse biotas of exceptionally preserved plant and animal body fossils (Bonde et al. 2008;Pedersen et al. 2011). The local stratigraphic succession comprises the Fur Formation, a world-renowned Konservat-Lagerstätte, and the underlying, less known Ølst Formation (Rasmussen et al. 2016, fig. 5). Sediments of these rock units accumulated in a restricted marine basin that covered much of what is now Denmark during and immediately after the most extreme greenhouse event of the Cenozoic: the Paleocene-Eocene Thermal Maximum (PETM) (Heilmann-Clausen et al. 1985;Heilmann-Clausen 1995;Willumsen 2004). The fossil content is noteworthy because it comprises a selection of early representatives of many modern animal groups, including birds and bony fishes (Pedersen et al. 2011). Osteichthyans are a particularly conspicuous element of the vertebrate fauna; a diverse bony fish assemblage, dominated by pelagic subtropical and tropical forms, flourished in the western Limfjord region during the earliest Eocene (Bonde 1987(Bonde , 1997Pedersen et al. 2011). These fossils are typically preserved as articulated skeletons with body outlines comprising integumentary remnants, such as phosphatized scales, along with organic residues of eyes (Bonde et al. 2008). However, other soft parts, such as internal tissues, are normally absent. Over the last few years, Eocene-age Limfjord fossils have been intensively investigated, and it has been shown that the preservation often extends to both sub-cellular and molecular levels (e.g. Lindgren et al. 2012Lindgren et al. , 2017Lindgren et al. , 2019. Although most studies have dealt with specimens from the Fur Formation (e.g. Vinther et al. 2008;Lindgren et al. 2012Lindgren et al. , 2017Lindgren et al. , 2019Gren et al. 2017) fossils of the underlying Ølst Formation also retain original biomolecular components (Lindgren et al. 2015a).
In the present study, we assess the ultrastructural and biochemical inventory of remnant soft tissues in larval-stage fish fossils from the Stolleklint Clay of the Ølst Formation. These specimens display structures composed of dark matter that were subjected to high-resolution imaging, elemental and molecular analyses. Our investigation shows that these remnant soft parts are consistent with originally eumelanin-containing internal and integumentary tissues of extant marine fish larvae.

GEOLOGICAL SETTING
The fossils analysed in this study were buried in finegrained detrital sediments belonging to the Stolleklint Clay, an informal rock unit that crops out locally in the Limfjord region of north-western Jutland, Denmark (Heilmann-Clausen 1995). The Stolleklint Clay is of earliest Ypresian (earliest Eocene) age and constitutes the lowermost part of the Ølst Formation (Heilmann-Clausen 1995), a sedimentary succession that occurs sporadically across the Danish Basin, from northern Jutland (Heilmann-Clausen et al. 1985) to Femern Baelt (Sheldon et al. 2012). In the Limfjord area, the Ølst Formation is represented only by the Stolleklint Clay (Heilmann-Clausen et al. 1985;Heilmann-Clausen 1995). The sediments are thought to have accumulated beneath deep, oxygen-depleted waters within an enclosed marine basin during the PETM (Heilmann-Clausen et al. 1985;Bonde 1997;Bonde et al. 2008) forming grey laminated clays rich in organic matter, with occasional interbedded ash layers that derive from volcanism associated with the opening of the North Atlantic (Heilmann-Clausen 1995;Larsen et al. 2003).

MATERIAL AND METHOD
We selected five fossil fish larvae (Fig. 1;FUM-N-12779, FUM-N-12781, FUM-N-13476, FUM-N-16240 and NHMD 625460) that were mechanically prepared without the addition of either preservatives or consolidants. Four specimens (FUM-N-12781, FUM-N-13476, FUM-N-16240 and NHMD 625460) were split along their long axis to produce opposing part and counterpart samples, whereas one individual (FUM-N-12779) is represented only by a part section. All fossils were examined and photographed using an Olympus SZX10 stereo microscope equipped with an Olympus SC30 digital camera. In addition, the dark matter in NHMD 625460 (Fig. 1A) was analysed by field-emission scanning electron microscopy (FEG-SEM), energydispersive x-ray spectroscopy (EDS), and time-of-flight secondary ion mass spectroscopy (ToF-SIMS).

