One foot out the door: limb function during swimming in terrestrial versus aquatic turtles.

Specialization for a new habitat often entails a cost to performance in the ancestral habitat. Although aquatic lifestyles are ancestral among extant cryptodiran turtles, multiple lineages, including tortoises (Testudinidae) and emydid box turtles (genus Terrapene), independently specialized for terrestrial habitats. To what extent is swimming function retained in such lineages despite terrestrial specialization? Because tortoises diverged from other turtles over 50 Ma, but box turtles did so only 5 Ma, we hypothesized that swimming kinematics for box turtles would more closely resemble those of aquatic relatives than those of tortoises. To test this prediction, we compared high-speed video of swimming Russian tortoises (Testudo horsfieldii), box turtles (Terrapene carolina) and two semi-aquatic emydid species: sliders (Trachemys scripta) and painted turtles (Chrysemys picta). We identified different kinematic patterns between limbs. In the forelimb, box turtle strokes most resemble those of tortoises; for the hindlimb, box turtles are more similar to semi-aquatic species. Such patterns indicate functional convergence of the forelimb of terrestrial species, whereas the box turtle hindlimb exhibits greater retention of ancestral swimming motions.

Diversity of limb-bone safety factors for locomotion in terrestrial vertebrates: evolution and mixed chains.

During locomotion over land, vertebrates' limb bones are exposed to loads. Like most biological structures, limb bones have a capacity to withstand greater loads than they usually experience, termed a safety factor (SF). How diverse are limb-bone SFs, and what factors correlate with such variation? We have examined these questions from two perspectives. First, we evaluated locomotor SF for the femur in diverse lineages, including salamanders, frogs, turtles, lizards, crocodilians, and marsupials (opossums). Comparisons with values for hind-limb elements in running birds and eutherian mammals indicate phylogenetic diversity in limb-bone SF. A high SF (∼7) is primitive for tetrapods, but low magnitudes of load and elevated strength of bones contribute to different degrees across lineages; moreover, birds and eutherians appear to have evolved lower SFs independently. Second, we tested the hypothesis that SFs would be similar across limb bones within a taxon by comparing data from the humerus and femur of alligators. Both in bending and in torsion, we found a higher SF for the humerus than for the femur. Such a "mixed chain" of different SFs across elements has been predicted if bones have differing variabilities in load, different costs to maintain, or high SF values in general. Although variability in load is similar for the humerus and femur, a high SF may be less costly for the humerus because it is smaller than the femur. The high SFs of alligators also might facilitate differences in SF among their limb bones. Beyond these specific findings, however, a more general implication of our results is that evaluations of the diversity of limb-bone SFs can provide important perspective to direct future research. In particular, more complete understanding of variation in SF could provide insight into factors that promoted the evolutionary radiation of terrestrial locomotor function in vertebrates.

Mechanical properties of the hindlimb bones of bullfrogs and cane toads in bending and torsion.

When compared with most vertebrates, frogs use a novel style of jumping locomotion powered by the hindlimbs. Hindlimb bones of frogs must withstand the potentially erratic loads associated with such saltatory locomotion. To evaluate the load bearing capacity of anuran limb bones, we used three-point bending, torsion, and hardness tests to measure the mechanical properties of the femur and tibiofibula from adults of two species that use different jumping styles: explosively jumping bullfrogs (Rana (Lithobates) catesbeiana) and cyclically hopping cane toads (Bufo (Chaunus) marinus). Yield stress and strain values for R. catesbeiana and B. marinus hindlimb bones are within the range of values previously reported for other vertebrates. However, anuran hindlimb bones generally stand out as having higher yield stresses in bending than those of closely related, nonsaltatory salamanders, highlighting the importance of considering phylogenetic context in comparisons of bone functional capacity and adaptation. Stiffness values for both frog species tested were also high, which may facilitate efficient transmission of muscular forces while jumping. Elevated stiffness may also contribute to some discrepancies between determinations of bone properties via hardness versus bending tests. In comparisons between species, B. marinus bones showed significantly higher bending yield stresses than R. catesbeiana, whereas R. catesbeiana bones showed significantly higher torsional yield stresses than B. marinus. These differences may correlate with differences in jumping style and limb anatomy between ranid and bufonid frogs, suggesting that evolutionary changes in bone mechanical properties may help to accommodate new functional demands that emerge in lineages.

In vivo strains in the femur of river cooter turtles (Pseudemys concinna) during terrestrial locomotion: tests of force-platform models of loading mechanics.

