Diagenetic Changes on Bone Histology of Quaternary Mammals from A

Palaeogeography, Palaeoclimatology, Palaeoecology 537 (2020) 109372

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Palaeogeography, Palaeoclimatology, Palaeoecology

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Diagenetic changes on bone histology of Quaternary mammals from a T tropical cave deposit in southeastern Brazil ∗ Elver Luiz Mayera,b, , Alex Hubbec,d, Jennifer Botha-Brinke,f, Ana Maria Ribeirob,g, Paulo Miguel Haddad-Martimh, Walter Nevesi a Instituto de Estudos do Xingu, Rua Constantino Ferreira Viana, Quadra 8, Centro, São Félix do Xingu, PA, 68380-000, Brazil b Programa de Pós-Graduação em Geociências, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9500, Porto Alegre, RS, 91501-970, Brazil c Departamento de Oceanografia, Instituto de Geociências, Universidade Federal da Bahia, Salvador, BA, 40170-020, Brazil d Instituto do Carste. Rua Barcelona, 240/302, Belo Horizonte, MG, 30360-260, Brazil e Karoo Palaeontology, National Museum, Box 266, Bloemfontein, 9300, South Africa f Department of Zoology and Entomology, University of the Free State, Bloemfontein, 9300, South Africa g Setor de Paleontologia, Museu de Ciências Naturais, Secretaria de Meio Ambiente do Rio Grande Do Sul, Av. Salvador França, 1427, Porto Alegre, RS, 90690-000, Brazil h Instituto de Geociências, Universidade Estadual de Campinas, Campinas, SP, 13083-855, Brazil i Instituto de Estudos Avançados da Universidade de São Paulo, Rua da Praça Do Relógio, 109, São Paulo, SP, 05508-050, Brazil

ARTICLE INFO ABSTRACT

Keywords: Karst caves are suitable environments for the accumulation and preservation of fossils. Cave deposits are often Diagenesis complex and the environmental conditions within cave sites result from intricate interactions between various Bone preservation biological, physical and chemical factors. However, it is not fully understood how the complexity of the en- Cervidae vironmental conditions of caves influences bone diagenesis. The study of the initial stages of bone diagenesis Tayassuidae depends to a large extent on understanding the changes in the bone histology. To contribute to this issue, we Karst examine a set of postmortem changes affecting the bone histology of Quaternary mammals that accumulated Microstructure naturally in Locus 2, a pitfall site in Cuvieri Cave, located in the tropical region of Brazil. Our analyses show that bones deposited in caves may be subject to a peculiar set of environmental conditions that in tropical regions may prevent the preservation of bone histological structure. The effect of diagenetic processes on the bones differs depending on the taphonomic stage of the bone and the diagenetic alterations appear to haveinfluenced each other. The deposition of bioclasts following the entrapment of individuals in Locus 2 favours the proliferation of bacteria on bones and appears to be important in directing the diagenetic alteration. The hydrological regime of the cave, that is recharge with potential phases of higher humidity, also is important in directing the diagenetic alteration and further decreased the preservation potential of the bone microstructure. The formation of macroscopic and microscopic cracks related to bone weathering in caves shows that the taphonomic processes peculiar to these environments are poorly understood, highlighting the need for more research to be conducted on cave taphonomy.

1. Introduction decomposition and involving UV radiation, moisture and temperature, among others factors; Behrensmeyer, 1978; Trueman et al., 2004; Bone diagenesis consists of processes by which, after an individual's Pfretzschner, 2000; Pfretzschner and Tütken, 2011) and microbial de- death, bones are altered in the depositional environment. The under- composition (degradation of organic and inorganic elements of bone by standing of the diagenetic processes is important because this phe- direct and indirect effects of microorganisms; Hackett, 1981; Jans et al., nomenon determines if a bone deteriorates until it is completely dis- 2002; see also Kendall et al., 2017). The consequences of both processes integrated or if it is preserved as a fossil. Abiotic and biotic factors can be observed as macroscopic changes, such as weathering cracks on acting on bones influence diagenetic processes, such as bone weath- the external surface of bone (Behrensmeyer et al., 1978; Dirks et al., ering (physical and chemical destruction of bone enabling further 2015; Pokines et al., 2018) and microscopic changes, such as

∗ Corresponding author. Instituto de Estudos do Xingu, Rua Constantino Ferreira Viana, Quadra 8, Centro, São Félix do Xingu, PA, 68380-000, Brazil E-mail addresses: [email protected] (E.L. Mayer), [email protected] (A. Hubbe), [email protected] (J. Botha-Brink), [email protected] (A.M. Ribeiro), [email protected] (P.M. Haddad-Martim), [email protected] (W. Neves). https://doi.org/10.1016/j.palaeo.2019.109372 Received 27 September 2018; Received in revised form 23 July 2019; Accepted 11 September 2019 Available online 03 October 2019 0031-0182/ © 2019 Elsevier B.V. All rights reserved. E.L. Mayer, et al. Palaeogeography, Palaeoclimatology, Palaeoecology 537 (2020) 109372

