Bromalites from the Upper Triassic Polzberg section (Austria); insights into trophic interactions and food chains of the Polzberg palaeobiota

A rich assemblage of various types of bromalites from the lower Carnian “Konservat-Lagerstätte” from the Reingraben Shales in Polzberg (Northern Calcareous Alps, Lower Austria) is described for the first time in detail. They comprise large regurgitalites consisting of numerous entire shells of ammonoid Austrotrachyceras or their fragments and rare teuthid arm hooks, and buccal cartilage of Phragmoteuthis. Small coprolites composed mainly of fish remains were also found. The size, shape and co-occurrence with vertebrate skeletal remains imply that regurgitalites were likely produced by large durophagous fish (most likely by cartilaginous fish Acrodus). Coprolites, in turn, were likely produced by medium-sized piscivorous actinopterygians. Our findings are consistent with other lines of evidence suggesting that durophagous predation has been intense during the Triassic and that the so-called Mesozoic marine revolution has already started in the early Mesozoic.


Geologic setting
The Polzberg outcrop (710 m above sea level) is located on the western slope of Mount Schindelberg (1066 m), 4 km northeast of Lunz am See around Polzberg (postal code 3293) in the Lunz Nappe of Lower Austria. Hidden in a small mountain creek, the locality is accessible from the south over the Zellerrain street 71 via Mariazell or from the west over Lunz am See the Weißenbach street 25 and then street 71 (1:50,000, sheet 58 Baden 1996 21 ; Fig. 1). The exact position of the fossiliferous locality (Reingraben Shales) was determined by GPS (global positioning system): N 47°53′4.98″ and E 15°4′28.15″.
Excavation campaigns were organized by the Austrian Geological Survey in 1885 and 1909. During these years, two fossil mines were driven into the basal part of the Reingraben Shales yielding better-preserved fossils, not harmed by weathering of soft marly deposits on the surface. The historical abandoned and collapsed mines were located approximately at N 47°53′23.31″ and E 15°4′45.80″.

Fossil content in the Reingraben Shales
In the late 19th and early twentieth centuries, hundreds of fossils have been collected from Polzberg locality 29 . The fauna was dominated by ammonites of the ceratitid species Austrotrachyceras austriacum with original aragonite shell, hundreds of fish remains, and arthropods. The ammonites derive from several distinct ammonite layers. Rare ammonites of the genera Carnites, Sageceras and Simonyceras also occur. Over 100 fragments of coleoid proostraca and phragmocones of Phragmoteuthis bisinuata 30 , showing arm hooks and buccal cartilage have been recovered from the Polzberg deposits. The Reingraben Shales contain common remains of fossil fishes 11,17,[31][32][33] . The lungfish Ceratodus sturi 16 was also found in deposits from Polzberg 26 . A single conodont cluster with abundant Mosherella, assigned to Triassic jawless fishes, was reported. Noteworthy, Forchielli & Pervesler 15 re-described the arthropods consisting of dozens of thylacocephalan Austriocaris 11 and noted the presence of other arthropods (including crustaceans with decapods and isopods) from the Polzberg locality.

