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1 ZOOL 25434 1–9 ARTICLE IN PRESS

Zoology xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Zoology
journal homepage: www.elsevier.com/locate/zool

2 The regenerated tail of juvenile leopard geckos (Gekkota:

3 Eublepharidae: Eublepharis macularius) preferentially stores more fat
4 than the original
5 Q1 Anthony P. Russell ∗ , Sabrina E. Lynn, G. Lawrence Powell, Andrew Cottle 1
6 Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, AB, Canada T2N 1N4
7

8
23 a r t i c l e i n f o a b s t r a c t
9
10 Article history: The tail of many species of lizard is used as a site of fat storage, and caudal autotomy is a widespread
11 Received 30 July 2014 phenomenon among lizards. This means that caudal fat stores are at risk of being lost if the tail is auto-
12 Received in revised form tomized. For fat-tailed species, such as the leopard gecko, this may be particularly costly. Previous work
13 14 November 2014
has shown that tail regeneration in juveniles of this species is rapid and that it receives priority for energy
14 Accepted 3 December 2014
Available online xxx
allocation, even when dietary resources are markedly reduced. We found that the regenerated tails of
15
juvenile leopard geckos are more massive than their original counterparts, regardless of dietary intake,
16
and that they exhibit greater amounts of skeleton, inner fat, muscle and subcutaneous fat than original
17 Keywords:
18 Caudal autotomy
tails (as assessed through cross-sectional area measurements of positionally equivalent stations along
19 Compensatory regeneration the tail). Autotomy and regeneration result in changes in tail shape, mass and the pattern of tissue dis-
20 Energy reserves tribution within the tail. The regenerated tail exhibits enhanced fat storage capacity, even in the face of
21 Potential trade-offs a diet that results in significant slowing of body growth. Body growth is thus sacrificed at the expense
22 Tail regeneration of rapid tail growth. Fat stores laid down rapidly in the regenerating tail may later be used to fuel body
growth or reproductive investment. The regenerated tail thus seems to have adaptive roles of its own,
and provides a potential vehicle for studying trade-offs that relate to life history strategy.
© 2015 Published by Elsevier GmbH.

24 1. Introduction encounters (Bellairs and Bryant, 1985; Russell et al., 2014). Various 39

Q2 costs have been associated with autotomy (Higham et al., 2013), 40

25 The tail of many species of lizard serves as an important site including reduction of sprint speed (Daniels, 1983), reduced sta- 41

26 of fat storage (Avery, 1970; Pond, 1978; Dial and Fitzpatrick, 1981; bility in jumping (Gillis et al., 2009, 2013), compromised mid-air 42

27 Arnold, 1984; Doughty et al., 2003), and caudal fat stores may com- maneuverability (Jusufi et al., 2008), reduced body support while 43

28 prise half or more of the total lipid reserves of lizards (Vitt and climbing (Jusufi et al., 2008), and diminished reproductive out- 44

29 Cooper, 1986; Doughty et al., 2003). Caudal lipid deposits consti- put (Smyth, 1974; Dial and Fitzpatrick, 1981; Zera and Harshman, 45

30 tute a biochemically efficient means of storing energy (Derickson, 2001; Maginnis, 2006). In those species that sequester considerable 46

31 1976), and may be used for reproductive purposes (Jameson, 1974; amounts of fat in the tail, the loss of this structure also results in 47

32 Smyth, 1974; Derickson, 1976; Dial and Fitzpatrick, 1981), for the sacrifice of a primary energy store (Doughty et al., 2003), bring- 48

33 drawing upon when food becomes scarce (Bustard, 1967; Avery, ing about a significant reduction in body mass (Dial and Fitzpatrick, 49

34 1970; Smyth, 1974; Arnold, 1984), or a combination of these. 1981; Lynn et al., 2013). This is a far from trivial event and poten- 50

35 Caudal autotomy is a widespread phenomenon among lizards, tially impacts viability and survival (Bauwens, 1981; Arnold, 1984). 51

36 and is encountered in the majority of families (Higham et al., 2013). This combination of factors indicates that tail loss in many taxa is 52

37 It most often occurs in response to a predatory attack (Higham et al., a risky and potentially paradoxical strategy, involving the ready 53

38 2013), although it may also be prompted by intraspecific agonistic surrender (for the promotion of immediate survival) of a struc- 54

ture in which considerable energy reserves have been stored (for 55

the promotion of long-term survival and fecundity) (Clark, 1971; 56

Dial and Fitzpatrick, 1981). It is possible that this conflict may, to 57

∗ Corresponding author. Tel.: +1 403 220 5198; fax: +1 403 289 9311.
some extent, be tempered in species in which the majority of cau- 58
E-mail address: arussell@ucalgary.ca (A.P. Russell).
1 dal reserves are located far proximally in the widest portion of the 59
Present address: Department of Biochemistry and Molecular Biology, University
of British Columbia, 2350 Health Science Mall, Vancouver BC, Canada V6T 1Z3. tail (Doughty et al., 2003; Lin and Ji, 2005), whereas the majority 60

http://dx.doi.org/10.1016/j.zool.2014.12.003
0944-2006/© 2015 Published by Elsevier GmbH.

Please cite this article in press as: Russell, A.P., et al., The regenerated tail of juvenile leopard geckos (Gekkota: Eublepharidae: Eublepharis
macularius) preferentially stores more fat than the original. Zoology (2015), http://dx.doi.org/10.1016/j.zool.2014.12.003
 G Model
ZOOL 25434 1–9 ARTICLE IN PRESS
2 A.P. Russell et al. / Zoology xxx (2015) xxx–xxx

61 of tail breaks may occur further distally (Chapple and Swain, 2002; Lynn et al. (2013) hypothesized that the compensatory regener- 127

62 Doughty et al., 2003; Lin and Ji, 2005). For fat-tailed species, such ation of juvenile leopard gecko tails represents a potential trade-off 128

63 as the leopard gecko (Eublepharis macularius), however, lipid stores strategy, whereby a structure with enhanced energy storage capac- 129

64 are more evenly distributed along the length of the tail (Lynn et al., ity relative to the original tail is favored in association with its 130

