HERPETOLOGICAL JOURNAL, Vol. I, pp. 230-234 (1988) 230

EGGSHELL STRUCTURE OF LIZARDS OF TWO SUB-FAMILIES OF THE GEKKONIDAE

D. c. DEEMING

Pure and Applied Zoology. The University, Whiteknights, P. 0. Box 228. Reading RG6 2AJ, England

Present address: Department of Celland Struciura/ Biology, School of Biological Sciences. University of Manches1er, Coupland Ill Building. Coupland S1ree1. ManchesTer M 13 9PL, U. K.

(A ccep1ed 19.5.87)

ABSTRACT

The aim of this study was to describe and compare shell structure of parchment-shelled and rigid-shelled eggs of gekkonid lizards. Scanning electron microscopy was used to describe eggshells of two,species of gecko of the Eublepharinae and fo ur species of Gekkoninae. Eggshell fr om a lacert id lizard was also described. The crystalline nature of calcium carbonate deposits on eggshells was studied; an aragonitic chelonian eggshell was used as a control. Eublepharine eggshells consist mainly of a fi brous membrane with an external layer of calcite. They resembled lacertid and other lizard shells described previously. Gekkonine eggshells have thin, fibrous shell membranes overlain by a relatively thick layer of calcite and resemble other hard-shelled gecko eggs reported elsewhere. A layer of fibres in a matrix coats the external surface of the calcite layer of gekkonine shells. S h e ll structure is considered important in determining water loss from gecko eggs, nest local ion and embryonic development. A more detailed ex amination of the reproductive biology of gekkonid eggs could help in assessing the role of the shell in reptilian development.

INTRODUCTION hoped that this would provide information on the funct ional and taxonomic significance of shell In recent years interest in the structure of reptilian structure in the Gekkonidae. eggshells has increased for two reasons. First, the sheil has been used as a taxonomic character (Bustard, 1968) that may also shed some light on t he evolutionary relationships between reptiles and birds MATERIALS AND METHODS (Erben, 1970; Packard and Packard, 1980). Second, studies on gas exchange of reptilian eggs have led to Eggshells from six species of gecko were examined. research into the role of the shell during incubation For comparison, a single representative of the fa mily (Packard, Taigen, Packard and Shuman, 1979; Lacertidae and a single species of the Testudinidae were also studied. The details of the sources of these Packard, Packard and Boardman, I 982a; Packard, Burns, Hirsch and Packard, l 982b). eggs, the species represented and the number of specimens available are shown in Table I. Oviparous lizards and snakes (Squamata) typically lay soft, parchment-shelled eggs (Halliday and Adler, T.hepar ticular crystal morph of any CaC03 present 1986). Gekkonid lizards are exceptional in this respect; in the shell was ident ified by using M eigen 's reaction lizards in two sub-families of the Gekkonidae (Deer, Howie and Zussman, 1966). This involves (Gekkoninae and Sphaerodactylinae) lay rigid-shelled boiling shell fragments for two minutes in 0.5 mole.J-1 eggs (Bustard, 1968), whereas in the other sub-families cobalt nitrate (Co(N03)2). In this reagent aragonite (Eublepharinae and Diplodactylinae) soft-shelled eggs stains a pink-purple colour whereas calcite remains are laid (Kluge, 1967; Bustard, 1968; Werner, 1972). colourless. Published descriptions of eggshell struct ures have been Eggshell structure was studied by immersing limited to the Gekkoninae. Light microscopy was used fragments in liquid nitrogen for a few seconds so that to study Lepidodactylus lugubris eggshells (Schmidt, they could easily be fractured. Two pieces of each shell 1943) and scanning electron microscopy was used to were mounted on brass stubs using a conductive silver examine the shell structure of Tarento/a mauritanica paint and were sputter-coated in vacuo with gold (Krampitz, Erben and Kriesten, 1972; Erben and (Edwards S 1 SOB). The shells were then examined using Newesley, 1972) and Hemidactylus turcicus (Packard, a JEOL T300 scanning electron microscope at an et al., 1982a). This study was therefore undertaken to operating voltage of 25 or 30 kilovolts. The effect of provide information on the eggshell struct ure of acid treatment to remove any calcium carbonate was representatives of two sub-families of gekkonid assessed by etching fragments of Eublepharis and f lizards, the previously unstudied Eublepharinae with Chondrodactylus shef In I mole.J-1 hydrochloric acid for soft shells and the Gekkoninae with hard shells. I t was thirty seconds prior to examination. GEKKONID EGGSHELL STRUCTURE 23 1

Family Sub-family Species N Embryo Source of eggs

Gekkonidae Eublepharinae Eublepharis macularius 12 Hatched The Zoological Society of London, England. Hemi1heconyx caudicinc1us 8 Hatched The Zoological Society of London, England.

