Cecilia García-Campos

María Martinón-Torres

Laura Martín-Francés

Marina Martínez De Pinillos

Mario Modesto-Mata

Bernardo Perea-Pérez

Clément Zanolli

Elena Labajo González

José Antonio Sánchez Sánchez

Elena Ruiz Mediavilla

Elena Labajo Gonzalez

Jose Antonio Sanchez Sanchez

Claudio Tuniz

Jose María Bermudez De Castro

Contribution of dental tissues to sex determination in modern human populations

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche

INTRODUCTION

Determination of sex is an important aspect in forensic research, since accurate sex determination is one of the most important steps in the reconstruction of the biological profiles of human remains. Both archaeology and palaeoanthropology have also shown interest in this issue, not only to reconstruct ancient population demographic profiles, but because the variables traditionally used to determine sex have also been used to determine hominin taxonomy (Olejniczak et al., 2008a;[START_REF] Smith | Variation in enamel thickness within the genus Homo[END_REF]. On the other hand, sexual dimorphism studies also provide information about the life history, the evolution and behavior of a specific population and, as a result, of individuals (Alt, Rösing, & Teschler-Nicola, 1998;[START_REF] Greene | Advances in dental anthropology[END_REF].

In recent years, forensic specialists have developed different techniques to discriminate males from females since these allow them to exclude approximately half of the population during the identification processes. The pelvis has been long considered the most reliable skeletal remain for sex determination, on certain occasions with correct sexual diagnosis rates above 95% [START_REF] Ferembach | Recommendation for age and sex diagnoses of skeletons[END_REF][START_REF] Iscan | Determination of sex from the sacroiliac joint: A visual assessment technique[END_REF][START_REF] Luo | Sex determination from the pubis by discriminant function analysis[END_REF]. In addition, craniofacial and mandibular features have also shown high accuracy [START_REF] Ferembach | Recommendation for age and sex diagnoses of skeletons[END_REF][START_REF] Hu | Sex determination using nonmetric characteristics of the mandible in Koreans[END_REF][START_REF] Williams | Evaluating the accuracy and precision of cranial morphological traits for sex determination[END_REF]. However, the tendency of these bones to break down following deposition may complicate their assessment, particularly in the context of mass fatalities or catastrophic events, where these bones rarely appear complete. In this context, teeth provided a good alternative. Because of their chemical composition and the protection within the framework of the oral cavity, these anatomical structures are the best-preserved human remains.

Therefore, teeth can be considered useful research elements for such scenarios since there is a greater probability to recover them intact from skeletonized remains, and they take prominence when other skeletal parameters are unavailable.

Several researchers agree that the human permanent dentition is sexually dimorphic [START_REF] Feeney | Enamel thickness in Asian human canines and premolars[END_REF][START_REF] Harris | Tissue contributions to sex and race: Differences in tooth crown size of deciduous molars[END_REF][START_REF] Hillson | Dental anthropology[END_REF][START_REF] Saunders | Sexual dimorphism of the dental tissues in human permanent mandibular canines and third premolars[END_REF][START_REF] Schwartz | Sexual dimorphism in modern human permanent teeth[END_REF][START_REF] Stroud | Sexual dimorphism in mesiodistal dentin and enamel thickness[END_REF][START_REF] Zilberman | Sex-and age-related differences in primary and secondary dentin formation[END_REF]. In most contemporary human populations, males have larger teeth than females [START_REF] Alvesalo | The influence of sex-chromosome genes on tooth size in man: A genetic and quantitative study[END_REF][START_REF] Garn | Sex difference in tooth size[END_REF][START_REF] Harris | A principal components analysis of human odontometrics[END_REF][START_REF] Harris | Tissue contributions to sex and race: Differences in tooth crown size of deciduous molars[END_REF]Işcan and Kedici, 2003;[START_REF] Peckmann | Sex determination using the mesio-distal dimension of permanent maxillary incisors and canines in a modern Chilean population[END_REF], exhibiting the canine teeth the highest degree of dimorphism among the different tooth classes [START_REF] Acharya | Validity of the mandibular canine index (MCI) in sex prediction: Reassessment in an Indian sample[END_REF][START_REF] Acharya | Univariate sex dimorphism in the Nepalese dentition and the use of discriminant functions in gender assessment[END_REF][START_REF] Garn | Shape similarities throughout the dentition[END_REF][START_REF] Hillson | Dental anthropology[END_REF][START_REF] Lund | Gender determination by odontometrics in a Swedish population[END_REF][START_REF] Rao | Mandibular canine index-A clue for establishing sex identity[END_REF][START_REF] Schwartz | Sexual dimorphism in modern human permanent teeth[END_REF]. Mandibular canines are particularly interesting in sex determination of unidentified remains, since they are more resistant to periodontal disease and to severe trauma [START_REF] Roetzscher | Forensic and Legal Dentistry[END_REF]Nelson, 2014), which increases their chances of being recovered in a good state of preservations. For this reason, several researchers have employed mandibular canines to assess sex differences in size from samples of various geographic regions and diverse population groups [START_REF] Hillson | Dental anthropology[END_REF]Işcan and Kedici, 2003;[START_REF] Moorrees | Mesiodistal crown diameters of the deciduous and permanent teeth in individuals[END_REF][START_REF] Peckmann | Sex determination using the mesio-distal dimension of permanent maxillary incisors and canines in a modern Chilean population[END_REF]. In recent years, multivariate statistics, in general, and discriminant function analysis, in particular, have been explored with this objective in mind. Discriminant functions have successfully determined sex by using a combination of tooth sizes and root lengths [START_REF] Garn | Relationship between root lengths and crown diameters of corresponding teeth[END_REF], by using the ratio between canine width and intercanine distance [START_REF] Rao | Mandibular canine index-A clue for establishing sex identity[END_REF], or by obtaining diagonal tooth measurements (Cardoso, 2010;[START_REF] Ateş | Sexual differences in Turkish dentition[END_REF][START_REF] Peckmann | Sex determination using the mesio-distal dimension of permanent maxillary incisors and canines in a modern Chilean population[END_REF][START_REF] Zorba | Sex determination in modern Greeks using diagonal measurements of molar teeth[END_REF].

Moreover, other researchers have observed that dental tissue proportions in the permanent dentition also differ significantly between males and females [START_REF] Feeney | Enamel thickness in Asian human canines and premolars[END_REF]Harris and Hicks, 1998a;[START_REF] Saunders | Sexual dimorphism of the dental tissues in human permanent mandibular canines and third premolars[END_REF][START_REF] Schwartz | Sexual dimorphism in modern human permanent teeth[END_REF][START_REF] Stroud | Sexual dimorphism in mesiodistal dentin and enamel thickness[END_REF][START_REF] Zilberman | Sex-and age-related differences in primary and secondary dentin formation[END_REF]. Various studies support this idea, suggesting that dental tissue development is influenced by sex-linked genes [START_REF] Alvesalo | Sex chromosomes and human growth. A dental approach[END_REF][START_REF] Alvesalo | 47, XXY males: Sex chromosomes and tooth size[END_REF][START_REF] Alvesalo | Enamel thickness in 47, XYY males' permanent teeth[END_REF][START_REF] Guatelli-Steinberg | Dental crown size and sex hormone concentrations: Another look at the development of sexual dimorphism[END_REF][START_REF] Pentinpuro | Crown heights in the permanent teeth of 47, XYY males[END_REF][START_REF] Zilberman | Sex-and age-related differences in primary and secondary dentin formation[END_REF], which, in turn, to a certain extent, determines the amount of enamel and dentine contribution to total crown size. Crown tissue components have commonly been studied by looking at the two-dimensional physical cross-sections and X-ray projection radiographs of molars [START_REF] Grine | Enamel thickness of deciduous and permanent molars in modern Homo sapiens[END_REF][START_REF] Macho | Enamel thickness of human maxillary molars reconsidered[END_REF][START_REF] Martin | The relationships of the later Miocene Hominoidea[END_REF][START_REF] Schwartz | Sexual dimorphism in modern human permanent teeth[END_REF][START_REF] Stroud | Sexual dimorphism in mesiodistal dentin and enamel thickness[END_REF] whereas data on anterior teeth, and in particular on canines, are limited [START_REF] Feeney | Enamel thickness in Asian human canines and premolars[END_REF][START_REF] Saunders | Sexual dimorphism of the dental tissues in human permanent mandibular canines and third premolars[END_REF][START_REF] Schwartz | Sexual dimorphism in modern human permanent teeth[END_REF]. Some of these studies have identified a thicker enamel in female crowns was as the signal of sexual dimorphism [START_REF] Alvesalo | 47, XXX females, sex chromosomes, and tooth crown structure[END_REF][START_REF] Moss | Analysis of developmental processes possibly related to human dental sexual dimorphism in permanent and deciduous canines[END_REF][START_REF] Schwartz | Sexual dimorphism in modern human permanent teeth[END_REF]. However, other authors have pointed to variations in the amount of dentine as the main reason behind these differences between the sexes (Harris and Hicks, 1998b;[START_REF] Stroud | Sexual dimorphism in mesiodistal dentin and enamel thickness[END_REF]. The emergence of new techniques and methodologies, such as computed microtomography (micro-CT), might shed light on this issue.