65 2013), rendering such potential tempering less effective. loss of autotomic capacity. For this to be supported, differential 131

66 Tail autotomy has been shown to have positive value in preda- costs and benefits associated with the original and regenerated 132

67 tor encounters (Vitt and Cooper, 1986), enhancing the chances of tails would have to be evident. The original tail, in this context, 133

68 escape by directing the potential predator’s focus away from the has the benefit of being able to be easily cast off in an encounter 134

69 head and body (Dial and Fitzpatrick, 1981). Regeneration of the with a predator, resulting in enhanced potential for escape. The 135

70 tail restores this anti-predator function (Dial and Fitzpatrick, 1981). cost is the jettisoning of the energetic investment devoted to the 136

71 Tail regeneration may be rapid, indicating that the restoration of its formation of the tail, including any stored fat reserves. For the 137

72 functional roles has high priority (Meyer et al., 2002). Indeed, the regenerated tail, following maximal caudal autotomy, the cost 138

73 regenerating tail may divert energy away from other body regions, incurred is that the tail can no longer be voluntarily shed, thus elim- 139

74 resulting in a slowing of their growth (Arnold, 1984). Regenerated inating the protection against predation that the original affords. 140

75 portions of the tails of lizards are well-known to differ from the For a trade-off to be feasible, the regenerated tail would have to 141

76 originals in the replacement of the vertebrae by a hollow cartilagi- gain a benefit by serving as a more effective fat store than the 142

77 nous rod (Bellairs and Bryant, 1985; Arnold, 1988; Meyer et al., original. 143

78 2002; McLean and Vickaryous, 2011), the arrangement of the mus- We herein test the possibility that the regenerated tail of the 144

79 culature, and the reduced motility of the tail when cast off (Clark, juvenile leopard gecko, subsequent to maximal caudal autotomy, 145

80 1971; Arnold, 1984; Meyer et al., 2002). is a more effective fat store than the original. We do this by 146

81 Juvenile lizards tend to lose their tail at the proximalmost investigating the overall form and mass of the tail, and the rep- 147

82 autotomy plane, and thus frequently exhibit maximum cau- resentation of its major tissue constituents (skeleton, muscle and 148

83 dal autotomy (Webb, 2006) and complete caudal regeneration. fat) under controlled experimental circumstances. We employ the 149

84 Although it has been surmised that tail loss in juveniles may have animals studied by Lynn et al. (2013), consisting of juvenile leopard 150

85 little cost other than the temporary loss of the ability to autotomize geckos with original tails raised on high and low dietary regimes, 151

86 (Dial and Fitzpatrick, 1981), it is evident that, for leopard geckos at and juveniles with maximally autotomized tails undergoing caudal 152

87 least, considerable stored fat may be lost through this process, even regeneration raised on the same dietary regimes. Tail growth and 153

88 at very young ages (Lynn et al., 2013). In juveniles of this species, the regeneration were studied over a 14 week experimental period (see 154

89 energetic cost of regeneration is considerable, and regrowth follow- Lynn et al., 2013 for details). 155

90 ing maximal autotomy is accelerated at the expense of normal body Our predictions are as follows: (i) When body size is accounted 156

91 growth (snout–vent length and mass) (Lynn et al., 2013), the tail for, lizards with regenerated tails will have heavier tails than lizards 157

92 displaying compensatory regeneration (the preferential regrowth with original tails when raised on the same dietary regime. (ii) 158

93 of a lost structure at the expense of overall growth: Diaz-Guisado When tail length is accounted for, lizards with regenerated tails 159

94 et al., 2006; Barría and González, 2008; Lynn et al., 2013). Even in will have tails with a larger cumulative cross-sectional area when 160

95 the face of reduced food availability and intake, juvenile leopard raised on the same dietary regime. (iii) When body size is accounted 161

96 geckos of both sexes preferentially divert energy so that the regen- for, lizards with regenerated tails will have a larger fat compo- 162

97 erating tail grows at a substantially faster rate than the tail of their nent than lizards with original tails, regardless of dietary regime. 163

98 original-tailed counterparts (Lynn et al., 2013). An entirely new tail, (iv) When body size is accounted for, lizards with regenerated 164

99 having a greater mass than the original, was generated within 14 tails will not have larger tail skeletal or muscle components than 165

100 weeks of the autotomy event (Lynn et al., 2013). It attained a mean lizards with original tails, regardless of dietary regime. Should 166

101 of only 64% the length of the original, was much more bulbous, these predictions hold, the case for a trade-off between the costs 167

102 softer and was more yielding to the touch. Short, rotund tails have and benefits of the original versus the regenerated tail will be 168

103 been hypothesized to potentially be able to store more fat (Pond, strengthened, but will require further investigation to be fully cor- 169

104 1978), and regenerated tails have been reported to have a higher roborated. 170

105 fat content than originals (Arnold, 1984; Meyer et al., 2002).
106 If autotomy is maximal (occurring at the proximalmost auto- 2. Materials and methods 171
107 tomy plane), the regenerated tail will not have the capacity for
108 further autotomy. Bellairs and Bryant (1985) noted that the absence 2.1. Experimental animals and dietary regimes 172

109 of fracture planes in the regenerated tail means that a second

110 bout of autotomy is not possible for that part of the tail. If caudal The juvenile leopard geckos used in this study are those reported 173

111 autotomy is maximal, then no autotomy planes persist, and any on by Lynn et al. (2013), and all husbandry procedures are identical 174

112 further episode of tail loss must come about as a result of forcible to those reported therein. Thirty-two individuals were used in this 175

113 breakage (“passive rupture”; Bellairs and Bryant, 1985). This is a dif- investigation. Animals were maintained under Permit BI 2007-32 176

114 ferent phenomenon from the reflexively driven (Russell et al., 2014) issued by the University of Calgary’s Animal Care Committee. 177

115 self-amputation that characterizes autotomy. The force required to The high and low dietary regimes established by Lynn et al. 178

116 induce passive rupture is at least double that required to break the (2013) were employed in this study. At approximately 104 days of 179

