Effects of Incubation Temperature on Crocodiles and …aerg.canberra.edu.au/library/sex_reptile/1989_Webb_Cooper-Preston... · Effects of Incubation Temperature on Crocodiles and

AMER. ZOOL., 29:953-971 (1989)

Effects of Incubation Temperature on Crocodiles and theEvolution of Reptilian Oviparity1

GRAHAMEJ. W. WEBB

AND

HARVEY COOPER-PRESTON

G. Webb Ply. Limited, P.O. Box 38151, Winnellie, N.T., 0821, Australia andConservation Commission of the Northern Territory,P.O. Box 496, Palmerston, N.T. 0831, Australia

SYNOPSIS. Crocodylus porosus is a mound-nesting crocodilian in which incubation temper-ature influences the rate of embryonic development, the probability that embryos willsurvive to hatching, post-hatching growth rates and the probability of hatchlings survivingto 2 yr of age. Similar responses have been described in Alligator mississippiensis (Joanenetai, 1987) and C. niloticus (Hutton, 1987), and they reflect a suite of "non-sexual" effectsof incubation temperature. Temperature-dependent sex determination allocates sex onthe basis of these "non-sexual" effects. In C. porosus, it results in maleness being assignedto embryos with high probabilities of surviving and good potential for post-hatchinggrowth. Within the limits of survival, effects of the moisture environment on embryologicaldevelopment rate and hatchling fitness seem minor relative to those of the temperatureenvironment.

Reptilian orders have either obligate oviparity (chelonians, crocodilians and rhyncho-cephalians) or facultative oviparity (squamates), depending on the extent of embryonicdevelopment within the oviducts. The distinction is equally one between embryos whichare buffered from thermal effects within a female's body {facultative oviparity) and thosethat are not (obligate oviparity). Facultative oviparity and internal thermal buffering maybe the primitive condition within the Class Reptilia, and the "shell-less" eggs of extantsquamates may reflect the original amniote egg. Obligate oviparity, which also exists inbirds, appears to have been a specialized development, and is a blind end in the evolutionof viviparity among vertebrates. The significance of thermal buffering being lost in obligateoviparous reptiles remains unclear.

INTRODUCTION Wilhoft et al., 1983; Hou Ling, 1985; Ray-Incubation temperature influences the n a u d a n d p i e a u ' 1 9 8 5 ; Standora and Spo-

sex of crocodilians (Ferguson and Joanen, tlla> 1 985; Tokunaga, 1985, 1986; Webb1982, 1983; Webb and Smith, 1984; Fer- et aL> 1 9 8 6 : Bull> 1 9 8 7 ; Head et al, 1987).guson, 1985; Hutton, 1987; Lang, 1987a; Y e t s e x 1S o n ly o n e o f t h e embryologicalSmith, 1987; Webb et al, 1987a; Yama- parameters influenced by incubation tem-koshi etai, 1987; Lang et al, 1989) in much perature, and from a fitness point of view,the same way as it does in a number of l l maY b e o f secondary importance,chelonians and lacertilians with tempera- Among crocodilians, incubation temper-ture-dependent sex determination (TSD) a t u r e affects the probability of embryos(for examples see: Yntema, 1976, 1979; surviving (Webb and Smith, 1984; Lang etBull and Vogt, 1979; Bull, 1980, 1983; aL> 1989), the frequency of abnormalitiesMrosovsky, 1980; Mrosovsky and Yntema, among embryos and hatchlings (Webb et1980; Miller and Limpus, 1981; Bull et al., aL> 1983a, b; Ferguson, 1985), body size1982a, b; Vogt and Bull, 1982; Vogt etai, a t hatching (Hutton, 1987; Webb et al,1982; Yntema and Mrosovsky, 1982; Lim- 1987a), the weight of residual yolk atpus etai, 1983, 1985; McCoy et al, 1983; hatching (Webb et al, 1987a), hatchling

pigmentation patterns (Deeming and Fer-guson, 1989; Lang, unpublished data), post-

,_, . „. , ,.,. hatching growth rates (Hutton, 1987;1 From the Symposium on Biology of the Crocodiha T , , m o « , , , .

presented at the Annual Meeting of the American Jo anen et al., 1987) and post-hatching pat-Society of Zoologists, 27-30 December 1987, at New terns of thermoregulation (Lang, 1987a).Orleans, Louisiana. These effects are independent of sex

953

954 G. J. W. WEBB AND H. COOPER-PRESTON

(Smith, 1987; Webb et al., 1987a) and canbe considered "non-sexual" responses toincubation temperature. Like sex theyappear to be linked more proximally toembryonic metabolism and developmentrate than they are to temperature per se(Webb and Smith, 1984; Webb et al.,1987a), and thus they can be influenced bythe moisture and gaseous environments ofincubation. However, these influencesappear minor relative to that exerted byincubation temperature.

If there were selective advantages in allo-cating sex on the basis of the "non-sexual"responses to incubation temperature, TSDwould provide advantages that are unavail-able with genotypic sex determination(GSD) (Ferguson and Joanen, 1982, 1983;Bull, 1987; Head et al., 1987; Joanen et al,1987; Smith, 1987; Webb et al., 1987a),and which are consistent with the predic-tions of Charnov and Bull (1977). In Alli-gator mississippiensis such a situation exists;"maleness" is allocated to embryos withthe greatest potential for post-hatchinggrowth (Joanen et al., 1987). A similar sit-uation appears to exist in Crocodylus niloti-cus (Hutton, 1987), and the results we pres-ent, indicate that the relationship also existsin saltwater crocodiles (Crocodylus porosus).

