Muscle fiber-type variation in lizards (Squamata) and phylogenetic reconstruction of hypothesized ancestral states
1 Department of Ecology and Evolutionary Biology, University of Arizona,
P.O. Box 210088 Tucson, AZ 85721, USA,
2 Department of Integrative Physiology, University of Colorado, Boulder, CO
80309, USA
3 Department of Biology, University of California Riverside, CA 92521,
USA
* Author for correspondence (e-mail: kebonine{at}u.arizona.edu)
Accepted 28 September 2005
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Summary |
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Key words: comparative method, fiber type, fiber-type composition, histochemistry, Phrynosomatidae, phylogeny, skeletal muscle
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Introduction |
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We analyzed the iliofibularis (IF), a hindlimb muscle that has many
characteristics conducive to comparative study. It is parallel-fibered, or
unipennate, and spans both the knee and hip joints. It is active during the
swing phase (when the femur is being abducted and the knee bent) of both
graded and burst locomotion in lizards
(Jayne et al., 1990). The IF
is relatively easy to find in a cross-sectional segment of lizard limb,
contains discrete red and white regions, and has been extensively studied in
lizards (e.g. Gleeson and Dalessio,
1990
; Gleeson et al.,
1980
; Putnam and Bennett,
1982
; Gleeson,
1983
; Gleeson et al.,
1984
; Johnston and Gleeson,
1984
; Gleeson and Harrison,
1986
; Gleeson and Johnston,
1987
; Mutungi,
1990
; Mirwald and Perry,
1991
; see references in table 1 of
Bonine et al., 2001
). For
several lizard species the IF muscle has been characterized for fiber-type
composition (see Bonine et al.,
2001
and references therein) and for fiber-type recruitment
patterns (Jayne et al., 1990
).
In Varanus exanthematicus, the Savannah monitor lizard,
electromyographic studies show that the red region is active at both low and
high locomotor speeds, with regular bursts of activity, whereas the white
region is active only above a threshold speed and with often irregular
activity (Jayne et al., 1990
).
Moreover, this threshold speed is consistent with the maximal aerobic speed of
V. exanthematicus, indicating an important role for the red region
during sustained, aerobic activity, and increased recruitment of predominantly
anaerobic fibers of the white region at higher speeds
(Jayne et al., 1990
). A
logical prediction from these data would be that species with higher aerobic
locomotor capacities should have more red fibers, whereas species with greater
anaerobic burst capabilities should have a greater proportion of white fibers.
The IF is active during the recovery phase of hind-limb cycling and therefore
could be rate-limiting in stride frequency during sprinting, or partly
determine fatigue resistance during sustained locomotor bouts.
Within the lizard species we studied, many use bipedalism frequently and
some, like the horned lizards (Phrynosoma), are not known to use
bipedalism at all. Because we studied the IF, a muscle found only in the
hindlimb, quadrupedal versus bipedal locomotion may be important.
However, data on locomotor abilities within species that are known to run
using both two and four limbs indicate that bipedalism does not confer a speed
advantage relative to quadrupedalism
(Irschick and Jayne, 1999).
We, therefore, believe detailed examination of the IF is relevant for all the
species included herein to initiate detailed comparison of muscle morphology
and physiology across a broad taxonomic range, especially in the context of
potential explanatory relationships for variation in locomotor performance
abilities.
Here, we report data for 14 phrynosomatid species [11 first reported by
Bonine et al. (2001) and an
additional three] as well as 10 species from other lineages (outgroups). This
allows us to test the hypothesis that the negative FGFOG relationship
in the IF is a general characteristic of lizards (ignoring snakes, which lack
IF muscles). We also use phylogenetic methods to estimate ancestral states of
muscle-fiber composition, and then test whether large evolutionary changes
(high rates of evolution along particular phylogenetic branches) have occurred
within the Phrynosomatidae. For the latter analyses, we employ recently
developed methods that incorporate information on within-species variation
(Garland et al., 2004
).
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Materials and methods |
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Iguania
Phrynosomatidae
Autarchoglossa
Teiidae
Animal collection
To avoid complications from comparing widely divergent locomotor modes, we
focused on species that are largely terrestrial (as opposed to saxicolous or
arboreal) and occur in arid or semi-arid habitats. All lizard species included
in this study are diurnal, and the majority are insectivorous
(Stebbins, 1985;
Conant and Collins, 1991
). The
two representative crotaphytids are carnivorous, whereas Dipsosaurus
dorsalis (Iguanidae) is primarily herbivorous
(Stebbins, 1985
). To avoid
possible sex and ontogenetic differences, we studied only adult males.
