Comparative actualistic study hints at origins of alleged Miocene coprolites of Poland

Geological setting

The Turów lignite mine is located in the south-western part of the Lower Silesia Voivodeship (SW Poland) and covers the former village of Turów. It is located in the central part of the mesoregion Żytawa-Zgorzelec Depression located between the state borders of Germany and the Czech Republic (Fig. 1A). Turów lignite deposits are part of the Upper Lusatian Brown Coal Basin. This basin comprises a few tectonic sinkholes (Piątkowska, Kasiński & Graniczny, 2000) that developed in Paleogene at the junction of two regional zones of strong tectonic activity: Ore Mts. Graben (Ohrza rift) and the Lusatian-Elbe Tectono-Volcanic Zone (Jęczmyk & Sztromwasser, 1998). The most southern of these is the Zittau basin (Figs. 1A, 1B), which was filled mainly by limno-fluvial or limnic clays, silts, sands and thick layers of lignites exploited in the Turów mine (Kasiński, 2000; Kasiński et al., 2015). Furthermore, there are numerous vulcanite rocks of late Eocene, Oligocene and early Miocene age (Kasiński et al., 2015). The basal part of sedimentary section of the Zittau basin is not older than the early Oligocene, however most sediments were formed in the Miocene (Kasiński et al., 2015). At the base of the Zittau Basin, Precambrian and Palaeozoic metamorphic and igneous rocks are present (Marcinkowski, 1985).

The lithological profile of the Turów mine is ca. 250 m thick and consists of seven lithostratigraphic units of sedimentary rocks. Apart from the two youngest units (Gozdnica Fm. and glacial tills), all of them are mainly composed of clays and/or muds with intercalations of sands or sands with gravels. Additionally, there are coal seams, especially in Opolno and Biedrzychowice formations, which are the deposits mined at the Turów mine. The oldest Cenozoic sediments of the profile are Oligocene sediments (Egger age), forming the lower and middle part of the Turoszów Fm. (Kasiński et al., 2015). The youngest in the profile are sands and gravels of the Gozdnica Fm. and Pleistocene deposits, mainly represented by tills (Kasiński et al., 2015).

The Turoszów Fm. was formed in fluvial and limnofluvial conditions, while the Opolno and Biedrzychowice Fms have been formed in limnotelmatic environments (sediments formed in the terrestrial environment most often as a result of sea regression), and Porajów Fm. represents limnofluvial environment. Sediments of the upper part of Miocene profile (Rybarzowice and Gozdnica Fms) have alluvial origin (Kasiński, Badura & Przybylski, 2003). Biedrzychowice Fm., within which excrement-shaped ferruginous masses and a turtle remain have been documented, was formed in vast swamps and rushmarshes. However, palaeobotanical data indicate that during the deposition of the upper part of this formation terrestrial forests (mezophilus mixed forests and upland mixed forests) also grew locally (Kasiński et al., 2010). Especially in the upper part of this formation, there are numerous palaeosols levels with plant roots and trunks preserved in situ (Kasiński & Wiśniewski, 2003). Intercalations of sands and sands with gravels, representing a braided river environment, are also present (Kasiński et al., 2010). Marsh forests mainly composed of Cupressaceae and Taxaceae (Sadowska, 1995; Kasiński & Wiśniewski, 2003). Based on palaeobotanical analysis of the coal seam it was concluded that there was a humid warm temperate climate similar to that of south-eastern China today (Durska, 2008; Kasiński et al., 2010). The excrement-shaped ferruginous masses and the turtle shell fragment documented in this article (Figs. 2A, 2B, 2E, 2G, 2N) were collected in an inactive part of the excavation, at the top layer of clay complex, which is covered by quartz sand layer of the highest part of Biedrzychowice Fm. in the uppermost part of early Miocene (Burdigalian; see Fig. 1C). Two distinct morphotypes randomly distributed within clay and mudstone on the flat surface of the excavation were noted.

Fossil content in the Turów area and adjacent areas

No animal fossil remains have been documented so far from the Oligocene–Miocene of the Zittau Basin with exception of burrows in sediment produced by indeterminate sediment eating fauna (Kasiński et al., 2015). Apart from the excrement-shaped ferruginous masses, a fragment of a turtle shell was found in the Biedrzychowice Fm. This shell fragment (see Fig. 2N) can only be identified as an indeterminate testudinoid. This turtle lineage is otherwise abundant in Oligocene and early Miocene localities in Germany and Czech Republic (von Reinach, 1900; Młynarski & Roček, 1985), but had not so far been documented from coeval localities in Poland. In older, Eocene and Oligocene localities in the neighbouring north-western Czech Republic and south-eastern Germany (Saxony and southeastern Saxony-Anhalt), rich assemblages of terrestrial-aquatic tetrapod fauna have been documented, comprising frogs, salamanders, choristoderans, crocodiles (also crocodile coprolites, see Kasiński et al. (2015), and literature cited therein), turtles, lizards, and snakes (Table 1).

