Report of bioerosions and cells in Cainotheriidae (Mammalia, Artiodactyla) from the phosphorites of Quercy (SW France)

Abstract

The phosphorites of the Quercy from SouthWest France are well known for fossils preserved in 3D with phosphatized soft-tissues. Given that phosphatization is known to favor fine cellular preservation, the present study delves into the histological analysis of white and brown bones of Cainotheriidae (Artiodactyla) recently excavated from the DAM1 site near Caylus. Microscopy revealed that the white bones were completely filled with bacterial erosions, while the brown bones showed a pristine histology and intralacunar content resembling fossilized osteocytes in some areas. After decalcification, a brown bone revealed an abundance of blood vessel-like structures, innumerable osteocyte-like structures with canaliculi and a few chondrocyte-like structures, while a white bone revealed only blood vessel-like structures that looked eaten away. All the data combined suggest the brown bones were shielded from bacterial attacks and were filled with fossilized organic matter and original biological structures. The data taken all together do not support that these structures are casts, but indeed original and endogenous cells. This study encourages further histochemical and mineralogical analyses on Quercy fossils and the unique taphonomy of DAM1 to better understand fossilization processes and their impact on the color of bones, the chemistry of skeletal tissues, soft tissues, and cells.

Full text

Report of bioerosions and cells
in Cainotheriidae (Mammalia,
Artiodactyla) from the
phosphorites of Quercy (SW
France)
Qian Wu1,2, Romain Weppe3, Carine Lezin4, Yanhong Pan5 & Alida M. Bailleul2
The phosphorites of the Quercy from SouthWest France are well known for fossils preserved in 3D with
phosphatized soft-tissues. Given that phosphatization is known to favor ne cellular preservation,
the present study delves into the histological analysis of white and brown bones of Cainotheriidae
(Artiodactyla) recently excavated from the DAM1 site near Caylus. Microscopy revealed that the
white bones were completely lled with bacterial erosions, while the brown bones showed a
pristine histology and intralacunar content resembling fossilized osteocytes in some areas. After
decalcication, a brown bone revealed an abundance of blood vessel-like structures, innumerable
osteocyte-like structures with canaliculi and a few chondrocyte-like structures, while a white bone
revealed only blood vessel-like structures that looked eaten away. All the data combined suggest
the brown bones were shielded from bacterial attacks and were lled with fossilized organic matter
and original biological structures. The data taken all together do not support that these structures
are casts, but indeed original and endogenous cells. This study encourages further histochemical and
mineralogical analyses on Quercy fossils and the unique taphonomy of DAM1 to better understand
fossilization processes and their impact on the color of bones, the chemistry of skeletal tissues, soft
tissues, and cells.
Keywords Quercy, Bacterial erosions, Cells, Bone color, Phosphatization
e “ Phosphorites du Quercy ” are located in southwest France and consist of plenty of fossiliferous sites from
the Paleogene1,2. Since the 19th century, a large number of fossil animals have been excavated from the detrital
and phosphatic sediments lled in the karst cavities along with the development of mining activities1,2. ese
fossils encompass disarticulated vertebrate bone fossils3–7, as well as in some rare cases, amphibians, reptiles
and insect fossils with so tissues preserved in three dimensions (3D)2,8–12, representing a thriving Paleogene
fauna13.
ese Quercy fossils with 3D preserved so tissues were oen referred to as the external casts of
“mummies”8–10. However, computerized tomography (CT) later revealed that the skeleton and so tissue
anatomical structures were internally preserved8–10. e external casts were presumed to be made of phosphatic
calcium2,8,9, which was conrmed by X-ray diraction identifying phosphate minerals (i.e., apatite, francolite)
in fossil insects14. e environment of Europe in the Paleogene was similar to a tropical rainforest, with erosion
and karstication of Mesozoic bedrocks occurring due to carbon dioxide-enriched water15,16. Sediments rich in
clay, quartz, ferruginous oxides and phosphate were accumulated within karstic cavities, where animals fell into
karstic sink holes2.
1University of Chinese Academy of Sciences, Beijing, China. 2Key Laboratory of Vertebrate Evolution and Human
Origins, Institute of Vertebrate Paleontology and Paleoanthropology, Chinese Academy of Sciences, Beijing, China.