Fig. 1. Geographic location of the study area and of Cuvieri Cave showing the position of Locus 2 (map courtesy of Laboratório de Estudos Evolutivos Humanos and Grupo Bambuí de Pesquisas Espeleológicas). microcracks on bone histological structure (Pfretzschner, 2000, 2004; paleontological (e.g. Pfretzschner and Tütken, 2011; Trueman et al., Pfretzschner and Tütken, 2011), and tunnels formed by bacteria 2003; Tomassini et al., 2015) and archaeological contexts (e.g. Hollund (Hackett, 1981; Jans et al., 2004). et al., 2012; Jans et al., 2002; Dal Sasso et al., 2014) formed within The diagenetic processes are related to some extent to the hydro- different environmental settings. However, as pointed outby Kendall logical regime of a given environment. Moisture is one of the main et al. (2017), the range of climates and environments explored during elements of bone weathering. It facilitates microbial metabolism and the study of bone diagenesis should be expanded. This is especially the groundwater is the means by which chemical exchanges occur be- important for cave environments, which are known to preserve many tween bones and the environment (Behrensmeyer, 1978; Fernandez- fossils around the world (Lundelius, 2006; Klein, 2009), and for which Jalvo et al., 2010; Hedges et al., 1995; Hedges and Millard, 1995; investigations include uneven research efforts in different climatic Pokines et al., 2018; Trueman et al., 2004; Turner-Walker, 2008). Thus, zones (North Temperate Zone, Bocherens et al., 2008; Hedges et al., hydrology is a major factor that influences the preservation of bone 1995; Quattropani et al., 1999; Nielsen-Marsh and Hedges, 2000a, histology in different depositional environments. Diffusion, hydraulic 2000b; Rogóz et al., 2009; Farre et al., 2014; Monge et al., 2014; Marín- flow and recharge are categories used as reference models to under- Arroyo, 2015; North Tropical Zone, Robles et al., 2002; South Tropical stand how hydrology influences bone diagenesis (Hedges and Millard, Zone, Sillen and Parkington, 1996; Hanson and Cain, 2007). To con- 1995). The diffusion regime is typical of soaked soils, where there isno tribute to broadening the spectrum of conditions investigated for bone gradient in the hydraulic potential neither in time nor in space, whereas diagenesis, this study aims to examine a set of postmortem changes in the hydraulic flow regime the quantity of water is more orless affecting the bone histology of Quaternary mammals that naturally constant in time, but variable in space. In the recharge regime the hy- accumulated in a pitfall deposit in Cuvieri Cave, located in the tropical draulic potential is more or less constant in space, but variable in time region of Brazil. Our findings provide important information on the (Hedges and Millard, 1995). Fluctuations in the recharge regime are influence of depositional environments on skeletal remains preserved in considered to be the most harmful for the microscopic integrity of karst caves. skeletal tissues (Hedges and Millard, 1995; Nielsen-Marsh and Hedges, 2000a; Kendall et al., 2017). In contrast, the diffusion regime seems to 1.1. Study site: Cuvieri Cave cause relatively little microscopic change (Hedges and Millard, 1995; Nielsen-Marsh, 2000a; Kendall et al., 2017). The Cuvieri Cave is situated within the Lagoa Santa Karst, in the Among terrestrial ecosystems, karst caves are suitable environments state of Minas Gerais, eastern Brazil (Fig. 1; entrance coordinates: UTM for the accumulation and preservation of fossils because the interior 7846105 N and 0603756 E; fuse 23K; Corrego Alegre Datum). The generally consists of protected areas, presenting less variable conditions current entrance of Cuvieri Cave (around 1.5 m × 1 m) is at the base of than its surroundings (i.e. temperature and humidity) and sediments a doline. After the entrance the cave develops into a sub-horizontal with pH levels that can buffer the dissolution of bones (Lundelius, 2006; passage that ends in three vertical pits, with no noticeable connection Simms, 1994). Deposits in karst caves preserve fossils of many verte- between them. The pits have been named Loci 1, 2 and 3, and ap- brate groups from the Paleozoic, Mesozoic, and Cenozoic, from fish to proximate depths of 16 m, 4 m and 8 m, respectively (Fig. 1) (see humans (Lundelius, 2006 and references therein; Klein, 2009 and re- Haddad-Matrim et al., 2017 for details regarding the cave deposits and ferences therein). Additionally, cave deposits are often very complex, the major processes involved in their genesis and evolution). Below we showing successive events of erosion and deposition and some ex- present details on Locus 2 as it is the deposit of interest in our study. tremely localized events (Gillieson, 1996; Auler et al., 2009; Haddad- Locus 2 presents a temporal discontinuity between different fossi- Martim et al., 2017), resulting in a plethora of processes that can in- liferous sedimentary facies (Hubbe et al., 2011, Fig. 2). Two facies were fluence bone diagenesis. Similarly, the environmental conditions within distinguished, an upper dark brown silt loam (BSL) and a lower dark cave sites result from intricate interactions between various factors reddish brown silt loam (RBSL). BSL is richer than RBSL in carbonates (e.g., the size and number of cave entrances, the morphology of the and organic matter. BSL has abundant remains of medium-sized extant passages, the level of the water table, geographic location, etc.), and it mammals, and RBSL has remains of medium-sized extant mammals as is possible to observe considerable variation between different passages well as large extinct mammals originally deposited within this facies. in the same cave and between different caves (Tuttle and Stevenson, RBSL facies was probably transported into the cave through mass flows, 1978). Therefore, it is not fully understood how the complexity of the with sediments penetrating the cave through its main horizontal pas- environmental conditions of caves influences bone diagenesis. sage (Hubbe et al., 2011). BSL facies was mainly transported into the The study of the initial stages of bone diagenesis depends to a large cave by percolating through small fissures connecting the epikarst to extent on understanding the changes in bone histology. To date, such Locus 2, through infiltration promoted by descending water (Hubbe investigations have dealt with fossil bones recovered from et al., 2011). AMS C14 dates indicate that the BSL was deposited during

2 E.L. Mayer, et al. Palaeogeography, Palaeoclimatology, Palaeoecology 537 (2020) 109372

Fig. 2. Stratigraphical and chronological context of the sampled bone specimens and their location. Site map (above) and cross section (below) with two sigma calibrated Accelerator Mass spectrometry radiocarbon dates (cal yr BP) for Locus 2. Symbols represent the exact location of each specimen. Solid symbols represent bones found in the BSL facies whereas open ones represent bones found in the RBSL facies. More than 15.000 other bones and teeth recovered from both facies are not represented here. Modified from Hubbe et al. (2011).

