chemical composition of egg shell

7/23/2019 chemical composition of egg shell

1/35

Biol

Rm (1977).

52. p p

71-105

BRCPAH 52-3

7'

THE PHYSIOLOGICAL ECOLOGY

OF

REPTILIAN

EGGS AND EMBRYOS. AND THE EVOLUTION OF

VIVIPARITY WITHIN THE CLASS REPTILIA

BY GARY C

.

PACKARD. C RICHARD TRACY AND JAN J R O T H

Department of Zoology and Entomology. Colorado State University.

Fort Collins.Colorado 80523.

U S A

(Received

27

July

1976)

CONTENTS

PAGE

I

ntroduction

. . . . . . . . . .

1

I1

Morphology of reptilian eggs

. . . . . . .

2

I11 Water relations of reptilian eggs *

75

(I)

Uptake of water from soil . . . . . . .

5

a)

Parchment-shelledeggs 75

(b) Calcareouseggs 7

. . . .

. . . . . .

2)

Mechanism ofwater transport into eggs . . . . 8

(3)

Adaptive significance ofwater uptake . . . . .

1

82

V Sources of minerals for embryos

. . . . . . .

VI

.

Exchangeofgases

. . . . . . . . .

6

(I) Th e atmosphere adjacent to eggs

. . . . . .

6

2) Themechanismof gasexchange . . . . . .

6

VII Temperature and metabolism 8

(I) Effects of temperature

. . . . . . . .

8

. . . . . .

Patterns of nitrogen excretion

83

. . . . . .

2) Metabolism of embryos sg

VI II Egg retention and the evolution of viviparity

.

(I) Distribution of the live-bearing method

. .

2)

A model for the evolution of viviparity

(3) Adaptive value

of

egg retention

a)

Temperature benefits

(b) Water for embryonic development

c )

Food for emergent young

(d) Protection of developing embryos

. . . .

. . . .

. . .

(e)

Arboreal and aquatic habits of adults

.

(4) Evolution of placentation . . . . .

5 )

Why are there no viviparous turtles or crocodilians?

. .

go

90

. .

92

.

94

94

. 96

* 97

. .

98

98

99

99

IXSummary

. . . . . . . . . . .

9

X Acknowledgements . . . . . . . . . 00

XI References

. . . . . . . . . . . 01

I. INTRODUCTION

Natural selection presumably operates on all stages in the life-cycle of an animal. and

so embryos. as well as adults. can be expected to exhibit adaptations to the environ-

ments in which they occur

.

Moreover. the success of a natural population often


2/35

72

G.

C. PACKARD,

R.

TRACYND J.

J.

ROTH

Table I. HZgher

levels of classifiation of surviving

members

of the

cl ss Reptilia

Subclass Anapsida

Subclass Lepidosauria

Order Chelonia- urtles, terrapins, and tortoises

Order Rhynchocephalia

-

he tuatara, Sphenodm

punctatus

Order Squamata- izards, snakes, and amphisbaenians

Order Crocodilia- alligators, crocodiles, and gavials

Subclass Archosauria

depends as much upon adaptations of embryonic stages as upon adaptations of adults,

and thus a consideration of the problems of embryonic existence is an essential com-

ponent of any full account of the ecology of an animal.

Our objective in preparing the present review of the physiological ecology of

reptilian eggs and embryos is to seek a greater understanding of the adaptations of

these embryos (and therefore of reptiles generally) to their physical environments.

Unfortunately, the literature on this subject is relatively sparse, many published

accounts on the biology of reptilian embryos are anecdotal, and reported studies

frequently have suffered from the use of inappropriate experimental designs and/or

inadequate analytical procedures. Nevertheless, we shall draw some conclusions from

the available data, and shall also identify several testable hypotheses for future

research.

11.

MORPHOLOGY

OF

REPTILIAN

EGGS

Oviparity presumably represents the ancestral mode of reproduction in the Class

Reptilia (Table I), for even the embryos of viviparous snakes and lizards possess an

egg-tooth, thereby reflecting their derivation from oviparous ancestors (Bellairs, I

970).

Oviparous reproduction characterizes the tuatara, all living Crocodilia and Chelonia,

and most species of the Squamata (Bellairs, 1959, 970).Nevertheless, a substantial

number of lizards, snakes, and amphisbaenians have assumed a live-bearing habit

(Bellairs,

1959),

nd numerous other species of the Squamata exhibit modes of repro-

duction that may represent intermediate stages in the evolutionary transition from

oviparity to viviparity.

The amniotic eggs of reptiles have long been regarded as similar to those of birds

(Bellairs, I

970).

Unfortunately, this generalization may have obscured important

biological differences between the eggs of Crocodilia and Chelonia on the one hand,

and those of Lepidosauria on the other. These differences pertain to the eggshell, the

egg (or shell) membranes, and the albumen layer.

Eggs of Crocodilia are virtually identical to those

of

birds (Fig. I). The calcareous

eggshell is thick and hard (Voeltzkow, 1892; eese, 1915 Bigalke, 1931 Guggisberg,

1972;

ingletary & Ogden,

1973),

nd is comprised of crystals of calcite (Erben,

1970;

Jenkins,

1975)

etween which there exists

a

matrix of proteinaceous fibres (Erben,

1970;

enkins,

1975).

The crocodilian eggshell is penetrated by numerous pores

(Reese,

1915;

igalke,

1931

Erben,

1970)

hat apparently allow respiratory gases to


3/35

Physiological ecology of reptilian

embryos

73

AVES

membrane

CHELONIA AND

CROCODlLlA

Blastodisc

Vitelline

mem brane

membrane

LEPIDOSAURIA

Blastodisc

Albumen

Shell

Vitelline

membrane

Fig.

I.

Schematic diagrams illustrating the major morphologic features of the eggs of Aves

(upper), Crocodilia and Chelonia (middle), and Lepidosauria (lower). Although a space

is

shown

here between the tertiary membranes of avian, crocodilian, and chelonian eggs for clarity of

illustration, the egg membranes of avian eggs are actually adherent except in the region of the

air space, and homologous membranes

in

crocodilian and chelonian eggs are adherent through-

out. See teXt for references.

diffuse between the enclosed embryo and the nest environment (see SectionVI, 2).

A

pair of tertiary egg membranes separates the eggshell from a thick layer of albumen

(Clarke,

1891;

Voeltzkow,

1892;

Reese,

1915;

Bigalke,

1931;

Erben,

1970;

Jenkins,

1975).Bigalke(193I )reported that an air space forms between the outer egg membrane

and the eggshell during developmentof embryonic crocodiles(Crocodylusniloticzrs),but

Reese

(1915)

and McIlhenny

(1935)

contended that there is no air space in eggs of

alligators (Alligator mississippiensis)at any time during development. Since eggs of

crocodilians seem to absorb water and to swell during the course

of

natural incubation

(McIlhenny,

1934, 1935

;Guggisberg,

1972),

thereby precluding formation of an air

space (Romijn

&

ROOS,

938),

we suspect that the eggs examined by Bigalke

(1931)


4/35

74

G. C.

PACKARD,

.

R.

TRACY

ND J. J.

ROTH

were not developing normally at the time of study, and therefore predict that an air

space does not usually form in eggs of crocodilians.

The eggshell is also well developed in eggs of Chelonia (Fig.

I ) ,

but varies from

being hard and brittle, as in tortoises and some fresh-water turtles (e.g. Lynn & von

Brand,

1945;

Young,

1950),

to being soft and pliable (parchment-like) as in other

species (e.g. Cunningham, 1922; Tomita, 1929; Lynn & von Brand, 1945; Legler,

1960; Bustard, 1972). The calcium salts of the eggshell are in the form of aragonite

crystals (Young,

1950;

Erben,

1970),

between which there is a matrix of proteinaceous

fibres (Simkiss & Tyler, 1959; Erben, 1970). The outer surface of the shell of tortoise

(Testudo graeca)

and soft-shelled turtle

(Trionyx

euphraticus) eggs seems to be covered

with a thin cuticle similar to that which occurs on the eggs of many birds (Young,

1950;

Simkiss

&

Tyler,

1959),

but a cuticle probably does not occur on eggshells of

most Chelonia (Giersberg, 1922; Simkiss & Tyler, 1959). Both calcareous and parch-

ment-like eggshells are penetrated by a large number of pores that presumably provide

for a diffusive exchange of respiratory gases (Young,

1950;

Erben,

1970).

Two ter-

tiary egg membranes lie between the thick layer of albumen and the eggshell (Young,

1950; Simkiss, 1962;Erben, 1970; Ackerman & Prange, 1972). An air space has been

observed in the hard, calcareous eggs of the tortoise, Testudograeca (Young, 1950),but

the eggs of most Chelonia seem to absorb water and to swell during normal incuba-

tion (see p. 75), thereby precluding formation of an air space (Romijn & ROOS, 938).

Thus, an air space probably is not of general occurrence in the eggs of Chelonia.

Eggs of oviparous LepidosauriaFig.