Gekkoninae ChondrodaC1y/us angulife r 6 Hat ched The Zoological Society of London, England. Gehyra mutilarn Dead The Jersey Wildlife Preservation Trust, Channel Islands, U.K. Phe/suma agalegae ? British Museum (Natural History), London. (Specimen Number BMNH 1975. 1165). Phe/suma gue111heri Dead The Jersey Wildlife Preservation Trust, Channel Islands, U.K.

Lacertidae Lacerta lepida 4 Hatched The Zoologica l Society of London, England.

Chelonia Teswdinidae Geoche/one radia1a Tnfert. The Jersey Wildlife Preservation Trust, Channel Islands, U.K .

N = Number of specimens; Hatched = Embryo hatched successfully; Dead = Embryo dead in shell at late stage of incubation; 1nfert. = Infertile egg.

TABLE I: Details of the numbers and sources of lizard and tortoise eggshells used for structural examination.

RESULTS removed the mineralised layer from the surface of Eubfepharis shells exposing the upper surface of the Staining eggs he! Is with cobalt nitrate showed that a II fi brous layer (Plate l d). the shells in the study were calcitic except that of the tortoise, Geoche/one radiGTa, which was aragonitic thus serving as a control for the method. The structure of eggshells from eu blepharine geckos (Eublepharis macularius and Hemitheconyx caudi­ cinatus) were similar and resembled that of Lacena lepida. The bulk of the shell (90 per cent of the total thickness) consists of fi bres (Plates Ja and I b) with a thin layer of calcite, less than I Oµm thick, on the shell's outer surface. The externalappearance of the calcium carbonate differs among the three species; the calcite is nodular in Eub!epharis and Lacerta shells (Plate I a and I b) whereas plaques of calcite are seen on Hemitheconyx eggshells (Plate 1 c). Hydrochloric acid lb

la Jc 232 D. C. DEEMING

more numerous in Phelsuma guentheri shells and can be seen protuding from the surface layer in Plate 2d. The surface layer of Cehyra mu1ilata shell is not illustrated but it has few fi bres and consists mainly of matrix. The calcite layer of Gehyra has numerous crystals covering its internal surface (Plate 2f).

ld

2a

Plate I. Scanning electron micrographs of eggshells from geckos oft he Eublepharinae. a gecko of the Gekkoninae and a lizard oft he Lacenidae. (a) Eublepharis mac11/arius. Surface of a radial frac1 ure showing the fibrous membrane (SM) which forms 1he bulk of the shell. The ou1er shell surface is covered by a thin layer of ca lcite (CL). (b) Lacena lepida. Surface of a radial fract ure showing the fibrous shell membrane (SM) and 1he thin layer of calcile (CL) covering the outer shell surface. (c) /-lemi!l1econyx caudicina1us. Outer shell surface shwoing calcite deposits. (d) Eublepharis

macu/arius. Outer shell surface after elching in I mole. 1 ·t hydrochloric acid for 30 seconds. The fibres of the shell membrane are exposed. (e) Chondrodac1ylus anguli(e r. Radial fracture of the eggshell showing the fibrous shell membrane (SM) which is detached from the calcite layer (CL). The arrows indicate holes in the top ofthe calcite layer. There is an external covering of fi bres in a spherulitic matrix (SFL). Scale bars = SOµm.

The eggshell struct ure oft he four gek kon i ne lizards, ChondrodaC1y!us angulifer, Phelsuma agalegae, 2c P. guentheri and Cehyra mu1ila1a are shown tn Plates le and 2. Each shell consists of a fi brous shell membrane ( 10 per cent of total shell thickness) covered by an outer, continuous layer of calcite. A layer of fi bres in an unidentified matrix is found external to the calcite layer. The continuity of the calcite layer of Chondrodactyfus angu/1fe r shells is broken by series of holes near the externalsu rface of the shell (Plate le). The external surface of Chondrodacty/us eggshell is covered by numerous fi bres embedded in a matrix (Plate 2a). Etching the shell with hydrochloric acid simply removed a superficial layer of the matrix exposing the surface of the fi bres (Plate 2b). The surface layer of Phelsuma agalegae shell consists largely of the matrix (Plate 2c). Fibres appear to be 2d GEKKONlD EGGSHELL STRUCTURE 233