The significance of the "non-sexual"responses of embryos to temperature mayextend well beyond a plausible explanationfor TSD. Crocodilian embryos are partic-ularly sensitive to incubation temperatureduring the first half of development (Webbet al., 1987a; Whitehead, 1987a), which isthe period that most squamate embryos areretained within the maternal oviducts(Shine, 1985). Squamates may thus be buff-ering their embryos against temperatureextremes during the most sensitive stagesof embryological development.

If so, the distinction between reptileswhich retain eggs to the extent of viviparity(squamates), and those that do not (che-lonians, crocodilians and rhynchocephali-ans), may be partly related to the non-sex-ual effects of incubation temperature. Thetwo groups can be referred to as havingfacultative oviparity and obligate oviparityrespectively. Birds, which have retained a

fundamentally reptilian egg, have obligateoviparity.

Within the ecologically diverse reptilesand birds with obligate oviparity, no knownspecies allows its embryos to develop withinthe oviducts to the extent found in squa-mates (Shine, 1985; Blackburn and Evans,1986). As a consequence, obligate oviparityof the form seen in reptiles and birds todayis unlikely to have given rise to facultativeoviparity. A more plausible explanation isthat facultative oviparity is the primitivecondition, and obligate oviparity has beenderived from a form offacultative oviparity.

This paper presents field and laboratorydata on the incubation temperatures towhich Crocodylus porosus embryos areexposed, and the effects they have on sex,survival and growth, both before and afterhatching. The relative contributions of thetemperature and moisture environment arediscussed. When viewed in the context ofthe "non-sexual" effects of incubation tem-perature, TSD is demonstrated to haveadvantages that would be unavailable withGSD. Ways in which the amniote egg andthe different modes of reptilian oviparitymay have evolved are discussed. Incuba-tion temperature has no doubt been a con-straining influence on the evolution of both,but its exact role remains unclear.

SALTWATER CROCODILES—A CASEHISTORY

Crocodylus porosus nests, eggs and embryoswere examined in the Northern Territoryof Australia. Eggs were collected from thefield at various ages and incubated underartificial conditions until hatching. Nesttemperatures in the field were measuredwith calibrated thermometers 2-3 eggsdeep, and often, a second nest temperaturewas taken in the mound away from theeggs. An egg from each clutch was sacri-ficed, and the embryo was given an ageequivalent (in days) based on incubation at30°C (MA30; morphological age at 30°C[Ferguson 1985; Webb et al., 1987a]). Timeof nesting was approximated by correctingthe MA30 for nest temperature with tem-perature-dependent development ratecoefficients (Webb et al., 1987a).

INCUBATION TEMPERATURE AND REPTILIAN OVIPARITY 955

Some young clutches (<8 days MA30)were incubated at precise temperatures(±0.2cC) within water-jacketed incubators.Most eggs were exposed on racks withoutnesting media (Method B of Webb et al.[1987a]), but some were incubated withinplastic bags (Method A of Webb et al.[1987a]). Large scale incubation of wildcollected eggs was carried out at mean tem-peratures fluctuating between 31°C and32°C, in a constant environment raising pen(Webb et al, 1983c).

Eggs that obviously contained deadembryos, or which failed to hatch, wereopened. The age of dead embryos allowedtime of death to be assigned to either beforeor after collection. All hatchlings weremeasured, weighed and sexed by exami-nation of the cliteropenis (Webb et al.,1984a). The extent of residual yolk wasgauged crudely by the extent of abdominaldistension. In different clutches, high mor-tality before collection was associated withhigh mortality in the incubators, whereaslow mortality before collection gave neg-ligible mortality in the incubators (unpub-lished data). In the text "embryo survival"refers to the proportion of fertile eggs thatsurvived to hatching, regardless of the ageat which they were collected.

The effects of incubation temperatureon post-hatching growth and survival werequantified from numbered hatchlings(scute clipped) raised at a crocodile farm.They were housed in identical outdoorconcrete bays, in which cover and a radiantheat source were provided. Each hatchlingwas measured, weighed and sexed threetimes over 2 yr. These data allowed a size-age curve to be constructed for each indi-vidual, from which the size at exactly 1 and2 yr of age could be predicted. This pro-cedure overcame problems associated withthe different real ages of individuals at eachinventory.

The clutch of origin of the hatchlingswas only partially controlled. The two larg-est clutches (n = 35 and 24 hatchlings) wereeach subdivided between three tempera-ture treatments (29, 30, 33°C; and 31, 32,33°C). The remaining hatchlings camefrom 12 clutches (1-20 hatchlings per

clutch), each from a single temperature.The final distribution was: 29°C (16H, 6clutches); 30°C (17H, 2C); 31°C (51H, 5C);32°C (15H, 3C); 33°C (25H, 2C).

Unless otherwise stated, errors are ± 1standard deviation.

NESTS AND EGGS

Crocodylus porosus nest during the annualwet season, when ambient temperatures arehigh and reasonably stable (Fig. 1). Femalesselect a vegetated nest site close to per-manent water, and within it construct amound of vegetation and soil some 1.8 mlong by 1.6 m wide by 0.5 m high (Webbet al., 1977). They excavate a centrally sit-uated egg chamber and lay their completeclutch within a 1 hr laying period (unpub-lished observations). The eggs are coveredwith 8-28 cm of nest material (Webb et al.,1977), and they remain at the orientationat which they were laid throughout incu-bation (66 to at least 114 days [Kar, 1979;Magnusson, 1979a]). Females remain at ornear the nest, may effect minor repairs toit (Webb et al, 1977), and may attackpotential predators (Webb et al, 1983*).Their movements over the nest usuallyconsolidate its structure.

