Seasonal aspects of the reproductive biology of the gopher tortoise, Gopherus polyphemus

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Seasonal aspects of the reproductive biology of the gopher tortoise, Gopherus polyphemus
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Gopher tortoise, Gopherus polyphemus
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Taylor, Robert Wayne, 1950-
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Thesis:
Thesis (Ph. D.)--University of Florida, 1982.
Bibliography:
Includes bibliographical references (leaves 84-89).
Statement of Responsibility:
by Robert Wayne Taylor, Jr.
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Typescript.
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Vita.

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Full Text










SEASONAL ASPECTS OF THE REPRODUCTIVE BIOLOGY OF THE
GOPHER TORTOISE, GOPHERUS POLYPHEMUS















BY

ROBERT WAYNE TAYLOR, JR.


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR
DEGREE OF DOCTOR OF PHILOSOPHY



UNIVERSITY OF FLORIDA


1982















ACKNOWLEDGEMENTS


I wish to express my appreciation to the members of my supervisory

committee, Dr. Walter Auffenberg. Dr. John H. Kaufmann, and Dr. Donald

A. Dewsbury for their assistance. I am especially grateful to my

chairman, Dr. Auffenberg, for his advice, understanding, and encourage-

ment during the period of my graduate study. I would also like to thank

Dr. Archie Carr, Dr. Martha L. Crump, Dr. Carmine A. Lanciani, Dr.

Elliott R. Jacobson, and Dr. Frank G. Nordlie for their guidance and

criticism. Dr. Lewis Berner and Dr. James H. Gregg graciously allowed

me to use their photographic equipment. The Department of Zoology and

the Florida State Museum, University of Florida, provided me with office

and laboratory space and funds for needed supplies.

Support was also provided by Dr. David Crews, who offered valuable

guidance and criticism. I thank Dr. Harvey H. Feder and Dr. Chris

Reboulleau who are responsible for the many steroid hormone determi-

nations. Without their generosity that portion of the study could not

have been completed. I am also indebted to Dr. Jacobson for making the

resources of his histology laboratory available to me. Dr. Gerald E.

Gause provided advice and optical instruments for the stereological

portion of the study.

Mr. Leon Norris provided valuable field assistance during the

collection of tortoises.













I am deeply indebted to all the members of my family, and

especially my wife, Gloria, for their help, encouragement, and under-

standing throughout the preparation of this dissertation.











TABLE OF CONTENTS


ACKNOWLEDGEMENTS. . . ii

LIST OF TABLES. . . .. vi

LIST OF FIGURES . . .... vii

ABSTRACT. . . . ix

INTRODUCTION. . ... . 1

MATERIALS AND METHODS . . ... .. 5

Preliminary Hormonal Studies . 5
Collection and Housing of Animals . 5
Sampling Procedures ................ 5

Detailed Study of Annual Cycle . .. 7
Collection and Housing of Animals . 7
Processing of Animals .............. 8
Histological Procedures .............. 9

Analysis of Tissues. ................... 9
Female Reproductive Tract . .. 9
Male Reproductive Tract . 10
Chin Glands . . 11
Courtship Behavior. .. . 12

RESULTS .. . . . 14

Female Reproductive Cycle.. . ... 14
Development of Ovaries and Oviducts . 14
Vitellogenic Cycle .. . 16
Corpora Lutea ... . .. 20
Hormonal Fluctuations . .... 20
Chin Gland Structure. ... . 26
Female Chin Glands. .. .. .. 29
Maturity, Fecundity, and Young. . .. 32

Male Reproductive Cycle. . 34
Testicular Changes. ............... 34
Spermatogenic Cycle ................ 41
Testosterone Fluctuations . 49
Male Chin Glands. . .. 52

DISCUSSION. . .... . 55













SUMMARY AND CONCLUSIONS ................... 82

LITERATURE CITED. . ......... .. 84

BIOGRAPHICAL SKETCH ...................... 90















LIST OF TABLES



TABLE

1 Summary of data regarding vitellogenesis and egg production
in female Gopherus polyphemus .. .... ..... 17

2 Monthly summary of numbers and percentages of female
Gopherus polyphemus possessing oviducal eggs, corpora
lutea, and/or ovarian follicles in each size category 18