117 original tail when loaded in tension (Bellairs and Bryant, 1985), age lizards that had been raised on an ad libitum diet were assigned 180

118 and the latter requires much greater force than reflexively driven to one of two groups: original (O), being allowed to retain the orig- 181

119 autotomy, which does not require the application of any tensile inal tail throughout the experiment, or regenerating (R), being sub- 182

120 force. Our induction of maximal caudal autotomy in juvenile leop- jected to maximal caudal autotomy at the commencement of the 183

121 ard geckos resulted in a situation in which autotomy is no longer experiment and undergoing caudal regeneration over the 14 week 184

122 possible (either during the process of regeneration or after its com- duration of the experiment. Sex was taken into consideration when 185

123 pletion), meaning that any investment in the regenerated tail is the animals were assigned to experimental groups, so that each 186

124 not compromised by the potential for subsequent autotomic loss. group was equally comprised of males and females. Preliminary 187

125 In association with this it is possible that the storage capacity of the t-tests were performed on final data to test for significant differ- 188

126 regenerated tail may be able to be enhanced. ences between sexes for three parameters: body mass, tail mass, 189

Please cite this article in press as: Russell, A.P., et al., The regenerated tail of juvenile leopard geckos (Gekkota: Eublepharidae: Eublepharis
macularius) preferentially stores more fat than the original. Zoology (2015), http://dx.doi.org/10.1016/j.zool.2014.12.003
 G Model
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A.P. Russell et al. / Zoology xxx (2015) xxx–xxx 3

190 and snout–vent length (SVL). No evidence of sexual differences was a method that measured the cross-sectional area of each con- 252

191 found, so the data for males and females were pooled for analysis. stituent tissue (skeletal, muscle, and fat) at a series of positionally 253

192 Initial measurements (total mass and SVL) were taken for all equivalent stations along the length of the tail as a surrogate for vol- 254

193 individuals (week 0). Initial body size differences were tested umetric comparison. Our assessments, therefore, are based upon 255

194 for by ANOVAs, which indicated that mean initial body size did sampling at points along a longitudinal transect of the entire tail 256

195 not differ significantly among groups (SVL: F3,28 = 0.833, p = 0.487; (Fig. 1). Investigation of tail anatomy by dissection revealed that 257

196 total mass: F3,28 = 0.011, p = 0.998). Immediately thereafter, those these stations are representative of the distribution of each tissue 258

197 animals assigned to the regenerating group were induced to auto- component along the length of both original and regenerated tails. 259

198 tomize by pinching the tail at its base (resulting in maximal caudal
199 autotomy). Half of each group was then randomly assigned to one 2.4. Tail sectioning procedures 260

200 of two dietary regimes: 8 individuals assigned to high (H) and low
201 (L) diet categories for original (O) and regenerating (R) groups, for Prior to sectioning, frozen tails were thawed and the following 261

202 a total of 4 groups and 32 individuals. For our analyses our data data collected for a second time: volume (determined by fluid dis- 262

203 are thus grouped, at the end of the 14 week period, as follows: placement; ml), mass (g), and total length (mm). No differences 263

204 original-tailed animals raised on the high and low dietary rations were noted between data recorded for fresh (immediately after 264

205 (hereafter referred to as the OH and OL animals, respectively), and autotomy or amputation) and frozen tails. To determine the dis- 265

206 the animals with regenerated tails raised on the same high and tribution and amount of skeleton, fat, and muscle in the tail, it was 266

207 low dietary rations (hereafter referred to as the RH and RL animals, cut into five segments of equal length (Fig. 1d) from anterior to 267

208 respectively). Some animals did not thrive (they developed “mouth posterior, permitting positionally equivalent slices for all tails to 268

209 rot”, which was not related to the dietary treatment but resulted in be taken and compared. The anterior margin of each segment was 269

210 a cessation of normal growth in the latter part of the experimental demarcated using a permanent marker, and the segments refrozen 270

211 period) and were thus not maintained in the experiment for the to ensure that they were rigid when sectioning took place (to avoid 271

212 entire 14 weeks. Final group averages are based upon 7 OH, 5 OL, 8 compression of tissues during the cutting process). 272

213 RH, and 4 RL individuals. A fresh, single-edged razor blade was used to cut a thin 273

214 To correct for increasing body size throughout the duration (1–2 mm) transverse section from the anterior end of each frozen 274

215 of the experiment the dietary resources provided to the growing tail segment. Each such slice (Fig. 1e), labeled A–E, from anterior 275

216 lizards were adjusted as follows. At the beginning of each week of to posterior for each tail, was placed on a microscope slide, illumi- 276

217 the experiment the average total mass of all OH animals was cal- nated with transmitted light (Fig. 2a and b), photographed using 277

218 culated, and 5% (high diet) and 1.25% (low diet) of that was used to a Nikon Digital Sight DS-L1 camera attached to a Nikon SMZ800 278

219 determine the mass of mealworms offered (see Lynn et al., 2013 for stereoscopic zoom microscope (Nikon, Tokyo, Japan), and each 279

220 details). Each individual was fed on a daily basis, which ensured that image stored as a digital file. Wherever possible, a single image was 280

221 all animals were subjected to the same level of human interven- taken of the entire cross-section. Another was taken of the skeletal 281

222 tion, eliminating handling/disturbance frequency as an additional portion at higher magnification to ensure that its perimeter was 282

223 variable. Thus, dietary provisioning was maintained at a weekly discernible. If a tail slice was too large to be accommodated in the 283

224 mass-corrected constant level. This also ensured that regenerat- field of view, one half or one quarter of it was imaged at a time, 284

225 ing individuals had their dietary rations determined as per their and a composite image generated. A scale bar was included in each 285

226 original-tailed equivalents, so that tail loss (and its associated picture. Following imaging, slices were repackaged, refrozen and 286

227 reduction in total mass) did not influence dietary provisioning. Ani- stored, along with the remaining tail segments. 287

228 mals fed on the high diet rations consumed significantly more food
229 than those provisioned with the low diet regime, but within dietary 2.5. Determination of cross-sectional area of tail components 288

