1. Introduction
Shape and size of leg bones are widely used in both bioarcheological and forensic contexts as sex indicators, e.g., [
1,
2,
3,
4]. Generally, males display larger and more robust tibiae and fibulae, with more robust lower extremities than females [
5,
6] due to a differentiation in environmental responses, hormonal surges, and physical loading patterns, which contribute to sexual variations of body shape and size/proportions [
7].
While the human tibial shape has been extensively studied with biomechanical and morphometric (both traditional and virtual) approaches for sex assessment [
3,
8,
9,
10,
11], few studies include the fibula. Sex assessment based on multivariate analyses of fibular measurements in populations with different ancestries has reported accuracies of 80–86% [
12,
13,
14,
15,
16]. Such studies, however, focus solely on gross anatomical fibular features and linear measurements, such as maximum length and maximum diameters, not considering the shape of the extremities. The most often given reason as to why there are few studies focusing on the fibula is the scarcity of fibulae in both forensic and bioarcheological contexts, with only 18–50% of preserved fibular segments of the expected total [
17,
18,
19]. However, it has been reported that for fragments of distal and proximal fibular extremities, the percentage of preservation is between 57–90% and 61–91%, respectively [
20].
Singh and Singh [
21] have included in their analysis measurements of distal articular surfaces, suggesting that the width of the distal extremity, together with fibular length and mid-shaft circumference, correctly estimated sex with 99% accuracy. In another study, Sacragi and Ikeda [
12] focused on the talar articular surface, the malleolar fossa, and the lateral malleolus, and using a population-specific discriminant function, they were able to correctly classify males at 90.1% and females at 91.4%. Naidoo and colleagues [
22], in addition, examined different linear and angular dimensions of the talar facet, and found significant variations among males and females, and confirmed consistency among different populations in a South African sample.
Several works examined measurements associated with the fibular nutrient foramen in South African populations [
4,
22,
23]. Fasemore and colleagues [
4] found relevant sexual differences among measurements of diameters and circumference taken at the level of the fibular nutrient foramen, with discriminatory cross-validated accuracy of 73% with combined measurements. Interestingly, the authors also highlighted how measurements of distance between the most proximal end of the fibula and nutrient foramen were not sexually dimorphic. The proximal end of the fibula is in fact rarely studied in anthropology for sex determination purposes, and only studies accounting for three-dimensional fibular shape variations included the proximal extremity, along with other tibial and fibular regions, in their analysis [
24,
25,
26,
27,
28].
Virtual morphometric approaches have the advantage of accounting for the whole bone shape and size variations, overcoming the possible limitations that may arise when focusing on single measurements (e.g., [
29]). Prior studies involving three-dimensional statistical shape models (SSM) of fibulae (within the tibiofibular complex), considering both fibular extremities and diaphysis, have found that sex contributes only subtly to the differentiation of fibular morphologies [
28]. Similarly, Tümer and colleagues [
25] did not find a consistent pattern of shape variation in relation to sex for the fibula. Furthermore, three-dimensional geometric morphometric (3D GM) studies of the whole fibula highlighted little to no morphological differences between the sexes, with the few observed differences mostly occurring in fibular length, but not in fibular shape, which was not sexually dimorphic [
30]. Indeed, Maas and Friedling found that fibulae were smaller in females than males. Interestingly, they also describe patterns of sex variations in relation to different ancestries (White, Black, and Coloured individuals, the three main groups present in South Africa). Such differences mostly relate to the contribution of body size to fibular shape among the different ethnicities of their subsamples, since Coloured individuals’ smaller fibulae may possibly reflect the genetic influence of small-bodied Khoisan and Asian groups [
30]. While differences in fibular shape emerged among different ancestries, in their study, Maass and Friedling [
30] adopted an approach based on fixed landmarks, which captured the whole fibular length, masking more subtle differences in epiphyseal shape and form, and in general is highly influenced by the size component [
31,
32,
33].
In this work we applied for the first time a 3D GM approach that integrates fixed landmarks and sliding (semi)landmarks of curves and surfaces to investigate sexually dimorphic features in the fibular proximal and distal extremities. The study involves three samples from identified modern skeletal collections (19–20th century), two from Italian and one from South African populations. The main goal of this study is to provide a new method for sex assessment of the human fibular extremities based on 3D GM to be applied in forensic and bioarcheological investigations. Based on previous studies on sexual dimorphism in the fibulae, we tested the two following hypotheses:
- (a)
Fibular epiphyseal form (size + shape) will be sexually dimorphic, likely reflecting functional differences (body shape, size, and proportions), while shape alone will be less informative [
30].