In 1996 and 1997, we collected lizards from populations in southern Arizona
and western New Mexico while based at the Southwestern Research Station (SWRS)
near Portal, Arizona, USA. In 1999, lizards were captured in the field from
targeted populations throughout the United States, Guam and Israel, and
shipped alive to Madison, Wisconsin, USA. We restricted animal collections
from these northern-hemisphere localities to late May through to early August
because of potential seasonal differences in metabolism and physiology (e.g.
Garland and Else, 1987).
Individuals of a given species (with a few unavoidable exceptions) were
collected from a restricted geographic area because populations may differ in
physiological characteristics (Garland and
Adolph, 1991
). During captivity, we kept individual lizards
isolated in either plastic containers or cloth bags (depending on size), with
periodic access to water but no food. Individuals were sacrificed within 2
weeks of capture. Variation in time in captivity prior to sacrifice was
randomly associated with species and lineage, and preliminary analyses
indicated no apparent relationship between time in captivity and fiber-type
traits. Furthermore, attempts to train lizards have not been successful
(Gleeson, 1979
;
Garland and Else, 1987
; A.
Szucsik, personal communication), suggesting that detraining is also unlikely.
Relevant animal care and use committee protocols were followed in Boulder,
Madison, and at SWRS.
Morphometrics
In addition to quantifying muscle morphology of the iliofibularis, we
measured morphometric traits for all animals. Body mass was measured to the
nearest 0.001 g within a few days of capture using a Mettler balance (model
PM200) in 1996 and 1997, and a Sartorius balance (model L420) in 1999. In
order to include variation in the width of the pelvic and pectoral girdles
among species, our measure of limb span was from toe-tip to toe-tip (excluding
the nail) with each limb held perpendicular to the axis of the body in the
same lateral plane as the body (Garland,
1984,
1985
;
Bonine and Garland, 1999
). Limb
and body proportions were measured to the nearest 0.5 mm using a clear plastic
ruler.
Tissue preparation and histochemical analyses
Preparation of tissues and histochemical analyses were as described in
detail by Bonine et al. (2001).
Briefly, in accordance with established protocols and guidelines for
physiological research, lizards were decapitated after we warmed them to their
approximate field-active body temperature. Warming animals prior to sacrifice
served three functions: (1) facilitated collection of blood quickly and in
sufficient quantities; (2) allowed for examination of physiological variables
under more ecologically relevant conditions; and (3) facilitated comparison
with previous research using similar protocols. Each hindlimb was quickly
removed intact along with a portion of the pelvis. Limbs were mounted above a
Styrofoam block with knee and ankle joints flexed at 90° to ensure
comparable muscle lengths among individuals. Muscle and mounting block were
then plunged into isopentane cooled in liquid nitrogen. In 1996 and 1997,
animals were shipped alive from SWRS to T. T. Gleeson in Boulder in
preparation for muscle composition measurements. In 1999, lizards were
sacrificed and tissues prepared in Madison. Tissues were stored at
80°C.
Serial, 10 µm-thick sections from frozen limbs were taken at mid-thigh
and used for histochemical identification of fiber types (e.g.
Putnam et al., 1980;
Gleeson, 1983
; Gleeson and
Harrison, 1986
,
1988
;
Garland et al., 1995
;
Bonine et al., 2001
).
Histochemical activities of alkaline-stable myosin ATPase and succinic
dehydrogenase/NADH diaphorase (SDH) were used to identify fibers as
slow-twitch oxidative (SO; light mATPase, dark SDH), fast-twitch glycolytic
(FG; dark mATPase, light SDH), or fast-twitch oxidative-glycolytic (FOG; dark
mATPase and dark SDH). For further comments on fiber-type terminology and
comparison with `the mammalian standard' see the discussion in Bonine et al.
(2001
).