230 regime treatments there was no significant difference in food con-

231 sumption between original-tailed and regenerating groups (Lynn Digital images of the cross-sections of the tail were imported 289

232 et al., 2013). into ImageJ version 1.43u (Rohlf, 2010). The following areas (Fig. 2a 290

and b) were traced using a WACOM tablet interface (WACOM Tech- 291

233 2.2. Assessment of tail mass and volume nology, Tokyo, Japan): area occupied by the skeleton (skeletal area 292

(sk), which also includes the enclosed spinal cord of the origi- 293

234 After 14 weeks, the intact tails of the OH and OL animals nal tail); skeletal (sk) + inner fat (if) area; skeletal (sk) + inner fat 294

235 were harvested by inducing maximal caudal autotomy (as outlined (if) + muscle area (mu); and skeletal (sk) + inner fat (if) + muscle 295

236 above), whereas regenerating (RH and RL) animals were euthanized (mu) + subcutaneous fat (sf) area (= total area). The skin was not 296

237 and their tails amputated at the junction between the tail base and included because it is very thin and flaccid, and was distorted during 297

238 the regenerated portion of the tail (because no fracture planes per- sectioning. 298

239 sist to enable a further bout of autotomy). Harvested tails were Areas of each region (skeletal; skeletal + inner 299

240 labeled according to the donor animal, weighed (g), their volume fat; skeletal + inner fat + muscle; and skeletal + inner 300

241 determined (by fluid displacement: ml), measured (total length; fat + muscle + subcutaneous fat) were calculated using ImageJ 301

242 mm), wrapped in plastic film, placed in screw-cap vials, and frozen software, with the dimensions set using the scale bar included in 302

243 until needed. each image. Three replicates were calculated for each parameter, 303

and their means recorded. From these cumulative area means, the 304

244 2.3. Assessment of proportional distribution of tissues in the tail respective areas of skeletal, inner fat, muscle and subcutaneous 305

fat were calculated by subtraction. Tissue identification in the tail 306

245 We initially intended to evaluate tissue distribution by assess- sections was verified by comparison with histological sections of 307

246 ing the volumes of the constituents in the original and regenerated original and regenerated tails stained with hematoxylin and eosin, 308

247 tails. To do this we developed three potential methodological or Masson’s trichrome (Humason, 1967) (Fig. 2c and d). The areas 309

248 approaches (see supplementary Table S1 and Fig. S1 in the online of each tail constituent were calculated per cross-section, and the 310

249 Appendix). We found, however, that because of the irregular shape values for the cross-sections added together to give an estimate 311

250 of both the original and regenerated tails (Fig. 1a–c), all such for the combined area of each constituent for each tail. Mean areas 312

251 approaches yielded highly inaccurate estimates. Thus, we adopted of the four tissue constituents for each tail segment, with standard 313

Please cite this article in press as: Russell, A.P., et al., The regenerated tail of juvenile leopard geckos (Gekkota: Eublepharidae: Eublepharis
macularius) preferentially stores more fat than the original. Zoology (2015), http://dx.doi.org/10.1016/j.zool.2014.12.003
G Model
ZOOL 25434 1–9 ARTICLE IN PRESS
4 A.P. Russell et al. / Zoology xxx (2015) xxx–xxx

Fig. 1. Shape of the original and regenerated tail of juvenile leopard geckos and sectioning procedures. Dorsal view of the original leopard gecko tail (a) at the start of the 14
week experimental period and (b) at the end of the 14 weeks. (c) Dorsal view of the regenerated tail at the end of the 14 week experimental period. The vertical dashed lines
(labeled A–E in panel a) represent the locations of the sectioning planes that divide the tail into five equal-length pieces (1–5). The sectioning protocol is depicted in panels
(d) and (e). Upon autotomy or excision from the body the tail was measured and marked (d) for division into five equal-length segments (1–5) at planes A–E. Cutting at these
planes resulted in the exposure of five anterior faces (e) labeled A–E. Hand-cut transverse slices at A–E resulted in the sections used for determining the area accounted for
by skeleton, muscle, inner fat and subcutaneous fat at each station along the tail (see Fig. 2a and b).

Fig. 2. Structure of the original and regenerated tail of juvenile leopard geckos as seen in cross-section. Representative hand-cut sections of the (a) original and (b) regenerated
tail viewed with transmitted light and showing the contrast between skeleton (sk), muscle (mu), inner fat (if) and subcutaneous fat (sf). Heavy black lines demarcate the
boundaries of these compartments. From such sections the cross-sectional area of each component was calculated. The area of the skeleton (sk) was calculated directly. All
other areas were calculated by subtraction of an inner component from an outer component. Thus the area of the inner fat (if) was calculated by measuring the area within
the outer boundary of the inner fat and then subtracting the area accounted for by the skeleton (sk) from that (and so forth). Panels (c) and (d) are histological sections of the
regions represented in (a) and (b), respectively, and show tissue detail to confirm identification. Abbreviations in (c) and (d) are as for panels (a) and (b).

Please cite this article in press as: Russell, A.P., et al., The regenerated tail of juvenile leopard geckos (Gekkota: Eublepharidae: Eublepharis
macularius) preferentially stores more fat than the original. Zoology (2015), http://dx.doi.org/10.1016/j.zool.2014.12.003
 G Model
ZOOL 25434 1–9 ARTICLE IN PRESS
A.P. Russell et al. / Zoology xxx (2015) xxx–xxx 5

314 deviations, for each experimental group are provided (Table S2 in 3. Results 377

315 the supplementary online Appendix).