I ) (usually have a flexible, parchment-like shell

containing relatively small amounts of calcium carbonate (Dendy, 18993; Bellairs,

I

959, 1970).

However, gekkonid lizards of the subfamilies Gekkoninae and Sphaero-

dactylinae characteristically produce eggs with relatively hard, calcareous shells

(Bustard, 1968a), as do lizards of the family Dibamidae (Boulenger, 1912).

The parchment-like shell of lepidosaurian eggs seems to be formed from a single

shell membrane comprised of approximately five layers of minute fibres (Giersberg,

1922;

Jacobi,

1936;

Tracy, Roth

&

Packard, unpublished), with crystals of calcium

carbonate deposited between fibres in outer layers of this membrane (Giersberg,

1922 ;

Harris,

1964).

Pores, such as exist in the shells of chelonian and crocodilian eggs, seem

not to occur in eggshells of Lepidosauria (Tracy, Roth

&

Packard, unpublished).

No other membranes seem to be present on the inner aspect of the shell of lepido-

saurianeggs(Dendy,

1899b;

Giersberg,

1922;

Weekes,

1935;

Fisk &Tribe ,

1949).

The

albumen layer in eggs of these reptiles is exceedingly small at the time of oviposition

(Dendy,

1899b;

Giersberg,

1922;

Jacobi,

1936;

Clark,

1946;

Fisk & Tribe,

1949;

Harris,

1964;

Badham,

1971),

but seems to increase in volume during the course of

normal incubation owing to the absorption and storage of water (see Section 111,

I ) .

There is no air space in parchment-shelled eggs of Lepidosauria at any time in

development (Dendy, 1899b; Harris, 1964).

Calcium carbonate is not deposited in the shell of eggs produced by live-bearing

lizards and snakes (Weekes, 1935; Bellairs, 1959; Hoffman, 1968), but the shell

membrane usually persists albeit in varying degrees of evolutionary reduction

(Giersberg,

1922;

Jacobi,

1936).

Presence of the shell membrane frequently has been


5/35

Physiological ecology of reptilian embryos

75

used as the criterion to distinguish ovoviviparity from true viviparity (Hoffman,

1968, 1970),

but other criteria (e.g. mass of yolk present in the egg,

or

role of maternal

secretions in nourishing developing young) have occasionally been used to characterize

these modes of reproduction (Bellairs,

1959, 1970;

Salthe Mecham,

1974).

How-

ever, intermediate stages can be found between ovoviviparity and true viviparity

by any of the commonly applied sets of criteria, and so any attempt to distinguish

between these patterns of reproduction inevitably tends to obscure details that are

important in considerations of the evolution of the live-bearing habit. Therefore, we

prefer to use the term viviparous to describe any reptile giving birth to free-living

young.

Many viviparous lizards and snakes exhibit rather complex forms

of

placentation

(Weekes, 1935;Bauchot, 1965; Hoffman, 1968), but in most of these species the large

mass

of

yolk provided by the female parent at ovulation is the primary (or only) source

of nourishment for the developing embryo (Weekes,

1935).

No

species of viviparous

reptile is known to produce alecithal eggs such as occur in mammals.

111. WATER RELATIONS OF REPTILIAN EGGS

(I) Uptake

of

water

from

soil

(a) Parchment-shelled eggs

Brimley

(1903)

and Dendy

( 1 8 9 9 ~ )

ere among the first to observe that parchment-

shelled eggs of Lepidosauria and Chelonia swell during the course of incubation, and

observations confirming this phenomenon have been made repeatedly since their early

work (e.g. Giersberg,

1922;

Lynn

&

von Brand,

1945;

Shaw,

1952).

The swelling

is

attributable to absorption of water

by

eggs from the substrate upon which they rest

(Giersberg, 1922; Cunningham & Hurwitz, 1936; Cunningham, Woodward &

Pridgen,

1939;

Lynn & von Brand,

1945;

Clark,

1 9 4 6 , 1 9 5 3 ~ ;

Mulherkar,

1962;

E r s t , 1971).With few exceptions, however, these studies have been performed in the

laboratory on substrates of unknown water potential (see p. 78), and so there is some

question

as

to whether or not the conditions of experimentation simulated conditions

found in natural nests. For this reason, it seems that generalizations concerning water

absorption by parchment-shelled reptilian eggs may be somewhat premature (e.g.

Bellairs,

1959, 1970).

Few observations seem to have been made

of

parchment-shelled eggs incubating in

natural nests. Eggs of the sea turtle,

Chelonia mydas,

apparently absorb water and swell

during the first 24 h after oviposition (Bustard, 1972), but it is not clear whether the

eggs continue to imbibe water during the remainder of incubation, particularly since

most of the eggs are not in actual contact with the moist sand forming the walls of the

nest cavity (Hendrickson, 1958).Also, Whitaker (1968) indicated that eggsof the lizard,

Leiolopisma suteri, increase in volume during their natural incubation, so that the eggs

become firmly wedged inside the incubation chamber ;presumably, such swelling sterns

from absorption of water.

Eggs of oviparous reptiles seldom are deposited in situations where soil comes into

contact with all surfaces of the eggs; and even in those species using a subterranean


6/35

76

G.

C.

PACKARD,

.

R.

TRACYND J. J.

ROTH

chamber for oviposition, the volume of the nest cavity greatly exceeds that of the

contained eggs (Allard, 1935;McIlhenny, 1935;Cagle, 1950; Brown, 1956; Hendrick-

son, 1958; Muth, 1976). Since viable eggs lose water continuously, even when

incubated in an atmosphere saturated with water vapour (Clark,

1946,

1 9 5 3 ~ ; itch

& Fitch,

1967)~

ater must evaporate constantly from the surfaces of eggs into the nest

atmosphere. It may seem anomalous that water can be lost from reptilian eggs to a

saturated atmosphere, but the probable explanation of this phenomenon is relatively

simple (Adolph, 1932). Metabolic heat produced by the embryo developing within an

egg raises the temperature of the egg and eggshell slightly above that of the surround-

ing mass of air in the nest cavity. Thus, the vapour pressure of water in the eggshell

is

greater than that of the surrounding air, and this difference in vapour pressure drives

the evaporation of water from the egg surface to the atmosphere (see Monteith, 1973

:

chapter 9).

Eggs taken from natural nests seldom show signs of desiccation (i.e. shrunken or

flaccid appearance, dented or wrinkled surface). Nevertheless, the eggs must have

been losing water continuously to the nest atmosphere, and

so

the only possible expla-

nation for their generally turgid state is that they were absorbing water from the sub-

strate (Clark, 1946, 1953u . In so far as the rate of water absorption exceeded the rate

of water loss by evaporation, the eggs swelled; but even eggs exhibiting no change in

weight and/or volume during development must have absorbed significant quantities

of water from the substrate to compensate for evaporative water loss from the exposed

surfaces. This line of reasoning, while admittedly indirect, constitutes the strongest

evidence that parchment-shelled eggs absorb water during the course of natural

incubation.

In some laboratory studies, parchment-shelled eggs have been reported to increase

in size at rates which remain constant throughout development (e.g. Reynolds, 1959;

Pandha & Thapliyal, 1967; Ernst, 1971; Dixon, Staton & Hendricks, 1975), whereas

in other instances the rates of change in size have been claimed to increase (Cun-

ningham

&

Huene,

1938

;Harris,

1964;

Garg, Pandha

&

Thapliyal,

1967)

or to decrease

(Blanchard, 1927;Lynn & von Brand, 1945

;

Mulherkar, 1962; Dmiel, 1967; Badham,

1971

;Subba Rao & Rajabai,

1972)

during the course of incubation. Additionally, eggs

of

several species of Chelonia and Squamata have been observed to exhibit a sudden

decline in weight and/or dimensions in the days immediately prior to hatching (Blan-

chard,

1927;

Cunningham

&

Hurwitz,

1936;

Cunningham

&

Huene,

1938;

Reynolds,

1959;

Gordon,

1960;

Bustard

&

Greenham,

1968;

Whitaker,

1968;

Dixon

et

al.,

1975),

whereas eggs of other species have been observed to swell appreciably just before

hatching (Harris, 1964). Unfortunately, it is difficult to attach much significance to

these several observations, for information has not been reported on the water poten-

tial of substrates in natural nests or of those upon which eggs were incubated in the

laboratory. Thus, we cannot conclude that conditions of laboratory study simulated

conditions occurring in nature.


7/35


77

(b ) Calcareous eggs

Even the hard, calcareous eggs produced by certain reptiles may exhibit a net

uptake of water during normal incubation (McIlhenny,

1935

;Guggisberg,

1972).