FEG-SEM and EDS
FEG-SEM images were acquired in a Zeiss Supra 40VP FEG-SEM instrument using an electron energy of 1 keV and an Everhart-Thornley type secondary electron detector (SE2). Elemental analyses were performed with an energy-dispersive spectrometer (X-MaxN 80, 124 eV, 80 mm 2 ) from Oxford Instruments linked to a Tescan Mira3 High Resolution Schottky FEG-SEM at an electron energy of 15 keV. The fossil material was uncoated during analysis to preserve it for further studies and display. Sample charging was minimized by using low electron energy during image acquisition, and by performing the EDS analysis under low vacuum conditions.

ToF-SIMS
In ToF-SIMS, a focused beam of high-energy (primary) ions bombards the sample surface, causing the emission of secondary ions, which carry detailed information about molecular compounds and structures on the sample surface (Thiel & Sj€ ovall 2015). By scanning the primary ion beam over a selected analysis area on the sample surface and tracking the yield of secondary ions at different positions, spatially resolved molecular data is obtained and can be presented either as ion images (displaying the signal intensity of selected secondary ions over the analysis area) or as mass spectra from selected regions of interest (ROIs) within the analysis area. Identification of eumelanin by ToF-SIMS is based on the close mass spectral agreement between a sample and well-defined eumelanin standards (e.g. Lindgren et al. 2012Lindgren et al. , 2014. Although none of the peaks in eumelanin reference spectra by themselves are specific to this pigment, the detection of all major eumelanin-related ions at a relative intensity distribution similar to that of the reference spectra has been found to provide reliable evidence for the presence of eumelanin in fossil samples (Lindgren et al. 2012(Lindgren et al. , 2014(Lindgren et al. , 2015b(Lindgren et al. , 2019.
ToF-SIMS analyses were conducted in a TOFSIMS IV instrument (IONTOF GmbH, Germany) using 25 keV Bi þ 3 primary ions and low-energy electron flooding for charge compensation. Positive and negative ion data were acquired under static conditions (maximum primary ion dose density < 10 12 cm −2 ) with the instrument optimized for high mass resolution (m/Δm ≈ 3000-5000). Calibration of the mass scale was done using the C − , C À 2 , C À 3 and C À 4 ions in negative-ion mode, and CH þ 3 , C 2 H þ 5 , C 3 H þ 5 and C 4 H þ 3 ions in positive-ion mode. As a comparative dataset, ToF-SIMS analyses were undertaken on synthetic and natural variants of eumelanin, pheomelanin, several porphyrins and three microbial mats (see details in Lindgren et al. 2012Lindgren et al. , 2014Lindgren et al. , 2015a. We also examined a pyomelanin chemically derived via auto-oxidation of homogentisic acid (HGA; see Turick et al.

General description
The fish larvae from Stolleklint Clay measure between 8 and 14 mm in standard length (i.e. length from the tip of F I G . 1 . A-G, fossil fish larvae from the earliest Eocene Stolleklint Clay, compared with an extant sea bream larva (H). A, NHMD 625460 (Perciformes); arrowheads indicate areas analysed using ToF-SIMS, SEM and EDS (anterior and posterior part of the peritoneal structure, and the 'eye'). B, FUM-N-12781 (Perciformes). C, FUM-N-12779 (Scombridae); arrows indicate pigmentations along the dorsal and ventral body margins. D, higher magnification of the area demarcated in C, showing the sub-circular eye residue and, above it, dark pigmentations in the occipital region of the head (arrow), as well as an association of remnant melanophores (arrowhead) along the dorsal edge of the skull. E, FUM-N-16240 (Actinopterygii). F, FUM-N-13476 (Percoidei); arrow indicates pigmentations along the ventral body margin near the anal fin. G, higher magnification of the area outlined in F showing the elongate peritoneal pigmentation (arrowheads), together with numerous smaller stains in the pectoral fin area, as well as remnant melanophores located along the dorsal margin of the body (arrows). H, a 42-day-old sea bream (Sparus aurata) larva; note distinct eumelanic pigmentations that are virtually identical in size, shape, anatomical position and arrangement, to those seen in the fossil fish larvae; integumentary melanophores (arrows) occur along the body margins and on top of the head, whereas the peritoneal pigmentation (arrowhead) stretches diagonally across the abdomen. Scale bars represent: 2 mm (A-C, E, F); 400 µm (D); 500 µm (G). the snout to the distal end of the last vertebra) and are preserved as flattened, yet largely articulated skeletons that are accessible either in oblique lateral or ventrolateral view (Fig. 1). With the exception of FUM-N-16240 (which can only be assigned to Actinopterygii), all fossils belong to the clade Perciformes. In addition, FUM-N-12779 probably represents a scombrid-like fish based on the presence of finlets. Associated with the bones are distinct brownish to blackish stains. The largest and most prominent of these is located within the orbital cavity, where it forms a sub-circular patch that measures about 1.0-1.3 mm in diameter ( Fig. 1A-F). Furthermore, there is an elongate, gently curved structure that stretches from the anterior section of the vertebral column and then diagonally across the abdomen to the ventral margin of the body somewhat anterior to the anal fin ( Fig. 1A-C, E-G). While the dorsal border of this feature is reasonably well demarcated, its ventral side is more irregular with numerous fine projections and minute outliers (Fig. 1G). Scattered dark stains also occur on top of the head in both larger and smaller clusters (Fig. 1D, arrow and arrowhead), in close proximity to fins ( Fig. 1C, F, G) and along the dorsal and ventral margins of the body ( Fig. 1B, C, F, G). Notably, though, scales and other softtissue residues are absent.