3Institut des Sciences de l’Évolution de Montpellier, Univ Montpellier, CNRS, IRD, Montpellier, France. 4Observatoire
Midi Pyrénées, Géosciences Environnement Toulouse (GET), UMR 5563, CNRS-CNES-IRD-Université Toulouse
III, 14 Avenue E. Belin, 31400 Toulouse, France. 5State Key Laboratory for Mineral Deposits Research, School of
Earth Sciences and Engineering, Centre for Research and Education on Biological Evolution and Environment and
Frontiers Science Center for Critical Earth Material Cycling, Nanjing University, Nanjing 210093, China. email:
wuqian@ivpp.ac.cn; alida.bailleul@ivpp.ac.cn
OPEN
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Phosphatization is an important taphonomic process of exceptional fossil preservation17–20. Notable
phosphatized fossils worldwide exhibit preserved cellular or sub-cellular structures, such as the putative embryos
of the Neoproterozoic Doushantuo Formation from China21–23, sh muscle bers of the Mesozoic Santana
Formation of Brazil24,25, and the dermal pigment cells of a snake from the Late Miocene Libros Konservat-
Lagerstätte from Spain26, emphasizing the importance of studying Quercy’s phosphatized fossils for insights into
cellular and even subcellular preservation.
e newly excavated site DAM1 dating from the Upper Eocene revealed many Cainotheriidae (Mammalia,
Artiodactyla) fossils, notably the species Paroxacron valdense7. In this new site, the fossil bones showed dierent
colors, some were white, some were beige and some were brown with a shiny patina (Fig.1). Some bones also
showed all types of shades within the same element (Fig.1).
Here, we investigated six bones with standard ground-sectioning methods as well as scanning electron
microscopy (SEM) paired with Energy Dispersive Spectroscopy (EDS). Additionally, two other bones were
decalcied in EDTA (Ethylenediaminetetraacetic acid). For the rst time, we report (1) the histological
preservation of mammalian skeletons and (2) osteocyte preservation in the Quercy. We also demonstrate a
link between bone color and the preservation of original microscopic structures and give some insights on the
taphonomic histories of these bones. is study further encourages the comparison of the taphonomic processes,
histology, cellular paleontology and chemistry of dierent fossil sites from the Quercy.
Results
Histology of the white bones
e patella QU-1, femoral head QU-3 and the distal epiphysis of femur QU-6 are white in color. in-section
slices indicate that the bone tissue in these specimens has undergone extensive modications (Figs.2, 5 and
6). Patella QU-1 is made of cancellous bone with many trabeculae (Fig.2B). e matrix of the cancellous bone
tissue shows many dark brown areas with few light areas made of unaltered bone matrix (Fig.2B-D). e dark
brown areas are composed of channels and pores corresponding to Non-Wedl Microscopical Focal Destructions
(MFD) resulting from bioerosion27,28. Notably, the spaces between the trabeculae are partially lled with detrital
clay (Fig.2E). e Non-Wedl MFDs are the pores and channels le by bacterial colonies during post-mortem
bacterial invasions27. We will refer to them directly as ‘bacterial colonies’ for readability.
At high magnication under the SEM, we did not see any evidence of fossilized/mineralized bacteria within
the pores (Fig.3). However, the pores did show some brous material, possibly representing collagen bers
(Fig.3). ese bacterial colonies are dark grey under the SEM, distinct from the unaltered bone around them.
Within the bacterial colonies, osteocyte lacunae are notably absent, whereas the unaltered bone matrix retains
some osteocyte lacunae (Fig.2D, E). is absence of osteocyte lacunae in areas aected by bacterial colonies
indicates substantial damage to the bone matrix microstructure. Similar patterns are observed under transmitted
light and SEM images of ground section slices of the femoral head QU-3 and the epiphysis of femur QU-6, where
bacterial colonies occupy the cancellous bone matrix (Figs.5B and C and 6C; Fig. S1, S2).
In summary, this histological examination provides detailed insights into the altered structure of the white
bones highlighting that they were originally attacked by bacterial colonies, shows the absence of osteocyte
lacunae, and shows the impact on both organic and inorganic components of the bone matrix.