3 E.L. Mayer, et al. Palaeogeography, Palaeoclimatology, Palaeoecology 537 (2020) 109372 the Holocene, with dates ranging from 8580 cal yr BP to the present, whereas the RBSL deposition dates to the Holocene/Pleistocene tran- sition (13,600–10,660 cal yr BP; Hubbe et al., 2011)(Fig. 2). The available information suggests that the hydrological regime was

of recharge with potential moments of water flow regime, at least since d ~8 cal kyr BP. We support this interpretation based on the proposed greyish/whitish and blackish whitish/whitish yellowish/whitish yellowish/whitish greyish/whitish and blackish yellowish/whitish process involved in BSL facies formation, which involves the transport Colour of sediments by water. Current observations show that seasonal hydrological processes are present during most of the wet season (between December and February), when the walls may become very humid and water drips from the ceiling completely soaking the sediments and forming ponds. However, it is possible that past phases of elevated rainfall may have promoted the maintenance of high humidity levels in Locus 2 throughout the year. Therefore, we consider the predominant condition to have been characterized by the fluctuation of humidity, but with potentially constant moments of humidity.

2. Material and methods central radial and linking MFDhistological and structures histological structures central radial histological structures histological structures Micro cracks

The excavation procedures employed in Locus 2 were adapted from archaeological methods and consist of exposing successively the skeletal remains contained in the sediments (see Hubbe et al., 2011 for

details). The specimens selected for sampling are metapodia of extant Wedl artiodactyls representing Cervidae (n = 4, deer) and Tayassuidae (n = 2, peccary) excavated from the fossiliferous deposit in Locus 2 of 1 1 Wedl and non- Cuvieri Cave (Fig. 2). All the Cervidae samples plus one sample of 1 11 non- Wedl1 0 1 – 1 central radial non- and Wedl linking MFD 1 and non- Wedl central radial and linking MFD and central radial and linking MFD and Tayassuidae were recovered from the BSL facies, of Holocene age, and only one Tayassuidae sample was excavated from the RBSL facies, of Pleistocene/Holocene age. We selected specimens of these taxa because they are common in paleontological and archaeological sites in Brazil, e e representing good models for comparative studies. Additionally, Cer- vidae remains are the most abundant in the Locus 2 assemblage, and f between 5750 and 8400 8580–8400 age > 8580 Tayassuidae is represented by two individuals that have been directly 3060–2850 dated. Considering that part of the procedures adopted here implies partial destruction of the specimens due to thin sectioning the bones, we chose metapodia because they may have less diagnostic use compared to limb bones and they are abundant in the Locus 2 facies and allowed us to select bone samples with different characteristics. We sampled bones from articulated (n = 2) and clustered skeletons (n = 1), as well as isolated bones (n = 3) (Table 1). The selection of specimens for sampling also considered their spatial distribution re- lative to dated specimens, such as a bone sample from the same exposition as a dated specimen or a bone sample from an articulated dated dated expositions dated specimen dated exposition Relation bone sampled/dating Age (cal yr BP) WS HI MFD skeleton (Table 1; Fig. 2). dated specimen c To provide a general description of each specimen sampled, we performed a macroscopic analysis considering the bones before the Exp. section procedures and as thick sections (see details on section proce- b dure below). It helps with characterization of the weathering features and physical integrity and/or breakage pattern. The weathering features were recorded following the six stage scale defined by Behrensmeyer (1978). The stages are ranked from bone surfaces dis- playing no sign of cracking or flaking (stage 0) to very fragile bone that has fragmented (stage 5) (Behrensmeyer, 1978). The physical integrity of the specimens was recorded as follows: those comprising at least 90% of the respective skeletal element were noted as complete; specimens encompassing less than 50% were noted as fragments, and specimens of Taxon Facies Depth (m) intermediary condition were noted as incomplete. If the specimen was a not complete, the breakage pattern was recorded as a fresh fracture, if the bone was broken relatively soon after death, or as a weathered fracture, if the bone was broken when dry (Lyman, 1994). The changes in bone histology were evaluated on the basis of thin Element Side sections and considering: 1) the general integrity of the histological structure; 2) the presence of microscopic focal destructions (MFD); and 3) the occurrence of diagenetic microcracks. The integrity of the his- External surface/sectioned area. In relation to datum. L-left; R-right. Approximate age. Number of the exposition. Estimate based on ages obtained from an articulated skeleton and an isolated specimen.

tological structure was evaluated using the Histological Index (HI) a b c d e f CVL2-6711CVL2-12008 metacarpal metatarsal R RCVL2-14964 metacarpal Tayassuidae R Cervidae BSLCVL2-15251 metacarpalCVL2-15472 −0.86 R Cervidae BSL metatarsal R −1.07 BSL Tayassuidae 16 RBSL Cervidae −1.16 from −1.20 37 articulated skeleton dated BSL from 44 clustered skeleton 45 between −1.23 5990–5750 isolated, from the from same articulated exposition skeleton as dated 46 from articulated skeleton 12.670–12.140 below 0 0 non- Wedl central radial Specimen Number CVL2-1915 metatarsal L Cervidae BSL −0.72 6 isolated, from the same exposition as

(Hedges et al., 1995, Table 1 p. 203) and considering the stained areas Table 1 Descriptive, stratigraphic, chronologic and taphonomic data of the sampled specimens from Locus 2 of Cuvieri Cave. WS, weathering stage; HI, histological index; MFD, microscopical focal destructions.