3.1. Gross morphology of the tail 378

316 2.6. Statistical analyses As indicated in Section 2.5, cross-sections of the tail at the var- 379

ious stations sampled along its length (Fig. 1) reveal that deep to 380

317 Heteroscedasticity for all variables, for each combination of diet the dermis four zones are recognizable (Fig. 2) on the basis of the 381

318 and autotomy, was tested by Fmax tests (Sokal and Rohlf, 1995) dominant tissue expressed in each: skeletal (which includes carti- 382

319 within each ANCOVA, with a sequential Bonferroni adjustment lage, in the case of regenerated tails, and central nervous system 383

320 of the acceptance level. Normality of distribution for each vari- components in original tails), a deep deposit of adipose tissue (the 384

321 able for each combination of diet and autotomy was tested by a inner fat deposits located deep to the musculature), muscle, and 385

322 Shapiro–Wilk W test (Shapiro and Wilk, 1965), with a sequential a more superficial adipose tissue deposit (the subcutaneous fat, 386

323 Bonferroni adjustment of the acceptance level. The assumptions of located deep to the dermis and superficial to the musculature). The 387

324 normality of distribution and equality of variances were found to original and the regenerated tail exhibit all of these regions, but 388

325 hold for all variables in all subsequent analyses. All tests of normal- they are somewhat differently arranged in each iteration (Fig. 2), 389

326 ity were performed using PAST v 2.17b (Hammer et al., 2001). indicative of the substantial anatomical differences between the 390

327 Not all lizards completed the 14 weeks of the experiment (see original tail and its replacement (Arnold, 1984; Bellairs and Bryant, 391

328 Section 2.1). Initial least-squares mean SVL and total mass for 1985; McLean and Vickaryous, 2011). 392

329 lizards in the RL group were greater than those for the lizards in Transverse sections of the original tail (Fig. 2a and c) reveal a 393

330 the other diet/autotomy groups, which did not differ appreciably central vertebra, surrounded by four bundles of inner fat, config- 394

331 among themselves. Factorial ANOVAs of initial SVL and initial total ured as an “x”. Superficial to this lies the caudal musculature, which 395

332 mass for lizards from which data could be taken at the end of the is also divided into four distinct sectors. A band of subcutaneous 396

333 14 weeks, with diet and autotomy groups as factors and a term fat surrounds the outer aspect of the muscle, and this, in turn, is 397

334 for interaction between factors, indicate that there were no differ- covered by a thin layer of skin (Fig. 2a and c). The regenerated 398

335 ences in initial SVL (F1,20 = 2.289, p = 0.146), or in initial total mass tail harbors a notochord-filled cartilaginous tube (Jonasson et al., 399

336 (F1,20 = 0.273, p = 0.607) among groups; thus, initial body size was 2012) surrounded by the inner fat compartment (Fig. 2b and d), 400

337 not a significant factor in determining whether animals completed which extends further dorsoventrally than mediolaterally (Fig. 2b). 401

338 the experimental period. Numerous wedge-shaped units of muscle radiate around this, and 402

339 We tested the gross effects of diet and autotomy by subject- a band of subcutaneous fat is located between the muscle and the 403

340 ing the tail mass data to a factorial ANCOVA with the diet and dermis of the skin. 404

341 autotomy groups as factors, with body mass as a covariate, and

342 an interaction term between the factors included, followed by a 3.2. Tail mass 405

343 post hoc multiple Tukey test for significant differences among the
344 least-squares means as indicated by the significances of the fac- Least-squares mean final tail masses differ among the four 406

345 tors. Differences in total tail cross-sectional area were tested for by diet/autotomy groups, with the low diet groups displaying smaller 407

346 a factorial ANCOVA with the diet and autotomy groups as factors, tail masses than the high diet groups, and the regenerated 408

347 with tail length as a covariate, and an interaction term between the tail groups displaying larger tail masses than the corresponding 409

348 factors included. This was followed by a post hoc Tukey test for sig- original tail groups (Fig. 3a). Autotomy group effect is signifi- 410

349 nificant differences among the least-squares means as indicated by cant (F1,19 = 7.579; p = 0.013), but diet effect is not (F1,19 = 0.008, 411

350 the significances of the factors. Initial ANCOVAs, as described above p = 0.931), and there is no significant interaction between diet 412

351 but also incorporating an interaction term between the covariate and autotomy group (F1,19 = 0.949, p = 0.342). There is a signif- 413

352 and factors, supported the assumption of parallelism (tail mass: icant covariate effect for body mass (F1,19 = 8.408; p = 0.009); 414

353 F1,18 = 0.880; p = 0.361; tail total cross-sectional area: F1,18 = 0.102; tail mass thus changes with body mass, although the lack of 415

354 p = 0.753). covariate–treatment interaction (see above) indicates that this 416

355 Effects of diet and autotomy on tail composition were examined relationship is parallel among treatments, even if there is a ver- 417

356 by subjecting the total cross-sectional areas of the four components tical displacement of slope. Since diet did not have a significant 418

357 of the tail (skeleton, inner fat, muscle and subcutaneous fat) to uni- effect and there was no significant interaction effect between diet 419

358 variate factorial ANCOVAs, with SVL as a covariate, for each variable, and autotomy group, we restricted our post hoc Tukey test to auto- 420

359 followed by post hoc Tukey tests for significant differences among tomy groups. The difference between autotomy groups (3.45 g) is 421

360 the least-squares means for the factor or factors indicated as having significant (p = 0.010) and substantial; lizards with regenerated tails 422

361 significant effects in that ANCOVA. The assumption of parallelism have heavier tails when the effect of body mass is accounted for, 423

362 for each univariate ANCOVA was tested as above; no significant regardless of diet regime. Statistical power for this ANCOVA (0.750) 424

363 interaction between covariate and treatment was supported for any is slightly less than the convention for acceptance level. 425

364 of the univariate ANCOVAs (skeleton: F1,18 = 0.154, p = 0.699; inner

365 fat: F1,18 = 0.409, p = 0.531; muscle: F1,18 = 0.109, p = 0.745; subcu- 3.3. Total cross-sectional areas 426

366 taneous fat: F1,18 = 1.470, p = 0.241). All analyses of covariance and
367 variance were performed using Systat v. 10.2.01 (Systat Software Least-squares mean tail total cross-sectional areas differ lit- 427

368 Inc., Richmond, CA, USA). tle between both diet regimes of the original tail group and both 428

369 A posteriori statistical power estimates were made for each uni- diet regimes of the regenerated tail group, but the regenerated 429

370 variate ANCOVA using the a priori option of G*Power (Faul et al., tail groups display much greater total cross-sectional areas than 430