The

most convincing evidence on this point was gathered by McIlhenny

(1934) ,

who

periodically opened a natural nest of the alligator,

Alligator mississ@piensis,

to examine

eggs developing therein. McIlhenny noted cracks in eggs when he opened the nest 6

weeks after oviposition, and the cracks had enlarged and widened when the nest was

again visited weeks later. Thus, the eggs under observation apparently were swelling

during incubation, presumably owing to a net uptake of water from the moist walls of

the nest chamber. Calcareous eggs experiencing a net increase in water content prob-

ably would not have an air space (Romijn & ROOS,

938),

and so the reports that no air

space forms in alligator eggs can be regarded as further evidence that a net uptake

of water normally occurs in eggs of Crocodilia (Reese, 1 9 1 5 ;McIlhenny, 1935).

Bustard(1971 ) recorded an increase n weight of crocodile

Crocodylusnovaeguineae)

eggs incubated on a moist substrate, but seemingly normal young also hatched from

two eggs that experienced a net water loss during the course of development.

Unfortunately, it is difficult to draw conclusions from these observations, because we

cannot determine which of the sets of incubation conditions most closely approached

those to which eggs are exposed in natural nests; and the number of eggs used was

extraordinarily small in any case.

Evidence concerning the uptake

of

water by the calcareous eggs of certain Chelonia

is equivocal. Eggs of a soft-shelled turtle

(Trio nyx triunguis)

experienced a

10

in-

crease in weight when incubated on a moist substrate (Mendelssohn, cited by Dmiel,

1967),

but calcareous eggs of the turtle,

Kinosternm subrubrum,

seemed not to absorb

water when incubated under superficially similar conditions (Lynn & von Brand,

1945).

Information on water potentials

of

egg contents and of substrates was not

presented in either of the preceding investigations, and so we are unable to draw con-

clusions from these studies concerning the water metabolism of turtle embryos

developing within calcareous eggs. The observation by Young

(1950)

of an air space

in eggs of the tortoise, Testudo graeca, indicates that the eggs experienced a net loss

of water after oviposition (Romijn &

ROOS, 938),

but the eggs probably were not

developing normally at the time they were examined (Young,

1950).

Calcareous eggs of gekkoninine and sphaerodactylininegeckos are especially interest-

ing with regard to their water relations, for these eggs have been described as being

cleidoic (Bustard,

1968b).

Since a cleidoic egg experiences a mass exchange only of

gases with its environment, there is, by definition, no absorption of water from the

substrate. Therefore, water lost from the surfaces of such an egg cannot be replaced,

and must be drawn from water reserves present in the egg

at

the time of oviposition.

We would be interested to learn whether the calcareous eggs of geckos have

a

large

*

The term cleidoic, which is not equivalent to amniotic, seems to have been used first by

Needham, in 1931, in describing the avian egg: Th e avian egg is thus, in the strictest sense of the

word, a closed box,

with

walls which can only be penetrated by matter in the gaseous state. The term

cleidoic

..

s suggested for this state of

affairs...

(Needham,

1963:

p.

1615 .


8/35

78

G. C

PACKARD, R.TRACYND .

J.

ROTH

volume of albumen, in contrast to eggs of other squamate reptiles, or whether water

reserves are sequestered in other compartments of the eggs.

2 )

Mechanism

of

water transport into eggs

Movement of water from the substrate into an egg must occur by diffusion of

liquid water through the eggshell from a region of high water potential (the sub-

strate) to a region of low water potential (the contents of the egg). Two factors deter-

mine the water potential of an egg:

(i)

the pressure potential of the contained liquids,

and (ii) the osmotic potential of these same liquids (Salisbury & Ross, 1969; see also

Rose, 1966). For our purposes, pressure potential can be regarded as being equivalent

to turgor pressure of the egg, and will generally have a positive value. Osmotic poten-

tial, on the other hand, is the component of water potential that becomes increasingly

negative as embryonic liquids become more concentrated with solute. For an egg to

absorb water, the potential of water in the substrate must exceed the algebraic sum

of the pressure potential and the osmotic potential of the egg contents.

The rate of water movement into an egg is directly related to (i) the difference be-

tween the water potential of the substrate and that of the egg contents, (ii) the surface

area and hydraulic conductance of the egg, and (iii) the hydraulic conductivity of the

material comprising the substrate upon which the egg rests (Tracy, 1976). These

several factors affecting water transport into reptilian eggs can be expressed formally by

the following equation:

me

= (Yf3Oll - Ye g g )

I I

whereme= water uptake by the egg (g.min-l), Ysoil

Y e g g )

the difference in water

potentials of the egg and the substrate upon which it rests (bars),

A

= the surface

area of the egg in contact with the substrate (cm2),K = the hydraulic conductance of

the egg (g.cm-2. min-l .bar-l), and ksoil= the hydraulic conductivity of the substrate

(g.cmW2. in-l ar-l m-l). The term A s e epresents the total hydraulic conduc-

tance

of

the egg, whereas the companion term ~ ? T A , ) ~ ~ ~ A ~ , , ~ ~epresents the total

hydraulic conductance of the soil (Tracy, 1976).

Examination

of

this equation reveals several interesting properties of water transport

into reptilian eggs. If an egg has the ability to take up water faster than the soil can

supply it, the egg can

cause

the soil to become drier with time, and the rate of water

uptake by the egg will decline owing

to

the reduced ability of dry soils

to

conduct

water to the egg surface (Gardner, 1965). On the other hand, if both the egg and the

soil conduct water at high rates, the egg will tend

to

take up water until its water

potential approximates that of the soil. Thus, the complex array

of

determinants of

water transport into reptilian eggs provides many ways for different species to adapt

to

different environmental conditions (e.g. by selecting nest sites in different types of

soil or in soils with different water tables, by changing the conductance or geometry

of the eggshell, etc.). Therefore, we believe that a biophysical approach to the study of

water relations of reptilian eggs promises to yield important biological insights that

were not forthcoming from earlier work.


9/35


79

Conversely, consideration of the physical determinants of water transport reveals the

difficulty of designing and interpreting experiments concerned with the water rela-

tions of reptilian eggs. Clearly, a number of factors influencing water transport may

vary simultaneously, thereby complicating studies of water dynamics. Furthermore,

eggs of most reptiles apparently lose water by evaporation while simultaneously

absorbing liquid water from the substrate (p.

76),

and

so

the ratio of egg surface

exposed to the atmosphere relative to the surface in contact with the substrate may

substantially influence the equilibrium weight of the egg or its viability in different

experimental settings. These problems make it very difficult to interpret the findings

of the many laboratory studies that have been reported in the literature.

Developing reptilian embryos apparently influence the uptake of water from the

substrate (Giersberg,

1922).

For instance, eggs of the lizard,

Agama ugama,

that have

been killed by exposure to strong sunlight fail to swell when placed in contact with

liquid water, whereas viable eggs swell in the expected manner (Harris,

1964).

Additionally, parchment-shelled eggs containing dead embryos frequently can be

identified by a loss of turgidity (Mayhew,

1963;

Ortleb,

1964;

Bustard & Greenham,

1968).

Although Legler

(1960)

reported that infertile eggs of the turtle, Tewapene

urnata, may swell for as much as 2 weeks before beginning to shrivel, it is possible

that the eggs he observed contained embryos which died after completing only 14

days of development.

There is some evidence that water absorption by eggs is related to the temperature

at which they are incubated. Eggs of hognose snakes (Heterodon platyrhinos and

Heterodon

nasicus) incubated at 26.7 C absorb water much more rapidly than eggs

held at

23-3

C (Platt,

1969),

and similar observations have been made on eggs of the

snake, Carphophis vermis, incubated at different temperatures (Clark, D. R.,

1970).

Although the rate of swelling may therefore be related to the rate of embryonic meta-

bolism (Section VII,

I),

these findings do not provide indisputable support for the

idea that embryos influence the uptake of water from the environment. For instance,

an increase in temperature would cause hydraulic conductivity of the substrate also

to increase (Gardner,

1968),

and this factor alone could cause water uptake to proceed

more rapidly.

Fluctuations in the hydric environment to which eggs are exposed may also have an

important influence upon the overall uptake of water during incubation. Eggs of the

colubrid snakes,

Elaphe guttata

and

Spalerosophis cliSJordi,

absorb more water when

exposed alternately to moist and to dry substrates than when they are held continu-

ously on a moist substrate (Clark, I953a; Dmi'el, 1967)' again raising the possibility

that embryos exercise some form of control over the uptake of water from the environ-

ment. However, a complete biophysical analysis of water fluxes is required before

other, purely physical, explanations of this phenomenon can safely be ruled out.

Much of the water absorbed by eggs of lizards and snakes is incorporated into the

albumen fraction (Clark,

1953b;

Badham,

1971).

The volume of the albumen in-

creases progressively during incubation (Fig. 2 , and

at

the time of hatching the

albumen may comprise nearly 1 5 per cent of the mass of the entire egg (Clark, D. R.,

1970).

For water to diffuse through the eggshell and into the albumen, the water


10/35

80

10 -

9 -

8 -

-

7 -

2 6 -

J

W

G.

C .

PACKARD,

. R.TRACYND J. J.

