The diagnostic utility of microCT for assessing bioerosion in archaeological bone

Recent advances have broadened the application of palaeoradiology for non-destructive investigation of ancient remains. X-ray microtomography (microCT) in particular is increasingly used as an alternative to histological bone sections for interpreting pathological alterations, trauma, microstructure, and more recently bioerosion with direct or ancillary use of histological indices. However, no systematic attempt has been made to conrm the reliability of microCT for histotaphonomic analysis of archaeological bone. The objectives of this study are therefore to (1) compare thin sections of human femora rated with the Oxford Histological Index to microCT sections using a newly developed Virtual Histological Index, and (2) provide an accessible methodology for the evaluation and visualization of bioerosion in archaeological bone using virtual anthropology techniques. We provide detailed descriptions of virtual sections and volume renderings, and also assess the ecacy of the method on cranial and postcranial elements, cremated long bones, and faunal samples. Furthermore, the need for time-consuming image segmentation is reduced by applying two noise-reducing, edge-preserving lters, and rendering with a colormap chosen to visualize bioerosion along with canal structure and density in 3D. The histological and virtual methods showed a strong correlation, providing the rst systematic data substantiating lab-based microCT as a suitable alternative tool for reconstructing post-mortem history in the archaeological record, and for the reliable, non-destructive screening of samples for further analyses.


Introduction
As human remains offer unique perspectives on burial behavior in the past, the continued development and testing of bioarchaeological methods and techniques play an important role in re ning our understanding of long term taphonomic processes. In recent years there have been numerous histotaphonomic studies of archaeological human and faunal bone that have looked at bioerosion to understand the mechanisms by which a corpse turns into a skeleton (e.g., Booth, 2016 Human bone is a composite material consisting in its molecular structure of a mineral phase, namely hydroxyapatite (Ca 10 (PO 4 ) 6 (OH) 2 ), and an organic phase (~ 90 % collagen Type I, 5 % non-collagenous proteins, 2 % lipids) (Boskey, 2013), which presents macroscopically in two morphological forms: the compact cortical layer, and the loosely organized spongy matrix of cancellous (trabecular) bone. It can further be described histologically as mature (lamellar) and immature (woven), the latter of which is temporary, being replaced by mature bone during growth and by remodeling (White et al., 2012). Cortical bone, the focus of this paper, is the dense, hard shell that surrounds the medullary cavity of long bones and is bounded externally by the periosteum and internally by the endosteum. It is comprised of dense accumulations of lamellar type bone, which is-in cross-section under magni cation with polarized light-visualized as bundles of concentric lamellae between To date most research on microscopic taphonomic alteration to bone has been performed using histology via light microscopy or scanning electron microscopy (SEM) (e.g., Hackett, 1981;Garland, 1987;Grupe & Garland, 1993; Turner-Walker, 2012; Turner-Walker & Jans, 2008). However, recent technical developments enable high-resolution non-destructive 3D imaging of archaeological samples. X-ray microtomography (microCT, or XRM) is a non-destructive imaging procedure that computes a stack of cross sections through an object from a series of 2D radiographic projections. This 3D volume dataset is composed of volumetric pixels (voxels), each of which has a speci c grey-scale value representing local X-ray attenuation as well as accurate size calibration (Withers et al., 2021). The microfocus X-ray tubes used in lab-based microCT scanners provide excellent image resolution, down to a few µm or less, which permit the accurate description of the minute physical properties of both the internal and external skeleton (Moore, 2013;Scherf, 2013) without, in most cases, causing tissue damage (e.g., DNA degradation: Walton et al., 2015) Volume image data obtained from tomographic methods allow the rendering and analysis of 3D models using dedicated software. Studies in the elds of palaeontology ( Importantly, studies comparing microCT images with histology to evaluate skeletal trauma (Baier et al., 2019), pathological alterations (Rühli et al., 2007), bone morphometry (Müller et al., 1998;Uchiyama et al., 1997), and cortical bone microstructure (Particelli et al., 2012), identify strong correlations between conventional histological section methods and virtual assessments.
Therefore, comparing bioerosion in thin sections with virtual sections is a crucial rst step towards con rming the reliability of microCT for identifying microscopic taphonomic alterations to bone. The dual aims of this research are thus to (1) systematically compare transverse thin sections rated with the OHI to microCT scan images using a newly developed VHI, and (2) provide an accessible methodology for the evaluation and visualization of bioerosion in archaeological bone using virtual anthropology techniques. We describe bioerosion in unedited virtual cross-sections and in 3D volume renderings. Furthermore, we reduced the need for time-consuming image segmentation by sequentially applying two noise-reducing, edge-preserving lters and using an image-display transfer function (colormap) that visualizes bioerosion and canal structure and density in 3D.
In doing so we contribute a minimally destructive investigative tool for reconstructing post-mortem history in the archaeological record, and for the reliable screening of samples for further analysis.