371 2007). A value for partial Á2 was calculated from the sums of squares the corresponding original tail groups (Fig. 3b). Autotomy group 431

372 from that ANCOVA. Power for each ANCOVA was adjusted itera- effect is significant (F1,19 = 29.100; p < 0.001) but diet group effect 432

373 tively under the G*Power a priori option until the required sample is not (F1,19 = 3.637, p = 0.072); the interaction effect is not signif- 433

374 size corresponded to that for the experiment, through trial and icant (F1,19 = 0.936; p = 0.346). The covariate effect of tail length 434

375 error. An acceptance level of 0.80 for power was applied to any (F1,19 = 5.084; p = 0.036) is significant; lizards with longer tails have 435

376 univariate ANCOVA. tails with a greater total cross-sectional area. Since there was no 436

Please cite this article in press as: Russell, A.P., et al., The regenerated tail of juvenile leopard geckos (Gekkota: Eublepharidae: Eublepharis
macularius) preferentially stores more fat than the original. Zoology (2015), http://dx.doi.org/10.1016/j.zool.2014.12.003
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Fig. 3. Mass and cross-sectional area data for original and regenerated tails of juvenile leopard geckos. (a) Least-squares mean tail masses with standard deviations for the
four diet/autotomy groups at the end of the experimental period. (b) Least-squares mean tail total cross-sectional areas (CSA) (for sections A–E in Fig. 1e) with standard
deviations for the four diet/autotomy groups at the end of the experimental period. (c–f) Least-squares means and standard deviations of total cross-sectional areas (CSA)
(stations A–E in Fig. 1e) of the inner fat (c), subcutaneous fat (d), skeletal (e), and muscle (f) compartments (Fig. 2) for the four diet/autotomy groups at the end of the
experimental period. Note that in contrast to panels (c), (d) and (f), the ordinate for panel (e) is standardized to 20 mm2 rather than 400 mm2 because of the relatively small
size of the skeletal compartment. For panels (a–b) and (d–f), solid transverse lines bridging between the mid-points of columns indicate comparisons of means within diet
groups, with associated probabilities; the dashed transverse line bridging between the pairs of columns (original tails and regenerated tails) indicates comparisons of means
between autotomy groups, with associated probabilities; for panel (c), the interrupted dotted line indicates comparisons of means among the diet/autotomy groups it overlies
(OH, OL and RL), with associated probability.

437 significant interaction effect between the factors, and diet was not total cross-sectional inner fat area between the RH tails and both 455

438 significant, our post hoc Tukey test examines only the difference of the original tail groups, but not between either of the original tail 456

439 between autotomy groups. The mean difference between autotomy groups, between either of the regenerated tail groups, nor between 457

440 groups (282.3 mm2 ) is significant (p < 0.001) and large; lizards with the RL group and either of the original tail groups (Table 1 and 458

441 regenerated tails have a greater cumulative cross-sectional area Fig. 3c). Calculated statistical power for this ANCOVA is high (0.991). 459

442 when tail length is considered, no matter the dietary regime to

443 which they were subjected. The statistical power for this ANCOVA
444 is high (0.940), despite the small sample size; it is not likely that 3.4.2. Subcutaneous fat 460

445 real differences among groups were not discerned. Mean total cross-sectional subcutaneous fat area is greater for 461

both regenerated tail diet groups than for both original tail diet 462

446 3.4. Cross-sectional area by tissue type groups, which do not display a great difference (Fig. 3d). The uni- 463

variate ANCOVA shows a significant autotomy effect (F1,19 = 48.508, 464

447 3.4.1. Inner fat p < 0.001), but no significant diet effect (F1,19 = 3.267, p = 0.087) and 465

448 The mean total cross-sectional area for the inner fat of no significant interaction effect (F1,19 = 3.966, p = 0.061). The SVL 466

449 the RH group is greater than the means of the other groups effect is not significant (F1,19 = 1.030, p = 0.323). The post hoc Tukey 467

450 (Fig. 3c). The univariate ANCOVA indicates a significant diet test of the mean difference between autotomy groups (71.48 mm2 ) 468

451 effect (F1,19 = 6.705, p = 0.018) and a significant autotomy effect is significant (p < 0.001); lizards with regenerated tails have more 469

452 (F1,19 = 11.787, p = 0.003), but no significant interaction between the subcutaneous fat. The calculated effect size (1.911), even with the 470

453 factors (F1,19 = 0.968, p = 0.337). There is no significant SVL effect small sample size, results in a very high calculated statistical power 471

454 (F1,19 = 0.225, p = 0.640). There are significant differences in mean for this ANCOVA (0.990). 472

Please cite this article in press as: Russell, A.P., et al., The regenerated tail of juvenile leopard geckos (Gekkota: Eublepharidae: Eublepharis
macularius) preferentially stores more fat than the original. Zoology (2015), http://dx.doi.org/10.1016/j.zool.2014.12.003
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Table 1
Pairwise difference among total inner fat cross-sectional area means for the four diet/autotomy groups at the end of the 14 week experimental period.

Original tails, high dietary regime Original tails, low dietary regime Regenerated tails, high dietary regime

Original tails, low dietary regime 11.64; p = 0.658

Regenerated tails, high dietary regime 31.98; p = 0.009 43.62; p = 0.001
Regenerated tails, low dietary regime 6.10; p = 0.939 17.74; p = 0.432 25.88; p = 0.096

473 3.4.3. Skeleton in total tail cross-sectional area among groups have been identi- 525

474 The mean values for skeletal area indicate that this compo- fied. Tail length covaries significantly with cross-sectional area, and 526

475 nent of the tail is larger in lizards with regenerated tails (Fig. 3e). there is no slope heterogeneity; regenerated tails are thicker along 527

476 The univariate ANCOVA of total cross-sectional skeletal area indi- at least part of their lengths, when tail length is taken into account, 528

477 cates a significant autotomy effect (F1,19 = 11.630, p = 0.003), but than original tails, as is evident in Figs. 1a–c and 3b. 529