Mean clutch size within 416 nests was49.98 ± 11.43 (range 2-78 eggs), and meanegg dimensions (mean of clutch means)were: length 7.84 ± 0.41 cm (n = 385clutches; range 6.57-8.95 cm); width4.90 ± 0.24 cm (n = 383; range 4.18-5.50cm); weight 109.19 ± 14.97 g (n = 357;range 65.4-147.0 g).

NEST TEMPERATURES

Temperatures within wild nests areaffected by the siting of nests relative tothe sun, exposure to wind, the incidenceof rain and flooding, the heat produced bydecomposition of vegetation and the heatproduced as a byproduct of embryonicmetabolism (Webb et al, 1977, 19836;Magnusson, 19796; additional unpub-lished data). Once nests are consolidatedby female activity over them, diel variationin nest temperature is reduced (1.1 ± 0.7°Cin one sample of nests [Magnusson, 19796]),although temperature gradients of 1-3CC


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MONTHFie. 1. Time of nesting for 492 wild Crocodylus porosus nests examined between 1973 and 1987 (lower),compared with average monthly rainfall (middle; total = 1,661 mm), and mean monthly maximum, meanand minimum temperatures (upper) in Darwin, Australia (1941 to 1986; Bureau of Meteorology).

exist between the top and bottom eggs(Webb et al., 1977). Nest temperaturesgradually follow changes in mean ambienttemperature (Webb et al., 1977; Magnus-son, 1979ft).

Mean egg temperature within 246 wildC. porosus nests, with embryos of variousages, was 31.65 ± 1.94°C (range 25.3-36.6°C). In nests with embryos between 14and 52 days MA30, the range of ages duringwhich sex is determined (Webb et al.,1987a), it was slightly less (31.52 ± 1.92°C

[range 25.3-36.6°C; n = 145]). Nest tem-peratures between December and Aprilwere generally high (Fig. 2), with a markeddecline at the start of the cool-dry periodof the year (May to July). Independent ofseasonal trends, mean nest temperatureincreased with embryo age (Fig. 3), whichappears to reflect metabolic heat produc-tion from within the clutch (Fig. 4). Thattemperatures among the eggs exceededthose of the mound is inconsistent with heatof decomposition explaining the temper-


ature rise. The variation in nest tempera-tures between December and April (Fig. 2)reflects an interaction between rainfall (acooling influence) and embryo age (awarming influence).

SEX, SURVIVAL AND DEFORMITIES

The sex ratio of hatchlings from wildnests containing embryos older than 52days MA30, reflects the relationship be-tween "spot" nest temperatures measuredin the field and sex (Fig. 5; lower). Clearly,high and low nest temperatures are asso-ciated with a preponderance of females,whereas a peak of males comes from nestsbetween 31.1 and 32.0°C. Results fromconstant temperature incubation in thelaboratory (Fig. 6), closely parallel thosefrom the wild.

The primary cause of embryo mortalityin wild Crocodylus porosus nests is flooding,and it could well account for 50% of alleggs laid in northern Australia (Webb etal, 1977, 19836, 19846). In contrast, only0.9% of wild eggs examined (n = 12,241)had signs of desiccation (air spaces). Pre-dation of eggs containing living embryosoccurs even more rarely (Webb et al, 19836,19846), although varanid lizards areattracted to nests with rotting eggs (Mag-nusson, 1982). Unfavorable incubationtemperatures are perhaps the second mostimportant cause of embryo mortality (Webbet al., 19836). In a sample of 173 clutches(Fig. 5; middle), very high nest tempera-tures (>35.1°C) occurred rarely, butinduced a variety of deformities (Fig. 5;upper). Nest temperatures between 33.1and 35.0°C, and between 28.1 and 29.0°Cboth resulted in higher mortality than thosebetween 29.1 and 33.0°C.

In the laboratory, incubation at 34°C wasonly attempted once. Nine of 11 eggs pro-duced deformed embryos, which died dur-ing incubation, and two eggs contained liv-ing, apparently normal embryos when theeggs were opened. Eleven eggs from thesame clutch were incubated at 28°C, and 8of them produced premature but other-wise normal hatchlings; 1 died beforehatching and 2 at the stage of hatching.Although based on small sample sizes, the

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FIG. 2. Egg temperatures within 246 wild Crocodylusporosus nests, subdivided according to the month inwhich they were measured. Horizontal lines delimittwo standard errors on either side of the means (cir-cles); non-overlap signifies significant differences atapproximately the 5% level. Numbers are the sample

laboratory observations are consistent withthe field results (Fig. 5).

TOTAL INCUBATION PERIOD

In addition to the date of nesting relativeto peak rainfall, the probability of mortal-ity due to flooding is a function of the timethat eggs remain in the nest before hatch-ing. Laboratory results (Fig. 7) indicate thattotal incubation time is highly correlatedwith incubation temperature, regardless ofwhether eggs are incubated in bags(Method A) or on racks (Method B). A sim-ilar relationship exists in the field (Webbet al, 19836, d), but it has not been quan-tified precisely. Switch experiments, inwhich eggs are incubated at one temper-ature and then switched to another, indi-cate that the major effect of temperatureon total incubation time occurs during thefirst half of embryonic development (Fig.8).