After incubation, sections were mounted on microscope slides and magnified images were digitized. The IF, which is located posteriorly and dorsally in the hindlimb, was identified in each cross section, and images of both mATPase- and SDH-stained cells were simultaneously compared to determine muscle fiber type. We counted fibers of each type and measured fiber cross-sectional areas by tracing the perimeter of each cell in both the oxidative and non-oxidative regions within the muscle. In general, we tried to measure all of the fibers in the IF for a given individual. When it was not possible to measure all fibers (because some individual muscles were too large, or the entire muscle was not contained in the serial section being measured), we determined regions of like-composition and measured a large number of fibers within that region. Depending on the individual, we measured approximately 60100% of the fibers in the oxidative region (this appears red in fresh tissue) located medially. For the lateral and more homogeneous white portion of the muscle, we measured approximately 4090% of the fibers. We assumed that the remainder of a given region comprised similarly sized fibers in the same relative proportion of fiber types. On average, we measured 439 fibers per individual. Cross-sectional areas were measured for four individuals of each species using the mATPase images (not the SDH images) to control for variation in cell deformation caused by the two different histochemical procedures. To assess the relative size of the iliofibularis muscle, whole-thigh and iliofibularis muscle areas were also measured by tracing digitized, lower-magnification mATPase images. All measurements were made using NIH Image (version 1.62) software.
Literature values
Data on iliofibularis fiber-type composition are available for several
species (see references in table 1 of
Bonine et al., 2001).
Unfortunately, many of these data are not comparable because the same
morphometric traits are not consistently reported (e.g. Agama agama;
Abu-Ghalyun et al., 1988
;
Gekko gecko; Mirwald and Perry,
1991
). Appropriate data for Iguana iguana are from
juveniles (Gleeson and Harrison,
1986
), and significant ontogenetic changes can occur in lizard
muscle [e.g. in Ctenosaura similis, thigh lactate dehydrogenase (LDH)
activity increases ontogenetically
(Garland, 1984
), in
Amphibolurus nuchalis, thigh LDH and citrate synthase activities
increase ontogenetically (Garland,
1985
)]. Other comparable data are for much larger lizards
(Varanus spp.; Mutungi,
1990
; Gleeson,
1983
) than those studied herein, or for species that are not
typically terrestrial (Chamaeleo spp.;
Abu-Ghalyun et al., 1988
;
Mutungi, 1992
). Hence, the
only literature data we include in our current analyses are for
Dipsosaurus dorsalis (mean values for 20 individuals; from
Gleeson and Harrison, 1988
).
As some of the morphometric traits we measured, specifically hindlimb span and
forelimb span, were not reported by Gleeson and Harrison
(1988
), we calculated
regressions on body mass for these variables from a set of nine different
D. dorsalis individuals (Garland, unpublished data) and then
calculated, from the regression equation, the hindlimb and forelimb spans at
the mean body mass reported by Gleeson and Harrison
(1988
). Overall, the analyses
and results are restricted to comparable data (generated from the same
laboratory) for adult male lizards of 65 g or less with similar locomotor
modes (primarily terrestrial).
Phylogeny
We attempted to sample broadly among extant lizards, while restricting
species studied to those available that we felt would provide meaningfully
comparable results (i.e. among small, terrestrial lizards). The included
species span much of the range of phylogenetic diversity of lizards, yet are
reasonably similar to the Phrynosomatidae in terms of body size and ecology,
thereby removing some of the complications that would be inherent in comparing
fossorial, or strictly arboreal species, with terrestrial ones (cf. discussion
in Garland et al., 1997).
Direct comparisons with snakes or other limbless lizards is not feasible given
the absence of the IF muscle in these groups. Furthermore, their limbless mode
of terrestrial locomotion is not comparable to limbed locomotion in the
terrestrial lizards on which we focused. However, we did include two species
of Scincidae and one species of Anguidae; both groups that have repeatedly
lost limbs in several lineages (e.g. Pough
et al., 2001
; Wiens and
Slingluff, 2001
).
The evolutionary hypothesis presented here
(Fig. 1) is based on recent
and/or comprehensive available phylogenies (see also
Perry and Garland, 2002). The
large-scale tree topology follows Estes et al.
(1988
), rather than the
single-gene-based analysis of Harris et al.
(1999
). Within Iguania, we
followed the family-rich nomenclature of Frost and Etheridge
(1989
), although some
relationships were clarified by Wiens and Hollingsworth
(2000
) [iguanids (e.g.
Dipsosaurus) basal within Iguania], Schulte et al.
(1998
; agamid placement within
Iguania) and Macey et al.
(1997
; crotaphytids sister to
phrynosomatids). The general evolutionary relationships within Phrynosomatidae
are well supported (Etheridge and de
Queiroz, 1988
; Frost and
Etheridge, 1989
; Wiens,
1993
; Reeder and Wiens,
1996
; Schulte et al.,
1998
). Within the Sceloporus group (which is represented
here by species of Urosaurus, Uta and Sceloporus;
Reeder and Wiens, 1996
), we
used the most recent topology as described by Wiens and Reeder
(1997
). The topology within
the sand lizards is supported by several researchers
(Changchien, 1996
;
Reeder and Wiens, 1996
;
Wiens, 2000
;
Wilgenbusch and de Queiroz,
2000
). The Phrynosoma (horned lizards) topology follows
the synthetic analysis of both mitochondrial DNA and morphology of Reeder and
Montanucci (2001
).