ROTH

I I I

I

0 10

20 30

40 50

60 70

Age days)

Fig. 2. Volume of albumen in eggs of the oviparous snake,Colder constrictor, at different times

after oviposition. Data are from Clark (19536).

0

+

1

I I I I ,

I

I

0

10

20 30

40

50

60 70

Age days)

Fig. 3 . Cumulative plots of nitrogen excreted as ammonia(A) , urea

0)

nd uric acid (+) by

embryos of the oviparous snake,

Colder

constrictor, at different stages of development. Data are

from Clark (1953b).

potential of the albumen must be lower than that of water in the substrate. Packard

(1966)

suggested that the urea, which is the primary nitrogenous waste produced by

embryos of the oviparous snake, Coluber constrictor, and which is sequestered mainly

in the albumen fraction of eggs (Clark, 1953b), may play an important role in water

absorption. Since metabolism

of

developing embryos is a continuous source of urea in

this species (Fig. 3), the water potential of the albumen could be held relatively low

throughout incubation (Clark,

1953

b) , and a continuous uptake of water might there-

fore result

(Fig. 2 .

However, Badham (1971) recently presented evidence that un-

hydrated proteins are added to the albumen of developing eggs of the lizard, Amphi-

bolurus

burbatus,

thereby indicating that important interspecific differences may exist


11/35

Physiological ecology

of

reptilian embryos

81

in the means by which embryos of the Squamata influence the water potential of their

egg contents.

As a parchment-shelled egg swells during incubation, it becomes more and more

turgid (Dendy, 1899a; Cunningham & Huene, 1938; Reynolds, 1959; Clark, D. R.,

I

970), suggesting that pressure potential of the embryonic liquids increases through-

out development (Giersberg,

1922);

and such an increase in hydrostatic pressure

would tend to oppose movement of water into the egg. Simultaneously, however, the

surface area of the eggshell would increase, and the thinning of the eggshell (Jacobi,

1936; Platt, 1969) could lead to slight increases in the conductance of the egg to

water. While these factors doubtless have an influence on water transport into in-

cubating eggs, the major determinants of water movement are likely to be the osmotic

potentials of soil water and egg contents, and the hydraulic conductance of the sub-

strate (Tracy, 1976).

(3) Adaptive szjpificance of water uptake

In most instances, failure of an egg to swell during incubation should not be taken

to indicate that the egg was not absorbing water, as some workers have supposed

(Dmiel,

1967),

but only that an equilibrium was reached between water uptake and

water loss. On the other hand, swelling of an egg indicates that water was absorbed in

excess of that required to replace water lost by evaporation.

There is considerable disagreement concerning the possible adaptive significance

of water absoption and storage by reptile eggs. Some investigators believe that water

accumulated within an incubating egg constitutes little more than a buffer against

the dangers of potential desiccation (Fitch & Fitch,

1967;

Badham,

1971;

Bustard,

1971a, b), and that uptake and storage of water is not essential for normal development

of the embryo (Bustard,

1966;

Dmiel,

1967).

Conversely, other workers contend that

absorption and storage of water is of utmost importance to the developing embryo

(Gordon, 1960).

We believe the weight of the evidence supports the contention that water absorption

is a critical factor in the normal development of reptilian eggs. Hatchlings of the

lizard,

Anolis carolinensis,

were found to be larger and heavier when hatched from eggs

incubated continuously on moist substrates than when they emerged from eggs sub-

jected to mildly desiccating conditions (Gordon, 1960). Such data do not demonstrate

that water absorbed from the environment is incorporated into embryonic protoplasm,

but they do indicate that water absorption has survival value.

In some investigations, hatchlings have been reported to weigh less than freshly

laid eggs (Lynn 8 : von Brand, 1945; Mendelssohn, 1963, 1965; Bustard, 1967;

Dmiel, 1967; Clark, D.

R.,

1970; Badham, 1971), eading several authors tacitly to

conclude that water absorbed by eggs is not incorporated into forming tissue. Before

arriving at such a conclusion, however, it

is

essential to consider the mass of the egg-

shell and other extra-embryonic materials that are discarded at hatching, and to

take into account the oxidation of organic material in embryonic metabolism (Cun-

ningham

et al.,

1939; Lynn & von Brand, 1945). Furthermore, other studies have

shown that hatchlings

of

many species

of

lizards, snakes and turtles weigh more than

6 B R E 52


12/35

82

G.

C . PACKAFW, R.

TRACY

ND J.

J.

ROTH

freshly laid eggs (Cunningham & Huene,

1938;

Brown,

1956;

Mendelssohn,

1963;

Bustard,

1 9 6 5 ) ~

hich is possible only in the event that a portion of the water absorbed

by the eggs was incorporated into the protoplasm of the embryos.

A. V. Fitch (1964) discovered living embryos of the lizard,

Eumeces obsoletus,

in eggs

that had shrivelled from desiccation, prompting a later suggestion (Fitch & Fitch,

1967) that uptake of water by eggs is of minor importance to the developing young.

However, evidence presented by A. V. Fitch (1964) indicates only that there was a net

loss of water from incubating eggs, and not that water uptake was not occurring.

Additionally, the nature of her study precluded incubating eggs from hatching, and so

it remains to be shown that embryos of Eumeces obsoletus can withstand desiccation

without ill effect. Indeed, it is important to note that turtle eggs subjected to desic-

cation early in incubation experience extraordinarily high mortality, whereas eggs

subjected to dehydration later in development - while not experiencing high mortality

requently produce abnormal hatchlings (Lynn & Ullrich,

1950).

In summary, we tentatively conclude that water absorbed by eggs of reptiles is in-

corporated, in part, into the protoplasm of developing embryos, and that failure to

absorb water leads to smaller hatchlings and, possibly, to a higher incidence of develop-

mental anomalies and embryonic death. I n the case of Lepidosauria, the increase in

volume of the albumen accompanying water absorption may have further adaptive

value in preventing the invasion of the eggs by bacteria (Board

&

Fuller, 1974).

IV. SOURCES OF MINERALS

FOR

EMBRYOS

Earlier studies of the eggs and hatchlings of the sea turtles, DermocheZys cmiace a and

Lepidochelys olivacea, revealed that hatchlings contain

3-5

times more calcium than is

present in the yolk and albumen of eggs at the time of oviposition (Simkiss,

1962,

1967). These observations led several investigators

to

examine the possibility that a

portion of the calcium required for ossification of bones of embryonic reptiles is taken

up from the substrate incidental to water absorption. Evidence now available indicates

that all of the calcium (and magnesium) required by developing embryos is present in

eggs at the time of oviposition (Cunningham et

al. ,

1939; Bustard & Greenham, 1968;

Jenkins & Simkiss, 1968; Bustard, Simkiss & Jenkins, 1969; Jenkins, I975), but two

fundamentally different means for providing this element seem to exist.

Embryos of crocodilians and chelonians, like those of birds, recover large quantities

of calcium from the inner surface of the eggshell during the course of development

and in this way largely satisfy their requirements for this element in ossification of

bones (Bustard et

al . ,

1969; Jenkins, 1975). The yolk is a rich source of magnesium

for embryonic chelonians (Bustard et

al.,

1 9 6 9 ) ~ ut the source of magnesium for

embryonic crocodilians has yet to be determined.

In contrast, embryos of snakes and lizards, including those geckos producing

calcareous eggs, appear to obtain both calcium and magnesium from rich stores

present in the yolk (Jenkins & Simkiss, 1968), and recover little (if any) mineral from

the eggshell. Use of the yolk, rather than the eggshell, as the reservoir for calcium

may have been a critical factor allowing viviparity to evolve in many species of the

Squamata (see Section VIII,

5 ) .


13/35

O I.4

Physiological ecology of reptilian

embryos

+

A

O+

A t

0

A

t

0

+

A

83

0

6

A A

0

10

20

30 40

50

60 70

Age

days)

Fig. 4. Cumulativeplots of nitrogen excreted as amm onia (A), urea 0)nd uric acid (+) by

embryos of

the

oviparous

izard,

Calotes versicolor,

at different stages of development. Values

are estimated from data present ed

by

Athavale & Mulherkar

(1967).

V. PATTERNS

OF

NITROGEN EXCRETION

The various patterns of nitrogen excretion among adult vertebrates generally are

thought to represent adaptive strategies related to the availability of water in the

environment (Campbell,

1973).

Ammonia, which is the usual end product of protein

metabolism in aquatic animals, is detoxified by incorporation into urea or uric acid in

amphibious and terrestrial species. Urea is highly soluble and can be retained in-

ternally for variable periods of time with no apparent ill effects, but substantial

quantities of water are required intermittently as a vehicle for eliminating the stored

waste. Conversely, uric acid (or urates) is relatively insoluble and can be eliminated

with a minimal

loss

of water.

Unfortunately, few studies have dealt with the patterns of nitrogen excretion of

reptilian embryos, even though available data suggest the existence of a spectrum of

adaptive responses among embryos

of

different species. Additionally, contrary to the

oft-cited prediction of Needham

(1963:

1132 ff.), the patterns of nitrogen excretion

exhibited by reptilian embryos seem frequently to differ from those characterizing

adults.