Palaeoenvironment of the Reingraben Shales
The Reingraben Shales 28 are interpreted as deposits of a relatively deep marine environment within intra-platform basin as inferred from the dominance of a nektonic fauna 15,34,35 . There is some confusion in the designation to either Lunz or Polzberg localities in official collections of the Natural History Museum Vienn, the Geological Survey Vienna or the University of Vienna. As stated by Forchielli and Pervesler 15 the fauna of the Polzberg Lagerstätte were often referred to the Lunz Lagerstätte in the scientific past. Both localities are different in biostratigraphy, lithology, and depositional environments. Data from the outcrops in the Polzberg area show a nektonic dominated fauna with abundant fishes and cephalopods. The well preserved soft bodied fauna, the abundance of organic material in the sediment, the presence of common framboidal pyrite (e.g., Fig. 3D) crystals 15 , the absence of sessile organisms, and lack of bioturbation suggest dysoxic to anoxic bottom conditions during deposition of the Reingraben Shales. As noted by Griffith 17 the Polzberg basin was mainly normal marine with ephemeral and limited freshwater input.
Type B Elongated, cylindrical, small (< 15 mm); they contain loosely dispersed remains of the body macrofossils/microfossils. NHMW 2020/0033/0007 AS193: Small elongated bromalite, up to 5 mm thick, 8 mm in breadth, and 13 mm in length, with rare fish scales (Fig. 4G). XRD analysis shows that the host rock surrounding bromalite contains mainly clay minerals, represented by illite-smectite mixed layer structure and chlorite, however small amount of mica and kaolinite are also detected (Supplementary Fig. S2; Supplementary Data S3). The XRD spectrum shows numerous strong reflections indicating the presence of quartz, sodium feldspars (albite) and carbonate minerals-calcite, with an admixture of dolomite. Calcite reflections are doubled, which indicates the occurrence of two generations of calcium carbonate, differing in structure, which in turn may be related to the occurrence of a more Mg-rich variety. For example, the main calcite d 10.4 peak consists of two overlapped reflections-3.027 and 3.010 Å. The first peak, 3.027 Å, represents a purer calcite variety; the second peak indicates a Mg-rich generation. In the analyzed bromalite sample, some minerals found in the parent rock sample are also present (quartz, chlorite and illite), but they represent only a small admixture. The main minerals detected in the bromalite matrix are apatite and calcite. The obtained apatite reflections match with a high score the carbonate apatite reference patterns. For the Rietveld refinement the 01-073-9696 (ICDD, PDF4 +) carbonate-fluorapatite structure was used. The calculated structural parameters (lattice parameters) of apatite are a = 9.3202(5) Å and c = 6.9012 (5) Scientific Reports | (2020) 10:20545 | https://doi.org/10.1038/s41598-020-77017-x www.nature.com/scientificreports/  NHMW 2020/0033/0008 AS244: Small elongated bromalite, up to 3 mm thick, 6 mm in breadth, and 11 mm in length, with relatively abundant fish scales (Fig. 4H). It is characterized by dark grey matrix as visible in CT scans with radiologically dense filamentous shapes located randomly throughout the bromalite matrix in some cases reaching to the peripheries. The further observations of thin-sections under optical microscope revealed these objects are crushed and etched small vertebrate skeletal remains, especially actinopterygian fish scales (Fig. 4H). Interestingly, a small fish mandible was observed in the section (Fig. 3A). An individual fragment of aptychus/cephalic cartilage was also observed (Fig. 3B).