- (b)
Fibular epiphyseal form dimorphism will differ between populations due to size variation (different patterns of epiphyseal size dimorphism due to differentiation of body size among Italian and South African groups), but shape alone will not reveal differences in the sexual dimorphism between the populations [
30].
4. Discussion
The aim of this study was to highlight patterns of sexually dimorphic form variations in the fibular proximal and distal epiphyses in individuals of three different populations from Italy and South Africa belonging to identified modern skeletal collections (19th–20th century), utilizing a 3D GM approach [
54]. Our study provides a new method for sex estimation on the fibula, potentially applicable in bioarcheology [
20], where this method may find primary implementation, and forensics, offering moderate to high accuracy (80–93% for Italian populations) for identification in case of the recovery of isolated fragments of this bone.
We expected the presence of variations of fibular epiphyseal size in relation to sex. Results from our analyses support a distinction among sexes, mainly more robust epiphyses in males than in females and especially in the Italian samples. Our second hypothesis was that little sex variation would have been present in the shape of the fibular epiphyses in relation to sex, depending on different ancestries and mostly referring to different patterns of epiphyseal size in different populations, due to a differentiation of body size among Italian and South African groups. Our results support the former point and provide only partial support to the latter one. In fact, the form and size of fibular extremities significantly distinguish between sexes in the Emilia-Romagna (ER) and Sardinia (SAR) populations but not in the South African population. Overall, our results show that fibular extremities account for subtle shape changes, while significant form and size differences are present between sexes, even with differences related to ancestry. The sex determination method provided in this paper, based on cross-validation LDA on form PCs, provides accuracy above 80% when the samples are pooled and reaches accuracy of 80–93% when Italian populations are considered separately. However, the method was not successful for the South African sample (50–53% accuracy).
Our results are consistent with previous investigation on sex determination using the fibula, adopting both traditional metrics [
4,
12,
16,
22,
55] and virtual methods [
25,
26,
27,
28,
30]. Specifically, we obtained similar results as previous studies that utilized 3D GM methods on the whole fibula [
30], which highlighted different degrees of size dimorphism between males and females, but no significant shape differences between the sexes were observed once size information is removed.
In our study, morphological differences of both extremities in form space suggest that females possess smaller articular surfaces, shorter and narrower malleoli, and shallower malleolar fossas, with subsequently reduced areas of insertion of knee and ankle collateral ligaments, and less robust muscle insertions for the m. fibularis longus, m. biceps femoris, and interosseous ligament distal attachment (
Figure 3,
Figure 7,
Figure 8, and
Figure S2). This indeed may reflect a sex-specific pattern of knee [
56,
57,
58,
59] and ankle/foot morphology and posture [
29,
60,
61], and in general supports the existence of sex-segregated patterns of lower limb muscle activation in the different sexes [
62,
63,
64,
65]. Regarding the ankle, previous studies have demonstrated sex-related differences in anatomical and biomechanical features of the joint [
59]. Adjei, Nalam, & Lee [
66], congruently with Trevino and Lee [
67], found that ankle stiffness varied significantly between sexes, both along the sagittal and frontal planes (therefore concurring in dorsiflexion/plantarflexion and inversion/eversion, respectively), with greater joint stiffness in females while both quiet standing and muscle activation occurred. The authors explain their findings with the greater passive resistance of females to a greater range of motion, lower elastic modulus, and higher ligamentous laxity [
68,
69,
70] than males, which by contrast possess more leg muscle mass [
71] and a higher muscle and cortical bone cross-sectional area in the whole lower limb [
7,
9,
72] than females. Females have an increased range of motion in the sagittal plane of rearfoot and midfoot, peak plantarflexion angle of rearfoot, peak dorsiflexion and abduction angle of midfoot, as compared with males [
73]. Females also possess greater ligamentous laxity, resulting subsequently in this increased mobility of the ankle joint [
68,
74]. Indeed, our results show that females have shallower malleolar fossae with reduced area of insertion of ankle stabilizers (transverse tibiofibular and posterior talofibular ligaments), less protruding malleoli with reduced area of insertion of ankle collateral ligaments (anterior, posterior talofibular, and calcaneofibular ligaments), and less robust muscle insertions for the m. fibularis longus, the main evertor of the ankle (
Figure 3,
Figure 7,
Figure 8 and
Figure S2). This result seems also to indicate sex-specific differences in structural properties of ankle stabilizers, as differences in tendon tissue properties of the leg have already emerged [
70].