4 E.L. Mayer, et al. Palaeogeography, Palaeoclimatology, Palaeoecology 537 (2020) 109372 included in the score. The index value 0 represents < 5% of intact bone cervid metatarsal CVL2-12008 (BSL facies) and tayassuid metacarpal with no original features identifiable, whereas the index value 5re- CVL2-15251 (RBSL facies) because they were not fused to their represents > 95% of intact bone appearing very well preserved. The MFD spective bone shafts (Fig. 3A). Cervid metacarpal CVL2-14964 and were divided in two categories, Wedl MFD, and non-Wedl MFD metatarsal CVL2-15472 are fragmented and consist only of the prox- (Hackett, 1981; Jans et al., 2004). The identification of MFD follows imal and medial regions, which reveal weathered fractures (both from criteria based on morphology, size and shape, which was proposed by BSL facies; Fig. 3; Table 1). Hackett (1981) and described by Jans et al. (2004), Turner-Walker and The Histological Index indicates that the bone histology of the Jans (2008) and Fernández-Jalvo et al. (2010). The Wedl MFD consists sampled specimens is poorly preserved (Table 1). Both specimens of of irregular tunnels, branched or not, with a diameter ranging from 5 to Tayassuidae, CVL2-6711 (BSL facies) and CVL2-15251 (RBSL facies), 10 μm (Hackett, 1981). The non-Wedl MFD includes three types of scored an index value 0. The remaining four specimens that were ex- tunnels named linear longitudinal, budded and lamellate, which reflect amined, all representing Cervidae from BSL facies, have an index value the same phenomenon through different perspectives of bone micro- of 1 (Table 1). In these specimens, the few and small areas where os- architecture. The linear longitudinal tunnels appear in transverse sec- teons can be recognized are located in the mid- and outer cortical re- tions as small round foci 5–10 μm in diameter, however, foci of a wide gions. range of sizes to more than 50 μm are seen where tunnels present some The microscopic focal destructions (MFD) identified include non- widening and branching (Hackett, 1981). Budded tunnels present Wedl and Wedl categories (Fig. 5; Table 1). The generalized alteration frond-like tunnels developed from and around Haversian canals of of the bone tissue difficults distinguishing the different types of the non- secondary osteons, and range from 20 to 50 μm in diameter. They ap- Wedl MFD, but they are recognizable in the inner and mid-cortical pear in transverse section as rounded, stippled foci (Hackett, 1981). In regions of almost all the specimens (CVL2-1915, CVL2-12008, CVL2- transverse section the lamellate foci are round or curved structures that 14964 and CVL2-15472 from BSL facies; and CVL2-15251 from RBSL develop parallell to the circular pattern of the lamellae and range from facies; Fig. 5A,C-E). The Wedl MFD was documented in CVL2-15472 10 - 250 μm (Hackett, 1981). The diagenetic microcracks were de- where they were widespread throughout the inner, mid- and outer scribed morphologically and compared with those present in Haversian cortical regions (Fig. 5B), and possibly in CVL2-6711 (Fig. 5F). The bone tissue of many vertebrate fossils that had fossilized in aqueous or histological structure is extensively altered and the osteon limits and terrestrial arid climate conditions (Pfretzschner, 2000, 2004; MDF are difficult to distinguish in CVL2-6711 and CVL2-15251 Pfretzschner and Tütken, 2011). The diagenetic microcracks that (Fig. 5G). In CVL2-14964, several osteons are packed with linear formed under water are found peripheral to the osteon and oriented longitudinal tunnels with smooth round borders (Fig. 5A). In CVL2- radially across the cement lines of the secondary osteons. They cross the 12008, linear longitudinal tunnels appear to be stained black and some cement lines and connect with adjacent osteons (Pfretzschner, 2004). osteons display stained tunnels with irregular and cracked borders Under arid conditions, central radial microcracks form from the Har- (Fig. 5C–E). vesian canal outwards (Pfretzschner and Tütken, 2011). If desiccation is Central radial cracks are present in at least some osteons of all the prolonged, osteons may be isolated from adjacent structures by cir- specimens studied (Table 1). Tayassuid specimens have long central cumferential cracks and small peripheral cracks appear in the osteon radial cracks running bilaterally from the few recognizable secondary boundary (Pfretzschner and Tütken, 2011). To distinguish the occur- osteons through regions of poorly preserved bone tissue (CVL2-6711, rence of the features described above into specific regions, we used BSL facies and CVL2-15251, RBSL facies; Fig. 6A and B; Table 1). Cervid arbitrary divisions of the cortical bone histology: inner cortical region, specimens have more than two inconspicuous central radial cracks mid-cortical region and outer cortical region. running from the secondary osteons that are distributed across patches The sections were prepared following the methods described in of well-preserved bone tissue (CVL2-1915, CVL2-12008, CVL2-14964, Botha (2003) with some modifications. The midshaft region of each and CVL2-15472, all from BSL facies; Fig. 6 E, F; Table 1). In the cervid bone was embedded in epoxy resin, Technoresin® LR151 Epoxy, under specimens (CVL2-1915, CVL2-12008, CVL2-14964, and CVL2-15472, vacuum using a Struers® CitoVac to prevent the bone from fragmenting all from BSL facies; Fig. 6 C–H; Table 1) another type of microcrack during the process. After the resin had set, 2 mm thick sections of the conects groups of non-Wedl MFD and/or histological structures such as midshaft region were cut using a diamond-tipped saw in a Struers® osteocyte lacunae and canaliculli. CVL2-12008 shows that black stained Accutom-100 cutting and grinding machine. The cut surface of the longitudinal tunnels close to one another are transversally linked by sections was polished until smooth using sand paper and mounted onto microcracks (BSL facies, Fig. 6C and D). The histological structures in a petrographic glass slide using resin adhesive, Epolam® 2022. Pressure CVL2-1915 (BSL facies) have been altered by microcracks, and some of was applied to the sections to avoid bubbles. The 2 mm thick sections them are linked to non-Wedl MFD as follows: i) inconspicuous central were polished using a cup wheel in the grinding mode of the Struers® radial cracks in the Haversian canals of secondary osteons; ii) possible Accutom-100 until smooth to generate thin sections observable by enlarged osteocyte lacunae; iii) osteocyte lacunae with lateral micro- transmitted light microscopy. The thin sections were examined using a cracks; iv) enlarged lacuna-canalicular system resembling the letter “T”; Nikon® Eclipse 50i POL microscope and the bone histology was pho- v) bundles of enlarged canaliculi; vi) longitudinal tunnels with cracked tographed using a coupled Nikon® digital camera. borders; vii) longitudinal tunnels linked by microcracks; viii) enlarged osteocyte lacunae linked by cracks; ix) microcrack segments linking 3. Results histological structures (in this case osteocyte lacunae and canaliculi) to taphonomic features (i.e. longitudinal tunnels) (Fig. 6 E, G-H). The The macroscopic examination found weathering cracks compatible same alterations can be seen on a broader scale throughout the bone with stage 1 in almost all the sampled specimens, except the metacarpal tissue (Fig. 6 F). Several microcrack segments in this specimen also of Tayassuidae CVL2-15251 (RBSL facies), which is consistent with appear to run along the cement lines, but deviate from them in certain stage 0 (Figs. 3 and 4; Table 1). Cervid metacarpal CVL2-14964 (BSL places (Fig. 6 G, H). facies) presents blackish stained areas on the thick section surface that are small and narrowly distributed around the weathering cracks 4. Discussion (Fig. 3B). The blackish stained areas on the thick section surface of cervid metatarsal CVL2-15472 are larger than in CVL2-14964, and Before discussing our results it is important to present the back- distributed beyond the adjacent area that contains the weathering ground in which it is framed. Our study assumes that all postmortem cracks (both from BSL facies; Fig. 3B). The other sampled specimens do changes observed, either macroscopic or microscopic, were generated not present these characteristics. The distal epiphyses are lacking from on bones inside Locus 2. The main reason for this assumption is that we