Embryos of the oviparous snake, Coluber constrictor, are ammoniotelic for the first

10 days of development (Fig.

3),

producing and releasing relatively large quantities of

ammonia gas, but then begin to channel the bulk of their amino nitrogen into urea

(Clark, 1g53b). Uric acid is not produced in significant amounts until just before

hatching, at which time the embryos seem to experience a biochemical metamorphosis

'in anticipation' of the new suite of physiological demands attending a free-living

existence (Packard,

I

966).

Data reported for the oviparous snakes,

Psammophis

6 2


14/35

84

9 r

8 -

7 -

6 -

J 5 -

2

3

4 -

3 -

F

2 2 -

M

0

c

1 -

G. C. PACKARD,

R.

TRACYND . J. ROTH

8

AA

0

4,

0 :

? + .I.

A

A

A

0

A 0

o +

+

4- +

1 I I

I

I

0 10 20 30

40 50

60

Age days)

Fig. 5. Cumulative plots

of

nitrogen excreted as ammonia (A) , urea

0)

nd uric acid(+)

by

embryos of the American alligator

Alligator

Pn;ssippiensis) at different stagesof development.

Data

are

from

Clark

e t

al. (1957).

schokari and Psammophis sibilans, are an inadequate basis for generalization concern-

ing the patterns

of

embryonic nitrogen excretion in these species (Haggag, 1964),but do

not differ conspicuously from those summarized in Fig. 3 for Coluber

constrictor.

Embryos of the oviparous lizard,

Calo tes versicolor,

may exhibit a pattern of nitrogen

excretion different from that described for embryonic snakes (Athavale

&

Mulherkar,

1967).Ammonia seems not to be an important excretory product in embryos of this

species (Fig. 4; the high values for ammonia at 25 and 35 days of age are most likely

due to experimental error), and urea and uric acid account for about equal amounts

of

the nitrogen excreted at all stages of development. We should mention, however,

that it is not clear from the report by Athavale & Mulherkar (1967)whether the entire

content of eggs was used in their analyses.

In particular, the albumen fraction, which

is a major repository for urea in eggs of other reptiles (Clark, 1953b), may not have

been sampled in this study of

Calotes

eggs, and certain of the nitrogenous materials

may therefore have been underestimated.

Careful examination of the extra-embryonic membranes in eggs

of

the oviparous

lizard,

Agama agama,

failed to reveal any sign of crystals of uric acid (or urate),

indicating that embryos of this species probably produce large quantities of ammonia

and/or urea during development (Harris, 1964).

Embryos

of

the sea turtle, Lepidochelys olivacea, produce significant amounts of

urea throughout development (Tomita,

1929),

and the small quantities of uric acid

that accumulate in eggs of this species probably originate in purine catabolism.

Al-

though ammonia production was not measured by Tomita, the continuous decline in

nitrogen content of incubating eggs (Nakamura, 1929) indicates that ammonia (in the

form

of

a gas) may be the primary excretory product throughout embryonic develop-

ment of this species.


15/35

. 0.3

Z

0.2

o.l


0

0

0

0 0

0

0 0

0

0

A

A

A

0

+++ ++++ ?+

9

t 1 I t

I I

0

10 20 30 40 50 60 70

80

Age

days)

Fig. 6. Cumulative plots of nitrogen excreted

as

ammonia (A) , urea 0)nd uric acid

(+)

by

embryosof theviviparous snake,

Thammphistirtulis,

at different

stagesof

development.Data

are

from

Clark

&

Sisken

(1956).

The pattern of nitrogen excretion manifest by embryos of the alligator,

Alligator

mksz+piensis,

is of special interest, since development proceeds inside hard, calcar-

eous eggs. Contrary to expectation (Needham, 1963)) little uric acid is produced by

embryos of this species (Fig. 5 ) ) and it seems likely that the urate originates in purine

catabolism rather than in deamination reactions. Amino nitrogen seems to be divided

evenly between ammonia (in the form of ammonium ions) and urea for the entire

course of embryonic development (Clark, Sisken

&

Shannon,

1957).

Embryos of the viviparous snake,

Thamnophis

sirtalk, exhibit a pattern of nitrogen

excretion quite similar to that described earlier for embryos of oviparous snakes

(Clark

&

Sisken,

1956).

Small amounts of ammonia (in the form of ammonium ions)

and uric acid accumulate in the tissues and extra-embryonic membranes of develop-

ing embryos, but the embryos clearly are ureotelic throughout the period of develop-

ment (Fig.

6).

Although the production of urea and ammonia by embryos may have

been underestimated somewhat in consequence of transplacental movement of these

solutes (Clark

&

Sisken,

1956;

see also Clark, Florio & Hurowitz,

1955;

Hoffman,

1970)) we doubt that the placental exchange of these materials

is

so extensive as to

alter our conception

of

the pattern of embryonic nitrogen excretion in this species.

In summary, uricotelism

is

not common among reptilian embryos, even

in

species

developing within calcareous eggs. Ammonia may be an important excretory product

in embryos

of

some species, especially in early stages of development, but all species

studied so far rely heavily upon urea as an end product in protein catabolism. Since

urea

is

an osmotically active material, use of this compound as

an

end product in


16/35

86

G. C PACKARD,. R.

TRACY

ND J. J.

ROTH

protein catabolism has important implications concerning the water metabolism of

reptilian embryos.

VI. EXCHANGE O F GASES

( I )

The atmosphere adjacent to eggs

The eggs of oviparous reptiles usually are deposited in sheltered sites beneath rocks

and other objects, or are buried in sand, friable soil, or decaying vegetation. Unfor-

tunately, information is as yet unavailable on the atmosphere to which eggs of most

reptiles are exposed; but with the possible exception of eggs incubating in soils with

much humus where microbial respiration may reduce the oxygen content of the air in

interstitial spaces, we suspect that the composition of air adjacent to eggs usually does

not depart appreciably from that of the overlying atmosphere, even when the eggs are

deposited in subterranean nests (see Seymour,

1973).

A known exception to our prediction concerns those reptiles depositing large num-

bers of eggs in a single nest chamber that may be at a considerable depth below the

surface of the ground. For example, there seems to be a pronounced depletion of oxy-

gen, and an accumulation of carbon dioxide, in the atmosphere inside nests of sea

turtles (Ackerman & Prange, 1972;Prange & Ackerman, 1974).Apparently, oxygen is

consumed by the large mass of eggs more rapidly than it can be replenished by diffu-

sion through the interstitial spaces in the substrate (Prange & Ackerman, 1974). The

influence, if any, of such hypoxic and hypercarbic conditions on embryonic develop-

ment remains to be determined.

(2)

The mechanism

of

gas exchange

The exchange of gases between a reptilian embryo and its environment presumably

is by diffusion. In Crocodilia and Chelonia, oxygen apparently diffuses inward

through pores in the eggshell (Reese,

1915;

Bigalke,

1931;

Young,

1950;

Erben,

1970),

and then through spaces between fibres of the two egg membranes (Fig.

I).

In

Lepidosauria, however, molecules of gas seem simply to diffuse through spaces

between fibres of the shell membrane (Fig.

I).

Using methods developed in studies of gas exchange across shells of avian eggs,

Ackerman

&

Prange

(1972)

estimated the permeability of the shell and outer egg mem-

brane of sea turtle (Chelonia mydm) eggs to oxygen. The mean value for the

permeability of their preparations was

6-59

x

I

0 6

cm3.

sec-l

.

cm-2. torr-I,

a

value

approximately twice that reported for similar preparations from eggs of the domestic

fowl (Wangensteen, Wilson & Rahn,

1970/71).

Since the thickness of the shell (which

is taken as an approximation to the length of the diffusion pathway) does not differ

appreciably between eggs of sea turtles and those of the domestic fowl (Wangensteen

et al.,

1970/71;

Ackerman & Prange,

1972),

the shell of turtle eggs seems to have a

total pore area approximately twice that

of

chicken eggs.

Regrettably, the preceding value for the permeability of sea-turtle eggs to oxygen

may be of limited use in constructing models of the process of gas exchange. Acker-

man & Prange

(1972)

failed to consider the possibility that in turtle eggs,

as

in bird

eggs, the inner egg membrane constitutes a major barrier to the diffusion

of

oxygen


17/35


of reptilian

embryos

87

(Kutchai & Steen, 1971), and so their data probably cannot be used to estimate the

total barrier to diffusive uptake of oxygen by eggs from the surrounding atmosphere.

Rand (1968) studied rates of water loss from eggs of the domestic fowl and crocodiles

(Crocodylw acutus), and noted that the rate of water loss (expressed as a percentage of

the original egg weight) was only slightly greater in crocodile eggs than in chicken eggs.

Assuming that the eggs of these two species were of about the same size, the data

indicate that the permeability of the shell of crocodile eggs to water vapour (and

presumably to other gases) is almost indistinguishable from that of chicken eggs.