5 Mass and total length measurements of 4 hatchling gopher
tortoises from a clutch of 4 eggs removed from a female
13 June 1979. . ... . .... 35

4 Characteristics of the four stages of spermatogenesis found
in male Gopherus polyphemus .. . ... 42

5 Monthly number of male Gopherus polyphemus in each
spermatogenic stage . . 46

6 Plasma testosterone levels (ng/ml) in male gopher tortoises 51

7 The role of estrogen and progesterone in the control of
ovarian and oviducal events in female Gopherus polyphemus .. .. 62














LIST OF FIGURES



FIGURE

1 Geographical distribution of Gopherus polyphemus 2

2 Changes in female Gopherus polyphemus reproductive
tissue weights during the annual cycle . 15

3 Representation of the progress of vitellogenesis and
egg production throughout the year for female Gopherus
polyphemus . . .. 19

4 Regression in size of corpora lutea following ovulation. .. 21

5 Mean monthly estrogen concentration in female Gopherus
polyphemus . . 22

6 Mean monthly progesterone concentrations in female
Gopherus polyphemus .... ........ ........ 23

7 Diagram of section through one of the chin glands of a
gopher tortoise. .. . . 27

8 Section through one of the chin glands of a gopher
tortoise . . 28

9 Variation in chin gland volume of female Gopherus
polyphemus . .. .. 30

10 Section through a gopher tortoise chin gland in the
"active" condition .. . 31

11 Section through a gopher tortoise chin gland in the
"inactive" condition . .. .. 31

12 Changes in male Gopherus polyphemus reproductive
tissue weights during the annual cycle 36

13 Relationship between relative testis size and total length
of male Gopherus polyphemus. .............. 37













14 Change in relative proportion of testicular tissue
composed of interstitial tissue and seminiferous
tubules in male Gopherus polyphemus during the annual
cycle. ... . ...... 39

15 Change in diameter of seminiferous tubules and their
lumens in male Gopherus polyphemus during the annual
cycle. . . ... 40

16 Section through a gopher tortoise testis in the inactive
condition (Stage 1). ........... 43

17 Section through a maturing gopher tortoise testis
(Stage 2). . .. . 43

18 Section through a gopher tortoise testis undergoing
maximum spermiogenesis (Stage 3) . 45

19 Section through a gopher tortoise testis with abundant
sperm in the tubule lumen (Stage 4). . 45

20 Change in diameter of the ductus epididymis and the
mass of sperm contained within it for male Gopherus
polyphemus during the annual cycle . .. 47

21 Section through the epididymis of a gopher tortoise
in late fall . . .. 48

22 Section through the epididymis of a gopher tortoise
in early fall. . . 48

23 Mean plasma testosterone concentrations in male
Gopherus polyphemus. ............. ... ... 50

24 Variation in chin gland volume of male Gopherus
polyphemus .. . . ...53

25 Summary of plasma testosterone fluctuation data from
important studies of male turtle reproductive
endocrinology. .. . 69


viii
















Abstract of Dissertation Presented to the Graduate Council of the
University of Florida in Partial Fulfillment of the Requirements for the
Degree of Doctor of Philosophy


SEASONAL ASPECTS OF THE REPRODUCTIVE BIOLOGY OF THE
GOPHER TORTOISE, GOPHERUS POLYPHEMUS

By

Robert W. Taylor, Jr.

August 1982


Chairman: Walter Auffenberg
Major Department: Zoology


The reproductive cycles of male and female gopher tortoises

(Gopherus polyphemus) were investigated in detail between March 1979

and May 1980 following preliminary studies in 1977 and 1978.. Gross and

histological changes in gonadal and chin gland morphology were examined.

The role played by the endocrine system in the control of the reproduc-

tive cycles was investigated.