Importantly, the sand lizards and horned lizards are sister clades, yet
exhibit remarkable differences in general ecology, locomotor and antipredator
behavior (Norris, 1958
;
Sherbrooke, 1981
;
Dial, 1986
;
Middendorf and Sherbrooke,
1992
; Bulova,
1994
), sprint speed, relative hindlimb length
(Bonine and Garland, 1999
) and
muscle fiber-type composition (Bonine et
al., 2001
). For the other half of the lizard tree, within
Autarchoglossa, the placement of teiids, lacertids, scincids and anguids is
based, again, on Estes et al.
(1988
). As information on
divergence times or some other common metric was generally unavailable, we
used arbitrary branch lengths as described below.
Statistical analyses
In general, estimates of mean trait values for species cannot be considered
to represent statistically independent data points because of differing
amounts of shared phylogenetic history. Therefore, we employed the method of
Felsenstein (1985) of
phylogenetically independent contrasts, which is well understood and
mathematically equivalent to generalized least squares approaches in the
applications employed here (Garland et
al., 1999
; Garland and Ives,
2000
; Rohlf,
2001
).
We used arithmetic mean values for each species to compute independent
contrasts (always one less than the number of tip species included). Species'
mean values of morphometric traits were log10 transformed (except
for total thigh cross-sectional area, which was square-root transformed) prior
to analyses, but proportion traits were not. For computing contrasts, we
originally considered many arbitrary branch lengths, including Nee's
(Purvis, 1995), those
described below, and various modifications of each. After checking diagnostic
plots (Garland et al., 1992
;
Diaz-Uriarte and Garland,
1998
) of the absolute values of standardized contrasts versus
their standard deviations (square roots of sums of corrected branch lengths),
three different branch lengths were used for different traits in order to
satisfy assumptions of the independent contrasts analyses. Pagel's arbitrary
branch lengths (Pagel, 1992
),
as shown in Fig. 1, were used
for proportions of each of the three fiber types in the IF, log total
cross-sectional areas of SO and FOG, the percentage of the IF area consisting
of oxidative fibers, and the proportion of the thigh consisting of IF muscle;
Pagel's modified by Grafen's (Grafen,
1989
) rho [rho is a parameter that transforms branch lengths by
altering the depths of the nodes relative to the tips of a phylogenetic tree.
In the present case, a rho of 0.5 pulls the nodal depths down towards the root
of the tree, thus making the overall tree less hierarchical (more like a
star)] with a value of 0.5 were used for log hindlimb span, log forelimb span,
and for log total cross-sectional area of FG; constant (all=1) branch lengths
were used for log body mass, log snout-vent length, the log of mean individual
fiber size for each fiber type, log IF cross-sectional area, and the square
root of total thigh cross-sectional area. A recent review paper with worked
examples (Garland et al.,
2005
), as well as the references cited above, provides a more
detailed description of the use of independent contrasts, including selection
of branch lengths.
We used the MS-DOS computer program PDTREE (Garland et al.,
1993,
1999
;
Garland and Ives, 2000
) to
enter trees and to compute independent contrasts
(Felsenstein, 1985
). Contrasts
were analyzed either within PDTREE (available on request from T.G.) or
exported to a conventional statistical program (e.g. Statistical Package for
the Social Sciences; SPSS, Inc., Chicago, IL, USA) for analysis by correlation
or regression through the origin. We used
=0.05 as the critical value
in all statistical tests.
Ancestor reconstruction
Hypothetical ancestral trait values at nodes within a phylogeny can be
estimated while computing independent contrasts via a rerooting procedure
(Garland et al., 1999; see
Johnston et al., 2003
for an
example). The values calculated via this method in PDTREE are identical to
those calculated using generalized least squares models or the methods of
Schluter et al. (1997
). We
used the program PDTREE to calculate ancestral values and 95% confidence
intervals at the root of our entire 24-species tree, at the base of the
phrynosomatids, and at the base of the Sceloporus group for the two
most variable fiber-type proportions, FG and FOG.
In an additional analysis, we included the standard error for each species'
mean value (as calculated from the four individuals measured per species) to
assess the effect of intraspecific variation on estimated nodal reconstruction
values. Using the DOS-based computer program PD_SE
(Garland et al., 2004), we
altered the lengths of terminal (tip) branches to reflect relative certainty
of the species' mean values in relation to their standard errors. These trees
with altered branch lengths were then entered back into PDTREE for computing
ancestral values.