layer of calcite. Parchment-shelled eggs only occur in about a quarter of the total number ofgenera of geckos (Kluge, I 967; Bustard, 1968) and contrary to the impression given by some reports (Packard et al. , 1982b) calcareous, rigid-shelled eggs predominate within the Gekkonidae. The structures for gekkonine eggshells reported in the present study (Sch midt, 1943; Krampitz et al., 1972; Packard et al., I 982a) are similar to those reported previously. The gekkonine shell membrane fo rms a thin fibrous layer outside which the calcite layer fo rms the bulk of the shell making it comparable to the palisade layer of the avian shell (Erben and 2e N ewesley, 1972; Krampitz el al. , I 972). The surfa ce fi brous layer is unique to gekkonine eggshells (Packard e1 al., I 982a; present study). The appearance of the outer surface of Chondrodactylus eggs was fo und to be almost identical to that reported for Hemidac1ylus (Packard et al. , l 982a). Similar layers of fi bres in a matrix were fo und fo r all of the other species in the present study though the numbers of fi bres was variable. The role of this fi brous layer is not clear. Other reports of deposits on reptilian shells are limited to a single description of a thin amorphous layer on Chelonia mydas eggshells (Baird and Solomon, 1979). In contrast, thin, amorphous cuticular coverings are common on avian eggshells (Tullett, 1984). 2f Poorly calcified eggshells are generally thought to be Plate 2. Scanning electron micrographs of gekkonine highly permeable. Squamate eggshells, including eggshells. (a) Chondrodaoylus angulifer. The surface of a Eublepharis, have water vapour conductance values shell showing fibres embedded in a matrix. (b) Chondro­ 100 times higher than equivalent avian eggs - 1 dactylus ongulife r. Outer shell surface after etching in 1 mole.1 (Ackerman, Dmi'el and Ar, 198S). Pliable-shelled hydrochloric acid for 30 seconds. h l ma aga!egoe. (c) P e su Chelydra eggs had shells 1 hat were SS times more Radial shell fract ure showing the upper surface of the shell permeable than avian eggs of comparable size whereas membrane (SM). This is covered by a layer of calcite (CL) which is covered in turn by a surface fi brous layer (SFL). (d) rigid-shelled turtle (Trionyx) and Alliga1or eggs have shells five times more permea ble (Packard e/ al. , 1979). Phe!suma gue111heri. Radial eggshel l fracture showing the fi brous shell membrane (SM) beneath the calcite layer (CL). Jn contrast, rigid-shelled eggs of sphaerodactyline The arrows show the fi bres that protrude from the surface geckos have rates of water loss half that of predicted fi brous layer (SFL). (e) Cehyro muti/010. Radial fra cture of rates (Dunson, 1982). the shell showing the externalsu rface of the shell membrane Rept ilian nest sites are usually characterised by their (SM). The surface fibrous layer was lost in this preparation high humidity and are often fo und in soil or compost exposing the upper surface of the calcite layer (CL) which is (Packard and Packard, 1980). Gekkonine nest sites are covered with small spheres (arrowed). (f) Cehyro mutiloto. A radial shell fracture showing the under surface of t he calcite unusual in that the eggs are stuck to walls and crevices layer (CL). Numerous crystals can be seen (arrowed) on the (Arnold and Burton, 1978) and are exposed to low humidities during incubation. Water retention by the under surface of the calcite layer. Scale bars = SOµm. egg would be advantageous in such conditions and calcareous gecko eggs appear to have developed DISCUSSION relatively impermeable shells. This may be associated with the external fibrous layer which may act in a The presence of two types of eggshell in one similar way to the avian cuticle which reduces water squamate fa mily is unique. The parchment-like loss from the egg (Christensen and Bagley, 1984; eggshells ofEuble pharis and Hemitheconyx consist of a Peebles and Brake, 1986; Deeming, 1987). fi brous shell membrane with an outer covering of The rigid-shelled gecko eggs are an interesting calcite. This study confirms the similarities in the development in vertebrate eggshell structure. The structure of shells of eublepharine geckos, lacertid eggshell can be used as a good taxonomic character for eggshells (present study), several iguanid eggshells classifying different types of gekkonid lizards (A no/is sagrei, A. limifrons, A. carolinensis, Ca!Iisaurus (Bustard, 1968). The same is not true of chelonid draconoides, Dipsosaurus dorsa!is and Iguana iguana; eggshells as structure can vary within the class and Kriesten, 197S; Sexton, Veith and Phillips, 1979; particular families (Ewert, 1979). The importance of Packard and Packard, 1980; Packard et al., I 982a; the eggshell in gecko biology extends well beyond its Packard et al., I 982b), and teiid lizards ( Cnemidophorus taxonomic use. The similarities and differences sexlineatus; Trauth and Fagerberg, 1984) as these between the rigid eggshells of chelonians, crocodilians, shells are also fi brous membranes overlaid by a thin gekkonid lizards, dinosaurs and birds (Erben, 1970; 234 D. C. DEEMING