On one hand, microtomographic techniques are considered more accurate than conventional radiographic techniques since they allow for standardized measurement planes [START_REF] Grine | An evaluation of dental radiograph accuracy in the measurement of enamel thickness[END_REF][START_REF] Olejniczak | Assessment of the accuracy of dental enamel thickness measurements using microfocal X-ray computed tomography. The Anatomical Record[END_REF]. The anatomy of canine teeth makes it difficult to define the bucco-lingual plane traditionally used in this kind of studies, in whose obtaining process small variations in the plane of orientation can result in large changes in tissue measurements values. This problem especially arises when physical cross-sections are used, in which it is difficult to locate the dentine horn before the tooth is cut. On the other hand, micro-CT does not require the partial destruction of the tooth, which makes easier to obtain accurate and wide enough samples for statistical comparisons [START_REF] Grine | Enamel thickness of deciduous and permanent molars in modern Homo sapiens[END_REF][START_REF] Macho | Enamel thickness of human maxillary molars reconsidered[END_REF][START_REF] Olejniczak | Assessment of the accuracy of dental enamel thickness measurements using microfocal X-ray computed tomography. The Anatomical Record[END_REF][START_REF] Saunders | Sexual dimorphism of the dental tissues in human permanent mandibular canines and third premolars[END_REF][START_REF] Schwartz | Taxonomic and functional aspects of the patterning of enamel thickness distribution in extant large-bodied hominoids[END_REF][START_REF] Smith | Variation in hominoid molar enamel thickness[END_REF][START_REF] Smith | Modern human molar enamel thickness and enamel-dentine junction shape[END_REF][START_REF] Smith | Brief communication: Enamel thickness trends in the dental arcade of humans and chimpanzees[END_REF]. In summary, the application of micro-CT to anthropological samples allows us to reconstruct both the external and internal structures of teeth in a nondestructive manner, as well as the virtual separation, and volumes and surfaces measurement, of the different dental tissues with great precision.

In the present study, we have focused on the microtomographic volumetric measurement of dental tissue proportions of mandibular canines, variables which have not been previously assessed in this tooth class, despite their potential relevance in the study of sexual dimorphism.

Therefore, the aims of this study are to: (1) determine the degree of sexual dimorphism in the dental tissue volumes and surfaces areas of mandibular canines; and (2) explore its potential for reliable sex determination through the application of discriminant function analysis. In addition, we also assessed the traditional two-dimensional (2D) variables of dental tissue components of the crown, whose results were contrasted against those obtained from previous ones [START_REF] Feeney | Enamel thickness in Asian human canines and premolars[END_REF][START_REF] Saunders | Sexual dimorphism of the dental tissues in human permanent mandibular canines and third premolars[END_REF][START_REF] Schwartz | Sexual dimorphism in modern human permanent teeth[END_REF]. Moreover, we assessed the discrepancies between volumetric results and 2D measurements, which have been already reported in literature in other tooth classes [START_REF] Benazzi | Technical note: Guidelines for the digital computation of 2D and 3D enamel thickness in hominoid teeth[END_REF]Olejniczak et al., 2008bOlejniczak et al., , 2008a)).

MATERIALS AND METHODS

Sample

The teeth studied here derive from 69 individuals (33 females and 36 males) of known sex and age (Table 1) of different geographic origin. A part of the sample was selected from the anthropological collection housed at the Escuela de Medicina Legal of Madrid. The complete sample includes 84 skeletons (24 females, 60 males) all individuals are adults aged between 20 and 55 years. The skeletons belong to individuals originating from of different regions of Spain, all born during the first half of the 20th century. In addition, our sample also includes canines from the South African modern human the Pretoria Bone Collection, permanently stored at the University of Pretoria [START_REF] L'abbé | The Pretoria bone collection: A modern South African skeletal sample[END_REF]. This collection comprises 139 adult individuals (23 females and 116 males) aged between 22 and 76 years. Finally, mandibular canines extracted at dental clinics in Sudan were also included. Information on the Sudanese collection can be found in [START_REF] Elamin | Malnutrition has no effect on the timing of human tooth formation[END_REF]. In the present study only one antimere per individual was included for analysis. A total of 54 mandibular canines (25 females and 29 males) showing a degree of wear smaller than three [START_REF] Molnar | Human tooth wear, tooth function and cultural variability[END_REF] were selected, in which the different variables were measured.

Category three is characterized by the obliteration of the apex at the incisal border and the presence of a dentine point instead. Moreover, from this sample two subsamples were created for statistical purposes. On one hand, 41 slightly worn teeth were used to create the discriminant functions, whereas, 13 teeth were used as a hold-out sample. Additionally, in order to assess the accuracy of the equations on extensively worn teeth, we also included another sub-sample of 15 teeth showing a higher degree of wear. This last sample included teeth with minimal dentine patch (degree of wear four) and with a more extensive dentine patch (degree of wear five). In no case was lateral enamel loss observed on the occlusal surface.

Micro-CT image acquisition

The Spanish and Sudanese specimens were scanned in two facilities. One part of the sample was scanned using a Phoenix v/tome/x s (GE Measurement & Control) microtomographic system housed in the Microscopy Laboratory of the Centro Nacional de Investigación sobre la Evolución Humana (CENIEH) in Burgos (Spain). The rest of the sample was scanned using the CTP-Mlab microCT located in the Multidisciplinary Laboratory of the International Centre for Theoretical Physics (ICTP) of Trieste (Italy) [START_REF] Tuniz | The ICTP-Elettra X-ray laboratory for cultural heritage and archaeology[END_REF]. All scans were performed with two 0.1-mm

Copper filters, 100-120 kV voltage and 110-140 µA amperage. The output images had a voxels size ranging between 17 and 21 µm. The South African specimens were scanned at the South African Nuclear Energy Corporation (Necsa), Pelindaba, using Nikon XTH 225 ST equipment, with the following parameters: 100 kV voltage and 100 µA amperage. The final volumes were reconstructed with an isotropic voxel size ranging between 40.8 and 50.8 µm. The subsequent image processing of each of the teeth was performed using Amira 6.0.0 software (Visage Imaging, Inc.).

Three-dimensional model generation and dental tissue measurements

Dental tissues (enamel, dentine-pulp complex) were semiautomatically segmented using the watershed segmentation tool and through manual editing. A non-local means filter was also applied. Small fractures and cracks were virtually filled in.

We followed the criterion put forward by [START_REF] Benazzi | Technical note: Guidelines for the digital computation of 2D and 3D enamel thickness in hominoid teeth[END_REF] for the tissue components isolation, considering the cervical line as the fundamental morphological feature to isolate the crown from the root. However, in contrast to the protocol proposed by these authors, the separation of both structures was made by hand as described below. First, each tooth was placed in its correct anatomical position (Figure 1a,c). Then, using the mesio-distal plane, we drew a straight line between the enamel maximum cervical extensions in the entire images stack (Figure 1b). Finally, we corrected the tissue assignment in the bucco-lingual plane in order to create a continuous curve line (Figure 1d). In this way, only the dentine contained in the enamel cap (Figure 1a) was considered as coronal dentine (Figure 1b), which was limited on the base by a homogeneous and smooth curve (Figure 1c) and isolated from the root (Figure 1d). Lastly, 3D tissue measurements were obtained using Amira 6.0.0 software. We examined the crown variables and indices previously described by [START_REF] Kono | Molar enamel thickness and distribution patterns in extant great apes and humans: New insights based on a 3-dimensional whole crown perspective[END_REF], Olejniczak et al. (2008b,b), and Skinner, Gunz, Wood, & Hublin, (2008a); Skinner et al. (2008b). The basal surface of the crown (BS), the root volume (Vr) and the total tooth volume (Vt) were also considered. For detailed explanations of the variables used in this study refer to Table 2. We did not reconstruct the different tissues in worn canines due to the lack of a standardized protocol in the 3D literature.

Table 2. [OES/EDJS] Scale free

Two-dimensional dental tissue measurements

To assess the histological crown area from a bucco-lingual plane, a virtual cross-section for each tooth was obtained following the methodology outlined in [START_REF] Benazzi | Technical note: Guidelines for the digital computation of 2D and 3D enamel thickness in hominoid teeth[END_REF], which introduces modifications to the previous protocol developed by [START_REF] Feeney | Enamel thickness in Asian human canines and premolars[END_REF]. The bucco-lingual plane here passes through two points located on the widest bucco-lingual bi-cervical diameter (typically the maximum cervical enamel extensions) and the dentine horn tip. To isolate the crown from the root, the line between the buccal and lingual cervical margins was determined as the lower limit of the crown section (Figure 2b). As some canines presented occlusal wear, crown reconstruction was carried out using open-source software (GIMP 2.8) and following the protocol proposed by [START_REF] Saunders | Sexual dimorphism of the dental tissues in human permanent mandibular canines and third premolars[END_REF] (Figure 2c). Finally, 2D tissue measurements were obtained using ImageJ software (v. 1.49i). The definitions of the 2D metrics and indices used in this study are presented in Table 3. We measured the variables in the bucco-lingual plane described by [START_REF] Martin | Significance of enamel thickness in hominid evolution[END_REF], and followed the protocols described by [START_REF] Feeney | Enamel thickness in Asian human canines and premolars[END_REF]. Statistical analyses were performed using SPSS software (v. 18.0, SPSS Science). First, standard descriptive statistics were calculated for each variable. The normal distribution was assessed using the Kolmogorov-Smirnov one-sample test. Levene's Test was then used to confirm the equal variance assumption. An Analysis of Variance (ANOVA) was employed to examine the possible differences between males and females. Additionally, the Analysis of Covariance (ANCOVA) was used. ANCOVA can be compared with ANOVA, but has the added feature that for each group, variance that can be explained by a specified covariate of interest can be removed. In this particular case we controlled for the effect of size (the area of the entire crown section in the two-dimensional approach; and whole tooth volume in three-dimensional one).