Regarding the knee, important differences among the sexes have been established in morphology and kinematics [
75]. El Ashker and colleagues [
57] found that males have higher functional hamstring-to-quadriceps strength ratios than females, suggesting possible quadriceps dominance in females or greater contribution of the hamstrings in males than in females, either in motion or during foot contact with the ground, stabilizing the joint angle. Their findings seem congruent with our result, showing a protruding area of attachment of m. biceps femoris in the proximal fibular extremity in males (
Figure 3,
Figure 7,
Figure 8 and
Figure S2). While the specific relationship between a muscle and its tendon can change depending on several factors (e.g., muscle/tendon specificity, loading degree, age), in our sample, such variations are minimal, since all individuals possess similar activity levels (i.e., sedentary lifestyle [
76], and age variations in the samples are similar (
Table 1). Therefore, tendon size could be used to assess muscle size [
54]. Finally, we did not find significant shape/form sex-related variation regarding articular orientation in agreement with previous studies on sex variations of the bones of the leg [
28], but see also [
77].
Our results show only weak sex-related differences in fibular muscle and ligament insertions when size is excluded from analysis (
Figure 2,
Figure 5,
Figure 6 and
Figure S1). It is interesting to notice that studies have found higher mean torque of all major muscle groups in the lower extremity in men (women < 62–70% of men), but when measurements are standardized by size (i.e., bone mass index or body weight), no significant differences in strength, endurance, or torque between sexes have been found [
78]. However, when body surfaces and dimensions are evaluated aside from muscle attachments, the relationship between sex, shape, and size is more complex: Wunderlich and Cavanagh [
79] stressed that female lower limbs are not solely scaled-down versions of the male lower limb. Particularly, the lateral side of the foot, directly involved in eversion, showed marked sex-related differences even after standardization of size [
60]. Indeed, our analysis indicates consistent differences between males and females in size, as sexes differ according to CS in the two Italian populations and when the sample is pooled. According to our results, fibulae of males possess larger linear dimensions than females, due to a generally larger body size (regardless of body mass), possibly due to a relatively longer pubertal growth period, and the subsequent development of larger bones and muscles under the influence of sex genes, sex steroids (androgens and estrogens), and other hormones, such as growth hormone (GF) and insulin-like growth factor 1 (IGF1) which, concurring with mechanical loading, may further contribute to the development of skeletal sexual dimorphism [
80,
81,
82]. Indeed, bone growth is differentiated between sex groups, mostly evident during puberty but to a lesser extent also apparent in early childhood [
83,
84,
85], and this concurs with the observed greater cortical bone plasticity in males, modelled by greater muscle mass during ontogeny, determining greater long bone lengths and breadths, also already observed for the fibula in a similar sample [
86]. During puberty, males develop higher peak bone mass, greater bone size, and, ultimately, a stronger skeleton than females [
87,
88], with different skeletal maturation timing for both fibular extremities according to sex [
55]. Our results are also congruent with the already observed different sizes in males and females for the whole size of the tibiofibular [
27,
28] and with prior assessments on the fibula alone [
30].
The present study highlights the importance of a population-based approach for sexing the human fibula, as our results point to a different degree of sexual dimorphism in populations with different ancestries (
Figure 7,
Figure 8 and
Figure 9). Maass and Friedling [
30] found that Coloured males and females differed significantly from Black and White males and females, with the latter groups not differing from one another, and they interpreted their results in light of the genetic contribution of small-bodied groups within the Coloured group. However, in their case, males and females had significantly different size within the three populations, considered separately. Our results comparing different ancestries, on the other hand, show that sexual variations in fibular extremities are not strictly related to size: in fact, while Sardinians and South Africans have similar (smaller) fibular epiphyseal sizes, the former show a difference in size among sexes, while the latter do not (
Figure 9). Some fluctuations in the degree of sexual dimorphism among different populations are expected and could be ascribed to either proximity among individuals of the same population (i.e., response to nutritional stress or overall improvements in the environment) or ultimate causations (i.e., selection and genetic adaptation to a variety of ecological, social, or economic factors) [
89,
90].