On the other hand, in the lower Miocene clays and sands of North Bohemian Brown Coal Basin, a very rich fauna assemblage was reported (Klembara, 1979, 1981; Roček, 1984; Szyndlar, 1991a, 1991b; Szyndlar & Schleich, 1993; Szyndlar & Rage, 2003; Čerňanský, 2010a, 2010b; Dvořák et al., 2010). The latter mentioned and illustrated numerous vertebrates represented by osteichthyan fish, amphibians, reptiles, birds, and mammals. The reptile taxa are shown in the table below (Table 2).

Optical microscopy and thin-sectioning

Optical observation of several thin sections from four ferruginous masses representing two morphotypes, and two representative concrections were carried out using Leica SZ-630T dissecting microscope and Nikon Eclipse E100 light microscopy, while the microphotographs were collected using Olympus BX51 polarizing microscope equipped with an Olympus SC30 camera and a halogen light source, installed Faculty of Natural Sciences at the University of Silesia in Katowice (Sosnowiec, Poland) (Figs. 3, 4).

Thin sections were made in the Grindery at the Faculty of Natural Sciences, University of Silesia in Katowice, Sosnowiec, Poland. Specimens were embedded in Araldite epoxy resin, sectioned, mounted on the microscope slides and polished with silicon carbide and aluminium oxide powders to about 30 μm thick.

Scanning electron microscopy and microtomography

The chemical composition, morphology of ferruginous masses matrix and microstructures topography were investigated using the desktop scanning electron microscopy (SEM) Phenom XL, Phenom World (Thermo Fisher Scientific, Eindhoven, Netherlands) equipped with a fully integrated energy-dispersive X-ray spectroscopy (EDS) detector and secondary electron detector (SED) located in the Faculty of Natural Sciences at the University of Silesia in Katowice (Bankowa, Katowice, Poland) (Fig. 5). Measurements were performed with low-vacuum settings with accelerating voltage 15 kV. Virtual sections of a selected specimen (GIUS 10-3739/23; Movie S1) were made in the Faculty X-ray Microtomography Laboratory at Faculty of Computer Science and Material Science, University of Silesia in Katowice, Chorzów, Poland using the General Electric Phoenix v|tome|x micro-CT equipment at 160 kV, 70 μA and scanning time of 20 min. Projection images were captured using a 1,000 × 2,024 pxs scintillator/CCD with an exposure time of 250 ms and processed using Volume Graphics® VGSTUDIO Max software and analysed using Volume Graphics® myVGL viewer.

XRD and confocal Raman spectroscopy

Bulk mineral composition of two powdered specimens representing each morphotypes was determined by Debye–Sherrer X-ray method using Rigaku SmartLab diffractometer equipped with Cu Kα1 source radiation. Measurement parameters were: acceleration voltage: 45 kV; filament current: 200 mA; step size: 0.05° 2Θ. Analyses of the collected data were carried out by means of XRAYAN Software using the newest ICSD database (Fig. 6).

To determine the more detailed mineralogical composition, the WITec confocal Raman microscope CRM alpha 300M equipped with an air-cooled solid state laser (λ = 532 nm and λ = 457 nm) and an electron multiplying CCD (EMCCD) detector was used. The calibration of the instrument was verified by checking the Si position. The Raman scattered light was focused onto a multi-mode fiber and monochromator with a 600 line/mm grating. To collect spectra of the coprolite matrix phases, the 50×/0.76 NA and 100×/0.9 NA air Olympus MPLAN objectives were used. All spectra were collected in the 200–4000 cm⁻¹ range with 3 cm⁻¹ spectral resolution (Figs. 7, 8). A surface Raman imaging map was collected in a 140 × 25 μm area using 140 × 20 pixels with an integration time of 0.5 s per spectrum, and precision of moving the sample during the measurements of ±0.5 μm. The cluster analysis was performed to group spectra into clusters. K-means analysis with the Manhattan distance for Raman imaging maps was carried out. The data obtained was manipulated by WITec Project FIVE Software (cosmic rays removal procedure and cluster analysis) and GRAMS software package (baseline correction).

For comparison purpose, a bulk sample from concrection was also analyzed (Fig. 9). XRD scan was performed using a PANalytical X’Pert Pro X-ray diffractometer (PW3040/60), equipped with a cobalt anode tube (40 mA, 40 kV) and an X’Celerator detector. The analyses were performed over a wide range of 2Θ (from 5 to 90) to obtain the appropriate number of peaks for each phase, with a long counting time constant (300 s) and precise step size (0.02°2Θ). The obtained results (Fig. 9) were evaluated in the HighScore program, version 4.9 (Degen et al., 2014) linked to the ICSD (ver. 2015), COD(ver. 2021), and ICDD PDF4 + databases (ver. 2019).