Erben and Newesley, 1972) may be important in Deer, W. A., Howie, R. A. and Zussman, J. (l966). An Intro­ determining the patterns of eggshell evolution. Further duction to the Rock-fo rming Minerals. Longman, London. investigations may allow us to test the significance of Dunson, W. A. (1982). Low water vapour conductance of the use of shell structure in evolutionary analyses of the hard-shelled eggs of the gecko lizards Hemidactylus and Lepidodactylus Exp. Zoo!. , 219, major groups of reptiles (Packard and Packard, I 980). . .!. 377-379. The existence of two shell types in a group of closely Erben, H. K. ( 1970). Ultrastrukturen und mineralisation rezenter und fossiler eischalen bei vogeln und reptilien. related lizards provides a usefu l opportunity to Biomineralisation, 1, 1-66. investigate the fu nctional significance of the eggshell. Erben, H. K. and Newesley, H. (1972). Kristalline bausteine A detailed comparative study of the reproduct ive und mineralbestand von kalkigen eischalen. Bio­ biology of eublepharine or diplodactyline and minera/isation, 6, 32-48. gekkonine or shaerodactyline geckos could provide Ewert, M. A. (1979). The embryo and its egg: development information that would allow the relationships and natural history. In: 'Turtles: Perspeclives and between eggshell structure and various aspects of Research'. Eds. Harless, M. and Morlock, H. John incubation of the egg to be understood. In particular, Wiley & Sons, New York. initial egg composition has been shown to be related to Hal liday, T. R. and Adler, K. (1986). The Encyclopaedia of the degree of calcification of the eggshell (Tracy and Reptiles and Amphibians. George A lien and Unwin, Snell, 1985) and it could be predicted that rigid-shelled London. eggs have a higher initial water content. Nest location Kluge, A. G. (1967). Higher taxonomic categories of and incubation conditions will differ according to shel l gekkonid lizards and their evolution. Bull. Amer. Mus. permeability and shell structure may have some Nat. Hist., 135, 1-59. influence on the development of the embryo. A study Krampitz, G., Erben, H. K. and Kriesten, K. (1972). Uber of gas exchange across rigid and soft shells could aminosaurenzusammensetzung und struktur von provide useful information concerningthe role of shell eischalen. Biomineralisation, 4, 87-99.

permeability in the physiology of the em bryo . Kriesten, K. (I975). Untersuchungen uber ulrastruktur. proteinmuster und aminosaurenzusammensetzung der eischalen von Tes1udo elepha111opus, Caiman cro'codilus and- Iguana ig uana. 'Z_ool.. .lb. Anal. . 94, 101-122. ACKNOWLEDGEMENTS Packard, G. C., Taigen, T. L., Packard, M. J. and Shuman, R. D. ( 1979). Water-vapour conductance of test udinian and crocodilia n eggs (Class Reptilia). Resp. Physiol. , 38, I thank the University of Reading for fi nancial 1-10. support and Mr. A. Jenkins for invaluable assistance Packilfd, M. J. and Packard, 980). Evolution of 1 he in using the electron microscope. I thank Mr. D. Ball of G. C. (I cleidoic egg among rept ilian antecedents of birds. the Zoological Society of London, Mr. Q. Bloxam of Amer. Zoo/., 20, 35 1 -362. the Jersey Wildlife Preservation Trust and Dr. E. N. Packard, M. J., Packard, G. . and Boardman, T. J. ( l 982a). Arnoldof th e British Museum for generously providing Structure of eggshells and water relations of rept ilian the eggshells used in this study. Many thanks to eggs. Herpetologica. 38, 136- 155. Professor K. Simkiss, Mr. D. Unwin, Dr. A. Kit chener Packard, M. J., Burns, L. K., Hirsch, K. F. and Packard. and the referee constructive criticism of this paper. for G. C. (1982b). Structure of eggs of Cal/isaurus draconoides (Reptilia, Squamata, lguanidae). Zoo/. J. Linn. Soc .. 75, 297-3 I 6. Peebles, E. D. and Brake, J. ( 1986). The role of t he cul iclc REFERENCES in water vapour conductance by the eggshell of broiler breeders. PouIt. Sci., 65, I 034- I 039. Ackerman. R. A., Dmi'el, R. and Ar, A. (1986). Energy and Schmidt, W. J. (1943). Uber den bau der kalkigen eischale water vapour exchange by parchment-shelled reptile von Lepidodactylus lugubris D. & B. (Gekkonidae).

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