Cross-sectional morphology and biomechanical advantage
Our results demonstrate that the cross-sectional morphology of long bones can differ among specialized locomotor habits in Mustelidae, a functionally diverse and speciose lineage within Carnivora. These findings fit well with broader patterns throughout the vertebrate skeleton, indicating linkages of form, function and behavior or performance; in other words, that bone geometry grossly reflects loading patterns. For example, differences in long bone cross-sectional traits have been reported in birds of differing locomotor modes [34, 92] and primates differing in slow climbing, suspensory, and leaping locomotor habits [16, 44, 86]. While many prior studies have focused on external bone dimensions and their relationships with higher-level biological factors such as locomotion, we have contributed a new, focused analysis of Mustelidae using the valuable perspective that analysis of internal (i.e. cross-sectional) bone dimensions can bring. Our findings have some general correspondences with similar analyses by Doube et al. [18,19,20]; cf. shapes of curves in our Figs. 4 and 5) and others, but the Mustelidae-specific insights are important and novel.
In line with our prediction, natatorial and fossorial mustelids tended to have greater values of cross-sectional traits than remaining mustelids (Fig. 4 and Additional file 1: Figure S1), with natatorial and scansorial mustelids possessing respectively the highest and lowest values of cross-sectional traits. Natatorial mustelids significantly differed from scansorial (red circles) and generalized mustelids (bronze triangles) in all cross-sectional traits of the humerus and radius, with these differences being rather extensive along these bones’ lengths. Natatorial mustelids also significantly differed from scansorial and generalized mustelids in ulnar cross-sectional traits, though to a much lesser extent, apart from ulnar CSA. In contrast, fossorial mustelids tended to significantly differ primarily from scansorial mustelids (navy circles) in humeral and radial cross-sectional traits; however, differences in ulnar SMA and MOD were not prevalent across the entire length of the ulna. We found that significant differences between fossorial and generalized mustelids (red triangles) only occurred in humeral SMA and MOD at localized regions along this bone’s length. Thus in mustelids, any possible locomotion-distinct phenotypes associated with cross-sectional morphology do not necessarily encompass all forelimb bones or all cross-sectional traits. Moreover, the four locomotor habits within Mustelidae sampled here likely are not each characterized by a distinct cross-sectional morphology, because generalized mustelids only rarely differed in cross-sectional traits from both scansorial and fossorial species (bronze circles and red triangles, respectively).
The low values of cross-sectional traits of the forelimb skeleton in scansorial mustelids correspond to the greater gracility of their forelimb skeleton [27, 53] and the relatively elongate and lightweight limbs of scansorial mammals in general [51, 52]. The gracile and elongate forelimb skeleton of martens, though not as extreme as in other scansorial carnivorans , likely confers advantages in bridging discontinuities in supports (e.g., tree branches) while climbing . Moreover, Cartmill  argued that larger body sizes may hamper climbing ability; therefore it is also seems plausible that overly robust or more massive limbs may also be disadvantageous for a climbing lifestyle.
Greater values of cross-sectional traits strongly distinguishing natatorial mustelids from scansorial, generalized, and, to a lesser extent, fossorial mustelids (Fig. 4 and Additional file 1: Figure S1: red circles, bronze and navy triangles) indicate that otters have humeri, radii, and, to a lesser degree, ulnae with greater relative resistance to compression (i.e., CSA) and bending (i.e., SMA) and greater structural strength (i.e., MOD) than mustelids of other locomotor habits. The greater values of cross-sectional traits for natatorial mustelids would be advantageous for swimming by drag-based propulsion, though the degree to which forelimbs function in swimming varies among otter species. Notably, the forelimbs of sea otters (Enhydra lutris) do not play a role in swimming but are extensively involved in tool use and prey manipulation, such as hammering open or prying prey loose [50, 68]. It could be that the forces generated in this behavior could require a forelimb skeleton structurally stronger than other mustelids; however, there currently appears to be no published data on the mechanics of tool use in this species.
However, greater values of cross-sectional traits – and consequently the increased load resistance they offer – are likely not critical to swimming in mustelids. Recent work comparing bone loading in turtles, both during walking and swimming, found significantly lower bone strains during swimming than walking, likely due to buoyant forces removing the need for the limbs to support body weight despite their roles in providing thrust during locomotion [103, 104].
An alternative and more likely, though not mutually exclusive, explanation would be the need for thicker bones to help counteract buoyancy during subsurface swimming [42, 43]. Given that natatorial mustelids have the highest values of cross-sectional traits, it strongly suggests that the need to counteract buoyancy may have a stronger influence upon cross-sectional morphology than any increased resistance to the musculoskeletal loads imposed by specialized limb functions occurring in mustelids. An exception to the general trend among otters is the small-clawed otter (Amblonyx cinereus), which lies comfortably in the range of scansorial mustelids. Notably, this species possesses rather gracile long bones more comparable to scansorial mustelids [27, 53] than to other otters, with its humerus further lacking the strong anterior bowing characteristic of other otters (; pers. obs.). Moreover, this species forages somewhat more terrestrially where it occurs sympatrically with Eurasian otters (Lutra lutra) and smooth-coated otters (Lutrogale perspicillata) , and the webbing is incomplete between its digits , so it could be considered less aquatic than other otter species.