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Results |
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Within each IF muscle, we found an oxidative core and a fast
twitch-glycolytic perimeter, as has also been found in all other species of
lizards that have been examined (see references in table 1 of
Bonine et al., 2001).
Furthermore, the oxidative portion of the IF was always located medially
within the muscle, nearest to the femur, and the more lateral portion of the
muscle was the predominantly fast-twitch region. Descriptive statistics for IF
morphometric and histochemical data are presented in
Table 2. The percentage of the
thigh that is IF ranges from 5.3% [Gambelia (gw)] to 12%
[Aspidoscelis (ct), formerly Cnemidophorus; Crother et al.
(2003
)]. Within the IF, the
percentage of white muscle ranges from 43% [Phrynosoma modestum (pm)]
to 82% [Aspidoscelis (ct)], whereas the percentage of red-oxidative
muscle ranges from 18% to 57% (the same two species defining the ends of the
spectrum). FG and FOG fibers made up the largest percentage of the IF muscle
cross-sectional area for all 92 individuals. The percentage of FOG and FG
fibers was also the most variable. The total cross-sectional area of FG fibers
in the IF ranges from 0.2 mm2 [P. modestum (pm)] to 2.7
mm2 [Laudakia (ls)], whereas the percentage of the IF that
is FG fibers ranges from 22% [P. platyrhinos (pp)] to 76%
[Aspidoscelis (ct)]. Members of the sand lizard subclade had
percentages of FG between 64% and 70%; Dipsosaurus (dd) had 71% FG.
For FOG fibers, the total area in the IF ranges from 0.2 mm2
[Carlia (Cf)] to 3.3 mm2 [Laudakia (ls)] with the
percentage of the IF that is FOG fibers ranging from 22%
[Aspidoscelis (ct)] to 72% [P. platyrhinos (pp)]. Among
subclades, FOG fiber percentage was highest in the horned lizards
(56%72%). The SO fiber total area in the IF ranges from 0.02
mm2 [Callisaurus (cd) and Holbrookia (hm)] to
1.27 mm2 [Laudakia (ls)] and the percentage of the IF that
is SO fibers ranges from <1% [Callisaurus (cd)] to 17%
[Sceloporus magister (sm) and Laudakia (ls)]. The maximum
and minimum species' mean fiber cross-sectional areas for each of the three
fiber types range from FG: 21289111 µm2
[Podarcis (Ps) and Dipsosaurus (dd), respectively]; FOG:
13186013 µm2 [Aspidoscelis (ct) and
Laudakia (ls), respectively]; SO: 5164983 µm2
[Gambelia (gw) and Laudakia (ls), respectively]. Note that
one of our largest species, Gambelia (gw), had the smallest SO
fibers. Mean values of morphometric and histochemical variables for each clade
are also presented in Tables 1
and 2.
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Table 3 contains additional
details of individual muscle-fibers, including mean sample sizes examined per
species. We have published some of these data previously (for 11 phrynosomatid
species; Bonine et al., 2001).
We present the data again here for completeness, and with minor adjustments as
noted in the footnotes of Table
3.
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Occasionally, we found fibers that did not stain darkly for either mATPase or SDH [10 of 92 individuals; less than 1% in one individual each of Uta (us), Cophosaurus (cx), Phrynosoma cornutum (pc), Laudakia (ls); 3% in one individual P. modestum (pm); a second individual of P. cornutum (pc) was 6% tonic, but this may be a result of unusually light staining overall for that individual]. Fibers with these staining characteristics have been termed `tonic' by previous researchers. Because we only rarely found these fibers, because their diameter was consistent with SO fiber size, and because their twitch properties are undetermined, we grouped these fibers in with the SO category for analyses. The Acanthodactylus are an exception.
All four individual Acanthodactylus (As) examined had a large proportion of different fibers that should be classified as tonic fibers. These fibers are located medially, in the oxidative region of the muscle, but were very large and did not stain darkly for either mATPase or SDH. On average, 12.0% of the IF cross-sectional area was tonic in Acanthodactylus (As) and 3.2% was SO fiber type. The oxidative proportion of the IF, the SO proportion of the IF, and the total SO cross-sectional area reported (Table 2) include both tonic and SO fibers for the Acanthodactylus, as these two fiber types should have similar contractile characteristics as compared to the fast contracting FG or FOG fibers. However, these tonic fibers are not included in calculation of the mean individual SO fiber cross-sectional area (Tables 2 and 3). For each individual, we measured a mean of 39.5 of these tonic fibers and report a mean size (±S.D.) of 4404±544 µm2. Almost all fibers (of all types) in the oxidative region were measured in these four individuals.