Material And Methods
The dataset is based on 28 samples selected from ve archaeological sites in the temperate European environment of Lower Austria dating from the Early Neolithic to Late Iron Age (Table 1). Studies have demonstrated intra-skeletal variation for diagenetic alteration, and that the cortex of long bones tends to best describe bioerosion (e.g., Booth, 2017;Dal Sasso et al., 2014;Jans et al., 2004). To facilitate comparison with previous research (e.g., Booth, 2017;Jans et al., 2004;White & Booth, 2014), samples 1-2 cm thick were preferentially extracted from the anterior aspect of the proximal femoral diaphysis (n = 15) inferior to the surgical neck. Rib (n = 2), mandible (n = 1), skull (n = 2), and humerus (n = 1) fragments were also selected to evaluate the e cacy of the method on non-femoral samples (Supplementary Materials ESM1). Five cremated long bones and two faunal mammalian long bones were also sampled. Samples were extracted with a diamond wheel blade attached to a Dremel® 3000 electric drill. The histological investigation was conducted using the well-established OHI (Hedges & Millard, 1995;Millard, 2001), while the virtual assessment was completed using the newly developed VHI. Table 1 Provenance and number of 28 cortical bone samples by period: femora (n = 15 + 5 cremated), ribs (n = 2), parietal (n = 2), humerus (n = 1), mandible (n = 1), faunal long bones (n = 2 observers within a single rating unit. This approach is applied here where we performed inter-rater reliability (IRR) tests between the two OHIs, between the two VHIs, and between the averaged OHIs and the averaged VHIs using the Pearson correlation coe cient (PCC), joint probability of agreement (JPA), and limits of agreement (LOA). Overall IRR analysis, also known as interrater agreement or inter-observer reliability, is the degree to which there is agreement amongst independent observers rating the same phenomenon. The PCC is the linear correlation between two sets of data where it measures covariances, resulting in values ranging between -1 (highly uncorrelated) and +1 (highly correlated). JPA is the percentage of times that raters agree, and is the least robust measurement of IRR as it does not account for agreement based on chance. To compensate for this weakness, LOA was applied. This approach calculates the difference between each pair of observations with the mean value acting as the bias value ± 2*standard deviation (Bland & Altman, 1989). This type of analysis provides insight into how much random variation in uences the ratings, as depicted in the Bland-Altman plots. Furthermore, to assess the true scope of variation of agreement, the JPA calculations were evaluated for an exact match (identical observations), an approximate match (≤0.5 difference), and a general match (≤1.0 difference. This approach captures the degree to which observers entirely agree (disregarding the principle of equi nality), and with applied thresholds for when observers somewhat and generally agree.
Light microscopy (LM) and Scanning electron microscopy (SEM) Twenty-four samples were selected and embedded in a two-compound resin (Biodur® E1/E2) following a protocol for undecalci ed bone (Schultz, 2001 "white" colormap, for which intensity gradients below the colormap port minimum are rendered transparent, was found to be less informative than the "glow" colormap, whose arti cial shading and edge coloring provides a good contrast range Virtual Histological Index (VHI) The VHI (  Canals are smooth, more yellow than red, and in sharp contrast to void; no evidence for bioerosion between canals within the void.