5 E.L. Mayer, et al. Palaeogeography, Palaeoclimatology, Palaeoecology 537 (2020) 109372

Fig. 3. Specimens from Locus 2 of Cuvieri Cave. A, initial weathering cracks indicated by arrows, except on CVL2-15251. CVL2-1915, lateral view of Cervidae left metatarsal; CVL2-6711, mesial view of Tayassuidae right metacarpal; CVL2-12008, lateral view of Cervidae right metatarsal; CVL2- 14964, ventral view of Cervidae right metacarpal; CVL2-15251, dorsal view of Tayassuidae right metacarpal; CVL2-15472, ventral view of Cervidae right metatarsal. B, thick sections of the specimens in A positioned with the dorsal face upwards.

have solid evidence that the main process of bone emplacement in has destroyed parts of the bone tissue, causing the disintegration of Locus 2 was the accidental entrapment of individuals inside the pit overall structure. Consequently, the bone microstructure is very poorly (Hubbe et al., 2011). Additionally, we consider the bones to have been preserved (low values of the Histological Index) and the physical exposed on the surface inside Locus 2 for a relatively long period of properties of the specimens have been altered. time because the sedimentation rate estimated for at least the BSL facies The further diagenetic processes were influenced to some degree by is ~7 cm/1000 yr; (Hubbe et al., 2011). This sedimentation did not the increased porosity resultant from the action of microorganisms on occur at a constant rate, but there is no evidence that it happened in a bones. This implies that the specimens had greater contact surface single spike of sedimentation. Thus, it is reasonable to assume that available for the exchange with the environment, with consequences for because the bones were deposited subhorizontally and there is no evi- bone weathering and chemical changes. On the one hand, the increase dence of major vertical displacement, the roughly one cm thick bones of specimens’ porosity makes wetting-drying effects prominent and the would have required approximately 150 years to be buried. During the repeated swelling-shrinking has caused the cracking at macroscopic and deposition and burial of the bones the environmental conditions in microscopic scales. On the other hand, the increased porosity also fa- Locus 2 were influenced by the hydrological regime of recharge, with voured the flow of groundwater between the environment and thein- potential phases of prolonged humidity. terior of the bones because it usually begins limited to the channels naturally present in bones (Hedges and Millard, 1995; Pfretzschner, 4.1. Postmortem changes and bone microstructure 2000; Turner-Walker, 2008). Unlike the uncertainty about when the action of the microorganisms The degradation by microorganisms was extensive. We found began, bone weathering may have started to affect the specimens im- mediately after the death of each individual (Kendall et al., 2017). abundant non-Wedl MFD in all specimens, whereas the Wedl tunnels were recorded only in CVL2-15472 (Cervidae, BSL facies) and possibly Weathering tends to have a cumulative effect on bones (Behrensmeyer, 1978; Pokines et al., 2018) and the apex of exposure of the specimens in CVL2-6711 (Tayassuidae, BSL facies). However, it is important to note that our results may be biased due to the fact that non-Wedl MFD during this process may have occurred close to its burial, after some microbial degradation has already take place. Microbial degradation are easier to recognize than Wedl tunnels in extensively altered mi- crostructures, as is the case in Locus 2. It may be related to the possi- would have contributed to weathering as the increased porosity caused by microbial tunnelling aided in the development of weathering cracks. bility that Wedl and non-Wedl MFD are different expressions of bacterial tunnelling formed as a function of the tunnelled tissue On the bone surface the cracks appear as long and narrow longitudinal features (stage 1; Behrensmeyer, 1978), but they may extend as far as microstructure (see discussion in Kendall et al., 2017). We have no evidence to say exactly when bacterial decomposition the inner cortex. Together, the aperture length and depth of the weathering cracks disrupted different regions of the bone cortical began. However, it may have started before much time had passed after the death of each individual inside Locus 2. If the tunnelling bacteria structure, which was already weakened by the increased porosity. Desiccation stress also affected the bone microstructure by inducing come from the individual’s gut microbiome, bone decomposition probably started early. Alternatively, if the source was the depositional the formation of microcracks on histological structures (e.g. Haversian environment, bacterial attack could be delayed. Regardless of where canals of secondary osteons and osteocyte lacunae) and on previously they originated, microbial activity decreased as the availability of or- formed microbial tunnelling (e.g. non-Wedl MFD). In some cases, long ganic components in the bones decreased (Hedges, 2002; Kendall et al., microcracks link the histological structures and microbial tunnelling. 2017; Pfretzschner, 2004) and the complete decomposition of collagen Water loss in the area around the Haversian canals of secondary osteons usually requires thousands of years (Kendall et al., 2017). Thus, there is results in a volume decrease that causes the formation of central radial a relatively long time spam at which the bacteria began to act. cracks around the canals due to shrinkage (Pfretzschner and Tütken, The action of microorganisms on bones was important for altering 2011). In our study sample, this process seems to have also created the microcracks observed in other histological structures, such as osteocyte the histologic structure in general. The widespread distribution of non- Wedl MFD throughout the cortex of the bones contributed to the de- lacunae, and in the microbial tunnel borders. This resulted in the het- erogeneous dissipation of the tension in the bone tissue, depending on struction of many parts of the histology itself and to undermine the overall bone microstructure. The non-Wedl MFD formed a maze of tiny the distribution of the histological structures and microbial tunnelling. It is possible that the long microcracks represent a preliminary stage of tunnels that extended through the tissue in different regions of the specimens and result in increased porosity. The formation of tunnels circumferential crack formation, which is associated with prolonged

