Perch Associated Expression of Phenotypic Plasticity in Limb Development and Sprint Speed in Agamid Lizard Calotes versicolor: A Laboratory Study

Veena H. F. AMMANNA, Srinivas K. SAIDAPUR and Bhagyashri A. SHANBHAG

Department of Zoology, Karnatak University, Dharwad 580003, Karnataka, India

Abstract New born hatchlings of Calotes versicolor were reared in terrarium having narrow or wide perches for a period of 4-month and their snout vent length (SVL), tail, fore and hindlimb lengths were measured at monthly intervals.Limb postures (closer to the body or spread away from the body) were also recorded. The sprint speed was recorded in two and four-month old lizards on a 1 m long race track providing 45° or 60° slope. In both the groups, SVL and tail lengths were comparable but the limb lengths and their growth rates were significantly greater in lizards of wider perch group. The lizards reared with narrow perches positioned their limbs closer to the body; while those reared on wider perches spread their limbs away from their body. Further, the latter exhibited significantly higher sprint speed regardless of the slope of the race track over those of narrow perch group. Sprint speeds of lizards in both groups were correlated with the limb sizes. The study showed that the lizards reared on narrow or wide perches exhibited divergent adaptive responses (phenotypic plasticity) by developing longer or shorter limbs and corresponding changes in their sprint speeds. These findings support the idea that availability of perch structure during early development evokes adaptive plasticity in the limb development and associated locomotory performance in arboreal lizards like C. versicolor.

Keywords behavior, Calotes, hatchlings, limb, lizard, perch, plasticity, sprint speed

1. Introduction

Variation in life-history traits such as investment in growth, reproduction, and survivorship is common in species that are geographically widespread (Roff 1992;Stearns 1992). These variations are shaped and maintained by differences in the availability and quality of resources among habitats or by differences in biotic interactions among habitats and is often considered to be evidence of adaptive strategies for dealing with different environments(Lepiset al., 2000; Sultan 2001; Traviset al., 2014).Variation in a trait or phenotypic plasticity in relation to changes in environmental conditions has been reported in several groups of animals, both invertebrates and vertebrates. For instance, aquatic invertebratesDaphnia pulexandChaoborus americanusproduce elaborate armored carapaces when they are grown with cues of predators, and such armors do not develop in the absence of predators (Parejko and Dodson, 1991). Adaptive phenotypic plasticity is also reported in a number of fishes in response to predator or food and habitat(Chapmanet al., 1999; Muschicket al., 2011; Robinson and Wilson, 1996). Several species of amphibian larvae display extensive plasticity in life history, morphology,and behavior in response to variations in environmental factors such as predation, diet, or habitat (Bekhetet al., 2014; Mogaliet al., 2011; Newman, 1992; Skelly and Werner, 1990; Van Buskirk and Relyea, 1998).Phenotypic plasticity in morphology and life history traits is also reported in some species of lizards. The influence of perch diameter and humidity is shown to influence plasticity in the limb length of lizards. In anoline lizards,perch diameter has an influence on limb sizes (Herrelet al., 2011; Losos, 1990; Lososet al., 2000; Moermond,1979; Williams, 1983). In these lizards, a correlation between hind limb length and perch diameter is reported in both natural and experimental populations (Kolbe and Losos, 2005; Lososet al., 1994, 1997, 2000). However, a recent study showed thatSceloporus undulatusjuveniles provided with high perches and no perches and fire ants in both the set ups, the hind limb length of lizards that climbed higher to escape from fire ants did not differ much compared to those without higher perches (Rosier and Langkilde, 2012). Apart from habitat, for instance the climate has its influence on phenotypic plasticity inLacerta vivipara.Lizardsliving in humid habitats exhibited larger body lengths, lower age at maturity,higher fecundity, and smaller neonatal size compared to those living in dry habitats (Lorenzonet al., 2001).Similarly, Lygosomine skinks that are leaf litter-dwelling or in rocky habitat revealed that those from rocky habitat have flattened body and long limbs to suit rapid climbing on rocks (Goodman and Isaac, 2008) and those living in leaf litter have relatively short limbs and slower running and climbing speed (Goodmanet al., 2008). Yet another study on a skink,Carlia longicepsshowed that nest temperature of two different habitat types, one incubated at rock outcrop nest temperature (cool) had longer limbs and run faster whereas the hatchlings obtained from the eggs incubated at forest environment (warm temperature)had shorter limbs and ran slower (Goodmanet al., 2013).Thus, it is obvious that habitat structure and climatic conditions influences limb growth in these lizards.

