Muscle internal structure revealed by contrast-enhanced μCT and fibre recognition: The hindlimb extensors of an arboreal and a fossorial squirrel


In individuals of similar body mass representing closely related species with different lifestyles, muscle architectural properties can be assumed to reflect adaptation to differing, lifestyle-related functional demands. We here employ a fibre recognition algorithm on contrast-enhanced micro-computed tomography (uCT) scans of one specimen each of an arboreal (Sciurus vulgaris) and a fossorial (Spermophilus citellus) sciuromorph rodent. The automated approach accounts for potential heterogeneity of architectural properties within a muscle by analysing all fascicles that compose a muscle. Muscle architectural properties (volume, fascicle length and orientation, and force-generating capacity) were quantified in 14 hindlimb (hip, knee, and ankle) extensor muscles and compared between specimens. We expected the arboreal squirrel to exhibit greater force-generating capacity and a greater capacity for length change allowing more powerful hindlimb extension. Generally and mostly matching our expectations, the S. vulgaris specimen had absolutely and relatively larger extensor muscles than the S. citellus specimen which were thus metabolically more expensive and demonstrate the relatively larger investment into powerful hindlimb extension necessary in the arboreal context. We conclude that detailed quantitative data on hindlimb muscle internal structure as was gathered here for a very limited sample further lends support to the notion that muscle architecture reflects adaptation to differential functional demands in closely related species with different locomotor behaviours and lifestyles.

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  1. Aerts, P., 1998. Vertical jumping in Galago senegalensis: the quest for an obligate mechanical power amplifier. Philos. Trans. R. Soc. Lond. B Biol. Sci. 353 (1375) 1607–1620.

  2. Allen, V., Elsey, R.M., Jones, N., Wright, J., Hutchinson, J.R., 2010. Functional specialization and ontogenetic scaling of limb anatomy in Alligator mississippiensis. J. Anat. 216 (4) 423–445.

  3. Amson, E., Arnold, P., van Heteren, A.H., Canoville, A., Nyakatura, J.A., 2017. Trabecular architecture in the forelimb epiphyses of extant xenarthrans (Mammalia). Front. Zool. 14 (1) 52.

  4. Böhmer, C., Fabre, A.C., Herbin, M., Peigné, S., Herrel, A., 2018. Anatomical basis of differences in locomotor behavior in Martens: a comparison of the forelimb musculature between two sympatric species of Martes. Anat. Rec. 301 (3) 449–472.

  5. Botton-Divet, L., Cornette, R., Fabre, A.C., Herrel, A., Houssaye, A., 2016. Morphological analysis of long bones in semi-aquatic mustelids and their terrestrial relatives. Integr. Comp. Biol. 56 (6) 1298–1309.

  6. Chi, S.W., Hodgson, J., Chen, J.S., Edgerton, V.R., Shin, D.D., Roiz, RA, Sinha, S., 2010. Finite element modeling reveals complex strain mechanics in the aponeuroses of contracting skeletal muscle. J. Biomech. 43 (7) 1243–1250.

  7. Close, R.I., 1972. The relations between sarcomere length and characteristics of isometric twitch contractions of frog sartorius muscle. J. Physiol. 220 (3) 745–762.

  8. Daley, M.A., Felix, G., Biewener, A.A., 2007. Running stability is enhanced by a proximo-distal gradient in joint neuromechanical control. J. Exp. Biol. 210 (3) 383–394.

  9. Delp, S.L., Anderson, F.C., Arnold, A.S., Loan, P., Habib, A., John, CT., et al., 2007. OpenSim: open-source software to create and analyze dynamic simulations of movement. IEEETrans. Biomed. Eng. 54 (11) 1940–1950.

  10. Demes, B., Jungers, W.L., Fleagle, J.G., Wunderlich, R.E., Richmond, B.G., Lemelin, P., 1996. Body size and leaping kinematics in Malagasy vertical clingers and leapers. J. Hum. Evol. 31 (4) 367–388.

  11. Demes, B., Fleagle, J.G., Lemelin, P., 1998. Myological correlates of prosimian leaping. J. Hum. Evol. 34 (4) 385–399.

  12. Dick, T.J., Clemente, C.J., 2016. How to build your dragon: scaling of muscle architecture from the world’s smallest to the world’s largest monitor lizard. Front. Zool. 13 (1) 8.

  13. Dickinson, E., Stark, H., Kupczik, K., 2018. Non-destructive determination of muscle architectural variables through the use of dice CT. Anat. Rec. 301 (2) 363–377.

  14. Doube, M., Klosowski, M.M., Wiktorowicz-Conroy, A.M., Hutchinson, J.R., Shefelbine, S.J., 2011. Trabecular bone scales allometrically in mammals and birds. Proc. R. Soc. B: Biol. Sci. 278 (1721) 3067–3073.