Means were determined to be significantly different at α = 0.05 level. A discriminant function analysis was performed to generate a set of univariate and multivariate functions from threedimensional data belonging to a subsample of 41 slightly worn teeth (20 females, 21 males). We suspect this technique could be especially useful in the sexing of fragmentary skeletal remains, which is why we attempted to minimize the number of dental variables needed. We created a function for each variable and indices (D1-D4) and for all of them combined (D5). Additionally, we generated multivariant equations using the Lambda Wilks stepwise variable selection using all the variables and indices (D6) as well as only the variables and indices from the crown (D7).

The stepwise discriminant function analysis calculates the optimal combination of variables and weights them to reflect their overall contribution to sex determination. Discriminant function scores were calculated using the raw coefficients multiplied by the corresponding dental dimensions plus the constant. The resulting score was compared with the sectioning point. If the score was greater than the sectioning point, the sex was determined to be male.

Finally, the discriminant functions were tested in the original sample and the leave-one-out method was chosen to calculate the cross-validation error index. The percentage of correct allocation accuracy in determining sex using the equations developed was also calculated for the hold-out sample: females and males separately, and for the whole of the sample, as well as for slightly worn and extensively worn teeth separately. Only the equations with high percentage correct assignments were selected and included in this study.

Inter-and intraobserver error

To estimate the interobserver error, a subsample of seven teeth were analyzed by two authors (CGC and MMM). Each tooth was measured three times by both researchers, who orientated, segmented and isolated the crown of each tooth, as well as, following the 2D and 3D methods of measurement described above, quantified each variable and index in an independent manner. The degree of error was assessed by calculating the inter-and intra-observer mean differences for each crown component measurement. The average intraobserver error for 2D measurements was 1.71% (0.60-2.81%), whereas the average interobserver error was 1.58% (0.85-2.31%).

Moreover, the average intra-observer error for 3D measurements was 1.59% (0.69-2.48%), whereas the average inter-observer error was 2.43% (1.72-3.15%). All values are within the accepted range.

RESULTS

Intrapopulation variability: Three-dimensional measurements

Descriptive statistics for the variables measured on each mandibular canine are provided in Table 4. Only the values from the lesser worn teeth (wear stage 3 or less) were used in the descriptive analysis and means comparisons (n = 54), excluding the variables total root volume (Vr) and basal surface (BS), which are not affected by wear. In these cases, we used the total sample (n = 69). The Kolmogorov-Smirnov test showed that all dimensions were normally distributed within each sex category. The results of the homogeneity of variance test indicate that the sample is statistically homogeneous for all dimensions and indices. All tooth dimensions were larger in males and the ANOVA showed that, except for the volume of the enamel cap (Ve), the eight direct variables measured were statistically larger for this sex (Table 4).

In light of these results, we hypothesized that size differences could be masking those possibly present in other variables. Therefore, a covariance analysis (ANCOVA) was applied in order to control for the effects of size (Vt) (Table 4). Although the Ve mean for both groups was statistically similar (ANOVA) the ANCOVA results revealed that, when we controlled for size, females had significantly larger Ve than males (female mean: 106.84 mm 3 ; male mean: 87.69 mm 3 ). These results were corroborated by the 3DAET and 3DRET results, the values of which were also significantly larger in females. In the same way, the ANCOVA showed that, when controlling for the effects of size (Vt), females have significantly larger outer enamel surface area (OES) than males, but a similar EDJ surface area (EDJS), which agrees with the significantly larger values of the OES/EDJS index for this sex. Our results also showed that males have absolute (Vcdp and Vtdp ANOVA results) and proportionally (Vcdp/Vc ANOVA results) larger dentine-pulp complex volume than females. However, although ANCOVA results showed that the total dentine and pulp volume (Vtdp) was larger in males, in the crown it was similar in both sexes (Vcdp ANCOVA results).

Intrapopulation variability: Two-dimensional measurements

To determine intrapopulation variability in 2D crown tissue components, a subsample of 54 slightly worn canines were used. The results of the statistical analysis are presented in Table 5. The Kolmogorov-Smirnov test showed that all dimensions were normally distributed within each sex category. The results of homogeneity of variance test indicate that the sample is statistically homogeneous for all dimensions and indices. Once again, ANOVA showed that all crown dimensions (b, a, e, BCD) were significantly larger in males, except in the enamel cap area (c) which was similar in both sexes. Our results indicated not only that the lower canines of males had larger crowns than females (a), but also that they were wider buccolingually (BCD). On the other hand, the comparison of the mean values of the enamel thickness indices revealed that, even though the average enamel thickness (2DAET) do not differ significantly, the relative enamel cap area (2DRET) does (p value = .002), being thicker in females.

As well as the 3D analysis, when we subsequently carried out the ANCOVA test, monitoring for the effect of size (a), significant differences were found in the enamel cap area, being greater in females than in males (ANCOVA estimated means: females 15.964 mm 2 ; males 14.272 mm 2 ).

The ANCOVA results also corroborated those obtained from the ANOVA focusing on the coronal dentine and pulp area (b) and bicervical diameter (BCD), which showed that males had significantly greater values for both variables (Table 5). The relative dentine and pulp area (b/a) values are in agreement with this result, revealing a smaller dentine-pulp complex in females greatest correct sex allocation was D5. We also used the combination of the seven functions (D1-D7) in the hold-out sample. When at least four of the outcomes indicated either "male" or "female" the individual was assigned this specific sex. Using this methodology, we obtained 92.30% correct classifications in the slightly worn teeth. Overall, there were no large differences between the percentages of correct assignations from the original sample and those from the crossvalidation results. This indicates that the discriminant functions created classify the individuals accurately. The functions were also tested on a hold-out sample of slightly worn teeth and extensively worn teeth separately. D3 (based on basal surface) and D4 (based on root volume) functions were tested on the total hold-out sample given these variables were not affected by wear.

Overall, in all discriminant functions females were classified more accurately than males. The percentage of correct determinations in the original sample ranges between 76.20% and 90.50%

in the case of males, and between 80% and 100% in the case of females; the cross-validation ranged between 76.20% and 85.70% in the case of males and between 80% and 95% in the case of females; and the hold-out sample validation (slightly worn teeth) ranged between 62.50% and 75% in the case of males and between 80% and 100% in the case of the females. The combination of the results from the seven functions correctly classified 100% of the females, and 87.5% of the males.

The mean difference between the percentages of correct determinations in the hold-out sample of slightly worn and extensively worn teeth was 9.64%. The allocation accuracy in the extensively worn teeth ranged between 73.33% and 80%, which is not very different to the results for slightly worn teeth. However, in this case the differences between males and females increased, especially in the D1, D3, D6, and D7 functions, which raised doubt about their precision. Finally, the seven-function combination had an 80% correct classification rate in the case of extensively worn teeth (87.50% females, 71.43% males).

DISCUSSION

Sexual dimorphism of dental tissue volumes and surface areas of mandibular canines

Male mandibular canines have been found to be greater than those of females. Both their crown (a, Vc) and root (Vr, Vc/Vt) were larger and, therefore, the overall size of the tooth (Vt). These results are in agreement with those obtained in the literature on canine crown dimensions (mesiodistal and buccolingual widths) in modern humans [START_REF] Acharya | Validity of the mandibular canine index (MCI) in sex prediction: Reassessment in an Indian sample[END_REF][START_REF] Ateş | Sexual differences in Turkish dentition[END_REF][START_REF] Peckmann | Sex determination using the mesio-distal dimension of permanent maxillary incisors and canines in a modern Chilean population[END_REF][START_REF] Pereira | Contribution of teeth in human forensic identification-discriminant function sexing odontometrical techniques in Portuguese population[END_REF], as well as, they also support the conclusions reached by [START_REF] Zorba | Sexual dimorphism of root length on a Greek population sample[END_REF] and [START_REF] Garn | Relationship between root lengths and crown diameters of corresponding teeth[END_REF], who argued that root dimensions of canines offer a reliable method for determining sex. Although, most of researchers who have studied sexual dimorphism in teeth have pointed out the high dimorphism of canines [START_REF] Acharya | Univariate sex dimorphism in the Nepalese dentition and the use of discriminant functions in gender assessment[END_REF][START_REF] Acharya | Validity of the mandibular canine index (MCI) in sex prediction: Reassessment in an Indian sample[END_REF][START_REF] Garn | Shape similarities throughout the dentition[END_REF][START_REF] Hillson | Dental anthropology[END_REF][START_REF] Lund | Gender determination by odontometrics in a Swedish population[END_REF][START_REF] Rao | Mandibular canine index-A clue for establishing sex identity[END_REF][START_REF] Schwartz | Sexual dimorphism in modern human permanent teeth[END_REF], a number of them have also observed differences in size of male and female premolars and molars [START_REF] Prabhu | Odontometric sex assessment in Indians[END_REF][START_REF] Viciano | Sex estimation based on deciduous and permanent dentition in a contemporary Spanish population[END_REF][START_REF] Zorba | Sexual dimorphism in permanent teeth of modern Greeks[END_REF], and, to a lesser extent, in their incisors [START_REF] Garn | Sex difference in tooth size[END_REF][START_REF] Staka | Sexual dimorphism in permanent maxillary central incisor in Kosovo: Albanian population[END_REF]. The existence of a noticeable sexual dimorphism in some permanent tooth dimensions have caused several authors to be interested in quantifying the level of sexual dimorphism of deciduous teeth [START_REF] Moorrees | Mesiodistal crown diameters of the deciduous and permanent teeth in individuals[END_REF][START_REF] Harris | Mesiodistal tooth crown dimensions of the primary dentition: A worldwide survey[END_REF][START_REF] Viciano | Sex estimation based on deciduous and permanent dentition in a contemporary Spanish population[END_REF], however, without achieving conclusive results.