> 85
Only minor amounts of destructive foci, otherwise generally well preserved.
Only minor amounts of destructive foci, otherwise generally well preserved; small patches of MFD present (darker grey values). The canals and lamellae are easily distinguished throughout the stack.
Canals are smooth, in sharp contrast to void, and primarily yellow; small patches of orange-red bioerosion between canals, and streaks within canals are evident, which may present as small clusters.
Destructive foci evident throughout stack but more than half of the bone remains unaffected; bioerosion spreads beyond lamellae in patches but the foci are still visible.
Canals may be smooth and still yellow in regions of minimal to no bioerosion but are surrounded by large clusters of bioerosion; some void remains; in regions where bioerosion clusters, canals are redder and may be di cult to distinguish from tunnelling.
Destructive foci may still be present but bioerosion now primarily presents in wide swathes of dark grey values in which lamellae are nearly indistinguishable; in small unaffected or less affected regions lamellae remain distinguishable; there is more affected bone than non-affected.
Bioerosion is now pervasive throughout. In heavily affected areas canals are redder, no longer smooth, and nearly indistinguishable from the surrounding bioerosion. Canals that are still visible may retain some smoothness in small pockets where bioerosion is weaker. Erosion is pervasive throughout and hardly any unaffected bone remains; lamellae are indistinguishable, only the slightest outline remains within the erosion; a small number of canals may still be present or may be completely obliterated.
Canals are red, thin, eroded, stringy, melted-looking, and present as an indistinguishable spongy texture, nearly or completely indistinguishable from bioerosion.

OHI with LM
No MFD can be found in the entire thin section. Many lacunae resemble fresh bone in their shape and condition, and osteocytes are clear; however, the majority display enlarged canaliculi indicative of Wedl tunnelling Type 2. A minority of Haversian canals are lled with a matrix that was not further identi ed. The circumferential lamellae of the periosteal layer are eroded while a layer of sediment covers the endosteal surface. There is a super cial area of brown staining along the periosteal surface.
Collagen content is very good with weaker birefringence in areas along the midline and towards the periosteum. There is a small concentration of micro ssures along the endosteal layer that follow the anatomy of a small number of osteons.

Virtual cross-sections
The bone is in nearly perfect condition with no bioerosion, only micro ssures originating from the endosteal and periosteal surfaces, some of which travel around or through the concentric lamellae towards the midline, as seen in the thin sections. These lamellae are distinct, and grey values remain consistent throughout and between slices. The endosteal and periosteal circumferential lamellae have aked off; matrix with higher density inclusions is attached to the surfaces with some in lling of canals as described in the thin sections.

Volume rendering
Canals are abundant, clearly de ned, and more yellow than red, though not as yellow as canals of other unaffected samples.
This likely results from the lower mineralization (lower density) associated with the younger age of this specimen (late juvenile) in comparison to, as will be seen, SB01 (Supplementary Materials ESM 4), which is a late adult. The canals are surrounded by void. The periosteal and endosteal borders are ragged where the circumferential lamellae have aked off.

Virtual cross-sections
The bone is in nearly perfect condition with no visible bioerosion. Grey values remain consistent within and between slices. The lamellae around Haversian canals are de ned as are the cement lines that surround them. There are inclusions within a small number of canals and attached to trabeculae, as noted in the histology, which are not to be confused with bioerosion.

Volume rendering
The preservation is excellent. Canals are abundant, clearly de ned, more yellow than red, and surrounded by void. The canals of this sample are more yellow than those of GÖ04 (also OHI/VHI 5), plausibly because of the increased mineralization of this sample, which also has cement lines (more highly mineralized cuffs surrounding concentric lamellae).

OHI with LM
The section is mildly affected by bioerosion with the highest concentration of MFDs present along the midline.

Volume rendering
The canals are smooth and yellow but are surrounded by small, orange-red thread-like clusters of MFD; upon zooming into the canals, thin areas of reduced density that present as reddish trails can be seen traversing the length of the yellow canals. These threads of bioerosion can be seen encircling and passing between canals. There are small perforations to some of the canals.
Bioerosion clusters in the midline, though much void remains. There are some inclusions in canals near the endosteum.

Volume rendering
Though most of the canals have become more red than yellow, they remain distinct from the surrounding bioerosion. The canals in less affected areas are still rather yellow and easily distinguished from microbial tunnelling, such as towards the periosteum. Patches of void remain in these areas.   The bone has undergone bioerosion in large irregular patches. Canals are present throughout the stack; the circumferential lamellae of the periosteal and endosteal surfaces have been shed. Bioerosion is present along the midline towards the periosteum more so than the endosteum. Large patches of bioerosion present as darker grey values; in these regions concentric lamellae are di cult or impossible to distinguish. In areas where bioerosion is arrested, MFD can be distinguished, and concentric lamellae remain visible. There is a thin micro ssure moving from the endosteum into the midline.