  15. Dunham, N.T., McNamara, A., Shapiro, L., Phelps, T., Wolfe, A.N., Young, J.W., 2019. Locomotor kinematics of tree squirrels (Sciurus carolinensis) in free-ranging and laboratory environments: implications for primate locomotion and evolution. J. Exp. Zool. Part A Ecol. Integr. Physiol. 331 (2) 103–119.

  16. Epstein, M., Herzog, W., 1998. Theoretical Models of Skeletal Muscle: Biological and Mathematical Considerations. John Wiley & Sons), Chichester.

  17. Essner, R.L., 2002. Three-dimensional launch kinematics in leaping), parachuting and gliding squirrels. J. Exp. Biol. 205 (16) 2469–2477.

  18. Fischer, M.S., 1994. Crouched posture and high fulcrum), a principle in the locomotion of small mammals: the example of the rock hyrax (Procavia capensis)(Mammalia: Hyracoidea). J. Hum. Evol. 26 (5–6), 501–524.

  19. Fischer, M.S., Schilling, N., Schmidt, M., Haarhaus, D., Witte, H., 2002. Basic limb kinematics of small therian mammals. J. Exp. Biol. 205 (9) 1315–1338.

  20. Gambaryan, P.P., 1974. How Mammals Run. Anatomical Adaptations. J. Wiley, New York, NY.

  21. Gans, C., Bock, W.J., 1965. The functional significance of muscle architecture-a theoretical analysis. Ergeb. Anat. Entwicklungsgesch. 38, 115–142.

  22. Gaudin, T.J., Nyakatura, JA, 2018. Epaxial musculature in armadillos), sloths), and opossums: functional significance and implications forthe evolution of back muscles in the Xenarthra. J. Mammal. Evol. 25 (4) 565–572.

  23. Gignac, P.M., Kley, N.J., Clarke, JA, Colbert, M.W., Morhardt, A.C., Cerio, D., et al., 2016. Diffusible iodine-based contrast-enhanced computed tomography (diceCT): an emerging tool for rapid), high-resolution), 3-D imaging of metazoan soft tissues. J. Anat. 228 (6) 889–909.

  24. Guy, P.S., Snow, D.H., 1981. Skeletal muscle fibre composition in the dog and its relationship to athletic ability. Res. Vet. Sci. 31 (2) 244–248.

  25. Herrel, A., De Smet, A., Aguirre, L.F., Aerts, P., 2008. Morphological andmechanical determinants of bite force in bats: do muscles matter? J. Exp. Biol. 211, 86–91.

  26. Huq, E., Wall, CE., Taylor, A.B., 2015. Epaxial muscle fiber architecture favors enhanced excursion and power in the leaper Galago senegalensis. J. Anat. 227 (4) 524–540.

  27. Kilbourne, B.M., Andrada, E., Fischer, M.S., Nyakatura, JA, 2016. Morphology and motion: hindlimb proportions and swing phase kinematics in terrestrially locomoting charadriiform birds. J. Exp. Biol. 219 (9) 1405–1416.

  28. Kilbourne, B.M., Hoffman, L.C., 2013. Scale effects between body size and limb design in quadrupedal mammals. PLoS One 8 (11) e78392.

  29. Kinugasa, R., Yamamura, N., Sinha, S., Takagi, S., 2016. Influence of intramuscular fiber orientation on the Achilles tendon curvature using three-dimensional finite element modeling of contracting skeletal muscle. J. Biomech. 49 (14) 3592–3595.

  30. Kupczik, K., Stark, H., Mundry, R., Neininger, F.T., Heidlauf, T., Röhrle, O., 2015. Reconstruction of muscle fascicle architecture from iodine-enhanced microCT images: a combined texture mapping and streamline approach. J. Theor. Biol. 382, 34–43.

  31. Kuznetsov, A.N., 1985. Comparative Functional analysisof the fore limbsand hind limbs in mammals. Zoologichesky Zhurnal 64 (12) 1862–1867.

  32. Lagaria, A., Youlatos, D., 2006. Anatomical correlates to scratch digging in the forelimb of European ground squirrels (Spermophilus citellus). J. Mammal. 87 (3) 563–570.

  33. Lammers, A.R., German, R.Z., 2002. Ontogenetic allometry in the locomotor skeleton of specialized half-bounding mammals. J. Zool. 258 (4) 485–495.

  34. Lautenschlager, S., 2015. Estimating cranial musculoskeletal constraints in theropod dinosaurs. R. Soc. Open Sci. 2 (11) 150495.