Ultrastructural and elemental analyses
Initial macroscopic examination showed well-defined brownish-blackish matter that was distinct from the adjacent bones and sedimentary matrix in both texture and colour (Fig. 1D, G). Subsequent SEM imaging of the orbital stain in NHMD 625460 revealed a dense mass of morphologically heterogeneous microbodies (measuring about 0.7-2.5 µm in maximum dimension), ranging from sub-spherical to highly elongate ( Fig. 2A). Although the different morphotypes occurred close to one another, mixing was limited. In stark contrast to this morphological disparity, the abdominal structure contained only subspherical microbodies, measuring roughly 500 nm in width and 750 nm in length (Fig. 2B). SEM-EDS analysis showed that both the orbital and abdominal structure were enriched in carbon relative to the surrounding sediment, where silicon, oxygen and aluminium instead dominated (Figs S1, S2). In both anatomical features, some microbodies occurred partially embedded within a mineral precipitate ( Fig. 2B; Fig. S3).

ToF-SIMS
ToF-SIMS data obtained from the orbital and abdominal pigmentation provided evidence for remnant eumelanin in both structures. Negative-ion spectra acquired from the carbonaceous matter faithfully reproduced all major peaks in a reference spectrum of a natural (Sepia) eumelanin standard, both with regard to precise peak positions (mass-to-charge ratio (m/z) values at high mass resolution; Table S1; Heing ard et al. 2021) and their relative intensity distribution (Fig. 2G). Notably, distinct peaks at m/z 50, 66, 74, 90, 98 and 122 represent key nitrogenbearing ions derived from the eumelanin molecular structure (Lindgren et al. 2012). These peaks were equally prominent (compared to the other eumelanin-related peaks) in spectra obtained from the fossil structures as they were in the eumelanin reference spectrum, to provide strong evidence for the identification of this pigment in the fossil sample. Additional peaks in the spectra from the fossil originated from sediment-related ions, for example, SiO À 2 (m/z 60), SiO À 3 (m/z 76), FeO À 2 (m/z 88), FeO À 3 (m/z 104), AlSiO À 4 (m/z 119) and Si 2 O À 5 (m/z 136) (Table S2). Moreover, peaks at m/z 80, 81 and 97 correspond to sulfate-related ions (SO À 3 , HSO À 3 and HSO À 4 , respectively). These were localized primarily to the eumelanin-rich (pigmented) areas.
Positive-ion ToF-SIMS data from the pigmented structures revealed relatively weak but significant spectral features characteristic of proteinaceous materials (Samuel et al. 2001;Wagner & Castner 2001), including peaks corresponding to CH 4 N + (m/z 30), C 2 H 6 N + (m/z 44), C 4 H 8 N + (m/z 70), C 4 H 10 N + (m/z 72), C 5 H 12 N + (m/z 86), and C 8 H 10 N + (m/z 120) ( Fig. S4; Table S3). Furthermore, ion images showed co-localization of all of these Ncontaining organic fragment ions, suggesting a common origin (Fig. S5). Their spatial distribution was also distinct from those of other components, including polyaromatic and aliphatic compounds, as well as the surrounding sedimentary matrix (Fig. 3A-D). However, the relative signal intensity distributions of these Nbearing fragment ions in the spectra from the fossil were noticeably different from those of modern protein reference samples of collagen, keratin and haemoglobin, showing considerably higher relative intensities from the smaller protein fragment ions (CH 4 N + and C 2 H 6 N + ) and lower intensities from C 4 H 8 N + , for example (Fig. 3E). eumelanin-rich areas in the 'eye' and 'peritoneum' (D (blue outline) and F (green outline), respectively) and sediment (F (red outline)). G, negative-ion ToF-SIMS spectra of the ROIs indicated in (D) and (F), together with a reference spectrum of a natural eumelanin standard (from Sepia officinalis); stars denote sulfate ions (SO À 3 , HSO À 3 and HSO À 4 ). Scale bars represent 2 µm (A, B). Peak assignments and observed m/z values are provided in Table S1 (eumelanin) and Table S2 (sediment and sulfate) and in Heing ard et al. functional parts of the RPE, as well as other melanosomebearing tissues. Conversely, the microbodies of the abdominal pigmentation are exclusively sub-spherical in shape, consistent with internal melanosomes observed in extant fish (Goda & Fujii 1996, fig. 5), and without a tendency to secondary layering. This obvious difference and close agreement to melanosomes in modern tissues, suggest that the pigmentations derive from anatomically distinct structures.