Vitellogenesis began in September and ovarian follicles were fully

enlarged by December. Most ovulation occurred between early April and

late May, and nesting was completed by mid June. Following a summer

period of minimal ovarian size, recrudescence began in early fall. The

period of courtship and mating has been reported to include the months

of March, April, and May, but evidence indicates that lowered levels

of reproductive behavior may be exhibited until fall. Plasma estrogen











and progesterone levels in females showed peaks associated with ovula-

tion and vitellogenesis.

Male reproductive tissue weights reflected the stage of spermato-

genesis. Testicular weights were lowest when the seminiferous tubules

were inactive, and greatest during spermatogenesis. After sperm were

produced, they moved to the enlarging epididymides, and testes weights

returned to minimal levels. Male testosterone levels peaked prior to,

and declined during, the spring mating season. A second peak was

coincident with fall spermatogenesis.

Changes in chin gland volume were similar in timing and magnitude

for males and females. The size of this secondary sexual characteristic

was greatest for both sexes during the spring mating season, but pre-

viously unreported secondary enlargements were seen in September.

Increases in chin gland size were correlated with peaks in plasma

estrogen (female) and testosterone (male) concentrations.

It is concluded that the endocrine system plays an important role

in the control of gonadal events and the development of secondary sexual

characteristics in both sexes of the gopher tortoise. The results of

this study generally confirm and expand upon what is known about

endocrine control of reproduction in other turtles. Other data indicate

that the gopher tortoise is characterized by a very low reproductive

potential. Taken together, these data provide a basis for making more

informed decisions about the management and possible protection of

Gopherus polyphemus.















INTRODUCTION


Until recently, detailed studies of the reproductive biology of

reptiles have been uncommon. Although some knowledge had been gathered

for a large number of species, most of it was fairly cursory and was

usually limited to observations concerning mating, nesting, and

offspring. During the past few decades a few reptile species have been

examined more closely. In general, however, the reproductive cycles of

reptiles are still poorly understood when compared to those of mammals.

The gopher tortoise (Gopherus polyphemus) is a locally abundant

inhabitant of suitable habitats throughout the southeastern Coastal

Plain from extreme southern South Carolina to extreme eastern Louisiana

and peninsular Florida (Auffenberg and Franz 1978,. 1982; Fig. 1). The

species is restricted almost exclusively to areas with well-drained

sandy soils and vegetative associations that permit abundant sunlight to

reach the ground. Here the animals construct extensive underground

burrows and feed on low-growing grasses and forbs (Auffenberg 1969).

Gopherus polyphemus has experienced dramatic population size declines

throughout its range in recent years (Auffenberg and Franz 1982), with

most of the remaining animals concentrated in southern Georgia and

northern Florida.

Information regarding the gopher tortoise's reproductive rate

(Douglass 1976; Iverson 1980; Landers et al. 1980), growth and maturity
























































Figure 1. Geographical distribution of Gopherus polyphemus.
Open circles represent isolated populations.
Adapted from Auffenberg and Franz 1978.










(Auffenberg and Iverson 1979; Landers et al. 1982), and population

composition (Alford 1980) has been compiled. The composite drawn from

the known data is that of a species whose individuals grow slowly,

mature at a relatively late age, and once mature have a very low

reproductive potential. Therefore, G. polyphemus must be categorized as

one of the most K-selected vertebrates studied (Pianka 1970).

Data bearing on the reproductive biology of the gopher tortoise are

included in the studies of mating and clutch development by Iverson

(1980) and Landers et al. (1980). Auffenberg (1966) and Weaver (1970)

described the courtship sequence, Douglass (1976) studied the mating

system, and Rose (1970) and Rose et al. (1969) analyzed the subdentary

(also called chin or mental) gland exudate thought to play a role in sex

and species recognition. However, no data regarding the physiological

factors thought to be important in the reproductive cycle were

available, nor had any information about the male cycle, other than

courtship behavior, been gathered.

The present study of the reproductive biology of Gopherus

polyphemus was initiated for two reasons. First, there was a general

lack of knowledge concerning reptilian reproductive cycles in spite of

their possible evolutionary significance relative to those of mammals.

Second, the continuing decline in gopher tortoise population size

suggested that such data should be gathered as soon as possible in order

that appropriate management and conservation policies regarding the

species can be formulated.