Histology of the brown and beige bones
e patella QU-2, femoral head QU-5 and the diaphysis of femur QU-6 are brown in color and the femoral
head QU-4 is beige. Unlike the eroded bone matrix observed in QU-1, these bones show a very well-preserved
microstructure in their ground section slices (Figs.4, 5 and 6). e spaces between the trabeculae of cancellous
Fig. 1. e eight Cainotheriidae fossils bones from DAM1 analyzed here and showing dierent colors. e
colors range from white to brown. A, white patella UM-DAM1-278 (QU-1) and decalcied white patella UN-
DAM1-285 (QU-7); B, brown patella UM-DAM1-279 (QU-2) and decalcied brown patella UN-DAM1-286
(QU-8); C, three unfused distal femoral epiphyses UM-DAM1-280, UM-DAM1-281 and UM-DAM1-282
(QU-3, QU-4 and QU-5) ranging from white to brown; D, distal femur UM-DAM1-283 (QU-6) with white
epiphysis and a brown diaphysis.
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bone in QU-2, QU-4 and QU-5 are lled with clay as in QU-1 and QU-3 (Figs.4E and H and 5F and I).
However, the bone matrix of the trabeculae is notably well preserved, featuring numerous osteocyte lacunae,
several osteons, and vascular canals under both transmitted light and SEM observations (Figs.4 and 5D-I,
Fig. S3, S4). Chondrocyte lacunae are organized in line near and perpendicular to the external surface of the
bone (Figs.4F and 5F), as seen in the articular cartilage of extant mammals29. Notably, there are no signicant
histological dierences observed between the beige and brown samples (Fig.5D-I). e ground section slices
of the diaphysis of QU-6 showed good preservation of the bone matrix with signicant brown color with some
black areas (Fig.6E). No sign of bioerosion is observed in the corresponding transmitted light and SEM image
of the diaphysis of QU-6 (Fig.6E, F). EDS analysis performed on the same slice showed that these dark areas are
made of iron (Fig.6F). erefore, these dark areas probably represent a deposition of dark minerals containing
iron (i.e., some iron oxides).
In QU-2, some globular intralacunar structures are found in the osteocyte lacunae (Fig.4C), potentially
representing fossilized remnants of original osteocytes or osteoblasts with their nuclei. We attempted to nd
these intralacunar structures using the SEM directly on the same slide. In the SEM images, some osteocyte
lacunae are indeed lled with a material resembling fossil cells (which would align with previous studies30–32)
(Fig.4H-I). In QU-2, 4, 5 and 6 (in all beige and brown areas), similar material was easily found by SEM, while
such preservation was not found in the bacterially invaded QU-1 and QU-3 (nor in the bacterially invaded white
epiphyses of QU-6).
ese intralacunar material lled most of the lacunae in the SEM images, showing a signicantly dierent
picture from the dark globule observed under the light microscope (Fig.4C, I). e dark globule seen under the
light microscope is not visible with the SEM, most likely because SEM only shows surface data, whereas standard
microscopy shows light shining through a material with a thickness and a depth of eld. SEM data indicates the
transparent part of the lacunae (seen under the light microscope) is not empty but lled by a dierent material.
It is likely that the ‘dark globule’ is located deeper within this material. is material was further analyzed by EDS
analysis (see next section).
SEM-EDS chemical analysis of the intralacunar material of brown bones
EDS is a technique for elemental analysis, but it can directly indicate the most probable mineral found in the
sample based on its elemental composition. SEM-EDS shows that the intralacunar material are embedded within
a fossilized bone matrix made of uorapatite (as shown by the presence of uorine (F), indicating that the original
hydroxyapatite was converted to uorapatite (Data S1 to S3)). EDS analysis performed on the intralacunar
material directly revealed the widespread presence of phosphorus and calcium, but the concentration of the
elements is not the same in all lacunae, and even varies within the same lacuna (Fig.7). Beside from phosphorus,
four other dierent types of elements are characteristics of the intralacunar material: aluminum (Al), calcium
Fig. 2. Histology of the white patella QU-1. A, photograph of the patella QU-1 with the red line indicating the
direction of the section; B, histological cross section of QU-1 under transmitted light and its close-up (C) and
(D), showing the bacterial colonies; E, SEM image of the section slice indicated by the box in B and its close-up
(F), showing the bacterial colonies and decrease of bone matrix density. e white patella is heavily attacked
by bacterial invasions. Slide is 94 micron thickness. bc, bacterial colony; cl, clay; oc, osteocyte; ol, osteocyte
lacuna; tb, trabecula; ub, unaltered bone.