  35. Legreneur, P., Thévenet, F.R., Libourel, P.A., Monteil, K.M., Montuelle, S., Pouydebat, E., Bels, V., 2010. Hindlimb interarticular coordinations in Microcebus murinus in maximal leaping. J. Exp. Biol. 213 (8) 1320–1327.

  36. Leischner, C.L., Crouch, M., Allen, K.L., Marchi, D., Pastor, F., Hartstone-Rose, A., 2018. Scaling of primate forearm muscle architecture as it relates to locomotion and posture. Anat. Rec. 301 (3) 484–495.

  37. Lieber, R.L., Fridén, J., 2001. Clinical significance of skeletal muscle architecture. Clin. Orthop. Relat. Res. 383, 140–151.

  38. Loeb, G.E., Gans, C., 1986. Electromyography for Experimentalists. The University of Chicago Press, Chicago.

  39. Marchi, D., Hartstone-Rose, A., 2018. Functional morphology and behavioral correlates to postcranial musculature. Anat. Rec. 301 (3) 419–423.

  40. Marchi, D., Leischner, C.L., Pastor, F., Hartstone-Rose, A., 2018. Leg muscle architecture in primates and its correlation with locomotion patterns. Anat. Rec. 301 (3) 515–527.

  41. Martín-Serra, A., Figueirido, B., Palmqvist, P., 2014. A three-dimensional analysis of the morphological evolution and locomotor behaviour of the carnivoran hind limb. BMC Evol. Biol. 14 (1) 129.

  42. Metscher, B.D., 2009. MicroCT for comparative morphology: simple staining methods allow high-contrast 3D imaging of diverse non-mineralized animal tissues. BMC Physiol. 9 (1) 11.

  43. Michilsens, F., Vereecke, E.E., D’Août, K., Aerts, P., 2009. Functional anatomy of the gibbon forelimb: adaptations to a brachiating lifestyle. J. Anat. 215 (3) 335–354.

  44. Mielke, F., Schunke, V., Woelfer, J., Nyakatura, JA, 2018a. Motion analysis of non-model organisms using a hierarchical model: influence of setup enclosure dimensions on gait parameters of Swinhoe’s striped squirrels as a test case. Zoology 129, 35–44.

  45. Mielke, M., Wölfer, J., Arnold, P., van Heteren, A.H., Amson, E., Nyakatura, JA., 2018b. Trabecular architecture in the sciuromorph femoral head: allometry and functional adaptation. Zoological Lett. 4 (1) 10.

  46. Myatt, J.P., Schilling, N., Thorpe, S.K., 2011. Distribution patterns of fibre types in the triceps surae muscle group of chimpanzees and orangutans. J. Anat. 218 (4) 402–412.

  47. Nyakatura, J.A., Stark, H., 2015. Aberrant back muscle function correlates with intramuscular architecture ofdorsovertebral muscles in two-toed sloths. Mamm. Biol. 80 (2) 114–121.

  48. Payne, R.C., Hutchinson, J.R., Robilliard, J.J., Smith, N.C., Wilson, A.M., 2005. Functional specialisation of pelvic limb anatomy in horses (Equus caballus). J. Anat. 206 (6) 557–574.

  49. Payne, R.C., Crompton, R.H., Isler, K., Savage, R., Vereecke, E.E., Günther, M.M., Thorpe, S.K.S., D’Août, K., 2006. Morphological analysis of the hindlimb in apes and humans. I. Muscle architecture. J. Anat. 208 (6) 709–724.

  50. Polly, P.D., 2007. Limbs in Mammalian Evolution. Fins Into Limbs: Evolution, Development and Transformation., pp. 245–268.

  51. Regnault, S., Allen, V.R., Chadwick, K.P., Hutchinson, J.R., 2017. Analysis of the moment arms and kinematics of ostrich (Struthio camelus) double patellar sesamoids. J. Exp. Zool. Part A Ecol. Integr. Physiol. 327 (4) 163–171.

  52. Roberts, T.J., 2002. The integrated function of muscles and tendons during locomotion. Comp. Biochem. Physiol. Part A Mol. Integr. Physiol. 133 (4) 1087–1099.

  53. Rosin, S., Nyakatura, JA, 2017. Hind limb extensor muscle architecture reflects locomotor specialisations of a jumping and a striding quadrupedal caviomorph rodent. Zoomorphology 136 (2) 267–277.

  54. Sacks, R.D., Roy, R.R., 1982. Architecture of the hind limb muscles of cats: functional significance. J. Morphol. 173 (2) 185–195.

  55. Samaras, A., Youlatos, D., 2010. Use of forest canopy by European red squirrels Sciurus vulgaris in Northern Greece: claws and the small branch niche. Acta Theriol. 55 (4) 351–360.