The specific objective of the present study was to examine the

reproductive cycles of male and female gopher tortoises throughout an

annual period and to compare the information gathered to similar data

for other vertebrates in general and other turtles in particular. Data

were to be gathered from studies of gonadal morphology (including

vitellogenic and spermatogenic cycles), changes in the paired glands

located in the underside of the lower jaw of all individuals (a

secondary sexual characteristic), aspects of endocrine system

physiology, and behavior associated with mating. These results would

then be combined with related previously existing information to

describe in detail the reproductive biology of G. polyphemus.















MATERIALS AND METHODS


Preliminary Hormonal Studies


Collection and Housing of Animals

Preliminary hormonal studies were conducted from May to October

1977, and March to August 1978. Male and female tortoises were

collected in Alachua and Lake Counties, Florida, between April 1977 and

April 1978. Most of the animals were captured in bucket traps placed at

the mouths of burrows (Taylor 1982). A few were picked up after being

discovered away from their burrows, usually when they were crossing

roads.

Tortoises were housed in a 275 m2 outdoor enclosure 8 km NW of

Gainesville and allowed to construct and utilize burrows. The number of

animals in the enclosure varied between 20 and 30. Food consisted of

naturally occurring grasses and forbs supplemented with a wide variety

of fruits and vegetables obtained from the refuse of local markets.


Sampling Procedures

During each study period 3 to 7 individuals were periodically

selected from the penned population for blood sampling. During most of

the study some individuals were bled each week. After the tortoises

were weighed and their chin glands measured, 1.0 ml of blood was removed

via cardiac puncture. A sterile 1 or 1 1/2 inch 21 or 23 gauge needle











attached to a 3 ml disposable plastic syringe was inserted through a

1/16 inch diameter hole drilled through the plastron along the midline

at the junction of the pectoral and abdominal scutes. Insertion of the

needle at an angle perpendicular to the surface of the plastron

facilitated blood removal in the vast majority of cases. Prior to

drilling the hole in the shell, the area surrounding the site was

cleaned by scrubbing it with a toothbrush and Betadine scrub solution.

The area was then wiped with a sterile prep pad saturated with 70%

isopropyl alcohol. Immediately prior to sampling each individual, a

small amount of ammonium heparin (aqueous solution--1000 USP units per

ml) was introduced into each syringe as an anticoagulant. The needle

was inserted into the vial and approximately 1 ml of heparin was drawn.

Most of the solution was injected back into the vial, leaving only the

amount of anticoagulant that remained at the tip of the plunger and in

the lumen of the needle. The needle was inserted into the heart only as

far as necessary to allow blood to flow into the syringe.

The drawn blood was placed into heparinized Vacutainer (Sherwood

Medical Industries) blood collection tubes (7 ml; 13 x 100 mm; green

stopper). The tubes containing the blood were placed in a Junior Angle

Centrifuge (Model 1600) and spun at 3000 RPM for 10 minutes. During

centrifugation of the blood the tortoise was prepared for reintroduction

into the enclosure. A stick of dental compound was gently heated with

the flame of a cigarette lighter until the end became soft and began to

"droop". This softened end was touched to the 1/16th inch diameter hole

in the plastron of the tortoise so that a small amount of the compound











partially filled and completely covered the opening. In only a few

seconds the material hardened, providing a solid closure for the wound.

A piece of waterproof adhesive tape was placed over the dental compound

as an additional protective measure. The method worked well, for the

dental material never dislodged during subsequent activities of the

tortoises. It could, however, be easily removed later by prying it

loose with the blade of a pocket knife for additional blood sampling.

After centrifugation, the plasma was pipetted from atop the compacted

cellular-material and placed in a second heparinized Vacutainer tube for

storage at -20 C. Frozen plasma samples were shipped periodically to

the Institute of Animal Behavior, Rutgers University, Newark, New Jersey

for determination of steroid hormone levels by radioimmunoassay.