Volume rendering
Canals are still present, but their quality varies between regions analogous to what is found in the virtual cross-sections. The canals to the right and towards the endosteum are denser (more yellow) than those to the left of the image. However, the betterpreserved canals are patched with red, and it is di cult to clearly visualize their interiors. The canals that have undergone heavier microbial attack are less dense, redder, and withered. It is di cult to distinguish the eroded anatomical canals from microbial tunnelling. Although the circumferential lamellae have been heavily eroded, the layer that remains is still relatively dense (yellower), though patched with red. Some void remains, particularly towards the right. The bacterial attack is heavy throughout much of the section except for the periosteal surface. There is yellow staining along the periosteum along with enlarged canaliculi and lacunae. There is some remaining collagen in this area. The midline to the endosteal surface is heavily eroded. There is also some yellow staining along the endosteal surface. MFD is present along the circumferential lamellae. There are two embedding artefacts. Only Haversian canals are identi able.

Virtual cross-sections
The bone has undergone bioerosion in large irregular patches. Anatomical canals remain but bioerosion is pervasive throughout and between slices. Only the smallest islands and trails of unaffected bone remain; the bioerosion is now so heavy that individual MFDs cannot be distinguished. Lamellae surrounding Haversian canals can only be distinguished from the surrounding interstitial lamellae because they have been, presumably, preferentially attacked by bacteria and thus present as thick rings of darker grey values. Both the periosteal and endosteal circumferential lamellae have been sloughed. Embedding artefacts travel transversely along a section of the periosteum as noted in the histology.

Volume rendering
As found in the virtual sections, bioerosion is pervasive throughout the sample. It is di cult to discern anatomical canals from microbial tunnelling; both are visualized as red channels surrounded by brous red webs. Slightly less affected, slightly more yellow canals can be recognized towards the left of the image. Virtual cross-sections The sample has undergone extensive bioerosion with almost no unaltered bone remaining. Canals remain distinct but bioerosion is extensive throughout and between slices; however, the bone is also porous and demineralized due to osteoporosis. Many of the canals exhibit clear evidence of resorption such as Howship's lacunae and heavily irregular borders. During senescence remodeling of Haversian systems, including smaller ones, results in the focal enlargement of canals and their coalescence, with a concomitant reduction in concentric lamellae (Seeman, 2013) as found in this sample, which should not be confused with bioerosion. Patches of bioerosion (dark grey values) are present as are MFDs, which, upon closer inspection, appear to surround canals; however, because the concentric lamellae are indistinguishable, partly due to age, it is di cult to see in all cases that the MFDs are concentrated around the canals. The circumferential lamellae of the periosteum and endosteum have been sloughed.

Volume rendering
The bone is so heavily demineralized by both osteoporosis and bioerosion that very little is visualized by the volume rendering using the glow.col colormap speci cally, other than the faintest wisps of bone. Neither canals nor bioerosion are visible within the void. OHI with LM Macroscopic examination revealed the sample was cremated, which was con rmed by histology. It appears that there is no bioerosion, though discoloration makes the assessment di cult. Osteocytes are numerous and the lacunae are slightly enlarged. Howship's lacunae are also visible. There is recrystallization within the Haversian canals. The collagen signal is weak.
Overall, the classic characteristics of concentric lamellae are lost as the matrix becomes homogeneous due to the cremation (Hunger & Leopold, 1978;Schultz, 1986).

Virtual cross-sections
The analysis of the microCT images con rms that the bone was cremated; however, the evaluation was hindered by the inability to visualize certain anatomical features due to burning. Grey values are, save for high density (bright white) recrystallization, consistently a medium-dark grey throughout the stack. The periosteal circumferential lamellae are missing, though some remain along the endosteum. Canals can be distinguished throughout, though they appear smaller than usual by visual inspection. However, a morphometric assessment is required to verify this. It is demonstrated that burning at medium-to- all are dense (whiteish), which may be crystallization. The lamellae surrounding Haversian canals are obscured by burning but are more clearly visible towards the endosteum and along the midline than the periosteum. That they remain visible may be due to the slightly denser cement lines that are visible around some Haversian systems. What may be Howship's lacunae can also be seen.