Fossorial mustelids have high values of cross-sectional traits compared to scansorial and generalized mustelids (Fig. 4 and Additional file 1: Figure S1: brown curves), likely due to the limbs having to function in soil, which has a high density. Although the degree of fossoriality may vary among taxa , most badgers and other fossorial mustelids (e.g., zorilla, Ictonyx striatus) dig as a means of foraging and may dig their own burrows [56, 59, 73, 77, 93, 98]. However, some badgers display exceptional digging ability, including rapid digging , digging extensive burrow systems , digging a new den every night , and burying food items several times larger than themselves as a cache . Interestingly, significant differences in SMA and MOD between fossorial and other mustelids were noticeably not as widespread in the ulna as in the humerus and radius. This is surprising given the insertion of the triceps muscle group, which is highly specialized with an angular head in mustelids , onto the olecranon process, and the triceps’ highly integral role in exerting force during scratch digging [39, 40, 71]. However, these distinct results for the ulna may be due to the trochlea of the humerus and the trochlear notch of the ulna restricting its movement to flexion and extension relative to the humerus regardless of specializations in limb function. Thus, the ulna cannot exhibit long axis rotation unlike the radius, and thus may experience a lower diversity of loading regimes than the latter bone. Moreover, given that the distal articulation of the radius has much broader contact with the carpus than that of the ulna, it could be possible that the radius receives more of the mechanical loads transmitted proximally along the forelimb by the manus than the ulna, and, if so, this may be reflected in the differences in radial cross-sectional morphology among mustelid locomotor habits. This discrepancy in results among the humerus, radius, and ulna suggests that the loading of limb bones during digging may be complex, with differing bones operating at different loads and safety factors (e.g. perhaps fitting the “mixed chain” hypothesis; ).
In addition to function, size may be another factor influencing forelimb morphology in mustelids. In particular, greater values of cross-sectional traits are generally associated with larger body sizes in many mammals [18, 19] and birds . Plotting dimensionless values of mustelid cross-sectional traits against body mass reveals a complicated relationship with body size (Additional file 4: Figure S2). Otters, which include the most massive mustelids, appear to have an allometric trajectory distinct from other mustelids’. However, for a given body mass where multiple locomotor habits coincide, scansorial mustelids have smaller values of cross-sectional traits than either fossorial and generalist mustelids do. This differentiation of locomotor habits for a given body mass suggests that our results are not solely due to the influence of size (i.e. scaling). Rather our results appear subject to the mixed influences of locomotor habit and size.
Resistance to bending vs. compression
The ratio R revealed that, by and large, differences in locomotor habit are not associated with a trade-off in resistance to bending vs. compression (Fig. 5). Humeral RCC was an exception to this, with significant differences occurring among mustelids locomotor habits between 25 and 90% of humeral length. Notably, in contrast to our separate tests of individual cross-sectional traits, RCC distinguished fossorial and natatorial mustelids, with badgers having significantly greater values of RCC than otters. This result suggests that, in the case of badger humeri, possible selection with regards to fossoriality in mustelids may pertain more to the ratio of resistance to particular loading regimes than the absolute resistance to a single loading regime. Compared to otters, badgers exhibit humeri with relatively greater resistance to bending about the cranio-caudal axis (Fig. 1) relative to the total amount of bone tissue comprising their humeral cross-section. In other words, badgers have a wider distribution of bone tissue in their humeral cross-section than otters in spite of having a lower amount of overall bone tissue within their cross-section. This result concurs with our earlier finding of badgers having more robust forelimb long bones (in terms of external dimensions; ) and our current finding that badgers have lower values of CSA than otters.
Humeral RCC also distinguished natatorial mustelids from scansorial mustelids, with martens having greater SMACC relative to CSA, and fossorial mustelids from generalized mustelids, further suggesting that the humerus’ relative resistance to different loading regimes may distinguish mustelid locomotor habits. The low values of humeral RCC displayed by natatorial mustelids likely reflect the medio-laterally compressed humeral diaphysis of otters, with such compression being common for aquatic tetrapods . These differences in RCC suggest there may be differences in incurred loading regime as forelimbs conduct different functions in mustelids. While this is an exciting topic of investigation, it unfortunately is beyond the scope of our study.
Apart from humeral RCC, there is striking uniformity among other ratios of R (Fig. 5) in Mustelidae. This uniformity suggests that the relative resistance to different loading regimes is not fundamental to functional specializations of the limb and that a single ‘design’ of relative loading resistance allows for disparate limb functions. Furthermore, the uniformity in R values suggests that distribution of bone tissue (i.e., SMA) relative to the total amount of bone tissue (i.e., CSA) of a cross-section may possibly be phylogenetically conserved, or biomechanically or developmentally constrained, at least for the radius and ulna. A conserved internal morphology of the ulna is particularly surprising when considering mammals more broadly, given the variability of the ulna’s external dimensions in terms of its reduction, relative olecranon length, and robustness in relation to specialized limb functions [87, 88], though admittedly mustelids in and of themselves do not display such wide extremes in ulnar morphology. It remains unclear if our findings would, however, relate to the mesopodium or autopodium (carpus/manus).