RESIDUAL YOLK

Excluding maintenance costs, the size ofan embryo at hatching will depend largelyon how much of the yolk has been con-verted to tissue and how much has beenretained as residual yolk (Webb et al.,1987a). In 124 hatchlings from eggs incu-bated at different constant temperatures


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FIG. 3. Egg temperatures within 220 wild Crocodylus porosus nests (December to April; the warmer months),subdivided on the basis of embryo age (30°C age equivalent; MAM). Horizontal lines delimit two standarderrors on either side of the means (circles). Numbers are the sample sizes.

(Table 1), two (1 each at 29°C and 33°C)were given a residual yolk code of 1,because they hatched prematurely, beforethe residual yolk was fully internalised. Ofthe remaining 122, at 30°C, 6% of animalshad abundant yolk, whereas at 32°C, 73%did (Table 1). However, no consistent trendwas present at the other temperatures.

HATCHLING SIZE

Multiple regression analysis with thesample of 122 apparently normal hatch-lings indicated that egg weight (trans-formed in various ways) explained a max-imum of 38.6% of the variation in headlength, 40.3% of the variation in snout-vent length and 81.3% of the variation inbody weight (hatchling plus residual yolk).Clearly, hatchling size is determined by eggsize. Incubation temperature explainednone of the additional variation in head orsnout-vent lengths, but did explain anadditional 2.3% (P < 0.0001) of the vari-ation in body weight. The temperaturecoefficient (1.22 ± 0.30 [SE]) indicated alittle over 1 g increase in body weight

(hatchling + residual yolk) per 1°C increasein incubation temperature.

POST-HATCHING SURVIVAL AND GROWTH

Crocodylus porosus hatchlings raised oncrocodile farms usually start feeding andgrowing within two weeks of hatching,although some individuals are reluctant tofeed, become emaciated, and eventually dieof starvation. Among the 122 animals incu-bated at constant temperature (Table 1),survival to 2 yr of age was highly correlatedwith incubation temperature (Fig. 9), andwas thus correlated with sex.

Of the 81 survivors, mean snout-ventlength (SVL) after two years of age washighly variable, but trends with both incu-bation temperature and sex were apparent(Fig. 10; upper). Among females alone,mean size after two years increased withincreasing incubation temperature between29°C and 33°C. Although sample sizes ofmales were small, their mean size was ele-vated above the general relationship forfemales. Multiple regression analysis, pre-dicting SVL from both sex (coded as 1 and

INCUBATION TEMPERATURE AND REPTILIAN OVIPARITY

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FIG. 4. The difference between nest temperatures measured among the eggs and away from the eggs, as afunction of embryo age (30°C age equivalent; MASO), in 163 wild Crocodylus porosus nests. Metabolic heat raisesegg temperature during incubation. Horizontal lines delimit two standard errors on either side of the means(circles). Numbers are the sample sizes.

0) and temperature, explained 14.4% (P =0.003) of the total variation in SVL: tem-perature independently explained 8.2%(P = 0.009) and sex 6.2%, although it wasoutside the 5% rejection level (P = 0.071).

"NON-SEXUAL" TEMPERATURE EFFECTS

The effects of incubation temperatureon crocodilians have usually been studiedin conjunction with research into TSD, andit is only recently that they have been con-sidered independent entities (Webb andSmith, 1984; Joanen et al, 1987; Lang,1987a; Smith, 1987; Webb et al, 1987a).The effects remain poorly understood, andthe mechanism through which they oper-ate is essentially unknown.

Incubation temperature affects the sizeof hatchlings. In Crocodylus johnstoni, lowtemperature incubation gives relativelyheavy hatchlings with little residual yolk,whereas high temperature incubation givesrelatively light hatchlings with a lot of

residual yolk (Webb et al, 1987a). In C.niloticus (Hutton, 1984, 1987) short ani-mals with abundant residual yolk comefrom the highest incubation temperatures(34°C). Trends in this direction weredetected with C. porosus at 30°C and 32°C,but they were not consistent over the fulltemperature range. The extent to whichsize at hatching and/or the amount ofresidual yolk with which embryos hatchaffects fitness in unclear.

In contrast to size at hatching, post-hatching survival of C. porosus in captivitywas related to incubation temperature:maximum survival occurred at 32°C (87%)and minimum survival at 29.0°C (44%) (Fig.9). In A. mississippiensis, survival under cap-tive raising conditions is generally high andstable over the incubation temperaturerange 29.4-32.8°C (Joanen et al., 1987).Mean size of C. porosus after 2 yr increasedwith increasing incubation temperature,and there was an additional tendency for


28 29 31 32 33 34TEMPERATURE (PC)

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FIG. 5. The relationship between Crocodylus porosus nest temperatures (measured by spot nest temperature2-3 eggs deep in each nest) and the sex of hatchlings in nests collected after sex was determined (lower), themortality of fertile eggs in nests collected at various ages (middle) and the incidence of obvious deformitiesamong hatchlings from nests collected at various ages (upper). For quantifying sex, only clutches collectedfrom the field when older than 52 days MAS0 were used. The high baseline level of mortality is attributablemainly to flooding. Numbers refer to the number of clutches used to calculate a mean value for any 1°Ctemperature range.


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FIG. 6. The relationship between constant incuba-tion temperature and sex for Crocodylus porosusembryos incubated in the laboratory for the whole oftheir development. Numbers are sample sizes. Dataare the pooled results of Methods A and B incubation(from Webb et al. [1987a]).

males to exceed the sizes of females (Fig.10). Among females, the maximum size wasattained by animals from 33°C (47 cm SVL)and the minimum size by animals from 29°C(37 cm SVL). Within the incubation tem-perature range at which C. porosus embryosurvival was generally high (29.1-33.0°C),better post-hatching performance camefrom a more restricted range (32.0-33.0°C).