On the other hand, the overall tooth size dimorphism has been interpreted as a consequence of differences in the amount of enamel and dentine between sexes. Most studies about dental tissue proportions of males and females have commonly been focused on the permanent posterior dentition. Even though some of these studies have found significant differences in the amount of enamel [START_REF] Alvesalo | 47, XXX females, sex chromosomes, and tooth crown structure[END_REF][START_REF] Moss | Analysis of developmental processes possibly related to human dental sexual dimorphism in permanent and deciduous canines[END_REF], it is becoming increasingly clear that sexual dimorphism of premolars and molars is due to the males' greater dentine dimensions, and that sex differences in enamel do not make a large contribution to overall tooth size dimorphism (Harris and Hicks, 1998b;[START_REF] Smith | Modern human molar enamel thickness and enamel-dentine junction shape[END_REF][START_REF] Stroud | Sexual dimorphism in mesiodistal dentin and enamel thickness[END_REF]. Our results also support this hypothesis. From this study it was found that lower canines of males have absolutely (b, Vcdp, Vtdp) and relatively (b/a, Vcdp/Vc) more dentine than those of females, whereas females have been found to have relatively (3DAET, 3DRET, 2DRET), but not absolutely (c, Ve), more enamel than males. In the same way, EDJ length and surface are greater in males, likely because of the greater dentine dimensions.

In the light of these results, it seems clear that size is a factor to keep had in mind. The relative enamel thickness (RET) has been considered as the most appropriate index for interspecies comparison, where the size of each species' teeth must be taken into account [START_REF] Martin | The relationships of the later Miocene Hominoidea[END_REF].

However, an increase in relative enamel thickness could correspond to a reduction in the amount of dentine (b, Vcdp), an increase in the average enamel cup thickness (2DAET, 3DAET) or both.

This makes this index difficult to interpret in insolation, mainly when groups overlap as in the case of the intra-species comparison outlined above [START_REF] Smith | Variation in hominoid molar enamel thickness[END_REF]. The same was true for the percentage of the coronal dimension represented by dentine and pulp (b/a, Vcdp/Vc).

Therefore, supplementary to the AET and RET (2D, 3D) indices, as well as to the relative coronal dentine and pulp dimensions (b/a, Vcdp/Vc), in this study statistical controls techniques, such as ANCOVA, were applied. Through the ANCOVA test, we have been able to assess the effect of size on the intra-population variability of not only of the enamel cup (c, Ve), but also that of the other absolute variables considered. The ANCOVA results showed that differences in male and female variable means persisted in most of measurements even when tooth size was controlled.

Surprisingly, only when we controlled for the effect of size were we able to appreciate that females have greater enamel cap size (c, Ve) than males, which concur with the RET results (2D and 3D).

In addition to dental tissue volumes, we have also found it interesting to compare the outerenamel (OES) and the enamel-dentine junction (EDJ) surface areas. The EDJ, which underlies the enamel cap, carries information about the original shape of the tooth crown. Previous studies have indicated that many features of the tooth crown originate at the EDJ prior to enamel deposition (Skinner et al., 2008a,b). On the other hand, increases in the enamel thickness (3DAET, 3DRET) could be related to increases in the enamel cap dimensions and/or decreases in the volume of the dentine and the enamel-dentine junction surface [START_REF] Grine | Enamel thickness of deciduous and permanent molars in modern Homo sapiens[END_REF][START_REF] Smith | Modern human molar enamel thickness and enamel-dentine junction shape[END_REF]. Our results showed that both surfaces, OES and EDJS, are significantly greater in males, but it was also found that males have larger canines, so these results could be more related to the differences in size than to the complexity of the surfaces. When we equated both groups' canines size (ANCOVA) we found that males and females have similar EDJ surface areas, but females have significantly wider OES, which is also reflected significantly in the OES/EDJS index (female mean: 1.46; male mean: 1.39). Therefore, a significantly greater absolute average (and/or relative) enamel thickness in females seems to be the result of an increase in enamel cap complexity rather than due to a decrease of dentine surface. These results were also reported for molars: [START_REF] Smith | Modern human molar enamel thickness and enamel-dentine junction shape[END_REF] showed that females often have a greater mean molar enamel cap surface area and it frequently contributes to greater enamel thickness. New Geometric Morphometrics studies of the OES and the EDJ surface areas of canines could shed light on this issue.

The potential of dental tissue proportions for reliable sex determination

Our results allow for the identification of significant sex differences in the dental tissue volumes and surface areas of mandibular canines. They have also shown that these differences were large enough to determine the sex with a high percentage of accuracy. Overall, the accuracies obtained from the use of multiple variables equations were higher than those obtained using univariate functions. The percentage of correct determinations increased when we used all the functions together (92.30%). Nevertheless, in some forensic and archaeological contexts it may not be possible to measure all of the variables, therefore univariate functions may prove useful in these cases. The function with the greatest correct sex allocation was D5 (original: 95.10%; cross-validation: 85.40%; hold-out sample validation on slightly worn teeth: 84.62%; and hold-out sample validation on extensively worn teeth: 80.00%) which includes all the variables considered in this study.

According to the results obtained from this study, the accuracy rate of sex determination was higher for females than for males. Other studies based on the discriminant function analysis of the canine crown dimensions have also found that the classification accuracy was higher for females and lower for males [START_REF] Ateş | Sexual differences in Turkish dentition[END_REF][START_REF] Acharya | Univariate sex dimorphism in the Nepalese dentition and the use of discriminant functions in gender assessment[END_REF]Işcan and Kedici, 2003). [START_REF] Plavcan | Sexual size dimorphism, canine dimorphism, and male-male competition in primates: Where do humans fit in? Human Nature[END_REF] suggested that this might be due to the retention of certain vestigial features. Adult male primates exhibit two sexually mature morphotypes: whereas the dominant males achieve full body size, with the development of strong secondary sexual features, other males retain a subadult body form; nevertheless, females have a unique sexually mature morphotype. This leads to in a higher variability in male sexual features, which may make difficult their sexual allocation, reinforcing Işcan's and Kedici's observation that "The difficulty or the lack of dimorphism comes from male subjects" (2003, p 1).

Finally, the percentage of correct assignment of sex in the extensively worn sample only decreased to 9.64% compared to the slightly worn sample; nevertheless, in this case the correct assignment differences between males and females increased, which made us doubt their precision when the wear increased.

Traditional 2D variables of dental tissue components and their utility in sexual dimorphism assessment

The few studies in literature which have assessed the dental tissue proportion variation in canine teeth includes 2D analysis based on physical sections [START_REF] Saunders | Sexual dimorphism of the dental tissues in human permanent mandibular canines and third premolars[END_REF][START_REF] Schwartz | Sexual dimorphism in modern human permanent teeth[END_REF] and 2D analysis of virtual sections obtained from microtomographic 3D models [START_REF] Feeney | Enamel thickness in Asian human canines and premolars[END_REF]. Although these researchers have observed dental tissue patterns similar to those described in this study, it is worth highlighting the low homogeneity in the results obtained by the different research approaches, including the present one. Our 2D analysis results concur with those obtained by [START_REF] Saunders | Sexual dimorphism of the dental tissues in human permanent mandibular canines and third premolars[END_REF] when analyzing the physical sections of mandibular canines. However, [START_REF] Schwartz | Sexual dimorphism in modern human permanent teeth[END_REF], applying the same technique as [START_REF] Saunders | Sexual dimorphism of the dental tissues in human permanent mandibular canines and third premolars[END_REF], concluded that female mandibular canines have a significantly greater average and relative enamel thickness than those of males due to their larger enamel caps and smaller dentine areas. Nevertheless, statistically significant differences were not found in any of the absolute measurements considered. More recently, making use of microtomographic virtual images, [START_REF] Feeney | Enamel thickness in Asian human canines and premolars[END_REF] assessed the dental tissue areas of canine and premolars longitudinal planes.

These authors found that males have significantly greater values than females in terms of the dentine area and EDJ length in the maxillary canines, but, like [START_REF] Schwartz | Sexual dimorphism in modern human permanent teeth[END_REF], no significant differences were noted for the mandibular ones.

The discrepancies between the different studies results might have a methodological origin.

Despite our results of the 2D and 3D analysis of the crown tissue components have ended up being similar, three-dimensional measurements have been considered as a more precise way to evaluate tissue proportions since they counteract the effect of dimensional loss suffered by classic 2D estimations [START_REF] Benazzi | Technical note: Guidelines for the digital computation of 2D and 3D enamel thickness in hominoid teeth[END_REF][START_REF] Feeney | Microtomographic analysis of sexual dimorphism and dental tissue distribution in human molars[END_REF][START_REF] Molnar | Hominid enamel thickness: I. The Krapina Neandertals[END_REF][START_REF] Shellis | Variations in molar enamel thickness among primates[END_REF]. Our study reveals that when only 2D variables from the crown are considered [START_REF] Feeney | Enamel thickness in Asian human canines and premolars[END_REF][START_REF] Saunders | Sexual dimorphism of the dental tissues in human permanent mandibular canines and third premolars[END_REF][START_REF] Schwartz | Sexual dimorphism in modern human permanent teeth[END_REF]) some measurements are overestimated or underestimated (Figure 3). Olejniczak et al. (2008aOlejniczak et al. ( , 2008b) ) (2007), do not allow obtaining a bucco-lingual plane in an accurate manner, since it is not possible to locate the dentine horn before the tooth is cut. The anatomy of canine teeth makes that small variations in this plane may lead to important change in the measurements, which could explain the discrepancies in literature. Therefore, the above makes us conclude that, as enamel thickness distribution is not homogenously distributed in the crown, the conventional 2D measurements in the bucco-lingual ideal section planes may not be a reliable estimator of 3D whole-crown variability, as they do not adequately capture the morphology of the entire tooth.