Previous analyses of ground reaction force (GRF) and kinematic data from river cooter turtles (Pseudemys concinna) during terrestrial walking led to three primary conclusions about the mechanics of limb bone loading in this lineage: (1) the femur was loaded in a combination of axial compression, bending and torsion, similar to previously studied non-avian reptiles, (2) femoral shear stresses were high despite the possession of a reduced tail in turtles that does not drag on the ground and (3) stress-based calculations of femoral safety factors indicated high values in bending and torsion, similar to other reptiles and suggesting that substantial 'overbuilding' of limb bones could be an ancestral feature of tetrapods. Because force-platform analyses produce indirect estimates of bone loading, we sought to validate these conclusions by surgically implanting strain gauges on turtle femora to directly measure in vivo strains during terrestrial walking. Strain analyses verified axial compression and bending as well as high torsion in turtle femora, with peak axial strains comparable to those of other non-avian reptiles at similar walking speeds but higher peak shear strains approaching 2000 microepsilon. Planar strain analyses showed patterns of neutral axis (NA) of femoral bending orientations and shifting generally consistent with our previous force-platform analyses of bone stresses, tending to place the anterior and dorsal aspects of the femur in tension and verifying an unexpected pattern from our force studies that differs from patterns in other non-avian reptiles. Calculated femoral safety factors were 3.8 in torsion and ranged from 4.4 to 6.9 in bending. Although these safety factors in bending were lower than values derived from our stress-based calculations, they are similar to strain-based safety factors calculated for other non-avian reptiles in terrestrial locomotion and are still high compared with safety factors calculated for limb bones of birds and mammals. These findings are consistent with conclusions drawn from our previous models of limb bone stresses in turtles and suggest that not only are turtle limb bones 'overbuilt' in terms of resisting the loads that they experience during locomotion but also, across tetrapod lineages, elevated torsion and high limb bone safety factors may be primitive features of limb bone design.

Regulation of myosin expression during myotome formation.

The first skeletal muscle fibers to form in vertebrate embryos appear in the somitic myotome. PCR analysis and in situ hybridization with isoform-specific probes reveal differences in the temporal appearance and spatial distribution of fast and slow myosin heavy chain mRNA transcripts within myotomal fibers. Embryonic fast myosin heavy chain was the first isoform expressed, followed rapidly by slow myosin heavy chains 1 and 3, with slow myosin heavy chain 2 appearing several hours later. Neonatal fast myosin heavy chain is not expressed in myotomal fibers. Although transcripts of embryonic fast myosin heavy chain were always distributed throughout the length of myotomal fibers, the mRNA for each slow myosin heavy chain isoform was initially restricted to the centrally located myotomal fiber nuclei. As development proceeded, slow myosin heavy chain transcripts spread throughout the length of myotomal fibers in order of their appearance. Explants of segments from embryos containing neural tube, notochord and somites 7-10, when incubated overnight, become innervated by motor neurons from the neural tube and express all four myosin heavy chain genes. Removal of the neural tube and/or notochord from explants prior to incubation or addition of d-tubocurare to intact explants prevented expression of slow myosin chain 2 but expression of genes encoding the other myosin heavy chain isoforms was unaffected. Thus, expression of slow myosin heavy chain 2 is dependent on functional innervation, whereas expression of embryonic fast and slow myosin heavy chain 1 and 3 are innervation independent. Implantation of sonic-hedgehog-soaked beads in vivo increased the accumulation of both fast and slow myosin heavy chain transcripts, as well as overall myotome size and individual fiber size, but had no effect on myotomal fiber phenotype. Transcripts encoding embryonic fast myosin heavy chain first appear ventrolaterally in the myotome, whereas slow myosin heavy chain transcripts first appear in fibers positioned midway between the ventrolateral and dorsomedial lips of the myotome. Therefore, models of epaxial myotome formation must account for the positioning of the oldest fibers in the more ventral-lateral region of the myotome and the youngest fibers in the dorsomedial region.

Slow myosins in muscle development.

Myogenesis has been a system central to investigations on mechanisms of diversification within groups of differentiating cells. Diversity among cell types has been well described in striated muscle tissue at the protein and enzymatic-function levels for decades, but it is only in recent years that some understanding of the molecular mechanisms responsible for this diversity has begun to emerge. Study of the expression of the slow isoforms of the myosin heavy chain has contributed to our understanding of how cell diversity arises within skeletal and cardiac muscle. Slow MyHc isoforms are developmentally responsive to a number of cues provided by the nervous systems, the endocrine system and, later in development, to functional demands on these developing tissues. Perhaps most informative have been studies on the mechanism for regulation of slow MyHc expression in mammals and birds where studies on the calcineurin-NF-AT pathways and nuclear hormone action have been shown to control MyHC gene expression in skeletal muscle and in the developing heart. The mechanisms involved in cell diversification in myogenesis are undoubtedly more varied and complex than those currently offered to explain cell diversification, but these recent studies have broadened our understanding of the interplay between the nervous system, the endocrine system and cell lineages in controlling cell diversification. Greater focus on the first fibers and cardiomyocytes to form in the embryo are likely to bring additional insights into the mechanism crucial for establishing the patterns of diversity required for successful formation of embryonic tissues.