In Crocodylus niloticus hatchlings incu-bated at 34°C outgrew those from 28°C and31°C within three months, even thoughthey were shorter at hatching (Hutton,1987). Post-hatching growth rates of A.mississippiensis are affected by incubationtemperature (Joanen et al., 1987), butmaximum growth of females and malesoccurs in animals incubated at tempera-tures within the middle of the range thatproduces both sexes (30.6°C and 31.7°Crespectively) (Fig. 11).

There are thus data for Crocodylus poro-sus, C. niloticus and Alligator mississippiensiswhich indicate incubation temperature isexerting significant effects on post-hatch-ing growth and/or survival. Given thatclutch effects were not completely con-

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FIG. 7. The relationship between constant incuba-tion temperature in the laboratory and total incu-bation period (to pipping of eggs) for Crocodylus poro-sus eggs incubated on racks (open circles) and in plasticbags (closed circles). Data are means from Webb et al.(1987a). The line is a polynomial, least squares regres-

trolled and that raising strategies differedgreatly, at this stage we attach little signif-icance to the different forms of the rela-tionship (Fig. 10 vs. Fig. 11). However, itis clearly an area which merits experimen-tation under tightly controlled conditions.

"SEXUAL" TEMPERATUREEFFECTS AND TSD

The mechanism through which incuba-tion temperature influences sex within anycrocodilian species remains unknown (Ray-naud and Pieau, 1985; Standora and Spo-tila, 1985;Gutzke, 1987; Head et al., 1987).However, the rate at which embryosdevelop is as highly correlated with sex asis incubation temperature itself (Webb etal., 1987a), and thus by independentlyinfluencing development rate, there areavenues through which the moisture(Gutzke and Paukstis, 1983) and gaseousenvironments of incubation could influ-ence sex.

The non-sexual effects of temperature,


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FIG. 8. The relationship between embryo age, and the effect of temperature on total incubation time inCrocodylus porosus. Eggs were incubated at 30°C for varying periods and switched to 32°C for the remainderof incubation. Closed and open circles are males and females respectively. Vertical bars are the mean incubationtimes (±SD) at constant temperatures of 30°C and 32°C (reprinted from Webb et al. [1987a]).

like sex, are predictable from embryonicdevelopment rate before sex is allocated. InCrocodylus porosus the sex determiningmechanism allocates "maleness" to em-bryos developing at incubation tempera-tures around 32°C; these have the lowestprobability of survival and growth beingcompromised. As adults the maximum sizeof C. porosus (both sexes) is highly variable(Webb et al., 1978). Males (4-6 m) are muchlarger than females (2.2-3 m) (Webb et al.,1978, 19846), and they engage in territo-rial conflicts with other large males (Lang,19876; unpublished data). Small ordeformed males are unlikely to be able tomaintain a territory and breed successfully,yet each year a great range of differentsized females have their eggs fertilised andnest successfully (even though female C.porosus are themselves remarkably intol-erant of other females [Lang, 1987b]).

There thus seem to be growth advantagesin C. porosus having TSD rather than GSD,and they are consistent with the predic-tions of Charnov and Bull (1977), Head etal. (I987),]onnenetal. (1987), Smith (1987)and Webb et al. (1987a).

The allocation of "femaleness" at anincubation temperature of 33.0°C, whichproved to give both high survival (Fig. 9)and high post-hatching growth rates (Fig.10), is superficially inconsistent with thishypothesis. However, our observation maybe an artefact induced by constant-tem-perature incubation, where the "expected"increase in temperature does not occur(incubators were continually adjusted tocompensate for metabolic heat produc-tion). Metabolic heat production in wildnests increases nest temperature by 1-2CCduring development (Fig. 4), and the allo-cation of sex in a nest incubating at 33°C


TABLE 1. Mean hatchling size among 122 Crocodylusporosus hatchlings was largely unaffected by incubationtemperature. *

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egg weight for the sample (105.5 g). The increase inhatchling weight (hatchling + residual yolk) was sig-nificant, but reflects in part the weight of residualyolk. HL = head length, SVL = snout-vent length,weight = hatchling + residual yolk, % abundantyolk = the percentage of the sample which, on thebasis of abdomen appearance, had abundant yolk.

midway through development, must beconsistent with the potential fitness ofembryos coming from a nest at 34-35°C atthe end of development where survival iscompromised.

For the relationship between TSD andpost-hatching performance to be correct,the "non-sexual" effects of temperatureneed to be induced prior to or concurrentwith the allocation of sex; that is, duringthe first half of incubation (see also Bull[1987]). This is the period in which devel-opment rate is highly temperature depen-dent, and it is also the period in whichincubation temperature exerts a majorinfluence on total incubation time (Fig. 8).

In summary, hatchling fitness varies pro-foundly with incubation temperature,independent of sex, and quite probablyindependent of the sex determining mech-anism. TSD allows that variation to beexploited differently by the two sexes, giv-ing advantages that would be unavailablewith GSD. That these may be related tobody size is suggested by the largest reptiles(crocodilians and sea turtles) having exclu-sively TSD. No extant crocodilians havesex chromosomes (Cohen and Gans, 1970),and the group as a whole may be commit-ted to TSD (Ferguson, 1985); but this isnot the case in chelonians (Bull, 1983).Given that GSD is widespread among theinvertebrate and vertebrate ancestors of

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TEMPERATURE (°C)FIG. 9. The relationship between constant incuba-tion temperature in the laboratory and survival to 2yr of age for Crocodylus porosus hatchlings raised on acrocodile farm (hatching details are in Table 1). Theline is a polynomial, least squares regression.

reptiles, TSD would seem to be a derivedrather than a primitive trait in reptiles.