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(Ca), silicon (Si) and iron (Fe) respectively (Fig.7C-E). ey also present some carbon (C) and oxygen (O)
(Fig.7 and see full EDS raw data into Data S4-6).
Decalcication results
To further investigate the potential presence of cells within this material from the DAM1 fossil site, we
decalcied two additional patellae. EDTA decalcication has proven useful to reveal cellular fossilization in
many fossils29,30,32–35. Aer decalcication of a brown patella (QU-8) in EDTA for 4 days, the bony matrix
revealed the presence of many chondrocyte-like structures, osteocyte-like structures, and blood vessel-like
structures (Fig.8A-D). ey are consistent in morphology with cells and so tissues found in previous studies of
fossil samples and that of extant mammals29,30,32–35. ese structures were not observed in the white QU-7 aer
4 days in decalcifying solution (Fig. S5).
Aer 11 days in EDTA (with no solution change), one small drop of solution from the decalcied brown
QU-8 was lled in a much higher concentration of these osteocyte-like structures, blood vessel-like structures,
and some pieces of bony matrix with cell-like structures still attached to it (Fig.8F, H). All of these structures
were brown in color (Fig.8). ese decalcied products are very dierent from those of the white patella QU-7
aer 11 days in EDTA: they show no cell-like structures, only broken blood vessel-like structures, and a few
pieces of altered bone matrix (Fig.8E, G).
Fig. 3. SEM image of pores and channels made by a bacterial colony in QU-1, with brous material within the
pores. e white arrows indicate the brous material in the pores.
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Discussion
e histological investigations in six bones of the site DAM1 revealed that the white bones were completely
bacterially invaded (Figs.2, 5B-C and 6C), whereas the brown and beige bones were not (Figs.4, 5D-I and 6E).
In the bacterially invaded white bones, the SEM clearly revealed well-dened bacterial erosions that decreased
the bone matrix density (Fig.2E, F). Bone matrix density decrease is commonly observed during bacterial
invasions (e.g., during taphonomic experiments on extant bone36 or in other vertebrate fossils, such as fossils
from the Miocene of Northwest China31,37). Bacteria dissolve the bone matrix and make it spongier during their
invasion38–40. Sometimes a brighter hypermineralized rim can form around the colonies and corresponds to
a redeposition of bone minerals28,37,40 but such a hypermineralized rim was not visible in any of the samples
analyzed here.
EDTA decalcication of the brown patella QU-8 showed that it was completely lled with chondrocyte-like,
osteocyte-like and blood vessel-like structures, themselves also brown in color (Fig.8A, F,H). On the other hand,
the decalcied white patella QU-7 showed no cell-like structures at all and only a few pieces of broken blood
vessel-like structures (that looked partially consumed by bacteria) and broken pieces of bony matrix (Fig.8E,
G). From all of the data combined together (i.e., histological, SEM and decalcication results), it appears that
the bacterial colonies found within the white bones consumed the original organic matter and structures. One
logical conclusion to make is that the brown color of the bones (QU-2, QU-4; QU-5, QU-8 and the diaphysis
of QU-6) is due to the presence of organic matter/so tissue structures and cells. Somehow these brown bones
must have been shielded from bacterial attacks, because all the white bones analyzed here are full of bioerosions
and show no cells and only so tissues that are consumed by bacteria (Fig.8G). e data does not support that
these structures are casts41 but rather original endogenous structures that were not eaten by bacteria in the
brown bones, and remnants of endogenous structures that are partially consumed by bacteria in the white bones.