478 no significant diet effect (F1,19 = 0.002, p = 0.963) and no significant Gross tail shape (Fig. 1a–c and Table S2 in the supplementary 530

479 interaction effect (F1,19 = 0.289, p = 0.597). SVL is not significant as a online Appendix) and mass are thus related to autotomic status 531

480 covariate (F1,19 = 3.209; p = 0.089). A pairwise post hoc Tukey com- rather than dietary intake, at least over the range of dietary cir- 532

481 parison between mean skeletal areas for original and regenerated cumscription examined in this experiment. The data (presented 533

482 tails (mean difference = 3.30 mm2 ) is significant (p = 0.004); lizards in Table S2) indicate that in original tails (D and E) little fat (in 534

483 with regenerated tails have a greater total skeletal cross-sectional either compartment) and relatively little muscle is present in the 535

484 area than lizards with original tails, regardless of diet. Calculated posteriormost two sections (Fig. 1a and b). In the regenerated tails 536

485 statistical power for this ANCOVA (0.77) is relatively high. the muscle and fat extend further distally as substantive compo- 537

nents (Table S2), and the distal end of the tail (Fig. 1c) is much 538

486 3.4.4. Muscle less tapered than in the original; regenerated tails consequently 539

487 Mean total cross-sectional muscle area appears greater for both have a greater cumulative cross-sectional area than their dietarily 540

488 regenerated tail diet groups than for both original tail diet groups equivalent originals. Thus, our second prediction is supported. 541

489 (Fig. 3f). The univariate ANCOVA of total cross-sectional muscle area Regeneration of the tail appears to be associated with a dispro- 542

490 indicates that there is a significant autotomy effect (F1,19 = 7.706, portionate diversion of resources to it. The quantitative difference 543

491 p = 0.012), but no significant diet effect (F1,19 = 0.599, p = 0.449) and between regenerated and original tails is not compromised by 544

492 no significant interaction between factors (F1,19 = 0.011, p = 0.917). a reduction in available dietary resources; regenerated tails do 545

493 Again, there is no significant SVL effect (F1,19 = 2.717, p = 0.116). The not approximate the dimensions of original tails regardless of the 546

494 post hoc Tukey test of difference between the means of the two dietary regime experienced by the bearers. This suggests that the 547

495 autotomy groups (80.13 mm2 ) is statistically significant (p = 0.015); regenerated tail, with autotomy no longer available as an option, 548

496 lizards with regenerated tails have a greater amount of muscle. The becomes a more prominent repository for stored lipids (as sug- 549

497 calculated statistical power for this ANCOVA (0.65) is relatively low. gested by Pond, 1978), when compared to the original. The original 550

tail, even under conditions of constant and prolonged high food 551

498 4. Discussion availability, never assumed the shape or mass of an equivalent 552

regenerated tail, which further suggests a change in the role of the 553

499 4.1. Gross dimensions of the tail tail following autotomy. The RL animals displayed a marked slow- 554

ing of body growth overall (Lynn et al., 2013), but the impact on tail 555

500 Dietary effect did not significantly contribute to final tail mass growth was much less. 556

501 among the diet/autotomy groups, but autotomy status did. The
502 regenerated tail groups displayed a greater tail mass than the orig- 4.3. Cross-sectional area by tissue type and tail composition 557
503 inal tail groups when body mass was accounted for. That there is a
504 significant body mass effect, but lack of interaction between body The primary difference in tail composition among the groups 558
505 mass and either autotomy status or diet, indicates that there is no in this experiment is again between lizards with original tails and 559
506 difference in the allometric coefficient for body mass and tail mass those with regenerated tails. The effect of diet is generally not sig- 560
507 among treatment groups, although the increased absolute mass of nificant and does not interact with autotomy state. Lizards with 561
508 regenerated tails requires that there will be offsets among groups. regenerated tails display significantly greater total cross-sectional 562
509 Our first prediction is thus supported; lizards with regenerated tails areas for all components of the tail than do lizards with original 563
510 have heavier tails than do original-tailed lizards of the same body tails. This supports our third, but not our fourth, prediction and 564
511 mass. The calculated statistical power of this analysis is sufficiently suggests that regenerated tails are not larger simply because they 565
512 close to the conventional acceptance level of 0.8 to provisionally are used as lipid depots. Instead it could imply that the increased 566
513 allow the conclusion that there are no significant differences in tail mass and total cross-sectional area of the regenerated tail involves 567
514 mass that can be attributed to dietary regime; those apparent in some aggrandizement of the associated skeletal and muscle ele- 568
515 Fig. 3a suggest a dietary effect, but this is dwarfed by the autotomy ments as well. Alternately, the increased total cross-sectional area 569
516 effect. On the basis of the range of dietary intake examined, which is of the skeletal and muscular elements of the regenerated tail may 570
517 known to have a marked effect on body growth (Lynn et al., 2013), be an ancillary effect of the general increased investment in fat stor- 571
518 it is evident that lizards with autotomized tails prioritize resources age. Our data do not allow a decision to be made between these two 572
519 to support the growth of the regenerating tail regardless of dietary alternatives. 573
520 intake. The total cross-sectional area of the inner fat deposits is the only 574

element of tail composition that shows both an autotomy effect 575

521 4.2. Total cross-sectional area and a diet effect, as expressed by the RH lizards. These had a sig- 576

nificantly greater cross-sectional inner fat area than either of the 577

522 Dietary effect was not significant in determining total tail cross- original tail diet groups, although this was not significantly greater 578

523 sectional area, but autotomy status was. The calculated power of than that of the lizards in the RL group (Table 1 and Fig. 3c). There 579

524 this analysis allows confidence in the conclusion that differences is no significant interaction between diet level and autotomy for 580

Please cite this article in press as: Russell, A.P., et al., The regenerated tail of juvenile leopard geckos (Gekkota: Eublepharidae: Eublepharis
macularius) preferentially stores more fat than the original. Zoology (2015), http://dx.doi.org/10.1016/j.zool.2014.12.003
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581 this component, which suggests that high dietary level lizards with gecko (Figs. 2 and 3d), and potentially other eublepharids (Bustard, 645