TEMPERATURE VERSUSMOISTURE EFFECTS

Successful development of any crocodil-ian embryo depends on there being an ade-quate moisture, temperature and gaseousenvironment for incubation. Many studiesemphasise the effects of the moisture envi-ronment on hatchling fitness (for examplesee: Packard et al. [1981], Gutzke et al.[1987]), whereas we have clearly empha-sised the temperature environment. Somebelieve that the moisture, temperature andgaseous environments act synergistically(Miller, 1985) such that no one parametercan be considered more important than anyother. As explained below, we considermoisture and temperature to play equiva-lent roles in setting the survival limits withinwhich most embryos will develop. How-ever, within those limits effects of the mois-ture environment appear minor relative tothose of the temperature environment.

With Crocodylus porosus in Australia andPapua New Guinea (Cox, 1985), desicca-tion of eggs is rare and flooding is common,


6 Or

50

40

30

6 6

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LU>

Z>ozC/)

< 40LU

30

11

24

I24

20

9

13

22

I I29 30 31

TEMPERATURE C°C)

32 33

FIG. 10. The relationship between constant incubation temperature in the laboratory and mean snout-ventlength at 1 yr (lower) and 2 yr (upper) of age for Crocodylus porosus hatchlings, from mixed clutches, raisedon a crocodile farm. Horizontal lines indicate 2 SEs on either side of the mean for males (open circles) andfemales (closed circles). Numbers are sample sizes.

but both these extremes of the moistureenvironment are lethal. Incubation tem-perature has similar lethal limits (Fig. 5).Gas exchange within nest mounds varieswith the structure of the mound (Lutz andDunbar-Cooper, 1984) and probably withthe frequency of rainfall and flooding

(Whitehead, 1987a, b). Critical limits occurin flooded nests, and they appear to occur

nests containing a high proportion ofinmud; there are a few data with which tofully evaluate them.

As selection can be expected to act rap-idly on females which consistently choose


nests with a temperature, moisture or gas-eous environment in which embryonic sur-vival is compromised, it seems likely thatall three parameters are intimately involvedin maintaining the nesting strategy (moundor hole) and delimiting the period of theyear in which nesting takes place. For manycrocodilians, the nesting period corre-sponds to the only time of the year duringwhich adequate moisture and temperatureconditions are synchronised, given the con-straints of hole or mound nesting. The rel-ative roles played by moisture and tem-perature in delimiting time of nestingprobably fluctuate greatly among differentspecies nesting in different habitats, in dif-ferent geographic regions.

Within these broad survival limits, incu-bation temperature exerts a profoundeffect on the rate at which embryos develop,which in turn influences the fitness and sexof crocodilian embryos (Webb et al., 1987a).Effects of the moisture (Gutzke et al., 1987)and gaseous environments (Ackerman,1980) appear to reflect the same underly-ing effects of development rate (Miller,1985; Webb et al., 1987a). Two lines ofevidence suggest that temperature can bevalidly considered the primary environ-mental variable affecting development rate,and thus fitness and sex.

Firstly, total incubation time is highlycorrelated with developmental rate and forexample, it can be used to predict sex ratioswith more precision than can be achievedfrom spot nest temperatures (Webb andSmith, 1984; Smith, 1987). Whitehead(1987a, b and unpublished data) quantifiedthe extent to which total incubation timewithin 17 wild Crocodylus johnstoni nests(69-82 days), could be explained by thetemperature, moisture and gaseous envi-ronments within those nests. Fifty-eightpercent of the variation in total incubationtime was attributable to nest temperature(range 27-35°C), whereas none could beexplained (regardless of transformations orvarying orders of entry into the analyses)by either the levels of oxygen in the nestthroughout incubation (PO2 range: 118-151 torr initially, dropping to 71-144 torrat hatching), nor the extent to which eggslost or gained water during incubation

5 8 r

5 54Oi

w o 52

UJ-1 50

4829 30 31 32

TEMPERATURE (°C)

33

FIG. 11. The relationship between constant incu-bation temperature in the laboratory and mean snout-vent length at 2 yr of age for male (open circles) andfemale (closed circles) Alligator mississippiensis. Dataare from Joanen et al. (1987), with total lengths con-verted to snout-vent lengths (measured to the backof the cloaca) with the formulae in Chabreck andJoanen (1979).

(range +2.3% to - 8 . 1 % of initial eggweight).

A further example, with turtle embryos,is provided by Gutzke et al. (1987). Eggswere incubated in wet (—150 kPa waterpotential), moist ( — 300 kPa) and dry(—1,100 kPa) substrates at three temper-atures (22, 27 and 32°C). Survival was com-promised in the dry substrate, but the rangeof temperatures tested was within survivallimits. Mean incubation time varied from42.8 days at 32°C to 98.6 days at 22°C, achange of 130% attributable to tempera-ture. In contrast, no effect of substratemoisture was demonstrated at 22°C and27°C, and only an 11% increase in incu-bation time occurred at 32°C (41.0 days inthe dry to 45.4 in the wet substrate). How-ever, even this was partly attributable toegg temperature, as egg temperaturesincreased in the dry substrates relative tothe wet ones, because of greater retentionof metabolic heat (Gutzke et al, 1987).