Volume rendering
Cremated bone is more di cult to visualize with the glow.col volume rendering. The canals are red and have the appearance of translucent melted wax. Although there appears to be little to no bioerosion in the virtual section, the demineralization caused by cremation results in a melted appearance. Virtual cross-sections The bone has undergone moderate bioerosion. Some circumferential lamellae remain along the endosteum and periosteum; both surfaces have light grey, higher density bands as noted in the histology. Bioerosion manifests in patches relatively evenly throughout and between slices though it is slightly more concentrated along the midline. Although MFDs have spread throughout the sample, they remain distinct and do not generally present as undifferentiated dark grey patches. There are several embedding artefacts that primarily travel longitudinally from the periosteum. There are few canals, which is expected for plexiform animal bone, and the few Haversian lamellae that are visible are di cult to distinguish. There is potentially also osteon banding between two longitudinal micro ssures below the transverse crack along the midline.

Statistical Results
The following results (Table 3) show that exact matching within each histological index is at least 54 % but diminishes across all observations. However, it is useful to also assess approximate and general observational matches, as the histological indices are categorical across several criteria. This approach elucidates the rate of approximate agreement of minimal variation and minor variation. Assessing for these minimal disagreements (≤0.5) demonstrates a signi cant increase in observation agreement for both OHI and VHI ratings. Furthermore, a general matching with a threshold of ≤1.0 between observations demonstrates a signi cant increase of all categories, with a minimum of 93 % agreement. The results of our PCC analyses (Table 4) demonstrate a strong positive (0.94) correlation between the OHI as rated by two observers, a stronger positive (0.96) correlation between the VHI as rated by two observers, with a strong correlation overall (0.97) between the mean values of OHI and VHI observations. To account for observational agreement by chance, LOA analysis was performed. As histological indices are based on multiple criteria with multiple descriptive indicators, the problem of equi nality arises. This approach helps address the chance to which observers agree and assesses agreement based on genuinely similar observations using the same rating scale. The LOA analysis (Table 5) demonstrates that for the OHI, observer 1 measures slightly biased (0.25), whereas for VHI observer 2 measures negligibly biased (-0.02). These negligible differences indicate that both observers produce relatively similar results, with a trend of observer 1 rating marginally higher observations (4s and 5s) and observer 2 rating slightly lower observations (1s and 2s) (Fig. 8a & b). A 95 % con dence interval is used to determine the limits of agreement across observations (see Bland & Altman, 1989). For the OHI, the systematic difference is greater than the VHI observations ( Fig. 8a & 8b) as indicated by the upper and lower limit ranges. One observation (GÖ25) falls outside the LOA for the OHI assessment (Fig. 9a); however, this is the only differences where the observers rated a specimen signi cantly different. Similarly, VHI observations were consistently matching and observer bias is distributed evenly with 1 observation falling outside the limits of agreement: GE08 where observer 1 rated the sample higher than observer 2 (Fig. 9b).
Furthermore, the trend across observations is similar for both the OHI and VHI where observer 1 tends to rate more specimens higher and observer 2 tends to rate more specimens lower (Fig. 9c). However, the OHI illustrates a slightly more accentuated slope (y = 0.0639x + 0.0753) compared to the VHI (y = 0.0537x -0.1671), demonstrating the minor differences in observations between the extreme ratings. This further shows that the observers are more prone to rate at either end of the scale and with minimal inherent bias in one observer compared to the other. It has previously been noted that archaeological samples fall into a bimodal distribution on the OHI scale, between extensive microbial attack and its absence (Kendall et al., 2018;Millard, 2001), which "suggests that histological alteration tends to go more or less to ''completion'' if it happens at all" (Hedges et al., 1995, p. 203