Calotes versicolor(Agamidae), is an oviparous,multiclutched lizard that breed seasonally from late May to early October (Shanbhag and Prasad, 1993). Studies on growth rate, age at sexual maturity and breeding in captivity inC. versicolorhave shown that some lizards breed in the first year when they are ～7.5 months old with ～ 8 cm SVL and those lizards that skip reproduction in the first year, breed during the subsequent breeding season in the following year at a larger body size (Pandavet al., 2010). Hence, indiscriminate clearing of flora for human activities may have influence on their morphology to adapt to changed habitat. The present study was designed to elucidate possible influence of the habitat structure on certain chosen parameters like the lengths of the body, limbs, tail, and sprint speed inC. versicolor.Therefore, the newborn hatchlings (from day 1) were raised in terrarium provided with either wide or narrow perches for a period of four months. We hypothesized that rearing hatchlings with different perch provision would affect limb posture/sitting posture, thereby influencing the growth rate of limb length provided that the lizards have ability for phenotypic plasticity in response to the ecological context during early development. Since these arboreal lizards are agile climbers, influence of perch structure on tail length is also studied.

2. Materials and Methods

Laboratory born one day old hatchlings ofC. versicolor(n= 40, 8 hatchlings/ clutch) from 5 different clutches were selected arbitrarily for this study. Each hatchling was uniquely marked with paint dots (white or black)on different areas of their body for identification of each individual. Care was taken to mark individual lizard with same paint and area whenever shedding of their skin was observed in order to follow up their identity. They were divided into two groups of 20 individuals from five parental lines. They were fed daily with termites and silkmoth larvaead libitumfor first 4 weeks and thereafter with grasshoppers until the termination of the experiment.Water was made available in a dish placed in the terraria.

2.1. RearingGroup 1： Rearing with wide perches. The hatchlings in this group were placed in terraria (60 L ×30 W × 30 H cm3) with two wooden perches of 30 cm long × 15 cm wide × 5 cm thick placed against the wall inside the terrarium at 45º slope (Figure 1A). Four sibs were housed in a terrarium and 5 such terraria with five parental lines were maintained. Group 2： Rearing with narrow perches. The hatchings in this group were placed in terraria (60 L × 30 W × 30 H cm3) with four wooden perches of 30 cm long × 1.5 cm wide × 0.5 cm thick placed inside the terrarium at 45º slope (Figure 1B). Four sibs were housed in a terrarium and 5 such terraria with five parental lines were maintained. The experiment was terminated after 4-month and the specimens were released in the botanical garden of the university.

2.2. Morphometric measurementsSnout-vent length(SVL), tail length (TL) and forelimb (FLL) and hindlimb(HLL) lengths from posterior insertion of limb into the body to distal tip of the claw of IV digit were recorded using digital vernier caliper with accuracy to the nearest 0.01 mm. These measurements were made on day of hatching (day 1), and 1, 2, 3 and 4-month old lizards.The difference in relative lengths of tail and limbs at each interval of time was analyzed by ANCOVA with SVL as covariate and perch as factor. The SVL, relative lengths of tail and limbs were log-transformed before analysis to meet the assumptions of normal distribution. The growth rate for SVL and relative growth rates for TL, FLL and HLL at one, two, three, and four-month were calculated using the formula of Kolbe and Losos (2005). The data on growth rate of each parameter between the two groups on one, two, three and four-month old lizards were analyzed using Mann-WhitneyUtest.

2.3. Limb posturesWhen the lizards were two-month old, their limb postures were recorded for 15 consecutive days. On each day, the posture of the limbs on the perch was recorded at around 9：00, 12：00, 15：00, and 18：00. The postures of the limbs were scored as follows： limbs held close to the body (fore limbs close, hind limbs close) or away from the body (fore limbs away, hind limbs away).The mean number of lizards displaying a particular limb posture on wide and narrow perches for a period of 15 d was computed. Variation in the mean number of lizards exhibiting a particular limb posture between the two groups was analyzed by Mann-WhitneyUtest.

2.4. Sprint SpeedThe running performance of lizards were carried out when they were 2-month (n= 15 per group) and 4-month (n= 5 per group) old.The trials were conducted on an open terrace exposed to natural sunlight.A 120 cm long and 20 cm wide plank with coarse surface was used as the race track. Race track was marked with a line at 10 cm distance from both the ends to denote the start and finish lines. The distance between start and finish lines was 100 cm. The race track was provided with 20 cm high acrylic plates along its length on both sides to prevent escape of lizards during the trials. Each lizard was prompted to run on the race track placed at 45° and 60° slopes. The running trials were carried out as follows.A transparent open-ended plastic cylinder (9 cm dia. and 17 cm H) was placed behind the start line. A test lizard was gently released inside the cylinder and allowed to settle for 5 min. The cylinder was then lifted gently to free the lizard. The test lizard was then gently prodded on its tail with a paint brush which made it run along the track. The time to reach the finish line was recorded using a digital stop watch. The trials were conducted 3 times for each lizard on each slope with an interval of 30 min between the trials. During a trial, if a lizard turned around and moved in the reverse direction, the trial was discarded. But the same individual was tested again after a gap of 30 minutes. The sprint speed in cm/ sec (cm s-1)was derived from time taken by an individual to reach the finish line. Fastest run among the 3 trials were used for analysis (4 lizards out of 9 in narrow perch group did not run properly on race track and hence excluded from the analysis).