Unfortunately, the characteristics of oxygen transport into eggs of crocodilians

cannot be predicted from data on permeability of these eggs to water vapour because

the inner egg membrane probably constitutes an important barrier to diffusion of oxy-

gen, but not to water vapour (Kutchai & Steen, 1971). I n chicken eggs, the perme-

ability of this membrane to oxygen increases during the course of incubation, owing

to the withdrawal of water from spaces between the fibres

of

the membrane (Kutchai

& Steen, 1971). However, since eggs of crocodilians absorb and store water during

the course of normal development (Mcllhenny, 1934, 1935; Guggisberg, 1972),

there may be no change in water content of the inner membrane between oviposition

and hatching, and so

resistance to inward diffusion of oxygen may not change during

incubation of crocodilian eggs. The possibility that the permeability of egg membranes

to oxygen does not increase during incubation of crocodilian eggs raises important

questions pertaining to metabolism and acid/base balance of developing embryos (see

Erasmus, Howell & Rahn,

1970/71),

and therefore merits further study.

Since the eggs of lepidosaurians seemingly have no pores in the eggshell through

which respiratory gases can diffuse, gas exchange must occur by diffusion between the

fibres of the shell membrane. We predict that surfaces of eggs exposed to the nest

atmosphere are involved in gas exchange, with oxygen moving inwards through air-

filled spaces between the fibres of the shell membrane, but that surfaces in contact

with (or in proximity to) the substrate and which are involved in water transport into

the eggs - are of little importance in gas exchange because the interstices of the shell

membrane are filled with liquid water. A corollary of this hypothesis is that some frac-

tion of the egg surface must not be in contact with the moist substrate, for uptake of

oxygen would be inhibited if diffusion had to occur through water-filled spaces in the

shell membrane. Such a circumstance would lead to a reduction in the rate of meta-

bolism and development of the embryo, and could conceivably result in suffocation.

These contentions receive some support from a study of eggs of the lizard, Amphi-

bolurus barbatus, in which eggs that were completely covered with moist sand (and

therefore were wetted on all surfaces) took longer

to

complete incubation than eggs

incubated on the surface of the sand (Bustard, 1966). Further support for our con-

tentions comes from observations that parchment-shelled eggs of turtles suffer

extraordinarily high mortality when soil or sand comes into contact with all surfaces of

the eggs (Cagle, 1950; Simon, 1975).


18/35

88

G.

C PACKARD,

.

R. TRACY

ND

J. J. ROTH

VII. TEMPERATURE AND METABOLISM

(I )

Effects of temperature

Embryonic reptiles seem to behave like 'typical' ectothermic organisms in their

physiological responses to temperature. Metabolism increases with temperature

(Zarrow & Pomerat,

1937),

and development of embryos to hatching proceeds much

more rapidly in warm environments than in cool surroundings (Legler,

1960 ;

Yntema,

1960;

Dmi'el,

1967;

Goode

&

Russell,

1968;

Platt,

1969;

Clark,

D. R., 1970;

Vinegar,

1973;

Sexton & Marion,

1974).

For example, an increase in incubation temperature of

I C shortens the incubation period for embryonic hognose snakes (Heterodon

nasicus) by

7

days (or

1 0 ;

Platt,

1969),

and a rise of temperature from

27.8

C to

32.8 C leads to the completion of embryonic development of box turtles

(Terrapene

ornata)

in

59

days instead of

70

days (Legler,

1960).

Although development may proceed over a range of temperatures, there appears

to be a particular temperature for each species at which embryogenesis proceeds

optimally. Relatively small deviations in temperature from the optimum frequently

lead to an increase in the incidence of developmental anomalies, but the anomalies

usually are so minor (e.g. changes in number of scale rows) as to escape notice (Fox,

1948;

Fox, Gordon & Fox,

1961;

Osgood,

1968;

Bustard,

1969b;

Moll & Legler,

1971; Vinegar, 1973, 1974). However, relatively large departures of the incubation

temperature from the optimum usually result in the formation of chimeric embryos

(Yntema,

1960;

Osgood,

1968

; Vinegar,

1974),

and embryonic mortality generally

increases appreciably (Licht & Moberly,

1965

; Bustard,

1971

a , b ; Vinegar,

1973;

Sexton & Marion,

1974).

Embryos of tropical species are less tolerant of departures

from their thermal optimum than are embryos of temperate forms (Licht & Moberly,

1965; Vinegar, 1973).

Despite the fact that developmental abnormalities and embryonic death result from

prolonged exposure of eggs of temperate reptiles to temperatures falling several

degrees below the optimum, further reductions in temperature generally lead to arrested

development, which can be tolerated with no apparent ill effect other than a slight

delay in the time of hatching (Cunningham, 1922; Fitch, 1954;Legler, 1960;Yntema,

1960; Maderson & Bellairs, 1962). Indeed, the Australian tortoise,

Chelodina expan sa,

deposits eggs in the autumn, and the eggs lie dormant in the soil throughout the

winter months, during which time soil temperatures decline to 5 C (Goode

&

Russell,

1968) and embryogenesis presumably is fully arrested (Cunningham, 1922

;

Yntema,

I

960).

When temperatures increase in the following spring, development of the tor-

toise embryos begins (or resumes), and normal young subsequently hatch from the

eggs. If temperatures in the soil during the winter were not low enough to arrest

embryogenesis completely, developmental anomalies would be expected to occur

(Yntema, 1960).

The duration of exposure of eggs to temperatures departing from the optimum

seems to be important, but this subject has not been fully explored. Brief exposure

of lizard and snake eggs to high and low temperatures indicates that embryos of tem-

perate forms can withstand extremes similar to those withstood by adults of the same


19/35


of

reptilian embryos

89

species (Fitch, 1964; Fitch

&

Fitch, 1967), but the range of tolerable temperatures

seems to diminish as the length of exposure increases (Fitch & Fitch,

1967).

The

ability briefly to withstand high and low temperatures

is

clearly of adaptive value, in

that excursions in nest temperature during midday and at night will not alter the

course of development significantly.

However, most reptilian embryos are probably not exposed to wide fluctuations in

temperature, either during the course of a single day or during the entire course of

embryogenesis, owing to the moderating effects of the nest in which the eggs usually

are laid (Reese, 1915; Brown, 1956; Goode & Russell, 1968; Bustard, 1969a; Rand,

1972; Chabreck, 1973; Van Devender & Howard, 1973). Temperatures in nests of the

American alligator (Alligator mississtppiensis) vary during the course of a single day

through a range

of

only 1.2 C, whereas die1 variation in air temperature above the

nests may be as much as

9-3

C (Chabreck,

1973).

Also, nests

of

the tropical lizard,

Iguana

zjpana,

are reported to have temperatures of 31-32 C for extended periods, and

to exhibit virtually no daily variation (Rand, I

972) ;

hese observations are consistent

with data indicating a limited tolerance of embryos of this species to fluctuations in

temperature (Licht & Moberly, 1965).

Sea turtles may present

an

important exception to the preceding generality. Die1

variation in temperature

of

nests

of

the turtle, Chelonia mydas,

is

less than z C

(Hendrickson, 1958; Carr & Hirth, 1961;Bustard & Greenham, 1968;Bustard, 197z),

but the mean temperature in nests increases steadily from about zg C at the time of

oviposition

to

nearly 35

C

ust before hatching (Hendrickson, 1958). Also, a gradient

in temperature of as much as

2

C usually isestablished between the centre of a mass of

eggs and the periphery

of

the nest (Bustard,

1972).

The increase in mean temperature

of the nest, and the formation of a temperature gradient betweenthe inside and the out-

side of the nest, presumably stems from metabolic heat production of the large number

of embryos concentrated in the nest chamber (Hendrickson, 1958; Bustard, 1972).

Since eggs in the centre of a sea-turtle nest must undergo incubation at slightly

higher temperatures than are experienced by eggs at the periphery, it is reasonable to

assume that development of centrally located eggs is completed before that of other

eggs in the nest (Bustard, 1972). However, young in centrally located eggs apparently

delay hatching (or emergence) until litter mates located on the periphery of the nest

have completed development. The high degree of synchrony in the emergence of

young from a given nest results from a form of social facilitation in which the hatching

and digging movements

of

each individual are 'reinforced' by the activity of adjacent

animals (Carr & Hirth, 1961;Bustard, 1972).

2 ) Metabolism

of

embryos

At temperatures approaching the optimum for development, oxygen consumption

of turtle embryos increases with age in general conformity with the sigmoid growth

curve (Lynn

&

von Brand,

1945),

and metabolism of advanced embryos is virtually

identical to that of hatchlings (Lynn

&

von Brand

1945;

Prange & Ackerman,

1974).

Metabolism of snake embryos also increases with age, but the observed pattern of in-

crease

is

exponential rather than sigmoid (Clark,

I953C;

Dmi'el,

1970);

the failure

of


20/35

90

G. C .

PACKARD,

. R.

TRACYND J J.