Refer to Fig. 2 for bivariate scatterplots of morphometric variables with body mass. Clear relationships with body mass are evident for SVL, HLS and FLS. However, species such as Callisaurus (cd; long limbs), Eumeces (ef; short limbs), and Elgaria (gk; elongate body, short limbs) are consistently different from the other species examined. The ratio of HLS:FLS does not have a clear relationship with body mass. However, the total range of variation of this metric is bounded by non-phrynosomatid species. Among phrynosomatids, the horned lizards have low HLS:FLS ratios and the sand lizards have relatively high ratios. Fig. 3 shows the range of variation in the proportion of FG fibers in the IF cross-sectional area across the phylogenetic sample we studied. The range of variation across all 24 species is almost the same as that found just within Phrynosomatidae; only Dipsosaurus (dd) and Aspidoscelis (ct) exceed the FG fiber proportion found in the sand-lizard subclade of Phrynosomatidae. Fig. 4 shows the relationship between body mass and the size and proportion of each of the three fiber types. The proportion of the iliofibularis composed of a given fiber type does not vary with body mass; however, the size of individual fibers does increase with body mass. Note that the horned lizards have a large proportion of FOG fibers in the IF (Fig. 4C). The proportion of IF composed of SO fibers is consistently low across all the species we examined (Fig. 4E). Also apparent is the small size of fibers in Gambelia (gw; Fig. 4B,D,F); Aspidoscelis (ct) and Elgaria (gk) also had consistently small fiber cross-sectional areas for their body masses. Acanthodactylus (As), the only species to clearly contain large tonic fibers, also had large SO fibers (Fig. 4F).
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Table 4B contains results for 23 species analyses (data unavailable for Dipsosaurus). The square root of total thigh muscle area was correlated with log IF cross-sectional area as well as with the cross-sectional area of the IF consisting of each of the three fiber types. The log of IF cross-sectional area was also highly correlated with the cross-sectional area of each of the three IF fiber types (Table 4B). Other significant correlations were among the IF cross-sectional areas for each of the three fiber types. Overall, these results once again emphasize the important influence of body size on hindlimb muscle morphology. It is important to note that the proportional representation of the IF or the individual fibers is not related to body size; a relationship that would have complicated our efforts to separate the effects of body size and phylogenetic relatedness (see Fig. 5).
Ancestor reconstruction
Estimated ancestral values for FG and FOG fiber proportions, and their 95%
confidence intervals, are reported in Table
5 and depicted in Fig.
8. Note that the extant range of variation, especially as manifest
in the horned lizards (Phrynosoma), in the proportion of either FG or
FOG fibers falls outside the 95% confidence interval of the estimated
ancestral values for this sample of 24 species. Inclusion of the standard
errors of species' mean values (calculated from the four individual
measurements taken per species) changed terminal branch lengths in a
noticeable way (Fig. 7), but
had virtually no effect on the estimated nodal values
(Table 5). However, confidence
intervals around the estimates were narrowed in every case with inclusion of
standard errors (Table 5).
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Discussion |
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Whatever the origin of the apparent `constraint' on fiber-type distribution
in lizards, it is potentially an important mechanistic explanation for
hypothesized trade-offs in whole-animal performance abilities. Indeed, we
predict, based on the FG and FOG proportions, that our sample of 24 species
will exhibit a trade-off in speed and stamina (see
Bonine, 2001;
Bonine, in press
). Furthermore,
we hypothesize that examination of the 12 lacertids studied by Vanhooydonck et
al. (2001
; see also
Huey et al., 1984
) will reveal
variation in fiber-type composition that explains the negative relationship
between speed and endurance observed in those species (we have muscle data for
only two of the species they measured). Contractile velocities at 40°C for
each of the three fiber types in Dipsosaurus support the above
predictions: SO,
4 lengths s1; FOG and FG,
89 lengths s1 and 16 lengths
s1, respectively (data from table 1, fig. 2, and related
text of Johnston and Gleeson,
1984
). If FG fibers also contract about twice as fast as FOG
fibers in other small lizard species, then the observed negative relation
between proportion of FOG and FG fiber types across species may be a main
cause of the negative relation between locomotor speed and endurance
(Vanhooydonck et al.,
2001
).