  56. Scheidt, A., Wölfer, J., Nyakatura, JA, 2019. The evolution of femoral cross-sectional properties in sciuromorph rodents: influence of body mass and locomotor ecology. J. Morphol.

  57. Schilling, N., 2009. Metabolic profile of the perivertebral muscles in small therian mammals: implications forthe evolution of the mammalian trunk musculature. Zoology 112 (4) 279–304.

  58. Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., et al., 2012. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9 (7) 676.

  59. Schmidt, A., Fischer, M.S., 2011. The kinematic consequences of locomotion on sloped arboreal substrates in a generalized (Rattus norvegicus) and a specialized (Sciurus vulgaris) rodent. J. Exp. Biol. 214 (15) 2544–2559.

  60. Schmidt, M., Fischer, M.S., 2009. Morphological integration in mammalian limb proportions: dissociation between function and development. Evolution 63 (3) 749–766.

  61. Siebert, T., Leichsenring, K., Rode, C., Wick, C., Stutzig, N., Schubert, H., et al., 2015. Three-dimensional muscle architecture and comprehensive dynamic properties of rabbit gastrocnemius), plantaris and soleus: input for simulation studies. PLoS One 10 (6) e0130985.

  62. Stalheim-Smith, A., 1984. Comparative study of the semifossorial prairie dog), Cynomys gunnisoni), and the scansorial fox squirrel), Sciurus niger. J. Morphol. 180, 55–68.

  63. Stark, H., Schilling, N., 2010. A novel method of studying fascicle architecture in relaxed and contracted muscles. J. Biomech. 43 (15) 2897–2903.

  64. Stark, H., Fröber, R., Schilling, N., 2013. Intramuscular architecture of the autochthonous back muscles in humans. J. Anat. 222 (2) 214–222.

  65. Steppan, S.J., Adkins, R.M., Anderson, J., 2004. Phylogeny and divergence-date estimates of rapid radiations in muroid rodents based on multiple nuclear genes. Syst. Biol. 53 (4) 533–553.

  66. Sullivan, S.P., McGechie, F.R., Middleton, K.M., Holliday, CM., 2019. 3D muscle architecture of the pectoral muscles of european Starling (Sturnus vulgaris). Integr. Organ. Biol. 1 (1) oby010.

  67. Taverne, M., Fabre, A.C., Herbin, M., Herrel, A., Peigné, S., Lacroux, C., Lowie, A., Pagés, F., Theil, J.-C, Böhmer, C., 2018. Convergence in the functional properties of forelimb muscles in carnivorans: adaptations to an arboreal lifestyle? Biol. J. Linn. Soc. 125 (2) 250–263.

  68. Thorington Jr, R.W., Darrow, K., Betts, A.D.K., 1997. Comparative myology of the forelimb of squirrels (Sciuridae). J. Morphol. 234, 155–182.

  69. van Eijden, T.M., Koolstra, J.H., Brugman, P., 1996. Three-dimensional structure of the human temporalis muscle. Anatom. Rec.: Off. Publ. Am. Assoc. Anatom. 246 (4) 565–572.

  70. Wells, J.B., 1965. Comparison of mechanical properties between slow and fast mammalian muscles. J. Physiol. 178 (2) 252–269.

  71. Wickiewicz, T.L., Roy, R.R., Powell, P.L., Edgerton, V.R., 1983. Muscle architecture of the human lower limb. Clin. Orthop. Relat. Res. 179), 275–283.

  72. Wölfer, J., Amson, E., Arnold, P., Botton-Divet, L., Fabre, A.C., van Heteren, A.H., Nyakatura, JA, 2019. Femoral morphology of sciuromorph rodents in light of scaling and locomotor ecology. J. Anat., {rs url}/10.1111/joa.12980.

  73. Youlatos, D., Samaras, A., 2011. Arboreal locomotor and postural behaviour of European red squirrels (Sciurus vulgaris L.) in northern Greece. J. Ethol. 29 (2) 235–242.

  74. Zajac, F.E., 1989. Muscle and tendon: properties), models), scaling), and application to biomechanics and motor control. Crit. Rev. Biomed. Eng. 17 (4) 359–411.

  75. Zelditch, M.L., Li, J., Tran, LA., Swiderski, D.L., 2015. Relationships of diversity), disparity), and their evolutionary rates in squirrels (Sciuridae). Evolution 69 (5) 1284–1300.

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Nyakatura, J.A., Baumgarten, R., Baum, D. et al. Muscle internal structure revealed by contrast-enhanced μCT and fibre recognition: The hindlimb extensors of an arboreal and a fossorial squirrel. Mamm Biol 99, 71–80 (2019).

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  • PCSA
  • Fascicle length
  • Pennation angle
  • Muscle architecture
  • Spermophilus
  • Sciurus