Ultrastructural and chemical preservation
In addition to eumelanin, our ToF-SIMS analyses detected a number of positive ions characteristic of proteins, which could indicate the presence of endogenous proteinaceous moieties in the otherwise largely melanized structures. However, the relative intensities of the nitrogen-containing peaks differed from those of our modern reference samples (Fig. 3E), which might be explained by partial degradation of the proteinaceous matter in the fossil. Regardless, without further in-depth analyses, it is not possible to confidently identify the source(s) of these ions.
Sulfate ions were identified in both melanized residues, as well as in the adjacent sedimentary matrix (albeit at lower signal intensities; Fig. 2G). Sulfur is a natural component of pheomelanin; however, none of the peaks that represent S-bearing ions in ToF-SIMS spectra from pheomelanins (see Lindgren et al. 2014) were prominent in the fossil samples. In addition, there was no indication of melanin leakage into the surrounding sediments. Instead, it is more likely that the presence of sulfate in the pigmented structures indicates secondary incorporation of sulfur into the eumelanin molecular structure (see McNamara et al. 2016).

Rationale for assignment of ontogenetic stage
All stages of the ontogenetic series of teleosts have been documented in the fossil record (Cloutier 2010), however, the early developmental history is usually considerably less known than later stages, a condition that probably reflects the rather poor fossilization potential of fish larvae. Furthermore, although fossils are known (Thomson et al. 2003;Micklich et al. 2009;Mizumoto et al. 2019;Carnevale & Bannikov 2020), detailed records of specific larval characters, such as pigmentation patterns, are rare. In fact, we are only aware of a single publication describing fossil larval pigmentations (Marram a & Carnevale 2015, fig. 9A), but without the level of details shown herein.
In addition to their small size, the Stolleklint Clay fish fossils display several features consistent with relatively early ontogenetic stages. An upward caudal inclination of the notochord can be seen in all five fish fossils, indicating that the notochord flexion was ongoing and perhaps even completed at the time of death. This 45°dorsal bending is completed during the final stage of the larval phase, termed the post-flexion stage (Kendall et al. 1984). Additionally, both the fin rays and caudal fin appear to be well developed; these body parts normally grow rapidly during the flexion stage to become fully formed during the juvenile phase (Kendall et al. 1984). Furthermore, the . D, overlay image of A-C, displaying sediment in red, N-containing organics in green, and PAH in blue; note distinct spatial distribution of N-containing organic fragment ions relative to ions derived from the sediment and PAHs, as well as from eumelanin (Fig. 2E). E, signal intensity distribution of characteristic 'protein' fragment ions in ToF-SIMS spectra from the pigmented fossil structures ('peritoneum' (posterior and anterior) and 'eye') and three modern protein reference samples (collagen type I, alpha-keratin and haemoglobin). Peak assignments and observed m/z values of the N-containing fragment ions are provided in Table S3. fossils seemingly lack scales and a complete body pigmentation (Fig. 1). This is typical for marine fish larvae (Kendall et al. 1984;Urho 2002), and unlike the condition of adult teleosts from the same formation (Fig. 4). Instead, the fossil fish larvae display pigmentation patterns that are entirely consistent with melanic depositions in integumentary and internal tissues of extant teleost larvae ( Fig. 1H (Fig. 1H). These same features are preserved in great detail in the Stolleklint Clay fish larvae, potentially down to individual cells (Fig. 1D), strongly suggesting a larval origin. For instance, the elongate abdominal structure (indicated by arrowheads in Fig. 1G) corresponds perfectly with the distribution of peritoneal melanophores associated with the gas bladder and/or gut in modern fish larvae ( Fig. 1  H), an observation that is independently corroborated by the melanosome and eumelanin pigment residues detected in our analyses. In modern fishes, these internal melanophores are often visible through the otherwise semi-to fully transparent larvae before being lost or hidden by muscles and integumentary pigments during later ontogenetic stages ( Although many of these biochemical and ultrastructural alterations have low preservation potential, transparency