Twenty one metrics taken from radiographs of the forelimbs and hind limbs of the silver fox and PWD were used to construct separate anatomical principal component (PC) matrices of the two species (see Methods section). presents the percent variation of each of the 21 PCs for the dog and for the fox, together with their heritabilities (see Methods section).
Parameters of dog and fox limb Principal Components (PC)
For the dog, 15 of the PCs showed significant heritability and an additional four were marginally heritable (italics). Only two, PCs 19 & 20 (bold), showed no heritability. Similarly, 15 of the fox PCs were significantly heritable and another three (italics) were marginally heritable. Fox PCs 10, 16, and 20 were clearly not heritable (bold). Significant heritabilities ranged from 70% to 25%.
In addition, QTLs could be identified in the dog population. These are shown in . Significant PC QTLs were identified on 19 of the 38 autosomes and more may be expected as marker coverage is improved for the genotyped population.
Fig. 1 QTLS identified for limb PCs in the dog. The physical map positions (y-axis) for all SSR markers used (−), are identified on the 38 dog autosomes (CFA 1–38) arrayed on the x-axis. QTL positions are indicated at genome-wide threshold levels (more ...)
PC1, or size, accounted for 50–58% of the variation. Other components represented different aspects of shape that could be characterized by the trait loadings for each component. A comparison between the dog and fox demonstrates that many of the PCs represent variations in shape that have been maintained over more than 10 million years of evolution, despite the extremely stringent selective bottlenecks of domestication and selection of breeds.
In both species, PC 2 and PC 21 represent the largest (12–15%) and smallest (0.2–0.3%) components of variation other than size. One might expect PC 21 to be an artifact of statistical noise. Evidence that PC 21 represents variation of a real phenotype (as opposed to nonspecific variation or “noise”) comes from comparing values for PC 21 computed separately from metrics of the right and left fox limbs. presents this right versus left correlation both for fox PC 2 and fox PC 21. If PC 21 were an artifact, we would expect no correlation between right and left values. It can be seen that the correlation for PC 21 is almost as good as that for PC 2. Moreover, both PC 2 and PC 21 are highly heritable (ranging from h2 ~0.75 for PC 2 in the dog to h2 ~0.4 for PC 21 in the dog and fox).
Correlations between the PC values obtained using sets of metrics from the right or the left limbs of the fox.
presents loadings of PC 2 and PC 21 that characterize these PCs in the dog and fox. It can be seen that PC 2, in both species, represents a trade-off in which limb width is inversely correlated with limb length, accounting for about 12–15% of the total variation or ~30% of the variation in shape. The loading patterns that characterize this PC are extremely robust: bootstrap analysis indicates that these loading patterns will be obtained in more than 99 out of 100 trials, and five QTLs for PC 2 have been identified in the dog located on autosomes CFA 1, 7, 12, and 27 (). (See Fig. 4 of Lark et al. 2006b
, for details of phenotypic effects of CFA 12 QTL genotypes).
Trait loadings for dog and fox PCs 2 and 21
In contrast, PC 21 a trade-off between the length of the radius and the length of the tibia and humerus, represents a minor fraction of the total variation, ~0.2%. However, variation in the length of the radius occurs in >99% of subsamples in both the dog and the fox, and in the dog an inverse correlation with the length of the tibia is similarly robust. In the fox, a similar inverse correlation with the tibial length is observed but it is somewhat less robust, occurring between the radius and tibia length in subsamples somewhat more frequently than 95% of the trials. In both fox and dog, inverse correlations of the radius with the humerus are observed with a frequency of better than 95% of subsampling trials.
Finally, three QTLs have been identified in the dog for PC 21 on autosomes CFA 26, 27, and 38 () and the relationships between genotype and phenotype for two of these QTLs are presented in for the CFA27 and CFA38 QTLs, respectively. The phenotype of the QTL on CFA27 appears to be recessive (two copies of the marker allele with which the phenotype is associated are required for phenotypic expression). In contrast, the phenotype of the CFA38 QTL is dominant.
