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Biomechanics
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Research
Cite this article: Rivera ARV, Blob RW. 2013
Forelimb muscle function in pig-nosed turtles,
Carettochelys insculpta: testing neuromotor
conservation between rowing and flapping in
swimming turtles. Biol Lett 9: 20130471.
http://dx.doi.org/10.1098/rsbl.2013.0471
Received: 21 May 2013
Accepted: 20 July 2013
Subject Areas:
biomechanics
Keywords:
kinematics, locomotion, Apalone ferox,
Caretta caretta, Trachemys scripta
Author for correspondence:
Angela R. V. Rivera
e-mail: [email protected]
†
Present address: Rowland Institute at Harvard
University, Cambridge, MA 02142, USA.
Electronic supplementary material is available
at http://dx.doi.org/10.1098/rsbl.2013.0471 or
via http://rsbl.royalsocietypublishing.org.
Forelimb muscle function in pig-nosed
turtles, Carettochelys insculpta: testing
neuromotor conservation between rowing
and flapping in swimming turtles
Angela R. V. Rivera† and Richard W. Blob
Department of Biological Sciences, Clemson University, Clemson, SC 29634, USA
Changes in muscle activation patterns can lead to new locomotor modes;
however, neuromotor conservation—the evolution of new forms of locomotion through changes in structure without concurrent changes to underlying
motor patterns—has been documented across diverse styles of locomotion.
Animals that swim using appendages do so via rowing (anteroposterior
oscilations) or flapping (dorsoventral oscilations). Yet few studies have
compared motor patterns between these swimming modes. In swimming turtles, propulsion is generated exclusively by limbs. Kinematically, turtles swim
using multiple styles of rowing (freshwater species), flapping (sea turtles) and
a unique hybrid style with superficial similarity to flapping by sea turtles
and characterized by increased dorsoventral motions of synchronously oscillated forelimbs that have been modified into flippers (Carettochelys insculpta).
We compared forelimb motor patterns in four species of turtle (two
rowers, Apalone ferox and Trachemys scripta; one flapper, Caretta caretta; and
Carettochelys) and found that, despite kinematic differences, motor patterns
were generally similar among species with a few notable exceptions: specifically, presence of variable bursts for pectoralis and triceps in Trachemys
(though timing of the non-variable pectoralis burst was similar), and the
timing of deltoideus activity in Carettochelys and Caretta compared with
other taxa. The similarities in motor patterns we find for several muscles
provide partial support for neuromotor conservation among turtles using
diverse locomotor styles, but the differences implicate deltoideus as a prime
contributor to flapping limb motions.
1. Introduction
The evolution of vertebrates has produced a variety of appendage-based
locomotor modes (e.g. running, flying and swimming) and associated morphologies. Among swimming taxa, vertebrate appendages have been modified
for rowing or flapping. Although more accurately viewed as points along a
continuum, rowing is characterized by anteroposterior oscillatory motions of
paddle-shaped appendages, whereas flapping is characterized by dorsoventral
oscillatory motions of wing-shaped appendages [1]. Rowing and flapping have
been documented among diverse taxa, including fishes [1–3], turtles [4–6],
birds [7] and mammals [8,9]. Understanding how new locomotor modes arise,
whether through changes in morphology, muscle activity or a combination of
both, is a major focus of evolutionary studies of musculoskeletal function. In particular, the idea that new behaviours can arise solely through changes in structure,
without concurrent changes in the patterns of muscle activity that control movement of those structures, has been formalized as the ‘neuromotor conservation
hypothesis’ [10]. While evidence for neuromotor conservation is found across
terrestrial and aerial locomotor modes [11–13], few studies have examined this
for swimming, particularly between aquatic rowing and flapping.
& 2013 The Author(s) Published by the Royal Society. All rights reserved.
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(a) Animals
Access to two pig-nosed turtles, C. insculpta (carapace length ¼
23.8 + 1.8 cm), and nine Florida softshell turtles, A. ferox
(carapace length ¼ 15.1 + 1.1 cm), was provided by a commercial
vendor. The number of Carettochelys was limited owing to infrequent availability of this rare species. Turtles were housed in
stock tanks [15].
(b) Collection and analysis of electromyography data
Bipolar stainless steel fine-wire electrodes (0.05 mm diameter,
California Fine Wire Co., USA) were implanted percutaneously
into target muscles of the left forelimb of Carettochelys (see [6]
(c) Statistical analyses
To assess general patterns of muscle function for each species,
means and standard errors for each variable were calculated
across all swimming trials (see table 1 and electronic supplementary material, table A1). Muscle activity variables include,
for each muscle: (i) onset, (ii) offset and (iii) duration. Data for
Carettochelys and Apalone were compared with those previously
published for Trachemys [5] and Caretta [6] to assess how motor
patterns during swimming compare among the species. Interspecific differences ( p , 0.05) were evaluated using separate
two-factor nested ANOVAs (SYSTAT), with individual nested
within species, followed by Tukey’s post hoc tests to evaluate
each pairwise species comparison.