Fig. 4. Histology of the brown patella QU-2. A, photograph of the patella QU-2 with the red line indicating
the direction of the section; B, histological of slice one of QU-2 under transmitted light and its close-up (C),
showing the unaltered bone tissue and the intralacunar preservation; D, histological of slice two of QU-2 under
transmitted light and its close-up (E-G); H, corresponding SEM image of (E) and its close-up (I), showing the
inlled osteocyte lacuna; ese brown bone shows pristine histological preservation and no bacterial invasion.
cl, chondrocyte lacuna; oc, osteocyte; ol, osteocyte lacuna; vc, vascular canal.
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is is supported by the results of another study on Cretaceous-Paleogene vertebrates preserved in glauconitic
greensands42. In this study, the fossil specimens that were dark in color exhibited excellent microscopic bone
preservation and yielded a greater recovery of original so tissues, whereas light-colored specimens exhibited
poor microscopic preservation and yielded few to no so tissues. is paper even discussed that the color
variation in these bones was likely caused by microbial degradation42. e histological images provided in this
paper do show a bone that looks invaded by microorganisms42. e present paper therefore represents another
example of study that suggest bacterial invasions and bone color are linked. In parallel, it is also possible that an
enrichment in iron oxides in the samples analyzed here (e.g., Fig.6F) altered the original (non-fossilized) colors
of all the microstructures analyzed here (i.e., bone, osteocyte-like and chondrocyte-like structures, blood vessel-
like structures) into a brown color. Iron oxides have been proposed to help preserve so tissues and cells in deep
time, in some instances32,33.
Bioerosion is a common determinant of bone preservation and can give insights on the early post-mortem
history40,43. In our study, bioerosion was found in all white fossil bones of DAM1, but not in the brown bones.
Since not all of the bones analyzed here were bacterially invaded, it means something stopped the bacteria from
growing at some point aer burial. e bones are not obviously rounded, indicating that they have not been
transported over long distances. e intensity and distribution of bacterial colonies in the specimens suggest
soil attack as in the bones from Neuadd36: bacterial colonies appear scattered and dispersed, not associated with
vascular supply, and/or peripherally aecting the periosteal and endosteal cortical layers (Fig. S1-S2), leaving the
medial cortical layer almost unaected36. erefore, it is possible that the microenvironments of these fossils in
the early stages of burial were dierent.
Our interpretation is that, in DAM1, the bones that were attacked by bacteria remained on the surface of the
sediment for some time in an environment that was probably saturated with water, conditions that allowed the
Fig. 5. Histology of three distal femoral heads (unfused) ranging from white (QU-3), to beige (QU-4) and to
brown (QU-5). A, photograph of QU-3 to 5 with red line indicate the direction of section; B, histological of
ground section slice of QU-3 under transmitted light and its close-up (C), showing the bacterial colonies as in
the white patella QU-1; D, histological of ground section slice of QU-4 under transmitted light and its close-up
(E-F); G, histological of ground section slice of QU-5 under transmitted light and its close-up (H-I); D-I show
the unaltered bone and well preserved bone matrix as in the in the brown patella QU-2. ickness of slide B, D
and G is 89 microns, 98 microns and 95 microns respectively. bc, bacterial colony; ch, chondrocyte lacuna; cl,
clay; ol, osteocyte lacuna; vc, vascular canal.
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bacteria to attack the bones directly. At the same time, other bones may have been buried in the detritic sediment
-certainly saturated with water. is detritic sediment directly or indirectly lled the bone porosity. Indirectly,
this means that chemical attack on the detrital sediment provided the chemical elements that made up the
various mineral phases that precipitated and/or accumulated in the porosity, in particular within the osteocyte
lacunae (see next section).
It is the rst time that bacterial invasions are reported and histologically analyzed in any fossil of the Quercy
phosphorites, giving an interesting insight on the taphonomy of the newly discovered site of DAM1 and showing
that fossils in the Quercy are not always exceptionally preserved. It is however too dicult to tell when exactly
the invasions occurred aer death, nor how long they were going on until the death of all the bacterial colonies,
but it is plausible to hypothesize attacks occurred during early diagenesis rather than during late diagenesis.