The second line of evidence is the degreeto which embryos are buffered from thevagaries of the nest environment. Growthand differentiation is the net result of asuite of biochemical reactions, all of whichare temperature dependent to varying


degrees (Romanoff, 1967). The egg shelland its contents provide little insulation,and as a consequence, nest temperaturesexert a profound effect at the tissue level.In contrast, the shell and shell membraneare partial barriers to water and gasexchange respectively (Whitehead, 1987a,b), and the embryos have homeostaticmechanisms which provide additional buff-ering. For example, up to 20% weight lossfrom Crocodylus johnstoni eggs (Manolis etal, 1987; Whitehead, 1987a, b) and at least11 % from Carettochelys insculpta eggs (Webbet al, 1986) have little effect on theembryos; the water loss is compensated forby exchanges of fluids within egg com-partments.

In summary, there are critical levels ofthe temperature, moisture and gaseousenvironments at which embryonic survivalis compromised. But within those limits,development rate and the effects of incu-bation environment on fitness and sexappear to be largely dependent on tem-perature. More subtle influences of themoisture environment exist, but they needto be demonstrated with rigid control oftemperature. It seems likely that the rela-tionship between fitness and moisturewithin survival limits is a plateau ratherthan a peaked normal distribution (Grigg,1987).

INCUBATION TEMPERATURE AND THEEVOLUTION OF THE REPTILE EGG

It seems reasonable to assume that theprovision of adequate incubation temper-atures has always been a constraint withwhich reptiles have had to contend, and itseems equally likely that incubation tem-perature effects, such as those described incrocodilians, are more widespread amongextant reptiles. However, we found thatour consideration of the role that incuba-tion temperature may have played in theevolution of reptilian modes of reproduc-tion, was confounded by anomalies in theclassical view of how reptilian oviparityevolved.

It has long been accepted that reptilesevolved from amphibian-like ancestors(Romer, 1970; Archer and Molnar, 1984)and that their success in the terrestrial

environment was in part attributable totheir refinement of oviparity: the devel-opment of the amniote egg which allowedreptiles to nest away from water (Packard,1966; Packard et al, 1977; Archer andMolnar, 1984). Viviparity among reptilesis usually viewed as being derived from ovi-parity, through increasing retention of eggsin the oviduct, in many cases a response toadverse thermal conditions (see review byShine, 1985). The general picture is thatoviparity allowed reptiles to overcome theconstraints of the moisture environment,and later, viviparity the constraints of thethermal environment.

Egg-retention in squamates is really a sub-category of oviparity, regardless of theexisting levels of placentation (Packard etal, 1977;Yaron, 1985). It is fundamentallya period in which "shell-less" amniote eggsdevelop in the oviducts, for varying periods.Squamates can also be considered to havefacultative oviparity; most species retain eggsin the oviducts for half the total period ofembryonic development, and a fifth of allspecies retain eggs until development iscomplete (Shine, 1985). The extent ofretention is labile within the group, andselection has increased and decreased itfrequently and independently, often inresponse to cold and hot climates respec-tively (Shine, 1985). If post-hatchinggrowth of squamates, like that of croco-dilians, is affected by incubation tempera-ture, then it is likely that the effects areinduced during the first half of develop-ment, when egg retention allows femalesto exert some control over the thermalenvironment.

In contrast to facultative oviparity, che-lonians, rhynchocephalians and crocodil-ians have obligate oviparity. Birds, whichhave an almost identical egg and share acommon ancestry with reptiles, also haveobligate oviparity. Eggs are laid whenembryos are at an early stage of develop-ment (well before the allantois is formed),and that stage is remarkably consistentwithin any particular group. Most impor-tant, obligate oviparous reptiles and birdsare a diverse assemblage of different sizedaquatic, terrestrial and aerial vertebrates,existing in a wide range of climates. All the


factors which have been hypothesised asleading to viviparity mfacultative oviparousreptiles (Shine, 1985), should apply to obli-gate oviparous reptiles and birds—yet innot one species is there significant reten-tion of eggs. For example, Sphenodon punc-tatus exists in an intensely cold region, andhas a 13-15 month total incubation period;yet egg-laying still occurs when embryosare at a gastrula stage (MofFat, 1985). Pen-guins are obligate oviparous birds whichincubate eggs in an intensely cold environ-ment. They would not have their "flight"compromised by retaining eggs, and if theywere facultative oviparous reptiles, theywould be prime candidates for "egg-reten-tion" and viviparity under current theory(Shine, 1985); yet no such developmentsseem to have taken place (Blackburn andEvans, 1986).

If it is assumed that selection has at sometime tried to move obligate oviparous rep-tiles and birds in the direction of egg reten-tion, one can only conclude that it has beena consistent and total failure. Notwith-standing the fact that this could beexplained in terms of the many costs andbenefits of retaining eggs (Blackburn andEvans, 1986), the most plausible explana-tion is that it is a lethal pathway; each timeit has been attempted, embryonic survivalhas been compromised. If so, obligate ovi-parity of the form seen in reptiles and birdstoday could never have given rise to facul-tative oviparity nor to viviparity.

The possibility that facultative oviparityis the primitive condition among reptilesdoes not appear to have been considered(Shine, 1985) until now. The first reptiles,the captorhinomorphs, were small terres-trial animals with a body form, posture andniche remarkably similar to today's lizards(Archer and Molnar, 1984). Based on heartstructure, crocodiles and birds evolvedfrom an animal similar to small extant liz-ards (Webb, 1979). We suggest that thecaptorhinomorphs also mirrored extantlizards in having facultative oviparity, andthat the varying levels of placentation seenin squamates today (Yaron, 1985) have along antiquity.