3. Results
Forelimb motor patterns for Carettochelys and Apalone were
calculated and compared with those published for Caretta and
Trachemys (figure 1 and table 1); turtles of each species
swam using similar forelimb cycle frequencies [15]. Patterns
are very similar across species for humeral retractors. There
are statistically significant, but minor, differences in onset
of coracobrachialis (Apalone earlier than Caretta) and offset of
pectoralis (Caretta earlier than Trachemys and Carettochelys).
Trachemys also exhibits a variable (i.e. not always present) early
burst of activity in pectoralis. Among humeral protractors, the
pattern of activation for latissimus dorsi is remarkably similar,
with only a minor difference in offset between Apalone and
Caretta. However, the pattern for deltoideus shows marked
differences among the species. Flapping Caretta exhibit a single
discrete burst of deltoideus activity during humeral depression
and retraction (opposite to the predicted action [6,17]), differing
significantly in onset and offset from the three freshwater
species. Furthermore, deltoideus activity in Carettochelys starts
and ends significantly earlier than in Apalone and Trachemys,
resulting in significantly decreased duration of activity during
protraction in the former. Finally, triceps differs primarily
among the species in that Trachemys exhibits two discrete bursts
of activity (the second being variable and not always present).
Onset of the primary triceps burst occurs significantly later in
Trachemys, and while offset did not differ, duration in Trachemys
was significantly shorter than in Caretta and Carettochelys.
4. Discussion
Muscles were active during the predicted portions of the limb
cycle and showed similar patterns among species with few
2
Biol Lett 9: 20130471
2. Material and methods
for details) and Apalone (see [5] for details) to generate data for
comparison to other species. Protocols differed only slightly for
the species; in particular, Carettochelys received local anaesthetic
(lidocaine) at implant sites and were tested the same day, whereas
Apalone was anaesthetized with ketamine prior to implants
and tested the following day. Electromyography (EMG) data
(5000 Hz) were synchronized with kinematics (collected using biplanar high-speed (100 Hz) video with high-contrast joint markers;
detailed in [15]) and analysed in LABVIEW, where muscle timing
was determined visually.
We focused on five target muscles, covering all major planes of
forelimb motion during swimming. Predicted actions were based
on anatomical position: coracobrachialis (humeral retraction; not
collected for Carettochelys), pectoralis (humeral retraction and
depression), latissimus dorsi and deltoideus (humeral protraction
and elevation), and the triceps complex (elbow extension) [17,18].
rsbl.royalsocietypublishing.org
Turtles represent an ideal group in which to study
appendage-based locomotion because propulsive forces are
generated exclusively by the limbs [14]. Species of aquatic turtles swim via rowing or flapping: all but one freshwater species
(there are over 200) swim using asynchronous rowing of
paddle-shaped forelimbs and hindlimbs, whereas all marine
turtles (seven species) swim using synchronous flapping of
forelimbs that are modified into flippers. A single freshwater
species, Carettochelys insculpta (hereafter ‘Carettochelys’; family
Carettochelyidae), has converged on synchronous motions of
foreflippers that superficially resemble flapping in sea turtles,
but that are nevertheless best classified, kinematically, as
rowing (albeit synchronously). While the dorsoventral component of forelimb motion in Carettochelys is increased
relative to other freshwater rowers, the primary direction of
forelimb movement is still anteroposterior, and overall kinematics resembles rowing in many respects [15]. However,
angles-of-attack indicate that Carettochelys are hydrodynamically more concordant with flappers during protraction/
elevation [15]. A recent examination of forelimb motor patterns
in rowing Trachemys scripta (hereafter ‘Trachemys’; family
Emydidae) and flapping Caretta caretta (hereafter ‘Caretta’;
family Cheloniidae) showed remarkable conservation in the
activation patterns of several muscles (e.g. coracobrachialis
and latissimus dorsi), but marked differences in others (e.g. deltoideus and triceps), suggesting that the evolution of flapping
in sea turtles (Caretta) was achieved through modification of
structures (e.g. flippers), as well as motor patterns [6].