ROTH

Table

2 .

Summary of the modes of reproduction characterizing fam ilies of

the order Squamata

(From Fitch, 1970,with additional information from Boulenger, 1912,and Broadley, 1974)

Only

those

families are listed for which reliable information is available. Fo r convenience, the classification scheme

used here is the same

as

was used by Fitch

1970).

Family

Amphisbaenidae

Gekkonidae

Iguanidae

Agamidae

Chamaeleontidae

Xantusiidae

Teiidae

Lacertidae

Scincidae

Dibamidae

Cordylidae

Gerrhosauridae

Anguidae

Anniellidae

Xenosauridae

Helodermatidae

Varanidae

Typhlopidae

Leptoty phlopidae

Uropeltidae

Acrochordidae

Boidae

Colubridae

Elapidae

Hydrophiidae

Viperidae

Crotalidae

Both modes of

All species reproduction All species

oviparous present viviparous

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

investigators to detect an inflection point in plots of oxygen consumption against age

may simply indicate that metabolism of snake embryos does not begin to stabilize until

just before hatching (Dmiel, 1970).

In a study of five species of colubrid and viperid snakes, the average slope of double

logarithmic plots of oxygen consumption versus mass

of

the embryos was

0.65

(Dmiel

I

970),but the biological significanceof his value is not clear. Interestingly, embryos of

many snakes seem to exhibit circadian cycles in metabolism, with peak metabolism

occurring during the day-time in embryos

of

diurnal species and

at

night-time in

embryos of nocturnal forms (Dmiel, 1969).

VIII. EGG RETENTION AND THE EVOLUTION OF VIVIPARITY

( I )

Distribution of the live-bearing method

Fitch (1970) as recently compiled and analysed data on the incidence of viviparity

among living reptiles, and his monograph serves as an excellent source of information

on this subject.


21/35


91

Data summarized in Table z indicate that viviparity has arisen independently in

several different families of the reptilian order Squamata, and

so

the mode of reproduc-

tion characterizing different species of lizards, snakes and amphisbaenians has little

value as an index to their phylogenetic affinities. For instance, oviparity and viviparity

characterize different congeneric species of such lizards as

Eumeces

(Scincidae),

Gmrhonotus (Anguidae), and Sceloporus (Iguanidae), and of such snakes as Agkistrodon

(Crotalidae),Natrix (Colubridae), and

Vipera

(Viperidae).

Of special interest are those species in which some populations may be characterized

by oviparity and others by viviparity, for insight may be gained from these species

concerning the adaptive value of the evolutionary transition to the viviparous state.

For example, the skink,

Mabuya quinquetaeniata,

s believed to be viviparous in South

Africa, but oviparous elsewhere in its range (Fitch, 1970). Likewise, the Central

American iguanid, Scelopmus variabilis,which is oviparous in most areas where it is

found (Fitch,

1970),

may be viviparous at higher elevations in the Mexican state of

Veracruz (Werler,

1951);

and the vipers, Echis carinata and Aspis vipera, are reported

to be oviparous in some localities and viviparous in others (Mendelssohn,

1963, 1965).

Unfortunately, variation between populations of squamate reptiles in their mode of

reproduction is not a well-documented phenomenon, and many of the accounts in the

literature are suspect. For instance, the most frequently cited example is the lizard

Lacerta vivipara,

which is widely believed to be oviparous at lower elevations in the

Pyrenean Mountains in the south of France, and to be viviparous elsewhere in its range

(Fitch,

1970).

In early September of

1924,

Lantz

(1927)

discovered approximately60

parchment-shelled eggs beneath a large stone situated in relatively moist terrain.

When the eggs were handled, young lizards emerged from several, and these hatch-

lings were immediately recognized as

Lacerta vivipara.

The other eggs in the nest

contained embryos at various stages of development, including some that had com-

pleted only about half of their embryogenesis. Since more than

12

females must have

been involved in laying

so

many eggs, Lantz

(1927)

suggested that oviparity may

characterize certain populations of the species.

Lantzs observations have never been confirmed, despite the fact that nearly 5

years have passed since publication of his report (Bellairs, 1970). Since Lantz did not

actually observe any instance of oviposition, and since he did not state that the un-

hatched eggs from the nest contained living embryos,

it is

possible that he drew

incorrect conclusions from his observations. I n unfavourably cool years

-

of which

1924may have been one, judging from the late date on which the communal nest was

discovered (see Blanchard & Blanchard, 1940) young female

Lacerta vieripma

may

prematurely expel some of their developing young while retaining the remainder of

their eggs until the completion of embryogenesis (Panigel, 1956; see also Hoover,

1936).

Eggs expelled prematurely have a parchment-like appearance owing to the fact

that they do not have the usual layer of mucus on the surface of the shell membrane,

and they probably do not survive long because of rapid desiccation (Panigel, 1956).

Thus, it is possible that some of the parchment-shelled eggs discovered by Lantz

were moribund eggs deposited prematurely by females responding in an unusual

way to the generally inclement weather conditions, and that the remaining eggs-


22/35

92

G. C PACKARD,. R.TRACY

ND

. J. ROTH

containing full-term young - were coincidentally deposited beneath the same rock

by other lizards of the same species shortly before Lantzs arrival.

2) A model

for the evolution

of

viviparity

Doubtless there is variation within natural populations of most reptiles both in the

thickness of eggshells and in the normal interval during which eggs reside in the ovi-

ducts of the female before deposition. When both relatively thin eggshells and rela-

tively long retention of eggs in utero characterize the reproductive effort of certain

females, there is the potential for evolution of viviparity, the only requisites being

that the thickness of eggshells and the period of intra-uterine retention are heritable

characteristics, and that females exhibiting these traits contribute disproportionately

to succeeding generations.

The simplest model for the evolution of viviparity from oviparity therefore entails a

gradual increase (over many generations) in the length of time eggs are retained in the

oviducts of the females prior to deposition, during which time development of the

embryos proceeds at near-optimal rates. Thus, the evolutionary transition from ovi-

parity to viviparity leads to eggs being laid at progressively more advanced stages of

development, and consequently, the interval from oviposition to hatching is progres-

sively reduced. The final stage in this sequence occurs when females deposit eggs con-

taining fully formed young which then emerge from within the enclosing shell and

extra-embryonic membranes.

A

survey of the reproductive biology of contemporary reptiles yields information

that is consistent with the preceding evolutionary model. For instance, eggs of many

reptiles pass through the oviducts rather slowly (see Reese,

1915;

amlett,

1952;

Cooper,

1965

Clark, D. R.,

1970).

During transit, embryonic development usually

proceeds

-

imited only by the requirements of the embryos for favourable tem-

peratures, an adequate exchange of respiratory gases, a means for excreting nitro-

genous wastes, and a source of water. Consequently, at the time of oviposition, eggs of

many reptiles contain embryos in various stages of organogenesis. A known exception

to this generality pertains to Crocodilia and Chelonia, whose embryos seem not to

proceed past gastrulation before oviposition, regardless of how long the eggs are

retained in the oviducts (Cunningham,

1922;

McIlhenny,

1934, 935

Risley,

1944;

Lynn & von Brand, 1945; egler, 1960;Decker, 1967); nd we shall return to this

point shortly.

In certain species of the Squamata, eggs spend so much time in the oviducts of

females that the species appear to be in evolutionary transition from oviparity to vivi-

parity. Eggs of the lizard, Lacerta muralis, are carried by females for about one month

after fertilization occurs (Cooper,

1965),

nd approximately

42

of the incubation

period of worm snakes, Car phq his vermis, is spent in the oviducts of the female parent

(Clark, D. R., 1970).Embryos of the garden lizard,

CaZotes versicolor,

have advanced

to a stage of development comparable to a

55-60

h chick embryo by the time the eggs

are laid (Muthukkaruppan, Kanakambika, Manickavel & Veeraraghavan, I970). Eggs

of

the Australian skink,

Saiphos

equalis,

are reported to hatch within a few days of


23/35


of reptilian

embryos

93

laying (Fitch, 1970), a feat that is matched by the 4-day incubation period of some

eggs produced in northerly populations of the snake, Opheodrys

vernalis

(Blanchard,

Th e lizard,

h e r t a wiwipara,

retains eggs internally for the full period of develop-

ment. The eggs can be removed surgically from females and successfully incubated

in

vitro on gauze moistened with saline (Panigel, 1956; Holder & Bellairs, 1962;

Maderson & Bellairs, 1962). The young hatching from such eggs appear normal in

every respect (Panigel, 1956), and thus females of this species seem to do little more

than provide a favourable thermal, hydric, and gaseous environment in which their

eggs can develop.