Future research of 3D dental tissue proportions may support the sexual distinctive patterns observed in this study. Although it is unclear which genetic mechanisms are at play, and the processes by which sexual hormones influence dental tissue formation, there are numerous studies which support the idea that both factors could be the cause of the dental tissue dimorphism previously described. The extensive literature on the enamel and dentin patterns of the permanent dentition belonging to individuals with sex chromosome abnormalities, suggests the existence of a genetic influence of sex-linked genes on both dental hard tissues and overall tooth size [START_REF] Alvesalo | Human sex chromosomes in oral and craniofacial growth[END_REF][START_REF] Alvesalo | Enamel thickness of 45, X females' permanent teeth[END_REF][START_REF] Alvesalo | Upper central incisor and canine tooth crown size in 47, XXY males[END_REF][START_REF] Alvesalo | Enamel thickness in 47, XYY males' permanent teeth[END_REF][START_REF] Alvesalo | 47, XXX females, sex chromosomes, and tooth crown structure[END_REF][START_REF] Pentinpuro | Crown heights in the permanent teeth of 45, X and 45, X/46, XX females[END_REF][START_REF] Pentinpuro | Crown heights in the permanent teeth of 47, XYY males[END_REF][START_REF] Zilberman | Crown components of mandibular molar teeth in 45, X females (Turner syndrome)[END_REF].Whereas Y chromosome linked genes seem to increase the mitotic activity within the dental lamina [START_REF] Alvesalo | Sex chromosomes and human growth. A dental approach[END_REF], X chromosome appears to be able to influence on the enamel growth [START_REF] Alvesalo | Upper central incisor and canine tooth crown size in 47, XXY males[END_REF]. These studies did not employ a standardized method to image dental tissues from in patient X-rays, so their result should be treated with caution. Their findings, however, are supported by those obtained in genetic loci studies of human amelogenin; which appear to be the principal intermediaries of this interaction [START_REF] Fincham | Human developing enamel proteins exhibit a sex-linked dimorphism[END_REF][START_REF] Fincham | The structural biology of the developing dental enamel matrix[END_REF][START_REF] Salido | The human enamel protein gene amelogenin is expressed from both the X and the Y chromosomes[END_REF].The amelogenin is the main protein component of the organic matrix in enamel, and its genes are on both the X and Y chromosomes [START_REF] Fincham | The structural biology of the developing dental enamel matrix[END_REF][START_REF] Fincham | Human developing enamel proteins exhibit a sex-linked dimorphism[END_REF]. The quantitative and qualitative differences in their transcriptional products influence the proportions in which hard dental tissues are present [START_REF] Salido | The human enamel protein gene amelogenin is expressed from both the X and the Y chromosomes[END_REF][START_REF] Schwartz | Sexual dimorphism in modern human permanent teeth[END_REF], likewise genetic alterations of these genes cause different dental tissues defects [START_REF] Cho | Alteration of conserved alternative splicing in AMELX causes enamel defects[END_REF][START_REF] Hu | Amelogenesis imperfecta in two families with defined AMELX deletions in ARHGAP6[END_REF].

On the other hand, some authors have argued about the actual extent to which sexual hormones have an effect on dental sexual dimorphism [START_REF] Gingerich | Size variability of the teeth in living mammals and the diagnosis of closely related sympatric fossil species[END_REF][START_REF] Guatelli-Steinberg | Dental crown size and sex hormone concentrations: Another look at the development of sexual dimorphism[END_REF][START_REF] Kondo | Associations between Carabelli trait and cusp areas in human permanent maxillary first molars[END_REF][START_REF] Kondo | Sexual dimorphism of cusp dimensions in human maxillary molars[END_REF][START_REF] Smith | Modern human molar enamel thickness and enamel-dentine junction shape[END_REF]. [START_REF] Guatelli-Steinberg | Dental crown size and sex hormone concentrations: Another look at the development of sexual dimorphism[END_REF], in a study based on post-natal testosterone levels, suggested that sexual dimorphism in population-wide patterns is mainly related to the growth-promoting effect of the Y chromosome, and that sex hormones have only a minor influence during tooth development.

However, opposite-sex twin pairs studies, in which the influence intrauterine levels of testosterone were also considered, supported that intrauterine male hormone levels influence on different tooth dimensions and contribute to the sexual dimorphism of human teeth [START_REF] Dempsey | Increased tooth crown size in females with twin brothers: Evidence for hormonal diffusion between human twins in utero[END_REF][START_REF] Ribeiro | Intrauterine hormone effects on tooth dimensions[END_REF]Ribeiro, Sampson, Hughes, Brook, & Townsend, 2012). In terms of dental tissues, [START_REF] Zilberman | Sex-and age-related differences in primary and secondary dentin formation[END_REF] studied the hypothesis that odontoblast activity is influenced by sexual hormones, and it is expressed in differences in dentine thickness that change with age. They concluded that the origin of differences in dental tissues proportions is probably due to the different rates of dentine deposition taking place during the lives of males and females. The discovery of Growth Hormone (GH) receptors in dental tissue could also provide a further clue as to why these differences exist [START_REF] Zhang | Evidence for a local action of growth hormone in embryonic tooth development in the rat[END_REF]. This hormone is the major regulator of postnatal growth and development [START_REF] Giustina | Pathophysiology of the neuroregulation of growth hormone secretion in experimental animals and the human[END_REF], which has a strong influence on the metabolism of oral tissues [START_REF] Slootweg | Growth hormone and bone[END_REF]. Particularly, GH is able to induce proliferation of epithelial stem cells, preameloblast differentiation and dentine matrix formation [START_REF] Young | The influence of growth hormone on cell proliferation in odontogenic epithelia by bromodeoxyuridine immunocytochemistry and morphometry in the Lewis dwarf rat[END_REF].This hormone is influenced by oestrogens and others sex steroids [START_REF] Alonso | Oestrogens and puberty[END_REF][START_REF] Meinhardt | Modulation of growth hormone action by sex steroids[END_REF][START_REF] Saggese | Sex steroids and the acquisition of bone mass[END_REF]. While at higher oestrogen concentrations growth is inhibited [START_REF] Alonso | Oestrogens and puberty[END_REF], the testosterone induces growth [START_REF] Clark | Growth hormones and sex steroid interactions at puberty[END_REF]. It could be the reason why dentine thickness differences are more marked in puberty [START_REF] Zilberman | Sex-and age-related differences in primary and secondary dentin formation[END_REF], when both hormones levels are higher, and the degree of dentine formation appears to increase suddenly in females during their fourth decade of life, when oestrogen levels decrease [START_REF] Woods | Age-progressive changes in pulp widths and root lengths during adulthood: A study of American blacks and whites[END_REF]. New research on about how the different combinations of sex hormones and their varying concentrations levels affect odontoblasts activity would be of help to clarify the mechanism by which they influence sexual dimorphism, as well as to know their role within the complex adaptive system of dental development, in which intervene genetic, epigenetic, and environmental factors.

CONCLUSIONS

The present study has found that dental tissue volumes and surface areas can be used in sex determination as they present a high degree of sexual dimorphism. Our results support the hypothesis that the sexual dimorphism of mandibular canine size is mainly due to males having a greater amount of dentine, whereas sex differences in enamel volume do not make a large contribution to overall tooth size. Discriminant function analysis gave a high classification accuracy ranging between 78.00% and 90.20% for the cross-validation, and between 76.92% and 84.62% when tested on a hold-out sample of slightly worn teeth. When we used the combination of the seven functions (D1-D7), 92.30% of the individuals were correctly assigned. Although these functions have been tested on a sample composed of individuals of different origin, this method needs to be tested on other population groups, as sexual dimorphism in teeth may vary between populations. The results obtained show the importance of dental histology as a methodology in sex determination in archaeological and forensic contexts, especially when other skeletal elements are fragmented and/or other measurements are unavailable.

Figure 1 .

 1 Figure1. Crown isolation. Canine in its correct anatomical position: lingual view (a), mesial view (c). Line which defines the lower limit of the crown in a mesio-distal plane (b), and in a buccolingual plane (d). Isolated volumes: enamel cap (a), and coronal dentine and pulp complex (b), which was limited on the base by a curve with a smooth surface (c) and the root volume(d)

  Abbreviation Complete definition Dimension

Figure 2 .

 2 Figure 2. (a) Mesial view of the individual UCM_20 lower canine. (b) The virtual cross-sections were obtained following the method of Benazzi et al. (2014) and the crown reconstruction (c) was performed by applying the Saunders et al. (2007) methodology

  the crown bounded by the outer enamel perimeter and a straight line between the buccal and lingual cervical margins. mm 2 Length of the EDJ e The linear distance of the line separating the enamel cap area from the dentine and pulp area above the cervical margin. The percent of coronal area that is dentine and pulp. [(b/a) × 100]

  also noted differences in dental tissue patterns based on 2D and 3D data when they compared molars from different hominins species, likewise Benazzi et al. (2014) observed similar differences when assessed the 2D and 3D measurements in modern human premolars, canines, and incisors. Additionally to the loss of one dimension, we also have to consider that physical cross-sections techniques, like those applied by Schwartz and Dean (2005) and Saunders et al.

Figure 3 .

 3 Figure 3. Percentage of the crown which is enamel (light grey) and dentine and pulp complex (dark grey) based on the 2D (1) and 3D (2) results obtained in the present study, for females (a) and males (b)

Table 1 .