Discussion
Types of bromalites. Possible explanation for the origins of distinct accumulations of shells and/or shell hash (morphotypes A and B) reported herein is that they represent abiotic structures produced by post-mortem transport by bottom currents or waves or that they are ichnofossils (e.g., infills of decapod burrows). However, the occurrence of densely packed ammonoid shells and their fragments in distinct oval spots, the rarity of these shells in the surrounding host rock, the lack of size sorting (shell size from below 1 mm up to 20 mm) and preferential orientation, low degree of shell roundness and abrasion, lack of ripple marks, and the fact that they do not penetrate deep into the sediment, all suggest that these accumulations were produced by predator activity (i.e., they represent bromalites).
Distinguishing between different types of bromalites is not always easy to draw. However, a number of diagnostic criteria have been recently proposed [36][37][38][39][40] . More specifically, regurgitalites are thin, and commonly composed of randomly grouped and intermingled angular skeletal fragments of different size, revealing low degree of roundness and distortion of individual crystal fibers at the shell edges. Furthermore, they usually lack a phosphatic matrix and may contain debris, which are not significantly affected by gastric etching (including soft body tissues). By contrast, coprolites typically are massive, usually thick, possess a more or less regular shape, and contain a phosphatic matrix and fossil inclusions revealing signs of digestion. The gastric residues (fossilized stomach contents, i.e., consumulites) may reveal similar features to regurgitalites, however they are usually thicker and are associated with the body fossil of the producer.
The evidence suggests that Polzberg locality preserves two types of bromalites with regurgitalites (morphotype A) being the most common. An interesting feature is the presence of uncrushed ammonoid shells within some regurgitalites (NHMW 2020/0033/0003 AS93 (Fig. 4C); NHMW 2020/0033/0002 1910 A (Fig. 4B). In other accumulations, crushed ammonite fragments predominate. The presence of uncrushed shells may result either from the fact that the predator swallowed the entire ammonoids or from the fact that while swallowing it crushed only the body chamber containing the soft tissues. In this case, phragmocone remains ingested undamaged. Accumulations of ammonoid shells consisting of phragmocones, interpreted as a result of predation, have been described in the literature 42 . However, among ammonoids from studied bromalites from Polzberg inner moulds are unknown, thus it is uncertain if these shells represent entire specimens or if they are phragmocone parts of ammonoids.
Potential producers of bromalites. Based on the size of regurgitalites (up to 100 mm) and the fact that in some of them entire (uncrushed) ammonite shells (diameter up to 20 mm) can be also found, it seems that they were produced by large durophagous predators. Cephalopods and arthropods noted from Polzberg section appear to be too small to produce these bromalites. Furthermore, these invertebrates have a rather alkaline gastric pH, and thus they produce regurgitates commonly containing specific hardparts (e.g., aptychi of ammonites) without etching-related features 43 .
A rich inventory of Triassic ichthyofauna and lack of the reptile remains in the Polzberg section allow to search probable bromalite producers among predatory fishes.
There is evidence that Palaeozoic and Mesozoic shelled cephalopods have been preyed upon by sharks and actinopterygian fishes 41,[43][44][45] . Noteworthy, Recent sharks and actinopterygian fishes are known to attack on Nautilus 46 . Interestingly, predation experiments on living durophagous fish (Diodon) revealed that the critical size of the prey, i.e., the size above which this fish is incapable of crushing a given prey species is about 12% of the fish length 38,47 . We thus hypothesize that studied regurgitalites at hand (dispersed over 7 cm and containing  margins (A,B). Both photographs taken from NHMW 2020/0033/0001 Polz. Scale bar equals 1 mm.
Griffith 17 stated that the Upper Triassic ichtiofauna of Polzberg region is characterized by the large content of flying fish, which, according to this author, suggests a strong predation pressure in this marine ecosystem. Furthermore, 55% of the genera of marine fish known from Polzberg were predatory 17 . On the other hand, the specimens of predatory fish from the Reingraben Shales of Polzberg, in which the body size could have been reconstructed, usually reveal rather small body sizes (a few to 11 cm in length) 17 . The largest specimens of predatory fish described by Griffith 17 belonged to Saurichthys (up to about 40 cm in length), palaeoniscids, probably belonging to the family Acrolepididae (20-25 cm in length), and Gigantopterus telleri (18.6 cm in length). Apart from these taxa, remains of large sarcopterygian fishes are known, namely dipnoan (lungfish) Ceratodus sturi, with the length up to about 1.5 m 16 and coelacanth Coelacanthus lunzensis 16,48 ). However, Mesozoic dipnoans were restricted to freshwater environments and their remains found in marine deposits are commonly interpreted as a result of post mortem transport from freshwater ecosystems and/or as a result of redeposition from older, non-marine sediments 49  In particular, a typical durophagous dentition (crushing or grinding teeth) with blunt and broad teeth is observed in Acrodus 58,59 . The actinopterygian assemblage of Polzberg does not contain typial durophagous taxa 17,55 although durophagy sensu lato (the ability to consumption of hard prey 56,57 ) is possible with other dental types, especially when dealing with thin-shelled preys, such as small ammonites occuring in regurgitalites. A Late Triassic fish Legnonotus 55 with peg-like teeth is also considered a durophagous. Likewise, hybodont or ctenacant sharks with relatively narrow and long cusp (tearing-type of dentition sensu Cappetta 58 ) might have attacked ammonites 42,45 . Small, thin-shelled scaphites were noted in the stomach of Cretaceous plesiosauroids and pliosauroids, which displayed conical, quite long teeth with an acute, but rounded apex 60 . Furthermore, thinshelled ammonites might have been preyed upon by marine reptiles with crunching teeth (robust with a blunt apices conical teeth) 60 . Triassic species of Saurichthys are characterized by monognathic heterodonty-the teeth in one jaw have different size and shape. They possessed two types of conical teeth: robust with approximately circular in section small teeth, and larger, slender teeth with acute apex 61,62 . Triassic coelacanths commonly possessed robust, conically pointed teeth, however they also had small rounded teeth on the parasphenoid 63 . On the other hand, shells of ammonoids which have been attacked by predators having conical teeth commonly reveal shell damages in the form of indentations or holes of various size and shape, occuring both in the body chamber and in the phragmocone 42,45 . Similar damages were not recorded in ammonoid shells from Polzberg. Given the above, we argue that a durophagous shark Acrodus was likely a producer of regurgitalites studied, although other durophagous predators (until now not found in the Upper Triassic deposit of Polzberg), which were present in the latest Ladinian and early Carnian in the other areas of the east part of Alpine domain, including durophagous (placodonts) and semi-durophagous (thallatosaurs) reptiles (recorded in a few outcrops in NE Italy 64,65 ; Slovenia 66 ; Hungary (Bakony) 64 , some hybodontid sharks (e.g., Palaeobates, Asteracanthus; see 64,67 and some actinopterygian fishes (e.g., Colobodus 64,67 ), cannot be fully excluded.
Regarding coprolites, given their longitudinal shape, small size and occurrence of the fish scales, we hypothesize that they were likely produced by medium-sized piscivorous actinopterygians (such as, common in the fish assemblage from Polzberg, Elpistoichthys and Thoracopterus or rarer Gigantopterus, acrolepids and Saurichthys). These coprolites do not reveal a spiral morphology, which is characteristic for coprolites produced by chondrichthyans, dipnoans and some actinopterygian fishes (e.g. 3,68 ).