The data on the sprint speed of lizards of the two groups on each slope were analyzed by unpairedttest for 2-month old lizards, and by Mann-WhitneyUtest for 4-month old lizards. Comparison of sprint speed of lizards within the groups on different slopes was computed using pairedttest in case of 2-month old lizards, and by Wilcoxon signed rank test for 4-month old individuals.

3. Results

During the course of the experiment, 5 hatchlings from each group died around 92 and 100 days and 6 lizards from each group around 110 days due to unknown reasons. Therefore, data on 15 lizards in each group up to 3-month and 9 (wide treatment) and 5 (narrow treatment)lizards/ group for 4-month (total 14 lizards) were used in the statistical analysis.

3.1. Morphometric changesFigure 2 shows SVL,relative lengths of tail and limbs of lizards reared with wide and narrow perches. At the start of the experiment the relative lengths of the tail (F1,29=0.06,P＞ 0.05), fore (F1,29= 0.08,P＞ 0.05) and hind (F1,29= 0.01,P＞ 0.05; Figure 2) limbs of lizards of the two groups were comparable. Both the fore and hind limbs were significantly longer in lizards reared with wide perches than those reared with narrow perches recorded at 2, 3 or 4-month(2-month： forelimb -F1,29= 5.47,P＜ 0.05; hindlimb-F1,29= 7.68,P＜ 0.05; 3-month： forelimb -F1,29=8.10,P＜ 0.01; hindlimb -F1,29= 12.07,P＜ 0.01;4-month： forelimb -F1,17= 9.86,P＜ 0.01; hindlimb -F1,17= 5.26,P＜ 0.05; Figure 2). The GRSVLand GRTLwere also comparable between the two groups throughout the 4-month experimental duration (Figure 3). The growth rate of forelimbs (GRFLL) and that of hindlimbs (GRHLL)of lizards reared with wide perches was significantly higher (from 2-month onwards) than that of those reared on narrow perches (2-month： GRFLL-U= 54.0,P＜0.05;GRHLL-U= 52.50,P＜ 0.05; 3-month： GRFLL-U= 35.5,P＜ 0.01;GRHLL-U= 63.00,P＜ 0.05;4-month： GRFLL-U= 8.50,P＜ 0.01;GRHLL-U= 16.50,P＜ 0.05; Figure 3).

Figure 2 SVL and the relative lengths of tail, fore and hind limbs of C. versicolor reared with wide and narrow perches. *Above error bars denotes significant differences between the two rearing groups.

3.2. Limb posturesOverall limb posture display of lizards reared with wide perches revealed that they stretch their forelimbs (= 0.67,P＞ 0.05) and hindlimbs (=4.42,P ＞0.05) away (Figure 4A) from the body at all times observed on a given day. However, the lizards associated with narrow perches were found to keep their forelimbs (= 3.96,P＞ 0.05) and hindlimbs (= 2.52,P＞ 0.05) close (Figure 4B) to the body since they had no space to spread their limbs. Hence, the number of lizards reared with narrow perches with their limbs close to the body was significantly more than those reared with wide perches (Forelimbs：U= 0.00,P＜ 0.05; Hindlimbs：U=0.00,P＜ 0.05; Table 1). On the other hand, lizards using wide perches tended to keep their legs away from the body more often when compared to the lizards reared with narrow perches (Forelimbs：U= 0.00,P＜ 0.05;Hindlimbs：U= 0.00,P＜ 0.05; Table 1).

3.3. Sprint SpeedBoth two and four-month old lizards reared with wide perches ran significantly faster than those reared with narrow perches when tested on the race track placed inclined at 45º(2-month： d：t28= 4.86,P＜0.001; 4-month：U= 0.00,P＜ 0.01; Table 2) or at 60º(2-month：t28= 4.04,P ＜0.001; 4-month：U=0.00,P＜0.01; Table 2). There was no significant difference in the sprint speed with respect to the slopes tested (45º and 60º angles) in two-month old lizards of both the groups(Wide：t14= 0.62,P＞0.05; Narrow：t14= 0.18,P＞0.05;Table 2). Similarly, the sprint speed of four-month old lizards of wide perch groups were comparable on both the slopes tested (Z= 1.72,P＞ 0.05; Table 2). Whereas a significant difference in sprint speed was observed in four-month old lizards reared with narrow perches when running on a race track at two different slopes (Z= 2.02,P＜ 0.05; Table 2).Their sprint speed was significantly lower on more steeper slope (60º) compared to that on a shallower slope (45º).