 1 Distribution of the sample by sex, origin and degree of wear[START_REF] Molnar | Human tooth wear, tooth function and cultural variability[END_REF]

		European		African		Total
	Wear	Female	Male	Female	Male	Female	Male
	<3	9	13	16	16	25	29
	>3	5	5	3	2	8	7
	TOTAL	-	-	-	-	33	36

Table 3 .

 3 Definition of the two-dimensional measurements from the crown and associated indices

	used in this study
	Metrics	Abbreviation Complete definition	Dimension
	Enamel cap area	c	The area of the crown bounded by the outer
			enamel perimeter and the EDJ.

Table 4 .

 4 Descriptive statistics, analysis of variance (ANOVA), and analysis of covariance

	(ANCOVA) results of the three-dimensional variables measured in slightly worn mandibular
	canines
	Female				Male			ANOVA ANCOVA
	Measurement N	Mean	SD	N	Mean	SD	Sig.	Sig.	Female	Male
										mean	mean
	Vc	25 196.48 38.15 29 223.80	37.25 0.01	-	-	-
	Vr	33 271.73 57.49 36 374.31	71.87 0.00	-	-	-
	Vt	25 467.01 84.98 29 603.49	91.05 0.00	-	-	-

Dimensions in millimeters. Significant p values in bold.

Table 5 .

 5 Descriptive statistics, analysis of variance (ANOVA), and analysis of covariance

	(ANCOVA) results of the two-dimensional variables measured in slightly worn mandibular
	canines
	Female			Male			ANOVA ANCOVA
	Measurement N	Mean SD	N	Mean SD	Sig.	Sig.	Female	Male
									mean	mean
	c	25 14.92 2.70 29 15.21	2.70 0.70	0.01 15.96	14.27
	b	25 35.44 4.67 29 41.96	5.83 0.00	0.02 38.09	39.60
	a	25 50.36 6.69 29 57.17	7.30 0.00	-	-	-

Dimensions in millimeters. Significant p values in bold.

Table 7 .

 7 Sex allocation accuracy (%) for each discriminant function generated from data collected in this study, tested on the original sample and using a cross-validation

		Tested on equations sample					Tested on hold-out sample
		Original			Cross-validation		Slightly worn teeth		Widely worn teeth
	Discriminant	Female	Male	Total	Female	Male	Total	Female	Male	Total	Female	Male	Total
	functions
	D1	80.00%	81.00%	80.50%	80.00%	76.20%	78.00%	80.00%	75.00%	76.92%	87.50%	57.14%	73.33%
	D2	85.00%	81.00%	82.90%	85.00%	81.00%	82.90%	100.00%	75.00%	84.61%	87.50%	71.43%	80.00%
	D3	90.00%	85.70%	87.80%	90.00%	85.70%	87.80%	100.00%	62.50%	76.92%	87.50%	42.86%	66.67%
	D4	85.00%	76.20%	80.50%	80.00%	76.20%	78.00%	100.00%	87.50%	92.31%	87.50%	71.43%	80.00%
	D5	100.00%	90.50%	95.10%	90.00%	81.00%	85.40%	100.00%	75.00%	84.62%	87.50%	71.43%	80.00%
	D6	95.00%	85.70%	80.20%	95.00%	85.70%	90.20%	100.00%	75.00%	84.61%	87.50%	54.14%	73.33%
	D7	90.00%	85.70%	87.80%	90.00%	85.70%	87.70%	80.00%	75.00%	76.92%	87.50%	57.14%	73.33%
	All combined	-	-	-	-	-	-	100.00%	87.50%	92.30%	87.50%	71.43%	80.00%

ACKNOWLEDGMENTS

This study has been supported by the Dirección General de Investigación of the Spanish Ministerio de Economía y Competitividad (MINECO/FEDER) grant number: CGL2015-65387-C3-1, 3-P. to the authors acknowledge The Leakey Foundation for the personal support provided by Gordon Getty and Dub Crook to one of the authors (MM-T). CG-C, MM, and MM-P are funded by a doctoral grant from the Junta de Castilla y León financed in turn by the European Social Funds through the Consejería de Educacion. LM-F is the recipient of a post-doctoral research grant at Atapuerca Fundation. Some of the micro-CT images were obtained in the Laboratory of Microscopy of the CENIEH-ICTS (Spain) and in collaboration with CENIEH staff. The remainder of the dental samples were scanned in the Multidisciplinary Laboratory of the International Centre for Theoretical Physics (ICTP) of Trieste in collaboration with Federico Bernardini. The African sample from Sudan was provided by Dr Christopher Dean from the Anatomy Department at University College London. Part of the sample was analyzed in the Anthropology Department at University College London in collaboration with Dr Christopher Soligo. The authors are indebted to A. Oettlé, G. Krüger, and E.N. L'Abbé for kindly authorizing access to the Pretoria Bone Collection (PBC) of the University of Pretoria. They also deeply thank F. de Beer and J. Hoffman for carrying out the X-ray microCT imaging of the human specimens from the PBC included in this study.

-P; Consejería de Cultura y Turismo of the Junta de Castilla y León; The

(ANOVA). In contrast, differences in EDJ length disappeared when the effect of size was controlled for.

Discriminant functions

Table 6 presents the unstandardized discriminant function coefficients, constants, sectioning points and the main statistics values obtained from discriminant function analysis. To provide a discriminant function model applicable to the different preservation conditions of dental remains, several techniques were developed. Through different univariate discriminant analyses we obtained four functions with high percentages of correct determinations. These functions used the following variables: total tooth volume (D1), total dentine and pulp volume (D2), basal surface (D3) and total root volume (D4). Additionally, we also carried out a multivariate analysis using all the variables and associated indices measured (D5). We also created multivariate equations using the Lambda Wilks stepwise variable selection using all the variables and indices (D6) and only with variables and indices from the crown (D7). All functions were statistically significant in the discrimination between groups (F value, p < .01). Table 6. Discriminant function analysis results for a sub-sample of slightly worn mandibular canines (n=41). Table 7 shows the accuracy of classification of each of the functions. The percentage of correct determinations for univariate functions ranged between 80.50% and 87.80% for the original sample, between 78% and 87.80% for the cross-validated data, and between 76.92% and 92.32%

Unstandardized Coefficients

for the hold-out sample validation using slightly worn teeth. On the other hand, multivariate equations had a classification accuracy which ranged between 80.20% and 95.10% for the original sample, between 85.40 and 90.20% for the crossvalidated data, and between 76.92% and


Alessandro Urciuoli

Clément Zanolli

Josep Fortuny

Sergio Almécija

Burkhard Schillinger

Salvador Moyà-Solà

David M Alba
email: david.alba@icp.cat

| Clément Zanolli

| Josep Fortuny

| Burkhard Schillinger

Neutron-based computed microtomography: Pliobates cataloniae and Barberapithecus huerzeleri as a test-case study

Keywords: X-rays, neutron radiation, neutron imaging, fossil catarrhines

come

Materials and Methods:

We compare neutron with X-ray μCT scans of fossils from two Miocene catarrhines from the Vallès-Penedès Basin: the cranium (IPS58443.1, holotype) of the putative stem hominoid Pliobates cataloniae, to discriminate between bone and matrix; and two lower molars (IPS1724n,o, holotype) of Barberapithecus huerzeleri, to discriminate among dental tissues.

Results: X-ray μCT scans of these specimens fail to retrieve any contrast between matrix/bone and enamel/dentine, whereas neutron μCT scans deliver high-contrast images, enabling a proper evaluation of the specimens' internal anatomy.

Discussion: Low bone/matrix intensity difference with X-ray μCT scans in IPS58443.1 is due to the extreme similarity in chemical composition between the matrix and the fossilized tissues, and the presence of high-density elements. In IPS1724, it is attributable to the convergence of enamel and dentine compositions during fossilization. On the contrary, neutron radiation returns very different contrasts for different isotopes of the same element and easily penetrates most metals. Neutron-based μCT scans therefore enable a correct definition of the bone/sediment and enamel/dentine interfaces, and hence a better segmentation of the images stack. We conclude that neutron radiation represents a successful alternative for high-resolution µCT of small-sized fossils that are problematic with standard X-rays.

| INTRODUCTION

The use of computed microtomography (μCT) in paleontological research has dramatically increased during the last decade, in parallel to concomitant enhancements in image detectors and computing power. The success of computed tomography in paleontology is due to the fact that it enables the non-destructive study of internal anatomy, the virtual extraction of sediment-embedded fossils, the virtual reconstruction of damaged specimens (including the mirroring of antimeres), and even the retrodeformation of plastically deformed fossils [START_REF] Macchiarelli | Early Pliocene hominid tooth from Galili, Somali Region, Ethiopia[END_REF][START_REF] Olejniczak | Assessment of the accuracy of dental enamel thickness measurements using microfocal X-ray computed tomography[END_REF][START_REF] Olejniczak | Technical note: compatibility of microtomographic imaging systems for dental measurements[END_REF][START_REF] Abel | A palaeobiologist's guide to 'virtual' micro-CT preparation[END_REF][START_REF] Benazzi | Technical note: Individual tooth macrowear pattern guides the reconstruction of Sts 52 (Australopithecus africanus) dental arches[END_REF][START_REF] Faulwetter | Micro-computed tomography: Introducing new dimensions to taxonomy[END_REF][START_REF] Macchiarelli | From outer to inner structural morphology in dental anthropology: integration of the third dimension in the visualization and quantitative analysis of fossil remains[END_REF][START_REF] Benazzi | Technical note: Virtual reconstruction of KNM-ER 1813 Homo habilis cranium[END_REF][START_REF] Cunningham | A virtual world of paleontology[END_REF][START_REF] Lautenschlager | Reconstructing the past: methods and techniques for the digital restoration of fossils[END_REF]. Modern X-ray μCT (especially based on synchrotron radiation) can reach a very high spatial resolution (up to less than 1 μm; [START_REF] Gren | Molecular and microstructural inventory of an isolated fossil bird feather from the Eocene Fur Formation of Denmark[END_REF], and the size of the specimens that can be scanned with a single acquisition has recently increased considerably (currently in the order of some decimeters; e.g., [START_REF] Tuniz | The ICTP-Elettra X-ray laboratory for cultural heritage and archaeology[END_REF].