Observations of extant excrements

External and internal features of feces of Recent animals (private farms and from wild animals from local forests or raised at the Silesian Zoological Garden in Chorzów, Poland) were analyzed (Figs. 2, 10). Over the course of 2 months, ca. 800 excrements belonging to modern fish, amphibians, reptiles, birds and mammals were collected (a detailed study on these feces is planned to be published elsewhere in the future). Lineages that had their representatives in the early Miocene sediments of North Bohemia, Czech Republic (see Table 3) were selected for a more detailed observation. Additionally, the animals had to be large enough to produce excrements with dimensions comparable to those currently documented in the fossil state. Thus, the feces of small fish, the remains of which are known from the Miocene sediments of North Bohemia, such as Chalcaburnus or Nemacheilus, toads and frogs (Bufo, Rana, Pelobates), birds (Upupa, Coturnix) and mammals (Chiroptera, Dryomys, Sciurus, Martes), were not taken into account.

A special attention was also paid to those animals that left at least part of their feces in the terrestrial environment (see Figs. 2H–2M, 10). These were snakes (king python (Python regius), tiger python (Python molurus), reticulated python (Malayopython reticulatus), common boa (Boa constrictor), king cobra (Ophiophagus hannah), Korean rat snake (Elaphe anomala), common European viper (Vipera berus)), lizards (komodo dragon (Varanus komodoensis)), and turtles (Mediterranean tortoise (Testudo hermanni), steppe tortoise (Testudo horsfieldii), Indian star tortoise (Geochelone elegans), Spanish pond turtle (Mauremys leprosa), and Nile soft shell turtle (Trionyx triunguis)). Excrements of the European beaver (Castor fiber), African lion (Panthera leo), cheetah (Acinonyx), Indian star tortoise (Geochelone elegans), and white-tailed Eagle (Haliaeetus albicilla) were additionally analyzed (Fig. 10).

General morphology

A total of 29 specimens of excrement-shaped ferruginous masses were collected. Among these specimens two different shapes and sizes were identified. More specifically, morphotype 1 (M1) is represented by small (mean length: 33.4 mm, see Table 4) sausage-shaped (=allantoid) specimens with smooth or rough surface and flared lower part (Figs. 2A, 2B). Morphotype 2 (M2), in turn, is represented by larger (mean length: 46.3 mm) and more rounded to oval, massive specimens with rough surface (Figs. 2E–2G). Rare specimens (Figs. 2F, 2G) included into M2, bear prominent pointed end covered by striate pattern (herein interpreted as a trace produced after closing anus, see Discussion below). Color of both morphotypes varies from pale orange, through greenish red, to burgundy-colored.

Optical microscopy, microtomographic and palaeontological studies

The thin sections from two specimens representing two morphotypes were studied both under transmitted and reflected light optical microscopy. The sections from both morphotypes look very similar. They are dominated by darker matrix almost not translucent making transmitting light observations difficult. The mineral matrix seems to be rather homogenous. Within the matrix of M2-type more translucent elongated straight or curly structures (up to about few mm long and 10–99 µm thick; mean: 52 µm) are visible (Fig. 3). The structures sometimes form arcs or are twisted. In the reflected light, they seem to be areas of light reduction, while surrounding matrix is oxidized. The dark (rusty-colored, brown to almost black), poorly translucent coloring of a matrix suggests iron-rich mineral which form the matrix. Therefore both mineral matrix as well as thin elongated straight to curly structures were studied in-depth under SEM and Raman imaging (see below). No other distinguishable microremains were noticed in thin sections. However, at the broken surfaces of some specimens of the first morphotype (M1) some tiny coalified debris were occasionally noticed (Figs. 2A, 2B).

Microtomographical studies of selected specimen (GIUS 10-3739/23; Movie S1) did not reveal any internal structures, which could have been eventually interpreted as undigested food remains.

The external appearance and internal structure of concretions from Turów mine are markedly different (Fig. 4). They are red to brown and irregular in outline. Locally they display a zonal structure and contain well-visible pyrite crystals.

Mineralogical, geochemical and structural analyses

XRD analyses of powdered fragments of two specimens from each morphotypes indicated that they both are composed of siderite with small admixture of geothite, and hematite.

Analyzed concretions are preserved as siderite (pure siderite and magnesian-calcian siderite) with major admixture of hematite and minor additions of goethite, pyrite and calcite (Fig. 9).