The primary goal of this study was to determine whether
forelimb motor patterns during swimming in Carettochelys
more closely resemble patterns of muscle activity in phylogenetically similar freshwater rowers or marine flappers,
with whose locomotor style Carettochelys shares some
similarities. To do this, we measured forelimb motor patterns
in swimming Carettochelys, as well as Apalone ferox (hereafter
‘Apalone’; family Trionychidae), a specialized rower and
member of the sister taxon to the monotypic Carettochelyidae
[16], thus providing a phylogenetic comparison. We compare
these results with previous measurements from a generalized
rower (Trachemys) and a flapping sea turtle (Caretta). By comparing motor patterns from four of the 13 families containing
aquatic species, we were able to test for neuromotor conservation across a broad range of taxa using a variety of locomotor
modes, including generalized and specialized rowing (which
differ in aspects of kinematics [15]), flapping and a unique
hybrid style of locomotion that displays similarities with
both freshwater rowers and marine flappers.
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variable
Trachemys
Apalone
Carettochelys
Caretta
scripta
ferox
insculpta
caretta
51 + 1.0
42 + 0.8
no data
F-value
d.f.
Tukey results
62 + 1.3
5.16*
2,9
AF–CC
coracobrachialis
onset
83 + 0.4
no data
84 + 1.3
0.83
2,9
42 + 1.1
no data
21 + 1.4
3.01
2,9
onset
62 + 1.5
51 + 0.6
57 + 0.9
57 + 1.7
1.19
3,11
offset
89 + 0.7
84 + 0.4
91 + 1.1
78 + 1.1
5.30*
3,11
duration
28 + 1.3
33 + 0.7
34 + 1.1
21 + 1.3
1.24
3,11
onset
83 + 1.0
88 + 0.8
94 + 1.1
91 + 0.9
1.60
3,7
offset
35 + 0.9
27 + 1.5
37 + 1.4
39 + 1.2
4.36*
3,7
‘Burst 1’ duration
35 + 0.9
27 + 1.5
37 + 1.4
37 + 1.2
3.70
3,7
‘Burst 2’ duration
16 + 1.0
12 + 0.8
6 + 1.1
8 + 0.9
1.67
3,7
total duration
51 + 1.3
36 + 2.5
41 + 2.0
44 + 1.6
1.89
3,7
onset
96 + 0.4
94 + 0.3
81 + 2.2
60 + 1.3
217.42***
3,10
all but AF–TS
offset
33 + 0.9
30 + 0.8
7 + 1.2
84 + 1.0
39.59***
3,12
all but AF–TS
pectoralis
a
CC– CI; CC –TS
latissimus dorsib
AF–CC
c
deltoideus
[216 + 1.0]
‘Burst 1’ duration
30 + 1.1
30 + 0.8
7 + 1.2
—
6.75*
2,10
AF–CI; CI –TS
‘Burst 2’ duration
4 + 0.4
6 + 0.3
18 + 2.2
—
26.94***
2,8
AF–CI; CI –TS
32 + 1.7
35 + 0.8
20 + 2.3
24 + 1.4
2.27
3,12
23 + 1.3
94 + 0.4
89 + 1.5
90 + 0.8
11.09**
3,11
total duration
tricepsd
onset
AF–TS; CC –TS;
CI –TS
[123 + 1.3]
offset
51 + 1.5
39 + 0.6
38 + 1.4
44 + 1.5
1.61
3,12
‘Burst 1’ duration
—
37 + 0.7
38 + 1.4
40 + 1.4
0.53
2,8
‘Burst 2’ duration
—
6 + 0.4
11 + 1.5
10 + 0.8
1.93
2,7
total duration
28 + 0.9
41 + 0.8
47 + 2.5
45 + 1.8
5.13*
3,12
CC– TS; CI –TS
a
Apalone, Carettochelys and Caretta exhibit one discrete burst that was compared with the presumptive homologous non-variable later burst in Trachemys.
b
Latissimus dorsi shows a continuous burst that spans the retraction/depression to protraction/elevation phase shift.
c
Apalone, Carettochelys and Trachemys each show one continuous burst (onset ¼ start ‘Burst 2’, offset ¼ end ‘Burst 1’) that was compared with the single discrete burst in
Caretta (offset ¼ [transposed value, by subtracting 100]).
d
Apalone, Carettochelys and Caretta exhibit one continuous burst (onset ¼ start ‘Burst 2’, offset ¼ end ‘Burst 1’) that was compared with the discrete non-variable early burst
observed in Trachemys (onset ¼ [transposed value, by adding 100]).
*p 0.05; **p 0.01; ***p 0.001.
exceptions, including two instances of variable bursts in
Trachemys ( pectoralis and triceps) and, most notably, activity
of the deltoideus in Caretta (figure 1).