Depending on the taphonomic processes occurring during fossilization, not all vertebrate fossil remains are amenable to internal anatomy analysis based on standard Xray μCT. The latter may fail to retrieve an adequate contrast between the fossil bone and the matrix or between different dental tissues because of two main problems: (1) the embedding matrix and the fossilized tissues have a very similar composition, due to element exchange resulting from permineralization [START_REF] Zanolli | The Early Pleistocene deciduous hominid molar FS-72 from the Sangiran Dome of Java, Indonesia: A taxonomic reappraisal based on its comparative endostructural characterization[END_REF][START_REF] Beaudet | Neutron microtomography-based virtual extraction and analysis of a cercopithecoid partial cranium (STS 1039) embedded in a breccia fragment from Sterkfontein Member 4 (South Africa)[END_REF]; (2) a considerable amount of high-density elements is present in the embedding sediment or in the fossil itself [START_REF] Spoor | Linear measurements of cortical bone and dental enamel by computed tomography: applications and problems[END_REF][START_REF] Abel | A palaeobiologist's guide to 'virtual' micro-CT preparation[END_REF]. Either of these problems hinders and might even entirely preclude the segmentation of the fossil, thus preventing the extraction of essential paleobiological evidence (e.g., [START_REF] Schwarz | Neutron tomography of internal structures of vertebrate remains: A comparison with X-ray computed tomography[END_REF][START_REF] Smith | Taxonomic assessment of the Trinil molars using nondestructive 3D structural and development analysis[END_REF]Zanolli et al., 2017a). For these reasons, an alternative to X-ray µCT is needed. Neutron radiography and tomography, respectively developed in the 1950s and 1970s [START_REF] Kardjilov | New features in cold neutron radiography and tomography Part II: applied energy-selective neutron radiography and tomography[END_REF][START_REF] Schwarz | Neutron tomography of internal structures of vertebrate remains: A comparison with X-ray computed tomography[END_REF][START_REF] Winkler | Applications of neutron radiography and neutron tomography[END_REF], constitute a potential alternative. However, so far neutron-based µCT (n-μCT) has only sporadically been used in paleontological and paleoanthropological research [START_REF] Schwarz | Neutron tomography of internal structures of vertebrate remains: A comparison with X-ray computed tomography[END_REF][START_REF] Sutton | Tomographic techniques for the study of exceptionally preserved fossils[END_REF][START_REF] Zanolli | Problems and limitations of X-ray microtomography for the endostructural characterization of fossil tooth tissues[END_REF]Zanolli et al., , 2017a;;[START_REF] Beaudet | Neutron microtomography-based virtual extraction and analysis of a cercopithecoid partial cranium (STS 1039) embedded in a breccia fragment from Sterkfontein Member 4 (South Africa)[END_REF][START_REF] Laaß | Evidence for convergent evolution of a neocortex-like structure in a late Permian therapsid[END_REF][START_REF] Schillinger | Why use neutrons? Restaurierung und Archäologie[END_REF]., Although the reliability of n-μCT for investigating the internal anatomy of fossils has previously been demonstrated by previous researchers [START_REF] Schwarz | Neutron tomography of internal structures of vertebrate remains: A comparison with X-ray computed tomography[END_REF][START_REF] Beaudet | Neutron microtomography-based virtual extraction and analysis of a cercopithecoid partial cranium (STS 1039) embedded in a breccia fragment from Sterkfontein Member 4 (South Africa)[END_REF]Zanolli et al., 2017a), here we test further the applicability of this method to specimens that cannot be properly analyzed by means of X-ray µCT. In particular, by focusing on the fossil remains of two European Miocene catarrhines, we address two common problems in CT-based paleoprimatological research: difficulties in discriminating fossilized cranial bone from the surrounding or embedding matrix (as exemplified by the putative stem hominoid Pliobates); and the inability to discriminate well between enamel and dentine in fossil teeth (exemplified by the pliopithecoid Barberapithecus).

| MATERIALS AND METHODS

| Studied sample

The remains of two Miocene catarrhines from the Vallès-Penedès Basin (NE Iberian Peninsula), housed at the Institut Català de Paleontologia Miquel Crusafont (Sabadell, Spain; ICP), were investigated: (1) the partial cranium of Pliobates cataloniae (IPS58443.1, holotype), a putative stem hominoid from the stratigraphic series of Abocador de Can Mata (ACM) locality ACM/C8-A4 (Alba et al., 2015: Figs. 1, 4) 2-10.3 Ma (late Miocene;[START_REF] Casanovas-Vilar | An updated biostratigraphy for the late Aragonian and the Vallesian of the Vallès-Penedès Basin (Catalonia)[END_REF]. These specimens are part of the holotypes of their respective species, that of Pliobates consisting of a partial skeleton, and that of Barberapithecus consisting of associated upper and lower teeth of a single individual. The former is thus far the only known individual of this species, which represents the only small-bodied ape currently known from the Miocene of Europe [START_REF] Alba | Miocene small-bodied ape from Eurasia sheds light on hominoid evolution[END_REF], whereas the hypodigm of Barberapithecus is restricted to two additional isolated teeth and a fragment o radius from its type locality [START_REF] Alba | A new pliopithecid genus (Primates: Pliopithecoidea) from Castell de Barberà (Vallès-Penedès Basin, Catalonia, Spain)[END_REF][START_REF] Moyà-Solà | A proximal radius of Barberapithecus huerzeleri (Primates, Pliopithecidae) from the Miocene site of Castell de Barberà (NE Iberian Peninsula)[END_REF]. The cranium of Pliobates consists of two main parts that are very crushed but not plastically deformed, enabling the virtual reconstruction of its external appearance based on X-ray µCT scanning. However, the poor discrimination between cranial bone and the embedding matrix precludes a clear ascertainment of the morphology of inner cranial structures such as the carotid canal, which in Pliobates apparently displays an orientation uniquely shared with extant hylobatids [START_REF] Alba | Miocene small-bodied ape from Eurasia sheds light on hominoid evolution[END_REF]. In turn, the teeth of Barberapithecus are well preserved; however, unlike for other Vallès-Penedès pliopithecoids (Zanolli et al., 2017b), standard X-ray µCt do not enable to adequately discriminate between enamel and dentine. This precludes ascertaining the endostructural dental morphology-in particular, the enamel-dentin junction (EDJ) morphology-or to compute 3D relative enamel thickness in this taxon, with potential implications for further clarifying its taxonomic/phylogenetic affinities as well as its paleodietary adaptations, respectively.

| Neutron-based computed microtomography

Radiographic contrast is generated using the attenuation and scattering of a beam that passes through an object. The main difference between X-ray and neutron-based tomography lies on the particles that interact with matter (photons produced by the kinetic variation of the electrons in the former, and neutrons in the latter). X-rays and synchrotron radiation interact with the electron cloud that surrounds the atoms, and thus are more scattered or attenuated by elements possessing a large number of electrons. Being uncharged, neutrons only interact with the nuclei via very short-range forces [START_REF] Schwarz | Neutron tomography of internal structures of vertebrate remains: A comparison with X-ray computed tomography[END_REF]. The probability of absorption depends on the number of nucleons, thus showing major differences between neighboring elements or even isotopes of the same element [START_REF] Schillinger | Why use neutrons? Restaurierung und Archäologie[END_REF]. The attenuation coefficient of X-rays rises monotonously with the number of protons of the elements, while neutrons show a decreasing trend [START_REF] Schillinger | Why use neutrons? Restaurierung und Archäologie[END_REF]. This allows high penetration power for heavy mineral elements, which are commonly present in paleontological specimens [START_REF] Beaudet | Neutron microtomography-based virtual extraction and analysis of a cercopithecoid partial cranium (STS 1039) embedded in a breccia fragment from Sterkfontein Member 4 (South Africa)[END_REF]. On the other hand, neutrons are strongly scattered by hydrogen and some other light elements, such that hydrogen-rich materials (i.e., organic materials, glues and resins) are easily detected [START_REF] Schwarz | Neutron tomography of internal structures of vertebrate remains: A comparison with X-ray computed tomography[END_REF][START_REF] Winkler | Applications of neutron radiography and neutron tomography[END_REF][START_REF] Schillinger | Why use neutrons? Restaurierung und Archäologie[END_REF].

Most samples become activated if irradiated with neutrons. The standard decay time for the radioactivity ranges between some days and few weeks, after which the specimens can be released [START_REF] Schillinger | Why use neutrons? Restaurierung und Archäologie[END_REF][START_REF] Schulz | Die neue Anlage ANTARES für Neutronenbildgebung am FRM II[END_REF]. In addition, specific elements (such as europium and cobalt) may achieve hazardous levels of radioactivity if activated by the neutron beam [START_REF] Sutton | Tomographic techniques for the study of exceptionally preserved fossils[END_REF]. However, this issue can be easily avoided by running preliminary tests of short-time irradiation and consecutive gamma scan [START_REF] Schillinger | Why use neutrons? Restaurierung und Archäologie[END_REF].