4. Discussion

Expression of phenotypic plasticity in any of the characteristic(s) needed for survival is important for the individuals. The study shows thatC. versicolorreared with wide or narrow perches from their birth exhibit plasticity in both fore and hind limb growth and is associated with the perch structure. The findings of the present study are in agreement with those of Lososet al.(2000).C. versicolorprovided with wide perches tend to spread their limbs away from the body most of the times and this behavioral posture perhaps promoted the limb growth. On the other hand, the lizards that were grown on narrow perches could not spread their limbs fully and were forced to keep them closer to the body.This limb posture adapted from the time of hatching until termination of the experiment (four-month) seems to hamper limb growth inC. versicolorreared with narrow perches. In anolines, it is suggested that smaller limbs are best suited in habitats with narrow perches since longer limbs are unsuitable for holding narrow perches (Lososet al., 2000; Lososet al., 2001). Developmental plasticity is essential since it has advantages for the individuals to quickly adapt to varying environment. Incidentally, the present study is the first of its kind to demonstrate that the provision of different habitat or perch type from the time of hatching influence the growth of limbs in a lizard(C. versicolor). Incidentally, the present study is the first of its kind to demonstrate that the provision of a different habitat or perch type from the time of hatching influence the growth of limbs in a lizard (C. versicolor).

The present study onC. versicoloralso revealed an association between sprint speed and limb length (both fore and hind limbs). The lizards reared with wide perches had longer limbs and ran faster than those reared with narrow perches with smaller limbs. A correlation between sprint capacity and hind limb length is reported in anoline lizards (Losos, 1990; Losos and Sinervo, 1989;Vanhooydoncket al., 2006) and three phrynosomatid subclades, sand lizards (Uma,Callisaurus,Cophosaurus,Holbrookia), horned lizard (Phrynosoma) and theSceloporusgroup (UtaandUrosaurus) (Bonine and Garland, 1999). Downes and Hoefer (2007) have also shown thatLampropholis delicataraised in enclosures with high coverage of plants climbed more frequently and grew longer hindlimbs and had greater sprint speed than did those individuals reared in enclosures where climbing provision was not provided.

The present study onC. versicoloralso revealed an association between sprint speed and limb length (both fore and hind limbs). The lizards reared with wide perches had larger limbs and ran faster than those reared with narrow perches with smaller limbs. The present study also showed that at two months of age,C. versicolor,regardless of their rearing condition, ran at comparable speed on two different slopes tested. But 4-month old lizards with shorter limbs reared (with narrow perches)ran slower on race track placed at 60° than the lizards of comparable size and age but with larger limbs (reared on wider perches). A study onStellio stellio(also an agamid lizard) reports that large-sized individuals run slower than the small-sized individuals with an increased slope (Huey and Hertz, 1982). The study, however, does not mention about limb size in these groups of lizards. The present study shows thatC. versicolorinduced to have shorter limbs runs slower than those lizards with longer limbs.However, the perch type has no significant influence on tail growth and length. This may be because there was no variation in perch heights in both the groups. SinceC. versicoloris a good climber, provision of perches of different heights may influence tail development. Hence,further studies are needed to establish relationships between perch height and tail size, if any.

Figure 3 The growth rate of SVL and relative growth rate of tail,fore and hind limbs of C. versicolor reared with wide and narrow perches. *Above error bars denotes significant differences between the two rearing groups.

Figure 4 Typical limb postures of C. versicolor (2 months old) on wide (A) and narrow (B) Perches.

Table 1 Display of limb posture of 60 days old C. versicolor reared with wide and narrow perches. The data consists of observations made for 15 consecutive days and 3 times a day for each lizard.

Table 2 Sprint speed of C. versicolor reared with wide and narrow perches on slopes at 45° and 60°.

In summary, the present study shows thatC. versicolorexhibits plasticity in limb growth in association with available perch structure/ dimensions during their early development period. The study also reveals that longer limbs help the lizards to be swifter in locomotory performance than those with shorter limbs, which is similar to that reported in earlier studies (Kolbe and Losos, 2005; Losos and Sinervo, 1989; Lososet al.,2000). However, lizards with shorter limbs (reared with narrow perches) perhaps offer them stability to cling to narrow perches. To the best of our knowledge, this is the first report documenting habitat structure influencing fore-and hindlimb growth. The study also suggests thatC. versicolorsubjected to local selection pressure such as change in habitat structure neonatally has the ability to exhibit plasticity in limb growth to suit the prevailing environment.

AcknowledgementsThe work was carried out following the guidelines of CPCSEA, New Delhi (Reg. No. 639/GO/02/a/CPCSEA).