6 E.L. Mayer, et al. Palaeogeography, Palaeoclimatology, Palaeoecology 537 (2020) 109372

humidity inside Locus 2, the specimens were not exposed to conditions compatible with flooded environments. In these conditions, the cement lines that delimit the secondary osteons are broken by many microcracks that run perpendicularly to the central canal of the osteon due to swelling caused by the absorption of large amounts of water (Pfretzschner, 2004), but we observed no such features in our sample. Due to the low sedimentation rate inferred for the BSL facies deposition (i.e. ~7 cm/1000 yr; Hubbe et al., 2011), the bones remained exposed inside Locus 2 for prolonged periods of time prior to burial. Thus, microbial activity most likely had its greatest development and decrease before the complete burial of bones. On the other hand, although the weathering process also occurs primarily before burial, it may persist after burial in the Locus 2 deposit. Since skeletal remains were not exposed to weathering factors outside of the cave (i.e. solar radiation, and broad temperature oscillation between day and nigth), the bone weathering present in our sample is interpreted as being due to swelling and shrinkage stresses of the bone material as consequence of wetting and drying cycles (Pokines et al., 2018). The occurrence of these cycles is in agreement with the hydrological conditions inferred for Locus 2 (see Introduction). Weathering conditions in caves differ from those on the surface, particularly for the bones buried inside caves. Thus, it is possible that the occurrence of wetting and drying cycles contribute to maintaining the process of bone weathering active after the burial of the specimens on Locus 2 (Pokines et al, 2018). Similarly, it is plausible that microcracks, as microscopic postmortem changes associated with bone weathering, may also have continued to form, even after burial (Fernández Jalvo et al., 2010; Pfretzschner and Tütken, 2011; Kendall et al., 2017). This interpretation agrees with the available information on the hydrology of Locus 2 fluctuating humidity levels should have led to wetting and drying cycles. The sediments and bones in the top and subsurface layers of the deposit (i.e. first few centimetres) may have been drained between periods of high humidity as these layers drain the most quickly due to the percolation of water to deeper levels and evaporation through the main conduit of the cave that communicates Fig. 4. Transverse sections of specimens from Locus 2 of Cuvieri Cave showing with the outer surface and the small passages in the ceiling. This may the extension of the weathering cracks. A, normal light image of Cervidae have occurred differentially between the upper and lower parts of specimen CVL2-1915 showing two weathering cracks reaching the boundary bones; the former was placed above the air/sediment interface and area between the MCR and ICR. B, normal light image of Tayassuidae specimen CVL2-6711 showing one weathering crack reaching the MCR. C, normal light presented greater potential for desiccation than the later. As the de- image of Cervidae specimen CVL2-12008 showing one weathering crack position of the BSL facies was gradual, the layers currently positioned reaching the boundary area between the MCR and ICR. D, normal light image of more deeply in the deposit represented the surface and subsurface Cervidae specimen CVL2-14964 showing two weathering cracks, one limited to layers in a given moment of the past. Hence, the bones and sediments the MCR and the other reaching the boundary area between the MCR and ICR. found in different depths were subject to desiccation, as mentioned, E, normal light image of Cervidae specimen CVL2-15472 showing two weath- more intensively during its turn in surface and subsurface position ering cracks, one reaching the boundary area between the MCR and the ICR and through the formation of the deposit. the other entering the ICR. ICR, inner cortical region; MC, medullary cavity; Based on our analyses, we found that the bone histological structure MCR, mid-cortical region; OCR, outer cortical region. of the specimens from different depths of the deposit, and different ages, were similarly altered. This was due to an alike set of postmortem exposure of the bones on the surface under dry conditions (Pfretzschner changes associated with processes involving mainly biological and and Tütken, 2011). If water loss continues via the Haversian canals of physical factors. After each individual died in Locus 2, the factors re- secondary osteons after the formation of central radial cracks, the lated to the diagenetic processes addressed were increasingly influential complete secondary osteon builds up shrinkage stress and separates by before the burial, probably decreasing during the rest of bones’ ta- a circumferential crack from the surrounding bone (Pfretzschner and phonomic history. These processes may have been exacerbated since Tütken, 2011). In our study, the depositional environment did not they appear to be linked to one another, such as verified elsewhere present arid conditions as verified by Pfretzschner and Tütken (2011). (Kendall et al., 2017), thus ultimately increasing the amount of diage- Additionally, the specimens were not kept dry for periods as long as the netic alteration. It is important to be clear that we analyzed a small materials analyzed by Pfretzschner and Tütken (2011). Therefore, we sample in this study. However, our results, although preliminary, will found that microcracks can form as a response to desiccation stress hopefully guide future endeavours on bone diagenesis in cave deposits. under environmental conditions that are different from the extreme represented by aridity. In spite of the fluctuating moisture and possible 4.2. Implications of caves as depositional environments periods of prolonged humidity inside Locus 2, other factors likely contributed to the loss of water from bones. As noted above, the water The fossiliferous deposit of Locus 2 of Cuvieri Cave presents char- present in the bones from Locus 2 could have evaporated through the acteristics that allowed us: i) to recognize that the animals died after cortical vascular canals, the enhanced porosity consequent of non-Wedl being entrapped in its interior; ii) to infer the hydrological conditions to MFD and, through the macroscopic weathering cracks. which they were exposed and; iii) to investigate in detail the diagenetic The microcracks suggest that, even during periods of prolonged alteration of the histology of fossil bones. Thus, it is possible for us to