Of particular interest, our new results reveal that some
differences in motor patterns (i.e. presence or absence of
variable bursts) observed between Trachemys and Caretta [6]
are not attributable to their difference in swimming mode
(i.e. rowing versus flapping) as previously proposed, because
rowing Apalone also lack variable pectoralis and triceps bursts
and differ in timing of triceps onset from Trachemys. Among
rowers, timing of peak elbow extension occurs significantly
later in Trachemys; additionally, the arm is held straighter
near the end of the limb cycle (i.e. retraction/depression) [15].
Thus, differences in triceps between rowers are associated
primarily with differing elbow kinematics. Furthermore, the
absence of a variable late triceps burst in the highly aquatic
Apalone, Caretta and Carettochelys versus its presence in semiaquatic Trachemys might be a constraint on motor pattern
associated with needing to move effectively over land, as
walking motor patterns in Trachemys exhibit two bursts [5].
The primary difference in motor patterns among species
occurs in the deltoideus. Rivera et al. [6] conclude that the
Biol Lett 9: 20130471
85 + 0.5
34.9 + 1.2
offset
duration
3
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Table 1. Mean values and standard errors for EMG timing variables, F-values for the main effect of species and Tukey pairwise mean comparison results.
Consistent with our previous work [5,6,15], ‘Burst no. 1’ and ‘Burst no. 2’ indicate early and late phase muscle activity of continuous muscles, rather than
discrete bursts. Values are means + s.e.m. Significant differences in pairwise comparisons are indicated. AF, Apalone ferox; CC, Caretta caretta; CI, Carettochelys
insculpta; TS, Trachemys scripta.
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Trachemys scripta (TS)
Apalone ferox (AF)
Carettochelys insculpta (CI)
Caretta caretta (CC)
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coracobrachialis
pectoralis
latissimus dorsi
Biol Lett 9: 20130471
deltoideus
triceps
10 20 30 40 50 60 70 80 90 100
percentage of cycle
protraction/elevation
retraction/depression
0
Figure 1. Bar plot showing mean (+s.e.m.) pattern of forelimb muscle activation during swimming in Carettochelys insculpta (black), Apalone ferox (white),
Trachemys scripta (grey, hatched ¼ variable) and Caretta caretta (diagonals). Vertical lines demarcate switch from protraction/elevation to retraction/depression
(solid, Apalone/Trachemys; dashed, Carettochelys/Caretta) based on maxima/minima of kinematics; a limb cycle is defined by the major plane of forelimb
motion (start of protraction to end of retraction for Apalone, Trachemys and Carettochelys, and elevation followed by depression for Caretta; detailed in [5,6,15]).
functional role of the deltoideus in Caretta has shifted during
the evolution of flapping in turtles to serve as a stabilizer,
minimizing anteroposterior humeral movements during the
downstroke through simultaneous activation with pectoralis.
Timing (though not duration) of deltoideus activity in
Carettochelys differs from that of flapping Caretta, as well as
rowing Apalone and Trachemys. Additionally, in comparison
to asynchronous freshwater rowers, the duration of deltoideus
activity in Carettochelys is significantly longer during retraction/depression and shorter during protraction/elevation.
Thus, the pattern of deltoideus activity associated with the
unique hybrid-style synchronous rowing by Carettochelys is
intermediate between rowing and flapping, showing a shift
towards the pattern observed in Caretta and concordant with
their exhibited bimodal pattern of elevation and depression
(see fig. 4b in [15]).
In conclusion, our data show a general trend of conservation of motor patterns among swimming turtles using a
variety of locomotor styles, ranging from generalized and
specialized rowing to flapping, and including the unique
hybrid-style synchronous rowing of Carettochelys. Some
variable muscle activity patterns found in more terrestrial
Trachemys were absent from highly aquatic species. Additionally, the deltoideus shows an evolutionary shift in timing that
is drastic in flapping Caretta and intermediate in Carettochelys.
Thus, this study provides partial support for the hypothesis
of neuromotor conservation across aquatic locomotor behaviours but with variation focused in specific muscles. These
data suggest that motor patterns may be conserved whenever
morphological modifications alone are not sufficient to produce new patterns of motion. Further, these results suggest
that evolutionary changes in muscle activation may occur
more readily for some muscles (e.g. deltoideus) while illustrating the potential for completely new forms of locomotion to
evolve through simple shifts in activation timing of a small
subset of muscles.
Acknowledgements. Jill Galan, Danielle Hulsey and Gabriel Rivera
assisted with data collection. Experiments were conducted under
Clemson University IACUC guidelines ( protocols 50110, 2008– 013
and 2008– 080).
Funding statement. Support provided by Sigma Xi (Grant-in-Aid-ofResearch to A.R.V.R.), NSF (IOS-0517340 to R.W.B.) and NIH
(2-R01-DC005063-06A1 to E. Peterson, subaward UT10853 to R.W.B.).
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