Aer just four days into a decalcifying solution, the brown patella QU-8 released many isolated osteocytes
with very clear canaliculi, chondrocytes and blood vessels (Fig.8). Aer 11 days in this same solution, the
solution was replete with cells and blood vessels (Fig.8). Light microscopy revealed dark clay and ferruginous
sediments in the trabecular gaps and in the vascular channels of another brown patella (QU-2); in this same patella
structures resembling cells with a globular nucleus were seen (QU-2; Fig.4C). is type of ne preservation was
expected due to the ne preservation potential of phosphatization18–20. We focused the EDS beams on these
same structures and found that they are composed of aluminum, silicon, calcium, phosphorus, and iron, which
most likely indicate the presence of at least partly authigenic minerals in the the osteocyte lacunae like silica,
calcium phosphate, clay and probably goethite (Figs.4I and 7). ese structures also contained some carbon
and oxygen (Fig.7; Data S4-S6). All of the evidence taken together suggests that the intralacunar content seen
under the light microscope (Fig.4C) and under the SEM (Fig.7) are fossilized cells (like those seen in Fig.8).
Additionally, although EDS is elemental, the EDS results suggest that the bones that were not invaded by bacteria
have cells that were mineralized by various mineralized phases, including calcium phosphate, alumino-silicates,
carbonates, silica and iron oxides (Fig.7). is has also been observed in other fossilized osteocytes30,31. For
example, alumino-silicied, silicied, and ironized chondrocytes were reported in Yan o r nis, Confuciusornis, and
Caudipteryx from the Early Cretaceous Jehol biota of China32,44 and ironized osteocytes were found in non-
Fig. 6. Histology of a distal femur (QU-6) with brown diaphysis and white epiphysis. A, photograph of QU-6
with red line indicate the direction of section; B, histological of ground section slice of QU-6 under transmitted
light and its close-up of epiphysis (C), metaphysis (D) and diaphysis (E), showing the bacterial colonies in the
epiphysis as in the white patella QU-1, and unaltered bone in the metaphysis and diaphysis as in the brown
patella QU-2; F, EDS of (E) showing the iron deposition in the bone matrix. Slide thickness is 120 microns. bc,
bacterial colony; md, mineral deposition; ub, unaltered bone.
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avian dinosaurs from Late Cretaceous of North America33. Ironized osteocytes-like structures were found in
Mongolemys from the Late Cretaceous of Mongolia30. Cases of calcied fossil cells are relatively less reported,
such as that of a royal fern from the Early Jurassic of Sweden45. Similarly to what is observed in this current study,
dierent types of mineralization occurred in cells within the same specimen of Caudipteryx and Mongolemys,
further supporting that having a dierent mineralogical composition in dierent cells within one same fossil is
not a rare phenomenon at all30,32. It is also important to note that some cell lacunae are highly likely also lled
with ‘pollution’ rather than in-situ mineralization.
As a famous phosphate mine, it is logical for the Quercy to be a favorable environment to preserve fossil
cells. Fine structures preserved by phosphatization require a rapid replacement of the structures by mineral
deposition46, which is consistent with our observation of rapid burial for the DAM1 specimens in situ. In the
present study, phosphorous was present- but was only one of the elements identied in the DAM1 fossilized
osteocytes and intralacunar structures (Fig.7). Phosphate is also favorable to subcellular preservation such as
nuclei preservation (e.g25). Further histochemical work is necessary to test whether or not the globules within
the cells seen under the light microscope (Fig.4C) are indeed remnants of cell nuclei. A similar type of cellular
preservation with fossilized nuclei has already been observed in previous studies in fossils of all ages and from
all around the world47. In fact, cell nuclei appear to be quite common in the fossil record with some recently
discovered examples in the Cretaceous dinosaur Hypacrosaurus from Montana or a Jurassic Royal fern from
Sweden45,48. In vertebrates, cartilage seems to be a good tissue for cellular and nuclear preservation (e.g48), and
chondrocytes in calcied cartilage are supposed to be more resistant to autolysis in general than osteocytes49.