Fibular extremities appear more dimorphic in the Italian samples than in the African one, likely as the result of the small sample size characterizing the South African sample or for other reasons which are not explored in this study. This result needs to be further investigated by increasing the sample size and including populations with different ancestry, subsistence strategies, and lifestyles that may lead to differences in the shape of the bones [
76]. However, the 80–93% accuracy in sex determination observed for the Italian samples shows the potentiality of the method presented in this study and is congruent with similar discriminant functions provided by studies on fibular sexual dimorphism [
4,
12,
13,
14,
15,
16,
22,
23,
91], mostly reaching the threshold of 80% suggested for sex estimation using long bones [
92]. Indeed, when form PCs do not differ significantly among the sexes, accuracy percentages are much lower. When size is considered aside from shape variations (CS), accuracy percentages are quite reduced (69–87%), suggesting that the interaction between size and shape (i.e., form) mainly accounts for sex differences in the human fibula.
The main limitation of this study is the low sample size of the South African sample, which may have influenced the lack of significance in all comparisons performed in our study. The small sample size is due to limited resource availability at the time of the sample collection.
Caution should therefore be taken in drawing conclusions on the lack of sexual dimorphism within this sample, as a larger number of individuals may indeed reveal sex-related patterns that are here undetected. A further confounding factor in the South African sample may be the mixed origin of the populations included in this study, whose different body may translate into masking the effect of size sexual dimorphism [
34]. Moreover, other factors may have contributed to the lack of sex variation in fibula, such as genetic background, environmental changes, and changes in socioeconomic and health conditions, as already observed in the larger bones of the lower limb [
93]. Even though previous GM results on the fibula indicate a lack of sexual dimorphism in the fibula [
30], the different methodology here employed (with sliding semi-landmarks instead of only fixed landmarks) could better detect subtle differences. Further studies are therefore necessary to evaluate in more detail this aspect in the South African population. Another limitation of the methodology here proposed is its time-consuming programming phase, which could limit its application in forensic contexts despite its accuracy.
5. Conclusions
In this study, we provided a novel methodology that can be used to determine sex in bioarcheological and, to a lesser extent, forensic investigations using the proximal and distal extremities of the human fibula, rarely analyzed in anthropology. To our knowledge, this is the first study that addresses fibular epiphyseal shape, form, and size in relation to sexual dimorphism, adopting a 3D GM method that includes both fixed landmarks and sliding (semi)landmarks, which together account for detailed morphological variations of fibular extremities. Our work included three different populations, two from Italy and one from South Africa, belonging to identified modern skeletal collections (19th–20th century).
In comparison to other sex determination methods based on fibular linear measurements, our method, despite being more time-consuming, provides an objective, more reliable, and repeatable tool for investigating this district in Italian populations when fibular extremities are found to be isolated and intact, which could be useful, especially for bioarcheological purposes but also in the forensic field. This method could also be integrated into a wider, accurate evaluation of the whole tibiofibular complex for sex determination, alongside the tibia.
The results of the 3D GM study of the proximal and distal extremities of the fibula showed that the fibula can be used to assess sex with accuracy ranging from 80% to 93% in the two Italian populations. The differences mainly consist in larger and more robust epiphyses in males than in females, with females showing narrower articular surfaces, shorter and narrower malleoli, shallower malleolar fossa and peroneal groove, and less protruding muscle insertions for the m. fibularis longus, m. biceps femoris, and interosseous ligament distal attachment. Such distinct form morphology may reflect a sex-specific pattern of knee and ankle/foot posture, with distinctive lower limb morpho- functional aspects in relation to sex. Our results also highlight the importance of a population-based approach to sex determination of the human fibula, as evidenced by our finding that sexual dimorphism can be determined for the two Italian populations but not for the South African one, reasonably related to its small sample size. This may suggest a different degree of sexual dimorphism in populations with different ancestries, which is not strictly related to size variations. However, when the sample is pooled, our sex estimation method based on cross-validation LDA on form PCs gives accuracy above 80%, which is lower when considering the size alone and the shape PCs, suggesting that the interaction of size and shape is the pattern that mainly reflects sexual dimorphism in the human fibula.