Discussion
The IRR results demonstrate that the VHI evaluation of virtual sections is congruent with the OHI rating of histological thin sections. As noted, certain structures taken into consideration by the OHI, such as individual concentric lamellae, cannot be differentiated, nor can structures smaller than 1 µm (e.g., osteocytes or their lacunae) be visualized without synchrotron-based microCT (Andronowski et al., 2017a,b), but undifferentiated concentric lamellae, and features such as shallow pits and tunnels can be seen with lab-based microCT. Moreover, our inability to visualize osteocytes and their lacunae may result from a contrast-to-resolution issue during the scan. Thus, the VHI is able to describe the level of bioerosion that a sample has undergone, speci cally that which results from bacterial attack. It may be possible that non-MFD bioerosion can be detected and differentiated with microCT images; however, the samples included in this study almost exclusively exhibited MFD, and not (4) presence of micro ssures, and (5) birefringence intensity (see Fig. 1b). We found that MFD can be clearly visualized both in virtual sections and in 3D volumes, and can, moreover, be virtually measured (as can anatomical features) to re ne their identi cation by size (Fig. 10). Such measurements are possible due to the pixel-size calibration inherent in microCT scans. In badly affected samples it is di cult to ascertain which type of tunnelling is present, as the bioerosion presents as large dark grey patches. However, this problem is also encountered with thin section micrographs of badly preserved samples, and, moreover, though morphologically distinct, different MFDs are very likely, "all aspects of the same type of bacterial attack, differing only in tissue microarchitecture and local hydrology" (Turner-Walker, 2019, p. 35). We were also unable to visualize the Type 2 Wedl tunnels diagnosed via histology. This is likely due to the small size of the tunnels, which although they may be long, are not thick enough to be visualized at the resolution we used. While it may be possible to detect a stain after its having been recognized through histology, it is not immediately apparent using virtual images alone that a change in grey values results from, for example, the impregnation of organic or inorganic substances as manifested macro-or microscopically as a stain, or if changes to the grey values result from demineralization caused by bacterial attack. Furthermore, while dissolution and recrystallization of HAp can be visualized as changes in grey values, it is not possible to determine the uptake or exchange of speci c materials like uranium or uorine, or nitrogen content as identi ed via energy dispersive X-ray spectroscopy, nor can microCT visualize the hypermineralized cuffs surrounding tunnels that result from mineral reprecipitation as is speci cally achievable with SEM ( As concerns qualitative descriptions of how bacteria invade bone, we once again nd congruence between microscopy and virtual imaging. Yoshino et al. (1991) described regions heavily affected by bacteria and fungi as potentially corresponding to low X-ray density. As noted, areas of low density (dark grey values) that result from mineral dissolution and redistribution (Hackett, 1981)  Further research is thus required to con rm that bacterial attack is curbed by cement lines.
The volume rendering depictions of bacterial attack are worth particular mention as we were able, using the "glow" colormap, to visualize in 3D what has previously only been reported in 2D. It is hypothesized that bacteria disseminate through and enlarge the canalicular network, accessing collagen via Haversian and Volkmann canals (Hackett, 1981 While it is not possible to visualize bacterial attack on the canalicular network due to restrictions imposed by scan resolution, we saw evidence of bacterial attack within the canals. Sample GÖ01 (Fig. 11) provided a particularly interesting volume in which ne red trails follow the interior length of well-preserved yellow canals, the colors re ecting differences in X-ray density.
These are arguably the demineralized traces of bacteria that have entered the Haversian and Volkmann canals, consumed their soft tissues (i.e., blood and lymphatic vessels, and nerve bers), and then attacked the more mineralized concentric and interstitial lamellae. Indeed, we see further evidence of bioerosion in the form of miniscule holes that penetrate some canals   Bell, 1990;Garland, 1987;Grupe & Dreses-Werringloer, 1992;Hackett, 1981;Piepenbrink, 1986;Schultz, 1986) that illustrate MFD, the magni cation was not high enough to clearly visualize the bioerosion and that the cross-sections these authors used resulted in a view that resembles grape clusters, which do not match what can be visualized using longitudinal sections such as those published by Bell (1990). These grape-like clusters are also visible in the 3D volume of GÖ01 (see Fig. 4). When the volume is viewed from different angles it can be seen that these clusters are actually tunnels that run longitudinally and at angles through the bone (Fig. 12). This visualization is similar to, but clearer than what is seen at very high magni cation (>600×) with microscopy. MicroCT is a valuable tool in this respect, as it permits the selection and visualization of any location within a sample in any orientation. Samples scanned, for example, in the coronal plane can be virtually resliced to the transverse plane. The virtual dataset can also be rotated to align with any anatomical plane using landmarks. We chose to reslice our samples for assessment in the orientation of traditional thin sections for two reasons (1) to facilitate comparison with traditional micrographs, and (2) 3D visualization of the canals and erosion in planes that do not follow the orientation of the Haversian systems made it di cult to assess bioerosion patterns in a three-dimensional sample. Furthermore, samples can be virtually cropped to visualize smaller volumes. The capacity to reslice, rotate, and virtually crop or dissect samples, repeatedly if desired, is speci cally achievable with virtual but not conventional histologic methods. microCT images and con rmed that within a given stack that individual slices are homogeneously affected by bioerosion. While it appears probable that this holds true for the entire midshaft, further research should assess different regions of the femoral diaphysis to con rm homogeneity within the entire shaft, particularly when staining is present in patches.
Effects of skeletal sample region: Using microCT Dal Sasso et al. (2014) found that skull, rib, and femur fragments present with different levels of bioerosion with femoral samples being the best preserved, and further suggest that the poorer preservation of rib and skull bone microstructure results from the higher volume of trabecular bone and porosity associated with the latter, which can be more heavily affected by inclusions and MFD. In the current study we had both femoral and rib samples from only two individuals (GÖ01/GÖ24 and GÖ04/GÖ25), none of which were badly affected by bioerosion; thus, we can neither con rm nor refute their ndings. Nevertheless, our research suggests that, although the femoral diaphysis, which has a thick cortical layer, is preferred for visualizing canal structure and Haversian systems, the VHI can also be applied to mandibular, cranial, and rib samples. However, the volume renderings can be more di cult to interpret. Ellingham & Sandholzer, 2020; Hanson & Cain, 2007). While we are able to visualize dense (bright white) in lling in the scans, which may be recrystallization, as well as cracking, morphometric analysis is required to con rm shrinkage.
Effects of pathological alteration: With any histological evaluation familiarity with microanatomical structures is required to identify their presence and destruction by bioerosion. It is also important to have an understanding of, for example, hematologic and metabolic bone disease, and how these features are visualized in microCT images. Pathological alterations affect the visualization and interpretation of skeletal remains, including, we found, in volume renderings using the "glow" colormap. While it was still possible to apply the VHI to the virtual sections of sample S04 (see Fig. 6), a late-mature female, it was immediately clear that this individual was severely osteoporotic. The volume rendering, using the "glow" colormap, of heavily demineralized samples such as S04 visualizes very little. Thus, for heavily demineralized, or samples characterized by pathological changes, it is worthwhile to try a variety of colormaps to test which can be used to best visualize canal structure and bioerosion in 3D.