Although chondrocytes were not well visible in the ground-section slices of the brown specimens studied
here, the decalcication method did help the identication of plenty of fossilized chondrocytes in the brown
patella QU-8 (Fig.4J-M). is highlights the importance of applying more than one method for the detection
of dierent cellular structures and/or tissues within one same sample. Our decalcication method showed that
chondrocytes are indeed preserved here in at least some specimens of DAM1 and suggests future histochemical
and cellular analyses on DAM1 cartilage are necessary.
Studies suggest that microbial activity may have a positive impact on the mineralization and on the
preservation of organic matter through the crystallization of microbial biolms on the decomposing organic
matter50. Studies also suggest the metabolic products of bacterial decay generate the conditions promoting
precipitation19,51. Previous research suggests that phosphate in bones may be the source of phosphorus for
Quercy phosphorylation2. Bacterial decomposition may release phosphorus from the bones into the surrounding
groundwater, providing a source of phosphorus for mineralization19. Here, our results do not support these
interpretations for the specic site of DAM1, it appears that instead, bacterial invasions (which apparently
Fig. 7. EDS analysis of the intralacunar content in brown QU-2. A, close up SEM image of the osteocyte
lacunae in Fig.4I and EDS analysis on the same image (B), showing the dierent chemical element content
of the dierent lacunae and dierent location in the same lacuna; Alumino-silicied (Abundant Si and Al,
probably component of clay) (C), calcied (Abundant Ca and P, probably components of hydroxyapatite or
uoroapatite) (C-D), silicied (Abundant Si, probably a component of Silica) (D). Another analysis in a brown
part of QU-6 (in the diaphysis) shows ironized intralacunar content (E).
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occurred early during burial rather than later during diagenesis) limited the preservation of organic matter
(i.e., blood vessels, and cells) in the bones of DAM1. e Quercy is highly likely lled with unique taphonomies
at each of its sites, even perhaps between two sites very close to each other and this requires further attention.
Conclusion and future research
Our results show that the dierent colors of the Cainotheriidae from DAM1 are linked with dierent
fossilization conditions, bioerosions, and most likely the presence of organic matter. e light-colored fossils
experienced obvious bioerosions, while the dark-colored fossils did not and preserved ne microstructures
including fossilized osteocytes. ese skeletal tissues with cellular fossilization might have undergone a very
dierent taphonomic and diagenetic process from the exceptionally 3D-preserved fossils with so tissues in the
Quercy. Here, the identication of cellular structures as an endogenous cellular preservation relies on histology,
SEM-EDS analyses, and decalcication. Histochemical and immunohistochemical analyses contributed to
the identication of fossil cells in diverse other taxa33,52–54. Future investigations should include more robust
histochemical and molecular analyses directly on isolated osteocytes that are released aer EDTA decalcication
to test for the presence of fossil cell nuclei at the chemical level and to better understand the overall chemistry
Fig. 8. Microphotographs of decalcied contents of white patella QU-7 and brown patella QU-8. Decalcied
contents of QU-8 aer 4 days in EDTA (A-D). Decalcied contents of white patella QU-7 aer 11 days in
EDTA in (E) and (G). Decalcied content of brown QU-8 aer 11 days in EDTA in (F) and (H). Images E and
F have the same scale. Images G and H have the same scale. In Qu-7, the ‘broken blood vessel’ (bbv) is clearly
eaten away (G); the red arrows in (H) are pointing at osteocytes in QU-8. bv, blood vessel; cc, chondrocyte; oc,
osteocyte.
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of the cells. In depth-mineralogical studies will also be necessary to better understand the full context of their
preservation and surrounding bone chemistry. ese rigorous examinations are essential for a comprehensive
understanding of these specimens and additional Quercy bones. Additionally, a recent study shows the 3D
preserved phosphatized fossil of Quercy also preserve bones, but the so tissues were hypothesized to be casts9.
It is unlikely that the so tissues are simply casts and future investigations should delve into comparing the
preservation of bones preserved with and without so tissues, and to better understand the preservation of so-
tissues and cells themselves.