The more detailed data on microstructure and elemental composition of excrement-shaped masses were collected by utilizing SEM/EDS. We found that the matrix is composed of irregular forms organized in net-system structures (Fig. 5). These forms are bound by thin walls that often consist of several layers (Figs. 5G, 5H). The chemical analyses showed that the walls consist of iron oxides and the interior is likely filled with iron carbonate. Two types of matrix occurring in both morphotypes (M1 and M2) were distinguished. The first one includes smaller (up to 10 μm in diameter) forms with a broad wall of iron oxides and an empty inner part (Figs. 5A–5F). Occasionally, larger forms with carbonate centers (up to 100 μm in diameter) can also be found in the vicinity of the large voids (Fig. 5G). Due to the presence of unfilled forms in the matrix, a distinct porosity pattern can be visible (Figs. 5A1–5F1). In this matrix type, within specimen of M2-type straight or curly elongated structures, which were also observed under optical microscopy, can be found (Figs. 3, 5A–5F). They can occur as thin (10–99 µm) lines with significantly limited porosity (Figs. 3, 5A–5B1, 5C–5F1). In the widest cross sections, some cellular structure is observed (Figs. 3H, 5A–5B1), while in the narrowest cross sections (Figs. 5C–5F1), characteristic scale-like pattern in observed (Figs. 3F, 3G, 5C–5F1). The second type of matrix consists of larger (up to 30 μm in diameter) forms characterized by thinner walls (Figs. 5I, 5J). Their center is always filled with iron carbonate, so there is no distinct porosity, as well as no elongated structures to be found.

To extend the observations and elemental analysis based on SEM, Raman spectra were collected. The data obtained from Raman spectroscopy allows to differentiate two iron oxides within the walls (Figs. 7A, 7B). The spectrum of the first one has bands at 1322, 662, 407, 296, and 227 cm⁻¹ (Fig. 6A), which are characteristic for the hematite (Hanesch, 2009). The second mineral forms only very thin (<1 μm) layers in the hematite. The main bands of its spectrum are 684, 553, 397, 299, and 242 cm⁻¹ (Fig. 5B), which allow identifying this mineral as goethite. The 1322 cm⁻¹ band at the goethite spectrum originated from the admixture of the hematite. The spectrum of the carbonate mineral reveals bands connected to the typical vibrations of the CO₃ group (Fig. 7C) indicative of siderite.

The Raman spectroscopy was also used to investigate the thin elongated structures in the matrix. There was no variation in the mineralogical composition of these forms in comparison to the matrix. However, during experiments with the 532 nm laser (green), we observed increased fluorescence in the area of the elongated structures with reduced porosity (Fig. 8). This may indicate that although these structures are composed of the same minerals as the matrix, their original chemical composition was different.

Comparative actualistic studies

For comparative purposes we investigated modern feces produced by a number of vertebrates, comprising all major groups (i.e., fish, amphibians, reptiles, birds, and mammals) (for details see Table 3). We compared our fossil specimens with excrements of extant fish, amphibians, birds and mammals, but they differ in size and shape, and therefore were not subject to further observations (Fig. 10). We noticed, however, that our excrement-shaped ferruginous masses ascribed to the morphotype 1 are very similar to sausage-shaped excrements produced by two testudinid turtle taxa (i.e., the Mediterranean tortoise (Testudo hermanni) and the steppe tortoise (Testudo horsfieldii)). Their surfaces are mainly smooth and rarely covered with cracks (e.g., Figs. 2C, 2D); the digested plant debris are sometimes visible on their surfaces (Figs. 2C, 2D). They do not reveal any internal layered structure and display bi—or unidirectional bends of 70–120°.

We also compared these fossil excrements with excrements of other extant turtles (another testudinid, i.e., the Indian star tortoise (Geochelone elegans), a geoemydid, i.e., the Spanish pond turtle (Mauremys leprosa), and a trionychid, i.e., the Nile softshell turtle (Trionyx triunguis)), but the feces of the Trionyx triunguis differ in size and shape.

On the other hand, our excrement-shaped ferruginous masses ascribed to the morphotype 2 are very similar to more or less rounded to oval, massive excrements produced by three snake taxa (the king python (Python regius), the common boa (Boa constrictor), and the king cobra (Ophiophagus hannah)). Their surfaces are rough, and often contain some remnants of etched hair and feathers (e.g., Figs. 2H, 2I). Moreover, the faeces of the Korean rat snake (Elaphe anomala) are also similar to the fossil morphotype 1; however, they differ in size (they are smaller, cf. Fig. 2L). The excrement surfaces of the latter species are covered by some hairs. We also observed three excrements of the common European viper (Vipera berus). They are different in shape and size one from another, their surfaces are covered by etched hair (Fig. 2M).