Detailed Study of Annual Cycle


Collection and Housing of Animals

Tortoises were collected in Alachua, Putnam, and Marion Counties,

Florida, between March 1979 and May 1980. All collecting sites were

within 40 km of each other. Animals were taken primarily by "pulling"

them from their burrows with a collecting hook (Taylor 1982). A few

individuals were bucket-trapped or found away from their burrows. No

animals were utilized that had been injured during the pulling process.

Tortoises were housed in an enclosure inside the Florida State Museum

until they were processed. All animals were examined within 48 hours

after capture. Water, but not food, was provided. Temperature was

maintained at 280C and the photoperiod was 12:12 L/D.











Processing of Animals

Weight and shell measurements were determined for each tortoise, as

well as the length, width, and height above the level of the skin of its

subdentary glands. Blood was then drawn for hormonal analysis following

methods similar to those used in the preliminary hormone study, with the

following exceptions: 1) blood sample size was 7 ml in order to

increase the plasma sample for radioimmunoassay. 2) the hole drilled in

the plastron was not plugged.

Each tortoise was killed by injecting 5 ml of 100% isopropyl

alcohol directly into its brain. This procedure rendered the animals

dead and completely relaxed within 10 to 20 seconds. Immediately after the

death of the tortoise its plastron was removed by cutting through the

bridge with a bone saw (Stryker Corp. autopsy saw, Model No. 9004-210).

The skin, muscle, and connective tissue were then separated from the

plastron with a knife, allowing the peritoneal cavity to be opened and

the viscera exposed.

The reproductive tissues were removed, excess liquid was drained,

and they were weighed on either an Ainsworth Type 21N Electronic Balance

or an Ohaus Dial-0-Gram Triple Beam Balance, depending upon their size.

The tissues were then fixed in either 10% neutral buffered formalin or

Bouin's Fluid. The intact chin glands were also dissected out and fixed

along with the other tissues. Tissues from 23 males and 31 females were

collected in this way. Six additional tortoises included in the study

were females obtained from a hunter who had butchered them. There was

no opportunity to obtain measurements of the live animals, blood











samples, or chin glands. Also, oviducal eggs may have been present and

removed from 3 of them.


Histological Procedures

The right testis and epididymis of each male and the right chin

gland of all individuals were prepared for microscopic examination.

Tissues were fixed for several days, rinsed in running water, and

transferred to 70% ethyl alcohol for storage. As time permitted, the

tissues were prepared for examination under light microscopy using

standard histological techniques (Humason 1979; Luna 1968). Paraffin-

embedded tissues were sectioned at 5 to 7 pm on an American Optical

rotary microtome with a steel knife. Sections were stained with

hematoxylin and eosin.



Analysis of Tissues


Female Reproductive Tract

In addition to the weights of ovaries and oviducts obtained upon

dissection, detailed analyses of ovarian follicles, corpora lutea, and

oviducal eggs were made from preserved tissues. The diameter of each

ovarian follicle greater than or equal to 5.0 mm was determined to the

nearest millimeter with dial calipers. Follicles were grouped into 5 mm

size categories for analysis of the data. Regardless of season, all

females had numerous undeveloped follicles less than 5 mm in diameter.

These were not counted. The number of corpora lutea and/or oviducal











eggs present was determined and the diameter of each one was measured

using dial calipers.


Male Reproductive Tissues

In addition to testes weights obtained upon dissection, several

groups of data were obtained from prepared microscopic sections using

stereological techniques (Chalkley 1943; Underwood 1970; Weibel and

Gomez 1962; Weibel and Elias 1967; Weibel et al. 1966). These pro-

cedures allowed conclusions to be drawn about the three-dimensional

testes based upon analysis of a two-dimensional section taken from those

tissues. A square grid reticle was placed in the ocular of the micro-

scope so that the grid was superimposed over the tissue section. The

grid used was composed of 11 horizontal and vertical lines, giving 121

points of intersection between those lines. Ten randomly selected

fields on a section from the center of each testis were examined using

the grid. The tissue types (seminiferous tubule wall or lumen, inter-

stitial cell of Leydig, vascular component, or connective tissue) under-

lying each of the 121 points was identified and recorded for each of the

ten random fields. From these data the relative proportion of each

testis made up of the component tissue types was determined and seasonal

differences described.