Conclusions
Discovery of bromalites at Polzberg locality not only proves for the first time the presence of large predators in the Reifling basin and provides insights into trophic interactions, and food chains of this Late Triassic ecosystem, but also constitutes another important evidence confirming previous hypotheses that the so-called Mesozoic marine revolution (a time of increased escalatory adaptions to shell-crushing predation 69 ) has already started in the early Mesozoic 70 . Not so long ago, it has been argued that marine durophagous predation was not intense during the Triassic 5 . For instance, McRoberts 71 pointed out that durophagous predators displayed low abundances and limited distribution during the Triassic. More recent reports, however, highlighted that innovations for durophagy appeared in many Triassic invertebrates (e.g., sea urchins) and vertebrates (including Chondrichthyes (hybodontids), some ichthyopterygian 72 and sauropterygian vertebrates (such as placodonts, pachypleurosaurs, some pistosaurs and nothosaurs) 70,[73][74][75][76][77] , which might have affected the evolution of shallow-marine benthic comunities. Nonetheless, the more direct evidence of durophagous predation in Triassic marine communities still remains limited 5 www.nature.com/scientificreports/ and together with other lines of indicators (i.e., predatory or defensive behaviors of predators and prey inferred from functional morphology, taphonomy and trace fossils) 5,8,70,73,78,79 confirm recent hypotheses about the early timing of Mesozoic marine revolution.

Material and methods
Among the several dozens of bromalite specimens obtained from the locality, 8 representative bromalites were selected for detailed investigation. These bromalites came from the ravin Schindelberggraben (Fig. 2)  Mineralogical studies. One specimen was examined using X-ray diffraction and Raman spectroscopy to determine its mineralogical composition. The powdered bulk X-ray diffraction analysis of coprolite matrix was performed using PANalytical X'Pert PROMPD PW 3040/60 diffractometer at the Laboratory of X-ray Diffraction, Faculty of Natural Sciences, University of Silesia in Katowice, Sosnowiec. Raman spectroscopy was used for in-depth characterization of carbonate phase of skeletal remnants observed in coprolite matrix. The analysis was performed using WITec alpha300 confocal Raman microscope equipped with a laser (λ = 532 nm), coupled with a CCD camera and with an Olympus MPLAN objective. XRD analyses were performed on powdered samples using a PANalytical X'Pert Pro MPD (multipurpose diffractometer) powered by a Philips PW3040/60 X-ray generator and fitted with a 1D silicon strip detector (X'Celerator). The measurements were performed using Co Kα-radiation with a wavelength of 0.1789010 nm, an acceleration voltage of 40 kV, a current of 40 mA, and with 0.02° 2θ step sizes between the angles of 5° and 90° 20 and a 200 s measurement time per step. The data obtained were processed using HighScore + software and the ICSD database and PDF4 + ICDD database. All XRD analyses were performed at the Faculty of Earth Sciences, University of Silesia in Katowice, Sosnowiec. The diffractometer was manufactured in the Almelo Malvern Panalytical B.V. factory (Holland). www.nature.com/scientificreports/ Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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