The suggestion raises the question ofwhether the extraembryonic membranes

of amniote embryos evolved in an oviductor an external egg. An oviducal origin hasnot been considered seriously (Szarski,1968), yet it is the most plausible expla-nation. Survival in an external reptilian eggis dependent on the extraembryonic mem-branes being in place, so it is likely thatthey evolved first (Szarski, 1968). The"shell-less" eggs of squamates may in factreflect the original amniote egg with theoriginal disposition of extraembryonicmembranes: the yolk sac surrounding thenon-cellular food supply; the allantois stor-ing waste products and facilitating gasexchange; the amnion enclosing theembryo within a fluid-filled sac and buff-ering it from mechanical shock; and thechorion, which completely surrounds theemb yo and yolk sac; thereby, containingin a single egg unit a series of separatechambers with different densities (Webb etal., 19876, c).

Facultative oviparity also seems to be anideal reproductive system within which torefine reptilian oviparity. It would allowthe amniote embryo to be modified in anenvironment controlled by the female,while simultaneously (but independently),selection could pursue the oviparous pack-aging for life outside the oviducts.

Obligate oviparity is best explained asbeing derived from facultative oviparity,specifically to meet the needs of voidingamniote embryos at an early stage of devel-opment. But it required a number ofembryological specialisations. For exam-ple, abundant albumen was needed for theimmediate supplies of subembryonic fluid(to store excretory wastes) and to facilitateyolk rotation (Webb et al. 19876, c). Thetiming of egg-laying with respect to embryoattachment to the shell membrane neededto be synchronised precisely. If attachmentoccurs before laying, embryos are unableto rotate to the uppermost part of the egg,and in large eggs they die. In the absenceof an allantois, their position at the top isneeded for gas exchange and access tosubembryonic fluid (Webb et al, 19876, c).These and a suite of other specialisationscould have evolved gradually in ̂ facultativeoviparous system. However, once inte-grated into an obligate oviparous system,


even minor deviations in the direction ofincreased egg retention in the oviductscould prove fatal.

A variety of costs associated with femalesretaining eggs (Shine, 1985; Blackburn andEvans, 1986) may have stimulated selectionfor obligate oviparity, but there were almostcertainly significant advantages obtainedby freeing development from the female.Selection for increased egg size, hatchlingsize, adult size and perhaps even body formare likely ones; the ability to combine largeclutch sizes, with large embryos and free-swimming hatchlings, and perhaps orna-mented hatchlings. Even in facultative ovip-arous reptiles, embryos must be main-tained above the yolk, which could wellpreclude having large clutches of large eggsunless they are aligned longitudinally inthe oviducts (such as in snakes).

If obligate oviparity was derived in thefashion suggested, then adaptations of theexternal egg for water (the shell) and gasexchange (the shell membrane) would havebeen achieved before the embryonic stageat laying was reduced. A significant prob-lem remaining may have been the expo-sure of embryos to the vagaries of theexternal thermal environment during theperiod when they are most sensitive to theeffects of incubation temperature. Thiscould be a basic cost of obligate oviparity,outweighed by advantages accrued fromother sources. That obligate oviparity is amechanism for avoiding high maternalbody temperatures (Anderson et al., 1987)may apply to birds, but is not a compellingargument for reptiles. However, if post-hatching growth is enhanced by fluctuat-ing incubation temperatures (relative tostable ones), which is suggested if Garnettand Murray's (1986) data on C. porosusgrowth are re-analysed with respect to theage at which eggs were collected, there mayin fact be advantages in large, thermally-stable, aquatic reptiles voiding their eggsinto a more labile thermal environment.

In summary, we believe the amniote eggevolved in the oviducts of a reptilian ances-tor, rather than in the external egg. Itevolved in an environment in which theeffects of incubation temperature could bemoderated by a female's behaviour. This

led to facultative oviparity in the first rep-tiles, where again temperature effects werecontrolled by the female. Obligate oviparityarose as a specialised development toreduce greatly the time eggs were retainedin females. Incubation temperature effectsbecame more dependent on the externalambient environment, although this wasnot necessarily a disadvantage.

The scenario would see the potential forviviparity being present in the earliest rep-tiles, which is consistent with some ich-thyosaurs being viviparous (Romer, 1945).It would also suggest that primitive stagesin the development of amniote eggs arelikely to be found in amphibians with vary-ing degrees of internal development ofembryos, rather than in those with speci-alised external eggs alone.

ACKNOWLEDGMENTS

This paper reports research results fromstudies that were primarily funded by theConservation Commission of the NorthernTerritory. Their continuing support forcrocodile research is very gratefullyacknowledged. Additional funding camefrom the University of New South Wales,and the Australian Research GrantsScheme (Grant No. D1/8316049). Wewould particularly like to thank CharlieManolis for assisting with every aspect ofthe paper, and Anthony Smith, PeterWhitehead and Jonathan Hutton for per-mission to quote data from their theses.Many people have helped with the salt-water crocodile nesting study, and I thankthem all. Drafts of the manuscript wereread and criticised by Jim Bull, Bill Free-land, Robert Jenkins, Rodney Kennett, ValLance, Jeff Lang, Charlie Manolis, RickShine, Anthony Smith and Sonia Tide-mann. Their comments did much to shapethe paper, and form the ideas currentlyexpressed in it, although full credit for anyof its remaining failures belongs clearly tous.

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