Materials and methods
Site DAM1
e studied osteological material comes from the karstic locality of Dams (near Caylus SW France), specically
from the DAM1 channel discovered in 2016 7. is cavity, partially emptied during 19th -century intensive
phosphate mining, still houses a large quantity of clay inllings and preserves several fossiliferous inllings of
dierent ages. In particular, two fossiliferous channels, DAM1 and DAM2, yielded an impressive number of
well-preserved Cainotherid remains due to low-energy transport7. Biochronological dating suggests an MP19
age (late Priabonian; ca. 34.1 My) for DAM1 and DAM2 inllings, based on rodent assemblages.
e Quercy phosphate inlling sites in SW France comprise over 200 localities recording local faunal
assemblages with a temporal resolution of approximately 1million years or less, covering a time span of more than
30million years55–57. is unique area shelters abundant fossiliferous caves with a continuous record spanning
the 42–24Ma interval (late middle Eocene–late Oligocene), capturing a dynamic history and signicant global
climatic changes.
Paroxacron valdense MP19 Upper Eocene
e fossil material examined in this study belongs to a small artiodactyl family, the Cainotheriidae, specically
identied as Paroxacron valdense7.is species represents over 90% of the fossil assemblage of both DAM1 and
DAM2 localities. Cainotheriids are small artiodactyls known from the late Eocene to the middle Miocene in
Western Europe7,58,59. is family is rather well diversied and is composed of around twenty species for at
least six genera58,59. Cainotheriid remains are particularly abundant in the fossil record and notably in karstic
inllings from the phosphorites of the Quercy7,57–59. Two isolated patellae (UM-DAM1-278, -279), three distal
femoral epiphyses (UM-DAM1-280 to -282), and a broken distal part of a femur (UM-DAM1-283) were selected
for histological analysis in this research, because they were thought to possess both bone and calcied cartilage.
Two additional patellae were decalcied in EDTA (UM-DAM1-285,-286). For readability, we assigned shorter
numbers (QU-1 to 8) for these specimens used in this study (Table S1).
Histology
e specimens were embedded in EXAKT Technovit 7200 resin and cured for 12h, cut into slices (Figs.2, 3,
4, 5 and 6, see the red lines) and polished until the desired optical contrast was reached. Ground sections were
observed under transmitted and polarized light using a Nikon Eclipse LV100NPOL and photographed with a
DS-Fi3 camera and the soware NIS-Element v4.60.
SEM-EDS
e ground sections were analyzed at the Chinese Academy of Geological Sciences (Beijing) using FEI Quanta
450 (FEG) at 20kV. Both BSE and SE modes (back-scattered electrons and secondary electrons) were applied to
the ground-sections. Both elemental mapping and specic points were targeted. Slides were un-coated.
EDTA decalcication
QU-7 and QU-8 (Fig.7) were decalcied in a solution of EDTA (0.5M; pH 8.0) for 11 days (without any solution
change). Two microscopic analyses were made, one aer four days in EDTA, and then a second one aer 11 days.
For photography, one drop of solution is pipetted on top of a glass slide which is then cover-slipped. ese slides
need to be rapidly photographed before evaporation.
Data availability
e data that support the ndings of this study are available from the corresponding author upon reasonable
request.
Received: 25 March 2024; Accepted: 25 September 2024
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Acknowledgements
We thank Zhang Shukang and Zhu Yuxia for preparing the ground section slices. We thank Maeva Orliac at the
ISEM (Institut des Sciences de l’Évolution de Montpellier) for providing all of the samples for this study and for
discussions.is work was supported by the Youth Innovation Promotion Association Grant of the Chinese
Academy of Sciences (2023078) and National Natural Science Foundation of China NSFC grant 42350610256
to AMB. NSFC grant 42302012 and the Beijing Natural Science Foundation (5224037) supported QW. e
collection of study material in the eld was nancially supported by the ANR DEADENDER and ENLIVEN
programs (grants ANR-18-CE02-0003-01 and ANR-22-CE02-0014-01)- PI Mr J. Orliac and the association
"Phosphatières du Quercy".
Author contributions
A M. B designed the project. Q. W. and A M. B collected the data. Q. W, R. W., C L., Y. P., A M. B analyzed the
data and wrote the paper.
Declarations
Competing interests
e authors declare no competing interests.
Additional information
Supplementary Information e online version contains supplementary material available at https://doi.
org/10.1038/s41598-024-74301-y.
Correspondence and requests for materials should be addressed to Q.W. or A.M.B.
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