Specimens

In September 2010 and September 2011, we captured a total of 22 adult specimens of three species of Anolis lizards by hand or noose in Cuba (Table 1). All animals were anesthetized and fixed in 100% ethanol, and stored in 70% ethanol. Since dry muscles are easily frayed and difficult to isolate, the samples were soaked in 30% ethanol overnight before dissection. The fore- and hindlimbs were dissected, and each muscle was isolated under a microscope (S240; Olympus, Tokyo, Japan). Nineteen muscles from forelimbs related to rotation of each joint and 22 muscles from hindlimbs were chosen for measurement (Zaaf et al., 1999; Herrel et al., 2008; Russell and Bauer, 2008; Anzai et al., 2014). These musculatures are illustrated in Fig. 1.

Measurements

Two parameters were measured in each muscle and compared. First, muscle mass was measured as an index of muscular force. Each isolated muscle was dried by blotting and weighed to the nearest 0.01 mg using a Shimadzu balance (AUW-220D; Kyoto, Japan). The length of each muscle moment arm was measured as a second trait. The length was defined as the distance from the center of rotation in each joint to the point of the muscle insertion, which theoretically represents the maximum moment arm (An et al., 1984; Fujiwara et al., 2011). Given the trade-off between torque and excursion, muscles with longer moment arms exert larger torque and muscles with shorter moment arms produce grater excursion. Lengths were measured using calipers to the nearest 0.05 mm.

Fig. 2.

Boxplots showing the normalized values for the forelimb muscles mass. The vertical axis indicates the residuals from ln-transformed muscle mass regressed against ln-total mass.

Fig. 3.

Boxplots showing normalized values for hindlimb muscles mass.

Statistical analyses

To remove the effect of body size among different growth stages and/or species, all the muscle measurements were corrected for size. For muscle mass, residuals from the regression of each ln-transformed muscle mass value against the ln-transformed total body mass were used. For the moment arm, the ratio of each measurement to the length of limb bone on which the muscle inserts was calculated. To analyze whether locomotion, display, and sex affect musculoskeletal traits, we used two tests of two-way analysis of variance (ANOVA). One test took locomotion (terrestrial runner or arboreal climber) and sex as crossed factors and other one used sexual display with dewlap (existence or absence) and sex as crossed factors. All statistical analyses were performed using R (version 2.15.0, R Foundation for Statistical Computing, Vienna, Austria) and P < 0.05 was the criterion for statistical significance.

Fig. 4.

Boxplots showing the normalized values for the forelimb muscles moment arm. The vertical axis indicates the ratios of the value to length of limb bone that the muscle inserts.

Differences among locomotion type

Although some studies indicated the relationship among appendicular musculoskeletal traits and locomotor behavior or habitat use in lizards, these studies analyzed only males, to exclude the effects of sexual difference (Zaaf et al., 1999; Vanhooydonck et al., 2006; Herrel et al., 2008; Anzai et al., 2014). Our data suggest that similar relationships between morphology and ecological traits in females may exist. In terms of muscle mass, the runner species A. sagrei and A. bartschi were equipped with well-developed extensor muscles in knee and ankle joints in both male and female (Fig. 3). Their large hindlimb extensors are suited for powerful kicking off from the ground or broad surfaces when running (Reilly, 1995, 1998; Herrel et al., 2008). Also, M. flexor tibialis internus lateralis and M. flexor tibialis internus medialis were expanded in runner species. Because these two muscles are apparently used by lizards when running on the ground through femoral adduction (Fieler and Jayne, 1998; Anzai et al., 2014), larger muscles are suited for rapid locomotion by both sexes in running species. In contrast, heavier retractor muscles in shoulder and hip joints and flexor muscles in the elbow were observed in the arboreal climber species, A. porcatus (Figs. 2 and 3). Since vertical climbing by lizards requires tension by the front legs to avoid backwards tumbling when their hind legs push for propulsion and countering gravity (Zaaf et al., 1999), enlarged elbow flexor muscles and proximal limb retractor muscles adapt lizards to scansorial locomotion in arboreal habitats. With regard to moment arm, A. porcatus possessed shorter moment arms in forelimb muscles (M. pectoralis, M. biceps brachii and M. brachialis anticus) and possessed longer moment arms in hindlimb muscles (M. puboischiofemoralis externus, M. iliofemoralis and M. caudifemoralis longus) than the two runner species (Figs. 4 and 5). A shorter moment arm provides a wider excursion angle in joints, thereby facilitating limb movement especially on narrow arboreal substrates (Peterson, 1973; Anzai et al., 2014). The greater flexibility of the elbow joint may be effective for stabilization in complex arboreal environments (Foster and Higham, 2012). In contrast, it is thought that larger torque is required for the hip joint to sustain the body when climbing (Zaaf et al., 1999). Although musculoskeletal traits of limbs in these three species showed different adaptive patterns in a habitat-dependent manner, the male and female tend to be equipped with similar muscular traits related to the locomotor style. This suggests that limb structures of males and females are similarly affected by ecological traits, as both males and females in each species reside in the same habitats (Schettino, 1999).