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Fig. 5. Examples of Microscopic Focal Destructions (MFD) present on the specimens from Locus 2 of Cuvieri Cave. A, cross-polarized light image of Cervidae specimen CVL2-14964 showing several non-stained secondary osteons packed with linear longitudinal tunnels (arrows) in the inner cortical region (ICRL). Normal light high magnification of the area in the dashed-line rectangle showing smooth round borders of the tunnels (arrows); B, Normal light image oftheCervidae specimen CVL2-15472 showing Wedl tunnels (arrows) widespread throughout the stained bone tissue in the mid-cortical region (MCR) with high magnification of the area in the dashed-line rectangle; C, cross-polarized light image of Cervidae specimen CVL2-12008 showing staining in the linear longitudinal tunnels (arrows) in the MCR and ICR; D-E, normal light high magnification of selected secondary osteons in C, showing stained tunnels with irregular and cracked borders (arrows). F,cross- polarized light image of Tayassuidae specimen CVL2-6711 showing histological structures extensively altered by indistinguishable MFD. G, cross-polarized light image of Tayassuidae specimen CVL2-15251 showing poorly preserved histological structure with many empty pores. discuss the influence of the depositional environment on the processes microorganisms, such as gut and soil bacteria (Mulec, 2008; Turner- that affected the skeletal remains which taphonomic history occurred Walker, 2008). Possible exceptions may be other entrapped vertebrates completely inside the cave. that survived the initial fall inside the pit (Kos, 2003a; Reed, 2006) or As the animals died after being entrapped inside Locus 2, their small vertebrates that eventually managed to access the bottom of the bodies were virtually protected from the action of processes that would pit (e.g. small rodents) that would have consumed part of the flesh of have occurred if they had died on the surface outside the cave, such as the carcasses. However, the vast majority of individuals deposited in necrophagy by large vertebrates (Lundelius, 2006; O'Brien et al., 2017). Locus 2 represent herbivorous taxa. A few omnivorous skeletons and With the deposition of their carcasses inside the pit, the action of other only one carnivorous individual have been excavated to date. In addi- vertebrates on their bodies was greatly limited or completely pre- tion, those that eventually scavenged a carcass inside Locus 2 probably vented. In this sense, practically all the biomass that comprised the soft also died in this pit because the walls are too steep to climb out and tissues was consumed by organisms that were able to access the corpses there were no escape routes. In the absence of competition with large in the bottom of the pit. Similarly, the bones, and their organic com- vertebrate scavengers, which could rapidly remove large portions of pounds, probably remained protected from exploitation by vertebrates. flesh and other soft tissues from the carcasses (O'Brien et al., 2017), Each corpse represented a rich, but ephemeral source of biomass invertebrates and microorganisms would have found abundant sub- amidst the scarce nutrients available in cave ecosystem (Barton, 2006; strate for their consumption and proliferation. Hence, the entrapment Mulec, 2008). It is probable that the soft tissues of each carcass were of the animals in the depositional environment at the bottom of the pit consumed mainly by invertebrate scavengers that inhabited the cave may have favoured the intense action of microorganisms, at least bac- environment (Ferreira and Martins, 2010) and by decomposing teria, on carcasses, including its bones.

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Fig. 6. Specimens from Locus 2 of Cuvieri Cave showing microcracks. A-B, normal light images of Tayassuidae specimens CVL2-6711 and CVL2-15251 showing discrete central radial cracks in a few recognizable secondary osteons (arrows) in areas of poorly preserved, stained bone tissue. C, cross-polarized light image of Cervid specimen CVL2-12008 showing black stained longitudinal tunnels transversally linked by microcracks (the microcracks path is developing between the ends of the arrows in pairs); D, normal light image of the same area in C, showing that the stained tunnels are linked by microcracks. High magnification shows the area indicated by the dashed-line rectangle; E, normal light image of Cervid specimen CVL2-1915 showing recognizable histological structures, some of them altered as follows: 1, radial crack in the Haversian canals of secondary osteons; 2, possibly enlarged osteocyte lacuna; 3, osteocyte lacuna with lateral microcracks; 4, enlarged lacuna-canalicular system; 5, bundles of enlarged canaliculi; 6, longitudinal tunnels with cracked borders; 7, longitudinal tunnels linked by microcracks; 8, enlarged osteocyte lacunae linked by cracks; 9, microcrack segment linking histological structures to non-Wedl MFD; F, normal light image of secondary osteons near the area in E (dashed-line rectangle), showing the occurrence of the same alterations on a broader scale over the bone tissue of CVL2-1915. Haversian canals of secondary osteons with short radial cracks are indicated by arrows. G, normal light image from another region of the specimen in E (CVL2-1915), showing the enlarged lacuna- canalicular system resembling the letter “T” more clearly (4) and bundles of enlarged canaliculi (5). Some microcrack segments (9) running along the cement lines (indicated by CL and a dotted line parallel to the trajectory), but they deviate from them in certain areas (arrows). H, high magnification of the area in the dashed-line rectangle in G (bundles of canaliculi not indicated), showing a long microcrack on the right (indicated by CL and a dotted line parallel to the trajectory) that deviates from the cement line (arrow) and on the left, a short microcrack extending parallel to the cement line.