Fig. 3 Phenotypic expression of dog QTL genotypes that regulate expression of PC21 (A, B), PC14 (C) and PC13 (D). QTLs shown are identified in : (A) PC21 QTL on CFA 27 marker FH4001 bp9855710-9855969; (B) PC21 QTL on CFA 38 marker G02108 bp17539884-17540056; (more ...)
The heritability of PC 21 in both the fox and the dog, the correlation between the left and right PC 21 values, and the identification of regulating QTLs in the dog compel the conclusion that this small amount of skeletal variation represents an hereditary variation in shape that has been preserved over the evolutionary history that separates foxes and dogs.
Additional heritable effects contrasting the forelimbs and hind limbs
The inverse correlation between forelimb length and hind limb length that occur in both fox and dog PC 21 is found in other aspects of shape that characterize the front and rear limbs of dogs and foxes.
In another PC, the lengths of the metacarpal and metatarsal bones also are inversely correlated (). These constitute extremely robust loadings of dog PC 14 and fox PC 15. Both PCs are significantly heritable () and a QTL has been identified on CFA 1 of the dog (). presents details of this dog PC 14 QTL. Although the relevant allele cannot be recessive, the data do not allow distinguishing between a dominant or additive mode of inheritance.
Trait loadings for dog PC14 and fox PC 15
Cortical thickness also contrasts the fore and hind limbs in both the dog and the fox. PC 20 in the dog and PC 18 in the fox describe an inverse correlation between the cortical thickness of the humerus and the femur (). These account for between 0.4% and 0.8% of the variation in shape (0.2—0.4% of the total variation if size is included). In both species, the cortical thickness in the foreleg is inversely correlated with that of the hind leg. In the fox, however, this PC is heritable (22%; ) whereas in the dog it is not. Another PC in the fox, PC 13, characterizes a similar relationship of the distal rear limb and forelimbs. This inverse correlation between cortical thickness of the radius and the tibia is highly heritable (37%; ) and accounts for ~1% of the total variation. No such PC was found in the PWD.
Trait loadings for dog PC20 and fox PCs 13 and 18
Finally, inverse correlations were observed between lever arms on the fore and hind legs. presents results for PC 13 in the dog and PC 14 in the fox with inverse correlations between metrics (loadings) of the olecranon (i.e., muscle lever of elbow) and the in-lever or calcaneous (i.e., muscle levers of the ankle joints of the dog and fox, respectively). Again, these inverse correlations were highly heritable in both species () and three QTLs were identified in the dog, on autosomes CFA 6, 15, and 24. Variation between haplotypes of the dog PC 13 QTL on CFA 24 is presented in . In this case, a recessive mode of inheritance has been established. Whereas the fore–hind relationship in the dog involved the olecranon in the forelimb and the muscle in-lever in the hind limb, a somewhat different relationship was found in the fox, involving the calcaneous on the hind limb. The in-lever is, in fact, a part of the calcaneous, posterior to the ankle joint. The portion of the calacaneous anterior to the ankle joint is part of the out-lever, of which the main portion is the metatarsal. It is noteworthy that PC 17 in the dog and PC 19 in the fox ( and ) represent a trade-off between the in-lever and the calcaneous of which the in-lever is a part. Although the variation involved is small, it is heritable and represents a trade-off between the in-lever and out-lever functions of the calcaneous; like PC 2, this is a trade-off between generation of force (power) and speed (velocity). This suggests that in the fox, the olecranon is involved in a trade-off involving the ankle out-lever of the hind limb, in contrast to the dog in which the trade-off involves the in-lever.
Trait loadings for dog PCs13 and 8 and fox PCs 14 and 12
A separate relationship was identified between the ankle in-lever and the pisiform (i.e., muscle lever of wrist joint) (). PC 8 in the dog and PC 12 in the fox, both show this inverse correlation involving PCs different from those in which the olecranon is a participant. These PCs also are heritable in both species, although, as yet, we have not identified QTLs associated with PC 8 in the dog. Again the variation explained by these muscle-lever-arm PCs is small (1–2% of the total variation) but clearly significant.