in the fossil larvae described herein can nonetheless be inferred from their overall lack of body pigmentation and squamation, in combination with the highly specific pigmentation patterns that are identical to those observed in extant transparent pelagic fish larvae. Given that a full body pigmentation is preserved in adult teleosts from the Stolleklint Clay (Fig. 4), these features strongly suggest that the fossil fish larvae likewise were at least semitransparent in life, presumably as a means of crypsis in open-water settings. Few small pelagic animals are fully transparent but instead exhibit rudimentary colour patterns despite the apparent risk of detection by visually hunting predators, to suggest important functions of these pigmentations (for instance, melanin in vertebrate eyes is essential for visual capacity; Strauss 2005). Nonetheless, the functional aspects of specific pigmentation patterns in otherwise transparent fish larvae (as well as other zooplankton) are generally poorly understood (Moser 1981;Kendall et al. 1984). The peritoneal melanophores associated with the gut have been hypothesized to help conceal the larvae from predators by masking light refracted from gut content and/or screening bioluminescent prey in the alimentary canal (Herring 1967;Moser 1981;Eastman & DeVries 1997). Similarly, the shield of melanophores above the gas bladder could potentially reduce light refraction in this area (Moser 1981). It has also been suggested that specific pigmentation patterns might play a role in intraspecific recognition among fish larvae (Moser 1981). Furthermore, the transparent nature of many larvae means that vital parts, such as the developing internal organs, may be exposed to harmful ultraviolet solar radiation (UVR). UVR has been shown to be detrimental to fish, especially during their early life stages (Hunter et al. 1979;Kouwenberg et al. 1999;Browman et al. 2000;Lesser et al. 2001;Steeger et al. 2001) and it has been suggested that the primary function of pigments in young fish is protection against harmful UVR rather than camouflage (Mueller & Neuhauss 2014). Moreover, pigmentations in crustacean zooplankton, inhabiting similar niches to many ichthyoplankton, have been shown to reduce UVR-induced damage (Morgan & Christy 1996;Bashevkin et al. 2019). Thus, the taxonomically widespread development of melanophores arranged in certain patterns in fish larvae, shown here to be traceable at least back to the early Eocene, might also indicate an adaptive photo-protective function for these eumelanin deposits (Breder 1962;Moser 1981). Indeed, a recent study (Kapp et al. 2018) found that all teleosts develop melanophores that cover the haematopoietic niche housing stem cells during their larval phase. The stem cells received measurable DNA damage when the protective pigmentation was experimentally removed (Kapp et al. 2018).

CONCLUSION
The fossil fish larvae described in this study exhibit residues of both internal and integumentary tissues in the form of dark organic stains, shown here to be dominated by traces of the pigment eumelanin. These remnant soft parts correspond in size, composition and anatomical position to eyes, peritoneum and integumental melanophores, respectively, forming pigmentation patterns that are virtually identical to those of extant pelagic teleost larvae.
Modern fish larvae are known to use semi-transparency as a means of crypsis in featureless open water environments. Consequently, the close resemblance between our fossils and extant teleosts indicates that the Eocene fish larvae used a similar strategy to camouflage themselves. Thus, the preserved pigmentation patterns are likely to represent means for concealment and UV protection, but also requirements for visual capacity.
Acknowledgements. The photograph of the modern sea bream larva shown in Figure 1H is credited to Bernd Ueberschär. Niels Bonde provided taxonomic information on FUM- N-12779, FUM-N-12781, FUM-N-13476, FUM-N-16240, NHMD 625460 and FUM-N-14772. We thank Annie Marie Kaargaard for the discovery of NHMD 625460. Financial support for this project was provided by a Grant for Distinguished Young Researchers (Award No 642-2014-3773;Swedish Research Council) to Johan Lindgren, as well as a project grant (Award No 2019-03731; Swedish Research Council) to Peter Sj€ ovall. Rana N. S. Sodhi and an anonymous referee commented on an earlier draft of this manuscript.

DATA ARCHIVING STATEMENT
Data for this study are available in the Dryad Digital Repository: https://doi.org/10.5061/dryad.p5hqbzkp6