Reconstructed ancestral values (Fig.
8) suggest that rather extreme divergence of FG and FOG fiber
proportions have occurred in both the sand- and horned-lizard lineages, and
especially in the latter. Although not formally tested (see equation A10 of
Garland et al., 1999), the 95%
confidence intervals around the nodal values do not include the values found
in the living horned lizard species we measured, indicating a tremendous shift
in the IF composition toward FOG fibers, and away from FG fibers, during the
evolution of this subclade. Their sister group, the sand lizards, have
diverged in the opposite direction, with more FG than FOG fibers, but not to
the same extent. Evolution of fiber-type proportions has also resulted in a
dramatic shift in muscle composition of Aspidoscelis (ct) toward many
more FG fibers and fewer FOG fibers (although this apparent trend must be
interpreted with caution because we have data only for four individuals of one
species). Indeed, Aspidoscelis has the highest proportion of FG
fibers, and the lowest proportion of FOG fibers, of any species measured, and
was well outside the 95% confidence interval values for the root of all 24
species. Based on Fig. 8, we
hypothesize that natural selection has shaped the IF muscle composition of
sand lizards, horned lizards, and Aspidoscelis in accordance with
their differences in ecology, behavior and morphology. Horned lizards are
relatively slow (Bonine and Garland,
1999
) and often rely on crypsis to avoid predation
(Sherbrooke, 1981
). Sand
lizards live in open desert areas
(Stebbins, 1985
) and rely on
speed to escape predators (Bulova,
1994
). The Sceloporus group, sister to the sand and
horned lizards, is often considered intermediate in many aspects of its
biology (e.g. Bonine and Garland,
1999
). Aspidoscelis is the archetypal active, widely
foraging lizard and has exceptionally high stamina in addition to being a fast
sprinter (Garland, 1994
;
Dohm et al., 1998
;
Bonine and Garland, 1999
;
Bonine, 2001
).
Inclusion of standard errors (Fig.
7), based on the values from the four individuals measured per
species, did not change the node estimates except in the third decimal place
(Table 5). However, confidence
intervals around the estimates did narrow slightly, which will enhance
inferential power. It is also important to note that these confidence
intervals are relatively narrow as compared with some published examples,
which involved fewer species (e.g. see fig. 8 in
Schluter et al., 1997; fig. 2
in Garland et al., 1999
; fig.
3 in Losos, 1999
). Thus, our
study illustrates that reconstructions of ancestral states for
continuous-valued characters can indeed be useful for drawing evolutionary
inferences (see also Johnston et al.,
2003
; Espinoza et al.,
2004
). However, it should be kept in mind that these techniques
assume no phylogeny-wide directional trends have occurred in past character
evolution. If such trends have occurred, then even rather narrow confidence
intervals may be wildly misleading (e.g. see fig. 3 in
Garland et al., 1999
). In the
present study, it is hoped that the inclusion of several outgroup taxa has
countered this possibility with respect to inferring ancestral values at the
base of the Phrynosomatidae (see also
Garland et al., 1997
;
Schultz and Churchill, 1999
;
Polly, 2001
).
Although we did not determine twitch kinetics or analyze enzyme activity
levels in these species (but for this information from Dipsosaurus
dorsalis see Gleeson et al.,
1980; Gleeson and Johnston,
1987
), we usually found three types of fibers that were relatively
easy to characterize, based on histochemical assays: FG, FOG and SO
(Guth and Samaha, 1969
).
However, examination of the Acanthodactylus (As) IF revealed many
large fibers in the oxidative, medial area that did not appear to stain for
either SDH or mATPase activity. These tonic fibers, not commonly found in
mammalian muscle (Saltin and Gollnick,
1983
), are apparently common in some other species that we did not
measure. For example, some chameleon species have 50% tonic fibers in the IF,
and each tonic fiber is twice the size (cross-sectional area) of the other
twitch fibers in the muscle (Abu-Ghalyun et
al., 1988
). In contrast to the chameleon muscle, tonic fibers are
only about 10% larger than FG fibers in our Acanthodactylus.
Therefore, it seems that lizard IF fiber-type composition may not be
restricted to FG, FOG, and SO fiber types. Our sampling of small (
65 g)
lizards that are primarily terrestrial was intentional, to facilitate
meaningful comparisons among species, but larger species and/or other
locomotor modes, such as arboreality or fossoriality, may reveal more diverse
muscle-fiber type properties. Currently, comparisons with values from other
studies of lizard IF in the literature are problematic because of the
different methods used by other researchers and the different data reported
(see table 1 of Bonine et al.,
2001
).