One striking morphological change during the transition from oviparity to viviparity

among squamate reptiles appears to be a reduction in the degree of calcification of the

eggshell and in the thickness of the shell membrane. In a population of green snakes,

Opheodrys

vernalis,

characterized by retention of eggs within the oviducts of females

for most of the period of incubation, the eggshells are described as being unusually

thin and delicate (Blanchard, 1933). Also,eggs of the oviparous lizard, Lacerta ugiZis,

have a lightly calcified eggshell, whereas those of the viviparous congener, Lacerta

vivipma, are completely uncalcified (Jacobi, 1936); and the shell membrane is much

thicker in eggs of the former species than in eggs of the latter (Jacobi, 1936). Indeed,

while the shell membrane persists in eggs of most viviparous lizards, snakes and am-

phisbaenians, in no instance is the membrane known to be impregnated with calcium

salts (Weekes, 1935; Bellairs, 1959).

While eggs are retained in the oviducts of the female parent, the embryos benefit

from the moderate thermal environment provided by the mother (see p.

94),

and

excretion of nitrogenous wastes probably presents no major problem since urea can

either be stored in embryonic and/or extra-embryonic fluids or be allowed to diffuse

out of the eggs into the oviducts.

Also,

water can be absorbed by eggs from the fluid

environment (Giersberg, 1922; Jacobi, 1936; Tinkle, 1973;Newlin, 1976), passing

directly into the yolk (Badham, 1971)rather than into an albumen fraction.

The major problem of intra-uterine existence is probably the exchange of gases,

and this problem may underlie the reduction in calcification and thickness of the egg-

shell attending the evolutionary transition from oviparity to viviparity. Since res-

piratory gases apparently diffuse through the liquid environment of the oviducts,

through the calcareous layer of the eggshell, and between the fibres of the egg or shell

membranes, the diffusion pathway may be relatively long, and the metabolism of the

developing embryos could be inhibited by a low rate of uptake of oxygen by the eggs.

Consequently, a reduction in the extent of the diffusion barrier imposed by the egg-

shell and associated membrane(s) may be essential if the metabolism of the embryos

is

to be sustained, particularly in later stages of development when oxygen requirements

are li el y to be high (Lynn & von Brand, 1945; Clark, 1 9 5 3 ~ ; miel, 1970; Prange

& Ackerman, 1974).

Support for the contention that adaptive thinning of the eggshell and associated

membrane(s) is necessary if intra-uterine development of reptilian embryos is to be

sustained comes from studies of the metabolism of developing snakes. Embryos of the

1933).


24/35

94 G. C. PACKARD, R.TRACYND J. J.

ROTH

oviparous snake, Elaphe guttata , either accumulate an oxygen debt during later stages

of

intra-uterine existence, or the rate of embryonic metabolism (and growth) is

depressed, owing to inadequate uptake of oxygen from the immediate environment

(Clark, 1952, 1953a);either of these alternatives suggests that intra-uterine develop-

ment of embryos would not proceed much further without some reduction of the

diffusion barrier imposed by the eggshell and shell membrane. Furthermore, it is

important to note here that embryonic development of Crocodilia and Chelonia is

arrested while eggs are retained within the oviducts of the female, possibly because

uptake of oxygen from the immediate environment is insufficient to sustain embryo-

genesis (Risley, 1944; Lynn

&

von Brand, 1945); and thick shells characterize eggs

of

both the Chelonia and the Crocodilia.

(3)

Adaptive value

of

egg retention

a) Temperature benejits

We conceive of egg retention as conferring special advantage to relatively K-

selected* reptiles (eggs cannot be retained for long intervals by animals producingmore

than one clutch per year) dwelling in thermally seasonal environments, particularly at

high latitudes and altitudes. Assuming that the enzymes of developing embryos have

the same thermal optima as the enzymes of the female parent, the adult animal can,

by regulating her body temperature behaviourally at an optimal level for physiological

functions, incidentally provide an optimal thermal environment for the offspring

developing within her body. Thus, during the warmth

of

the day, she may move

into warm parts of the environment and maintain a relatively high and constant body

temperature, whereas at night she may retreat to sheltered sites where neither she

nor her eggs will be exposed to particularly low temperatures. Were

this

female

to

deposit her eggs in a nest where daytime temperatures rose sufficiently high to support

embryogenesis at high rates, the eggs presumably would be exposed to nocturnal tem-

peratures sufficiently low to induce anomalous development (Vinegar, 1973, 1974).

Conversely, were she to deposit the eggs in a more protected site (the usual cir-

cumstance), where they would not be exposed to potentially harmful nocturnal

temperatures, diurnal temperatures in the nest presumably would be suboptimal for

development, and hatching would be delayed accordingly.

Retention of eggs internally for brief periods would enable embryos to attain a given

stage of development more rapidly than embryos of conspecific females that

do

not

retain eggs for the same length of time. Thus, if all embryos have similar rates of

development following oviposition, those eggs subjected to longer intervals of reten-

tion inside the female parent would hatch earlier than other eggs of the species. Early

hatching may have adaptive significance in allowing young more time to feed and to

grow before entering hibernacula for the first time (see Cagle, I IJ~O) ,r early hatching

may assure that young are not trapped (and killed) in the nest by early winter storms

(Risley, 1933). Thus, genetic variation among females in the length

of

time for which

* The term K-selected, which comes from the logistic model for population growth, refers to

populations of organisms characterized by relatively

long

Iife-spans, delayed maturity, and iteroparity

(Tinkle,

1969;

Pianka,

1970).


25/35


of

reptilian

embryos

95

eggs are held in the oviducts could provide the basis for an evolutionary transition to

viviparity stemming from thermal benefits to the embryos.

Evidence in support of the preceding evolutionary hypothesis is largely circum-

stantial, but persuasive nonetheless. As indicated earlier, relatively high and constant

temperatures, closely approaching the optimum for a species, provide for rapid

embryonic development (Blanchard & Blanchard,

1940;

Legler,

1960;

Yntema,

1960;

Dmi'el,

1967;

Goode & Russell,

1968;

Platt,

1969;

Clark,

D. R., 1970;

Vinegar,

1973;

Sexton & Marion, 1974) that is relatively free of thermally induced anomalies (Fox,

1948; Yntema, 1960; Fox

et

al., 1961; Osgood, 1968; Vinegar, 1973, 1974). It is

therefore important to note that pregnant female garter snakes

(Thumnophis sirtalis,

a viviparous species) have somewhat higher body temperatures in nature than either

males

or

non-gravid females (Stewart, 1965). Moreover, gravid female water snakes

(Nutrix fm ciata and Natrix taxispilota, which also are viviparous) seem to maintain

body temperatures within a narrower range than other individuals

of

these species

(Osgood,

I

970). Furthermore, when spiny lizards

(Sceloporuscyanogenys,

which again

are viviparous) are placed in a thermal gradient in the laboratory, pregnant females

cease basking at slightly lower body temperatures than do other animals; and these

females change position in the thermal gradient less often than other lizards, suggest-

ing that their body temperatures fluctuate relatively little during the course of a day

(Garrick,

1974).

These several observations on viviparous reptiles are indicative of the

kinds of benefits that might accrue to embryos of oviparous species at intermediate

stages in the evolutionary transition to viviparity.

The green snake (Opheodrys

vernalis)

provides a possible example of the evolution-

ary transition to viviparity in response to low temperatures and/or a relatively short

growing season. In northern Michigan

(ca. 45-5

N latitude), the thin-shelled eggs of

this species are laid in advanced stages of development, and hatching occurs about 2

weeks after oviposition (and in some instances in as little as

4

days; Blanchard,

1933).

In contrast, in a more southerly region

ca. 42O N

latitude), eggs of

this

species require

about 3 0 days between deposition and hatching (Stille,

1954).

Apparently, cooler con-

ditions and/or a shorter growing season in northern Michigan have led to the evolution

of longer periods of egg retention by females in that population because of the thermal

benefits accruing to the developing embryos (Packard, 1966).

In some species of oviparous lizards producing two clutches of eggs a year, eggs of

the second clutch are retained longer in the oviducts of the female than eggs of the

first clutch (Shaw,

1952;

Sexton & Marion,

1974).

Consequently, at the time of ovi-

position, eggs of the second clutch contain embryos in more advanced stages of deve-

lopment than were present in eggs of the first clutch at the time of oviposition; and the

interval from conception to hatching presumably is reduced for embryos of the second

clutch. Eggs of the first clutch have ample time for development to proceed to com-

pletion, even if they are deposited in nests where temperatures are slightly below the

optimum for embryogenesis. However, eggs of the second clutch must complete

development within a shorter span of time, and must hatch before the advent

of

un-

favourable weather in the autumn of the year (Sexton & Marion,

1974).

Thus, reten-

tion of eggs of the second clutch may be adaptive in providing for accelerated growth


26/35

96

G.

C . PACKARD,.

R.

TRACYND J. J. ROTH

of those embryos in the relatively favourable thermal environment afforded by the

maternal oviducts, thereby assuring completion of embryogenesis in what effectively

constitutes a short growing season.

In the past, patterns of geographic distribution of oviparous and viviparous Squa-

mata have been cited as evidence that viviparity often has arisen in response to tem-

perature stresses on embryos (Weekes, 1933, 1935 ;

chemical composition of egg shell

Documents