Evolution of cross-sectional morphology
Within Mustelidae, locomotor habit is intimately linked with phylogeny. Notably, natatorial species evolved from a single ancestor within our sampled mustelids, as is the case for scansorial species (Fig. 1). Among our sample, there is one instance of convergence in fossorial limb function (Ictonyx striatus), though until recently there was thought to be more convergence in fossoriality in Mustelidae . The preponderance of non-significant results for phylogenetic ANOVAs further underscores that phylogeny is a strong component of the observed morphological variation in Mustelidae. However, our lack of significant findings with phylogenetic ANOVAs goes against the known biomechanical relevance of cross-sectional bone dimensions for many mustelid species, particularly otters (e.g., ). While phylogenetic ANOVAs are vital to address the influence of shared ancestry upon trait variation, such analyses by themselves could lead to faulty interpretations of how morphology relates to biomechanical function. In turn, while standard ANOVAs are able to discern morphological differences relevant to biomechanics, they obviously fail to address the role of phylogeny in trait variation. Thus, the pairing of both ahistorical and historical analyses is required for a more comprehensive view of the evolution of biomechanical traits.
Fitting models of trait evolution uncovered that the most likely pattern of evolution with regards to the cross-sectional traits of the humerus, radius, and ulna was either a multi-rate Brownian motion model (BM3/BM4) or a multi-optima Ornstein-Uhlenbeck model (OU3/OU4) (Fig. 6). These models distinguish either distinct rates of evolution (Brownian motion models) or evolution towards distinct phenotypic optima (Ornstein-Uhlenbeck models) for the differing locomotor habits within Mustelidae. Both of these models propose that natatorial and scansorial mustelids morphologically diverged from one another and remaining mustelids, either by evolving under differing rates of Brownian motion or towards distinct adaptive optima. Moreover, finding BM4 and OU4 as the best-fitting model indicates that each locomotor habit within Mustelidae is tied to a divergence in forelimb cross-sectional traits. This result is in line with these two locomotor habits being the extremes of cross-sectional morphology in mustelid long bones.
The prevalence of OU models as the best-fitting models would suggest that the locomotor diversity among mustelids is the result of evolution towards distinct phenotypes ‘optimal’ for the biomechanical demands of a given locomotor habit. However, inspection of α, commonly interpreted as the strength of selection in OU models , is crucial prior to accepting an OU model as the most plausible mode of evolution for a given trait . When α does not significantly differ from 0.0, then the OU model is equivalent to a Brownian motion model . Inspection of α-values in instances where OU models were the best-fitting models revealed numerous instances where α could not be distinguished from 0.0 (Fig. 6 and Additional file 3: Tables S4-S6). The outperformance by the OU model vs. the BM model in these instances was due to the additional parameters of the OU model affording the best description of the data’s variance outside of the model’s biological relevance .
It thus appears that the cross-sectional morphology of the mustelid humerus, radius, and ulna has evolved predominantly due to a multi-rate Brownian motion process. Under such a mode of evolution, the distinct cross-sectional morphologies of mustelid locomotor habits are associated with a distinct rate of phenotypic evolution, and it is possible that these differences in rate are associated with different constraints upon the evolution of cross-sectional morphology in mustelids (see [74, 96]). Such constraints regarding mustelid limbs could be the biomechanical benefits of thinner, and presumably more lightweight, bones associated with a climbing lifestyle or more robust bones associated with an aquatic lifestyle (see above). Alternatively, cross-sectional morphology may have been under selection at one point during mustelid evolution, with resulting changes in morphology being conserved among later divergences of mustelids (i.e., phylogenetic inertia in the trait). This would be in contrast to a continuous selective regime acting across the branches associated with those later divergences (as is the case in an OU model).
This overall result contrasts with the likely mode of evolution for the external dimensions of the forelimb skeleton (e.g., lengths, diameters, and muscle in-lever lengths). The external dimensions of the forelimb skeleton likely evolved adaptively, with adaptive peaks distinguishing scansorial from remaining mustelids in terms of the length of muscle in-levers and long bone gracility . Then again, the contrasting results of the current study and those of Kilbourne  might be due to sample size. In the current study, we restricted our sample size to seven taxa per locomotor habit for a total of 28 taxa, whereas Kilbourne  sampled as many mustelid species as possible for a total of 41 taxa. However, another possible explanation may be that the different, functionally relevant traits within a single functioning organ may evolve by different processes in mustelids. These results also raise the question of how do differing traits, with different biomechanical functions (e.g., the mechanical advantage offered by muscle in-levers vs. the bending resistance offered by SMA), contribute to the overall adaptations occurring in a limb? This question merits future focus in trait evolution studies combining different kinds of traits, though current methods may be ill equipped to address it .