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The analysis of the specimens and their sedimentary context reveal 5. Conclusions the occurrence of contrasting hydrological circumstances (i.e. bones presenting macro and microcracks due to desiccation and sediments The specimens from Locus 2 of Cuvieri Cave present a set of post- deposited by descending water from the cave ceiling) that lead to the mortem changes affecting the histological structure of the bones found occurrence of bone weathering inside Locus 2. Bone weathering is a within the cave. The changes follow a sequence of diagenetic events complex phenomenon, and humidity, temperature, and solar UV ra- related mainly to biological and physical processes. The effect of each diation have been cited as the main environmental conditions involved process on the bones vary at different stages in the taphonomic history (Behrensmeyer, 1978; Lyman and Fox, 1989; Pokines et al., 2018, and the diagenetic alterations appear to be interlinked causing each to Kendall et al., 2017). Concerning cave environments, the issue of bone effect the other, such as verified by previous studies (e.g. Kendall et al., weathering is even less understood. In spite of a few attempts to in- 2017 and references therein). The bacterial decomposition altered the vestigate this topic, there is increasing fossil (e.g. Kos, 2003b; Lam, bones extensively, creating a maze of non-Wedl MFD that altered the 1992; Lyman, 1988; Mayer et al., 2016 and Mayer et al., 2018; Reed, bone mechanical properties by the increase of porosity during early 2006; Dirks et al., 2015; this study) and experimental (Pokines et al., diagenesis. Bone weathering can occurs inside caves and, even if lim- 2018) evidence favouring the idea that bone weathering occurs in cave ited to the initial stages, caused degradation on both the macro- and environments. Recent experimental findings greatly contributed to this microscopic scale, where deep macroscopic cracks and abundant mi- issue, by demonstrating that repeated wetting and drying cycles, with crocracks allowed water to be absorbed into the bones resulting in bones not being subject to direct solar UV radiation, freeze-thaw cycles, swelling and shrinkage. Diagenetic microcracks were formed under or high degrees of thermal expansion-contraction, can lead to the de- environmental conditions different from the extremes for which they velopment of subaerial weathering features on bones (Pokines et al., have already been described (i.e. arid and aquatic environments; 2018). Pfretzschner, 2000; Pfretzschner and Tütken, 2011). Continued swel- As inferred from these findings, a plausible depositional scenario ling and shrinkage can lead to further alteration where long microcrack could be depicted for bones exposed on the ground in environments link Haversian canals of secondary osteons and osteocyte lacunae as with similar conditions to those of the experiment, such as karst fea- well as the tunnels formed by bacteria. tures (Pokines et al., 2018), as is the case at Locus 2. This is because Bones deposited in caves are subject to specific sets of environ- bones deposited inside caves may experience subaerial weathering due mental conditions that in tropical regions may be very destructive to to seasonal fluctuations in moisture, causing wet-dry cycles (Pokines bone microstructure. The deposition of bioclasts during the entrapment et al., 2018). However, it is currently unknown if fluctuations in hu- of individuals in caves and the entering of microorganisms via the se- midity inside karst features can result in enough wet-dry cycles to cause diments transported from the cave ceiling favours the proliferation of bone weathering (Pokines et al., 2018). This seems plausible in caves bacteria on bone and is important in directing the events of diagenetic located in tropical regions and having a recharge hydrological regime alteration. The formation of macroscopic and microscopic cracks re- and a low sedimentation rate, as seems to be the case in Cuvieri Cave. In lated to bone weathering in caves shows that the taphonomic processes addition, in spite of speculation that bone weathering inside caves oc- that occur in these environments are poorly understood, highlighting curs at a slower rate than would be expected from bones deposited in the need for more research on cave taphonomy. The conclusions ob- surface terrestrial ecosystems (e.g. Mayer et al., 2016; Mayer et al., tained may be valid for other Quaternary fossiliferous deposits formed 2018), it is important to keep in mind that environmental conditions in caves and that share similar characteristics. can vary considerably in different locations of the same cave, and among different caves. For example the size and number of caveen- Acknowledgements trances and passages, as well as the level of the water table can cause great environmental variation (Tuttle and Stevenson, 1978). We would like to express our gratitude to José Hein (Matozinhos, The depositional scenario deduced from the weathering experiment Minas Gerais, Brazil) for kindly allowing us to conduct our field ex- conducted by Pokines et al. (2018) appears similar to what is inferred cursions on his property. IBAMA and IPHAN provided the permits ne- for Locus 2. Although the frequency of rainfall events that occurred in cessary for our investigation. We also thank the Fundação de Amparo à Locus 2 cannot be predicted and potentially took longer than a season Pesquisa do Estado de São Paulo for long-term research support in to occur, the wet-dry cycles would still have affected the bones de- Lagoa Santa region (grant 04⁄01321-6) and scholarships to AH (06/ posited in the cave. Thus, the presence of stage 1 weathering cracks in 51406-3 and 08/58554-3), PH (06/61297-7) and EM (07/53185-7 and bones of articulated and clustered skeletons found in Locus 2, inter- 09/03753-4); Conselho Nacional de Desenvolvimento Científico e preted as coming from entrapped individuals, possibly represents an Tecnológico) for financial support to EM (CNPq 140577/2014-9); the empirical finding of what was postulated from experimental evidence National Research Foundation (UID 98819), the Palaeontological about wet-dry cycles as a component of bone weathering in karst en- Scientific Trust (PAST), Johannesburg, South Africa, and the DST-NRF vironments (Pokines et al., 2018). Centre of Excellence in Palaeosciences (CoE-Pal) to JBB; and Danielle In general, the karst cave environment is chemically favourable for Eloff (Bloemfontein, South Africa) for the guidelines in preparing the the preservation of bones (Lundelius, 2006; Simms, 1994), both mac- thin sections. roscopically and microscopically (Hedges and Millard, 1995; Turner- Walker, 2008). However, depending on the temperature, pH, humidity, Appendix A. Supplementary data among other variables, which are determined by many factors such as the geographic location of the cave and sediment composition, the Supplementary data to this article can be found online at https:// microstructure of a bone may be lost (Hedges and Millard, 1995; doi.org/10.1016/j.palaeo.2019.109372. Turner-Walker, 2008). Thus, although karst caves located in tropical regions may be suitable environments for preserving the gross mor- References phology of bones, they may not be suitable for preserving the microscopic structure of bones. This scenario differs from that observed for Auler, A.S., Smart, P.L., Wang, X., Piló, L.B., Edwards, R.L., Cheng, H., 2009. Cyclic se- bones from dry cave sediments deposited under temperate climatic dimentation in Brazilian caves: mechanisms and palaeoenvironmental significance. Geomorphology 106 (1–2), 142–153. conditions, which present good histological preservation (Hedges and Barton, H.A., 2006. Introduction to cave microbiology: a review for the non-specialist. J. Millard, 1995; Nielsen-Marsh and Hedges, 2000a). Cave Karst Stud. 68 (2), 43–54. Behrensmeyer, A.K., 1978. Taphonomic and ecologic information from bone weathering. Paleobiology 4 (2), 150–162.

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