Ideally for the generality and reliability of our results, the properties
of the IF would be indicative of the physiology and function of many of the
muscles involved in lizard locomotion. We chose to focus on the IF for several
reasons, including its variable fiber-type composition, its parallel-fibered
structure, and its consistent location across species. Published examination
of other lizard hindlimb muscles indicates important properties in common with
the IF. Putnam et al. (1980)
examined 12 muscles, including the IF, involved in D. dorsalis
locomotion, and they all contain similar fiber-type proportions. However, only
two of the twelve muscles were parallel-fibered (IF and extensor digitorum
longus). In another study of D. dorsalis, muscle fiber
cross-sectional areas were similar across different locomotory muscles within
individuals, but individuals varied in mean fiber size
(Gleeson and Harrison, 1988
).
In four distantly related species of lizard, Putnam and Bennett
(1982
) reported that the IF
and gastrocnemius have similar contractile properties within each species.
Some of the variables reported here were not formally analyzed [for
example, using ANCOVA; but see Bonine et al.,
(2001) for more in-depth
analysis of data for 11 phrynosomatids] as our sample size in families other
than Phrynosomatidae was so small (1 or 2 species). However, we can point out
a few interesting observations. Gambelia (gw), a large crotaphytid
that often lives in sympatry with many of these other species and also feeds
on them, has surprisingly small iliofibularis muscle fibers for its body size
(Tables 2,
3;
Fig. 4). The reasons for this
are unclear, and the other crotaphytid measured, Crotaphytus (cc),
does not display this pattern. However, both of these species have a
relatively small proportion of iliofibularis muscle in their thigh
cross-sectional area (Fig. 5A).
The autarchoglossa species all have longer snout-vent lengths for a given body
mass than phrynosomatids and other iguanians
(Fig. 2A). Other potentially
important differences in body plan between iguanians and non-iguanians include
the short hindlimb span of Elgaria [gk; Anguidae; see also Wiens and
Slingluff (2001
)] and
Eumeces (ef; Scincidae; Fig.
2B). The forelimbs of all autarchoglossa are relatively short as
compared with iguanians (Fig.
2C). Within autarchoglossa, the relatively longer hindlimbs of
Aspidoscelis (ct), Acanthodactylus (As) and
Podarcis (Ps) may be related to their increased ecological reliance
on speed. Indeed, of the species measured by Bonine and Garland
(1999
), including some of the
phrynosomatids represented here, members of the genus Aspidoscelis
were the fastest [up to 6 m s1; Elgaria (gk) were
the slowest (1 m s1)]. When comparing the ratios of hindlimb
span to forelimb span, Aspidoscelis (ct) and Crotaphytus
(cc) have the highest ratios, and Elgaria (gk) has the lowest
(Fig. 2D). Most of the fast
species (Bonine and Garland,
1999
) have a relatively high ratio of hindlimb span to forelimb
span, consistent with the importance of limb length, and therefore stride
length (e.g. Irschick and Jayne,
1999
; Vanhooydonck et al.,
2001
), especially for these species that tend to run bipedally
(all measured except for horned lizards, scincids and anguids; personal
observation).
Greater research emphasis on muscle variation, and related traits, across
lizard species should improve our ability to explain differences in
performance abilities. For example, calculations of the power input required
to reach burst sprint speed in one lacertid species (Acanthodactylus
boskianus, not tested by us here) indicate that the force produced by the
muscle fibers is sufficient; no elastic enhancement from tendons is required
(Curtin et al., 2005). This
finding suggests that the links between muscle traits and whole-animal
performance may be relatively direct. Examination of sprint speed and
iliofibularis muscle composition in D. dorsalis revealed significant
negative relationships between fiber cross-sectional area and both sprint
speed and enzyme activities (Gleeson and
Harrison, 1988
). Attempts to include performance breadth across a
wide range of temperatures may further enrich our understanding of the
mechanistic drivers of variation in these well-studied ectotherms (e.g.
Johnston and Gleeson, 1984
;
Marsh and Bennett, 1985
). We
predict that analysis using multiple morphological and physiological
variables, including those pertaining specifically to muscle such as
fiber-type composition and individual fiber cross-sectional area, will provide
a more complete understanding of the observed variation in locomotor
performance abilities across lizard species. Moreover, these kinds of
synthetic analyses should allow us to infer what sorts of selective regimes,
active in the past, gave rise to the existing diversity of species and
phenotypes.
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