Even if both X-ray and neutron-based radiography produce a shadow image of the sample, the beams used for the analysis differ considerably. X-ray tubes generate a cone beam that magnifies the projection of the sample, while a neutron beam is approximately parallel and does not magnify [START_REF] Schillinger | Why use neutrons? Restaurierung und Archäologie[END_REF]. The quality of the image thus depends on the collimation ratio and on the distance between the detector and the sample, while the limit for the resolution of a parallel beam is constrained by the detector's resolution.

Using thinned detector screens (5-20 μm), a resolution of 10-20 μm is achieved [START_REF] Schillinger | Why use neutrons? Restaurierung und Archäologie[END_REF][START_REF] Schulz | Die neue Anlage ANTARES für Neutronenbildgebung am FRM II[END_REF].

| Scanning settings

The dimensions and scanning parameters for the studied samples have bee reported in Table 1. They were scanned with n-μCT at the imaging facility ANTARES, which is located at the cold neutron beam port of the reactor of the Forschungs-Neutronenquelle Heinz Maier-Leibnitz (FRM II; Garching bei München, Germany). It allows different detector positions and two different chambers, according to the requirements of the sample size, beam size, neutron flux and spatial resolution (for the specifics of the facility, see [START_REF] Schulz | Die neue Anlage ANTARES für Neutronenbildgebung am FRM II[END_REF]. Specimens were placed in chamber two, on a XY-Phi-table with an additional high precision 5-axes HUBER table. Measurements were carried out using the parallel neutron beam originated from the cold source of the FRM II reactor with an energy range of 3-25 meV and a collimation ratio of 500. Three different scans were obtained for IPS58443.1: a general one (877 projections for 2,400 slices) with a final isotropic voxel size of 19.90 μm; and two close-ups, for the temporal (1,105 projections for 2,518 slices) and maxillary (876 projections for 2,486 slices) areas, with a voxel size of 14.22 μm. In turn, due to the reduced size of the specimens (< 1 cm), IPS1724n,o were scanned with a single cumulative acquisition (2,221 slices) that yielded a final isotropic voxel size of 17.98 μm. The histograms of the images stacks were computed with Fiji [START_REF] Schindelin | Fiji: an open-source platform for biological-image analysis[END_REF]. X-ray μCT were performed for the same specimens. IPS58443.1 was scanned at the American Museum of Natural History (New York, USA) using a Phoenix v|tome|x s180 system, using 160 kV voltage, 1.4 mA current, 0.2 mm Cu filter, and magnification of 2.10, obtaining 1,600 slices (virtual cross-sectional images) of 0.2 mm in thickness and a pixel size of 95.23 μm. In turn, IPS1724n,o were scanned in a single acquisition at the TomoLab of the Multidisciplinary Laboratory of the International Centre for Theoretical Physics (Trieste; ICTP), with 13 kV voltage, 72 μA current, and a 1 mm Al filter, obtaining 1800 slices and a voxel size of 7.56 μm.

| RESULTS

The resulting images of X-rays and neutron radiography are very different. Standard radiography provides sharper air-specimen boundaries and uniform dark background, whereas neutron radiography shows more diffuse background noise, especially for IPS58443.1, probably due to the presence of the acrylic resin (Paraloid ® B72, 5% diluted in acetone) and nitrocellulosic glue (Imedio ® Banda Azul) used in the preparation process.

However, the contrast provided by X-ray μCT for IPS58443.1 is insufficient to perform an accurate analysis of the internal anatomy.

The partial cranium of Pliobates is filled with a mudstone matrix that is firmly stuck to the fossilized tissues and was only partially removed during the preparation process. Electrondense elements fill some of the fractures along the specimen or are found dispersed in the sediment, resulting in bright spots on the images stack that led to the underexposure of the radiographies. The histograms calculated for the whole images stack show a low-shifted distribution of the intensity curve with two extremely steep peaks (Fig. 2E). The lowest one belongs to the air background, while the other includes both sediment and fossilized bone.

Due to the spot-like accumulation of the denser minerals, their peak is diluted within the histogram and becomes visible only when cropping the image to the exact region of the bright spots. Even if contrast is very low, segmentation between outer sediment and fossilized tissues is possible [START_REF] Alba | Miocene small-bodied ape from Eurasia sheds light on hominoid evolution[END_REF]. However, contrast is insufficient to properly discern the inner matrix from internal bone boundaries of cranial cavities (Fig. 1E), especially close to the bright spots. In contrast, n-μCT produced more balanced images (Fig. 1F). This is clearly visible in the distribution of the intensities in the histograms (Fig. 2F), where the second peak (corresponding to the embeddingsediment/fossilized tissues compound) is lower and more distributed along the intensity axis. The fossilized tissues appear in lighter gray, as the permineralization process is likely not complete (or affects different areas of the fossil in a differential way) and their mineral composition still differs from the embedding matrix, which appears darker and richer in heavy elements (Fig. 1F). This enables the distinction and manual segmentation of the borders of the inner cavities, such as the bony labyrinth, the inner ear nerves, or the carotid canal, inter alia.

The two molars of Barberapithecus (IPS1724n,o) appear as a uniform gray-to-white mass in the X-ray scans, such that there is no discernible EDJ (Fig. 1A,C). Probably this is the result of deep mineralization of the dentine during the fossilization process, which caused it to converge in chemical composition with the enamel. The histograms calculated for each tooth, on the whole slices stack (Fig. 2A,C), show that there is a shift of the intensity curve towards higher intensity values for the standard X-ray μCT images stacks.

The right end of the curve shows a peak that corresponds to the brighter areas of the dentine, in which elements with a high attenuation coefficient have penetrated. The steep black background peak occupies the lowest intensity range. These two peaks flank a lower one (ranging from ca. 155 to 240 of the grayscale) that corresponds to the enamel-dentine compound, visible as an indistinct gray to white mass (Fig. 1A,C). In contrast, n-μCT images stacks have more balanced histograms, in which the dental tissues (i.e., dentine and enamel) are better differentiated (Fig. 2B,D). Apart from broader lower peak, due to a lighter background, the curves displays a different intensity for the enamel (darker) and the dentine (lighter). Inside the pulp cavity, the presence of lighter and hydrogen-rich minerals in the two molars (Fig. 1B,D) locally produces some noise (clearly visible in the histogram as an anomalous peak at the very end of the color map). However, it does not affect contrast in the EDJ, enabling the manual segmentation of enamel and dentine, and therefore a correct identification of the former (Fig. 3).

| DISCUSSION

Our results indicate that n-µCT provides a higher anatomical resolution for two specimens, whose internal anatomy could not be adequately segmented by means of X-ray µCT scans, due to several problems: an extreme similarity between the chemical composition of the matrix and that of the fossilized tissues (in the cranium of Pliobates); a high similarity between the chemical composition of different fossilized tissues (in the molars of Barberapithecus); and the presence of high-density elements (in the cranium of Pliobates).

Standard X-ray and synchrotron radiation μCT are preferable because of the greater sharpness of the images, the lack of activation, and the availability of the facilities in the case of the former. However, n-µCT provides a better contrast of different isotopes of the same element and more easily penetrates metals than X-ray µCT. These advantages provide a better definition of the bone/sediment boundary and between different dental tissues, thereby enabling a better segmentation of the images stack in fossils that are problematic with standard µCT. The complementarity of the contrasts obtained by n-µCT and its enhanced penetration power are particularly indicated for thick and heavy-element rich fossil material-two conditions that are commonly found in paleontological specimens.

We therefore conclude that neutron radiation represents an accessible and successful alternative to X-rays for the µCT of fossil specimens when the latter fail to obtain the desired outcome.

Zanolli, C., Alba, D. M., Dean, M. C., Fortuny, J., Macchiarelli, R., & Rook, L. (2017b).

Oreopithecus bambolii is still an "enigmatic anthropoid". American Journal of Physical Anthropology, 162 S64, 420.

  , with an estimated age of 11.6 Ma (middle to late Miocene boundary; Alba, Casanovas-Vilar, Garcés, & Robles, 2017); and (2) two lower molars (right M2 and left M3) of a single individual of Barberapithecus huerzeleri (respectively IPS1724n,o, holotype), a pliopithecoid from Castell de Barberà (Alba & Moyà-Solà, 2012: Figs. 4F-G, 5D, 10D-I, 12F, 13C), with an estimated age of 11.

FIGURE 1

 1 FIGURE 1 Selected images of X-ray and neutron microcomputed tomography (μCT) of two

FIGURE 2

 2 FIGURE 2 Whole images stack histograms from different sources: (A) X-ray

ACKNOWLEDGEMENTS

We thank Marta S. March and Jordi Galindo for assistance in collection managing, and J.

Thostenson and M. Hill for assistance with using the Microscopy and Imaging Facility of the American Museum of Natural History. This work has been supported by the Spanish Ministerio de Economía, Industria y Competitividad and the European Regional Development Fund of the European Union (MINECO/FEDER EU, project CGL2014-54373-P), the Spanish Agencia Estatal de Investigación and the European Regional Development Fund of the European Union (AEI/FEDER EU, project CGL2016-76431-P), and the Generalitat de Catalunya (CERCA Programme). We are also grateful to an annonymous reviewer for constructive comments that helped us to improve a previous version of this paper.

Spanish MINECO/FEDER EU, Project number: CGL2014-54373-P; Spanish AEI/FEDER EU, Project number: CGL2016-76431-P; Generalitat de Catalunya, CERCA Programme.