Conclusion
A strong correlation between histotaphonomy and virtual methods provides the rst steps towards substantiating microCT for assessing biogenic micro-taphonomy in archaeological bone. Though the evaluation of thin sections via light microscopy or SEM remains the best means to identify the presence and type of chemical and microbial diagenesis (e.g., collagen leaching using lambda lters, Wedl MFDs, and hypermineralized cuffs) affecting a sample, visualization with these methods entails time consuming, labor intensive, multi-step processes during any step of which artefacts may be accrued. Numerous 30-55 µmthick slices must be sectioned with a microtome from an embedded sample with small areas of each section assessed individually. Moreover, transverse histological thin sections permit visualization in only a single direction. Although SR-microCT (Caruso et al., 2020), which can provide superior resolution, has also been used, it is not as readily accessible as lab-based microCT scanners, the costs of which are within the reach of start-up grants. Using microCT several fragments of bone (or, if small enough, an entire skeletal element) can be scanned at once or in automated batches. While scan acquisition may take some hours, once scanned and reconstructed, the entire sample can be rather quickly and easily visualized in 3D using a colormap that reduces the need for time-consuming segmentation. Furthermore, specimens do not require embedding, sectioning, and polishing, though samples that have already been embedded can be scanned without a resultant decrease in the clarity of the visualization of microanatomy. The method can thus be applied, for example, to old samples from museum collections without the need for further destructive sampling.
This study has demonstrated that microCT can be used as a complementary method to histology for the rapid, accessible evaluation of bioerosion in archaeological bone, and for the screening of samples for further, more destructive analyses. The VHI method, like the OHI, remains subjective; however, the strong IRR results suggest that this is not a major problem.