582 regenerated tails display an increased tendency to sequester addi- 1967), appears to represent a particular adaptive response of this 646

583 tional resources in this compartment under conditions of high food structure. 647

584 availability. This would be expected if the greater tail size that is Priority of tail growth over body growth in juveniles that are 648

585 seen in lizards with regenerated tails is a function of the re-grown regenerating their tails (Lynn et al., 2013) provides the means of 649

586 structure being used as a storage organ; at some point, either in its rapidly sequestering fat stores that could later be used to fuel 650

587 regeneration or as an effect of food availability, preferential lipid growth of the body (Jameson, 1974). Capitalizing upon abundant 651

588 storage within the tail will begin. resources when available, and processing them for deposition in 652

a now permanent structure (the regenerated tail) could provide a 653

589 5. Conclusions repository to draw upon to fuel the growth of juveniles prior to 654

the energy demands of reproduction being manifested. Jagnandan 655

590 Our results are consistent with the premise that enhanced et al. (2014) found that for adult female leopard geckos the fully 656

591 fat storage in the regenerating tail has been strongly favored by regenerated tail, following maximal caudal autotomy, differed in 657

592 selection in this species, and points to an altered strategy for the size and shape from the original in a similar manner to that of juve- 658

593 regenerated tail when compared to the original. Indeed, because niles (Fig. 1b and c). It attained an average of only 61% the length of 659

594 our comparative data are drawn from the end of a 14 week period the original, but had a maximum girth of 131% that of the original. 660

595 of regeneration relative to continued growth of the original tail, it On average the regenerated tail accounted for only 15% of the body 661

596 is noteworthy that the animals that lost their tail at the start of the mass, versus 22% of the original. Thus, the regenerated tail of adults 662

597 experiment began from a value of zero for tail tissues that were has a significantly smaller relative mass and volume than the orig- 663

598 measured in this study. The final outcome for the regenerating ani- inal. Regeneration is thus not afforded the same level of priority 664

599 mals, regardless of dietary regime, was that they had heavier (but as it is in juveniles. If the original tail is retained into adulthood 665

600 shorter) tails when the effect of body mass was accounted for, and the stimulus required to initiate autotomy may be considerably 666

601 had significantly greater quantities (as assessed through cumula- higher than that for juveniles (Daniels, 1984). This indicates that 667

602 tive cross-sectional area) of all measured tissue components of the longer-term investment of stored reserves in the original tail may 668

603 tail. This indicates that not only is the regenerating tail a structure be accompanied by a diminished propensity for its sacrifice as a 669

604 that receives preferential allocation of available resources, but also predator distraction. Should autotomy of such tails occur, a rela- 670

605 that this allocation is extremely rapid when assessed against the tively diminished level of investment in its replacement is evident. 671

606 growth of the original tails, and against the growth of the body This indicates that if there is a trade-off in juveniles, the same may 672

607 (Lynn et al., 2013). not be the case for adults, for which economy of autotomy (loss of 673

608 Desert-dwelling eublepharid gekkotans are known to become only part of the original tail) may be a more frequently employed 674

609 inactive in hot, dry conditions and to employ their fat reserves to strategy. 675

610 endure long periods of fasting (Bustard, 1967). When active they Our data suggest that the regenerated tail may have adaptive 676

611 can be voracious feeders, and display the ability to generate suffi- advantages of its own. Given the high prevalence of tail loss and 677

612 cient fat reserves in a few days of feeding to allow survival for many regeneration among lizards, greater focus on the biological proper- 678

613 months of fasting (Bustard, 1967). The leopard gecko is a long-lived, ties of the regenerated structure may prove to be rewarding. For the 679

614 desert-dwelling, fat-tailed species that sequesters lipid reserves leopard gecko, for example, the full implications of the altered form, 680

615 in the tail (Cheek, 2005; Duscha, 2006). Juveniles exhibit greater mass and lipid-carrying capacity of the regenerated tail (when com- 681

616 capacity for fat storage in the regenerated tail when compared pared to the original) for locomotor biomechanics, reproductive 682

617 to their original-tailed counterparts. Since the original tail can no success, and long-term survival require further investigation. 683

618 longer be cast off after maximal caudal autotomy, and because the
619 regenerate is bulkier for all tissue components than the original, Acknowledgements 684
620 these aspects can be taken to be supportive of the contention that
621 a trade-off has occurred. The ability to deploy the tail as a detach- This work was supported by a Natural Sciences and Engineering Q3 685
622 able predator distraction (Higham and Russell, 2010, 2012; Higham Research Council (NSERC) Discovery grant (9745-2008) to APR. Tim 686
623 et al., 2013) is forsaken for a non-detachable regenerated body part Higham (University of California, Riverside) provided helpful com- 687
624 that restores some of the functions of the original tail (such as act- ments on an earlier version of the manuscript. Alex Tinius assisted 688
625 ing as a potential focus of attack by predators), and combines these with the assembly of the figures. 689
626 with the ability to accumulate greater amounts of stored reserves.
627 Whether there is a shift in the balance of total lipid reserves (so that Appendix A. Supplementary data 690
628 intra-abdominal fat reserves are lessened reciprocally in associa-
629 tion with an increase in caudal reserves), or whether lipid reserves Supplementary data associated with this article can be found, in 691
630 increase absolutely in those individuals with fully regenerated tails, the online version, at http://dx.doi.org/10.1016/j.zool.2014.12.003. 692
631 is not yet known.
632 An increase in the amount of fat stored in the regenerated tail, References 693
633 relative to the original, may indicate that individuals with fully
634 regenerated tails are better equipped to survive periods of resource Arnold, E.N., 1984. Evolutionary aspects of tail shedding in lizards and their relatives. 694
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macularius) preferentially stores more fat than the original. Zoology (2015), http://dx.doi.org/10.1016/j.zool.2014.12.003
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Please cite this article in press as: Russell, A.P., et al., The regenerated tail of juvenile leopard geckos (Gekkota: Eublepharidae: Eublepharis
macularius) preferentially stores more fat than the original. Zoology (2015), http://dx.doi.org/10.1016/j.zool.2014.12.003