However, in M. femorotibialis externus and internus, the female in all three species showed significant larger muscle mass than the male (Fig. 3). Male anoles tend to keep a wider territorial home range and to be more active than the female (Vanhooydonck et al., 2005; Johnson et al., 2010), and thus the male is predicted to exhibit morphological traits that are more adaptive for running or jumping, such as long hindlimb and highly developed extensor muscles. Although it is known that males are equipped with a longer hindlimb than females in some Anolis species (Butler et al., 2007), unexpectedly our data showed that females are equipped with larger knee extensors than males. Furthermore, the other two extensor muscles on the knee joint (M. iliotibialis and M. ambiens) showed no sexual differences and no interaction between sex and locomotion type in these muscles (Table 2). It means that the four muscles considered as “knee extensors” may have different roles. Although all four muscles are inserted through the same tendon on the head of the tibia, M. iliotibialis and M ambiens arise from aponeuroses from the pelvic bone and extend along the femur superficially, whereas M. femorotibialis externus and M. femorotibialis internus arise from femoral shaft and extend along the femur profoundly. Although several studies have measured electromyography in the hindlimbs of lizards (Reilly, 1995, 1998; Foster and Higham, 2014), no study has analyzed both the superficial knee extensor muscles and the profound muscles. Thus, these “knee extensor” muscles may not only extend knee joint, but also may be related to aspects of locomotion that differ in male and female.

Interspecies and sexual differences among existence/non- of display with dewlap

In forelimb, nearly half of the muscles were enlarged in species with dewlap (A. sagrei and A. porcatus) compared to A. bartschi, which lacks a dewlap. When males of these two species are displaying by extension of the dewlap, they raise their head and lift up their anterior body by their arms. Hence large forelimb muscles in two species may be related to sexual display. However, no sexual differences in mass of forelimb muscles were observed in the three species, with the exception of M. biceps brachii and M. triceps. The lighter forelimb muscles in A. bartschi are considered to be a specialization for cave habitats as both males and females show similar morphologies. In addition, knee extensors (M. ambiens, M. femorotibialis externus and internus) were enlarged in A. bartshci, thus this species may invest in locomotor behavior to maintain wider territory instead of sexual display behavior.

Elbow extensor muscle, M. triceps, showed statistical sexual differences in both muscle mass (Fig. 2; P < 0.05 with locomotion and P < 0.01 with display by two-way ANOVA) and moment arm (Fig. 4; P < 0.05 with locomotion and P < 0.01 with display); male A. porcatus in particular showed larger values than female in the boxplots. Anolisporcatus tends to keep its arms near the body as this species lives mainly in arboreal narrow habitats and hence when the male performs displaying behavior it needs to extend the elbow in order to raise the head and body. In contrast, A. sagrei mainly occurs on broad surfaces such as tree trunks or the ground (Schettino, 1999), they spread their arms widely to increase the stride (Foster and Higham, 2012). Thus, when male A. sagrei are displaying with the dewlap, they appear to raise the head and lift the anterior body by adducting their arms to make space for an expanding dewlap. However, no significant sexual differences were shown in humeral adductor muscles, M. pectoralis profundus, M. coracobrachialis longus and brevis. Also no statistical significance of interaction between sex and locomotion or display was indicated in most muscles (Table 2). Although differences in posture when lizards are displaying caused by differences in habitat may lead to sexual dimorphisms, dewlap behavior does not affect limb muscles, at least in A. sagrei.

However A. bartschi, which does not display with a dewlap, also showed sexual dimorphisms in the moment arm of elbow flexor muscles (Fig. 4), nonetheless non-significant by two-way ANOVA. There could be two explanations why this sexual dimorphism exists. First, the male can produce a larger torque than the female. Second, the female is equipped with larger excursion than the male in elbow flexion. According to Schettino (1999), no differences in ecological habits have been found between males and females in A. bartschi, but few ecological studies have been conducted on this species. Although we could not address further why the sexual difference is present, because of the lack of relevant ecological and behavioral data about A. bartschi, their sexual differences of body size suggest that male-male combat occurs in this species (Table 1; Lailvaux and Irschick, 2007; Thomas et al., 2009). A similar sexual difference in muscular traits of elbow flexion has been reported in Japanese toad (Oka et al., 1984). Male Bufo japonicusare equipped with powerful flexor muscles in the elbow, which relates to clasping behavior to hold females with their forelimbs during the breeding season (Oka et al., 1984). A larger torque of male A. bartschi may be related to holding the female during mating, although no extensive observation of mating behavior has been conducted in this species. Meanwhile, female A. bartschi bears enormous eggs compared to those of other Anolis lizards, and additionally lays eggs in crevasses, which differs from most Anolis species which oviposit in the soil (Schettino, 1999). These unique reproductive traits may affect the female-specific shorter moment arm on the elbow joint that we describe above. However, we need more detailed observations and ecological researches to discuss the significance of our data with respect to sexual differences in A. bartschi. In conclusion, we describe evident sexual dimorphisms in the appendicular musculature of three species of Anolis lizards and the possibility that these dimorphisms are affected by various sexual displays using the dewlap and ecological habitats.