Measurements of the diameter of the seminiferous tubules and

epididymides were made. A calibrated ocular micrometer was used to

measure 15 seminiferous tubules that were most nearly cut in perfect

circular cross section within the section from each testis. As many

circular cross sections through the ductus epididymis as possible were












measured (usually 5 to 8). Changes in the size of these structures were

determined for each of the stages of the spermatogenic cycle.

Approximately 15 measurements were also made of the distance

between adjacent seminiferous tubules within each testis. The number

varied slightly between individuals because the number and arrangement

of cuts through the tubules were not constant. Three representative

fields were selected for each testis. Within each field the distance

from a central tubule to its surrounding neighbors was recorded.

Fluctuations in these intertubular distances were related to changes in

the tubules themselves and the overall condition of the testis.

The process of spermatogenesis was divided into four stages based

upon the diameter of seminiferous tubules, thickness of and number of

cell layers in the wall of the seminiferous tubules, relative

proportions of cell types in the walls, and relative abundance of

spermatozoa within the tubules and/or epididymis of each testis. Each

male was categorized as having testes in one of these four stages.


Chin Glands

The volume of the gland on the right jaw of each tortoise was

calculated from the length, width, and height above the skin

measurements taken prior to dissection. The shape of the gland was

taken to be that of a "sphere" in which the "radii" of the 3 dimensions

are not necessarily equal. The formula used was V = 4/3 i abc; where a

= 1/2 the length measurement, b = 1/2 the width measurement, and c = the

height measurement. The calculated volumes were used as a measure of











gross morphological change in the glands during the annual cycle.

Histologically prepared sections of the glands were examined under a

light microscope to detect changes in structure or cellular development.


Courtship Behavior

Courtship and copulatory behaviors among captive (Auffenberg 1966)

and wild (Douglass 1976) gopher tortoises have been described. Previous

studies, however, have not attempted to quantify these behaviors or

relate them to known physiological conditions in specific animals.

Three attempts (1978, 1979, 1980) were made to gather detailed

information about behavioral sequencing of courtship and copulation, and

to relate these results to data concerning other reproductive parameters

in the same individuals. Unfortunately, these efforts did not result in

the accumulation of any useful information. I feel that the main cause

of this failure was the use of freshly caught tortoises in these

experiments. Animals that have been maintained in captivity for a

number of years are known to exhibit courtship periodically during the

breeding season (pers. obs.). It was my belief that the introduction of

animals recently obtained from the field into a restricted area would

cause increased levels of social interaction and facilitate the study of

mating behavior. Such was not the case, however. The tortoises'

primary interest was to hide themselves from view, and they spent

essentially no time interacting socially with each other. A lengthy

period of acclimation to captivity seems to be required before

individuals of this species can be expected to behave naturally. This

applies whether they are maintained indoors or out. Although no







13



behavioral information could be gathered, the other data regarding

gonadal development, chin gland morphology, hormonal fluctuations, and

the timing of the production of young could be correlated with the

results of previous studies of gopher tortoise reproductive behavior.















RESULTS


Female Reproductive Cycle


Development of Ovaries and Oviducts

Monthly mean weights of female reproductive tissue (Fig. 2) varied

during the year in a manner that reflected the progress of vitello-

genesis within the ovaries and the periodic presence of oviducal eggs.

Spring (March-April) and winter (December-February) weights were both

significantly greater than those of summer (May-September) (t = 5.42 and

5.43; p < 0.001 for both). Combined ovary and oviduct weights were

minimal in May. The single May female examined in detail had ovi-

posited. Three additional May females were obtained from a hunter, so

fresh tissue weights were not available. Their ovarian and oviducal

development was, however, similar to that of the more completely studied

individual. One of the 2 individuals in June contained 4 oviducal eggs,

causing the mean reproductive tissue weight for that month to be some-

what higher, but values in August and early September were consistently

low again. Dramatic increases in size occurred from late September

through November, but weights levelled off in December and remained

fairly constant through April. The combined weights of ovaries and

oviducts of females examined after vitellogenesis was complete but

before oviposition were 3 to 5 times as great as for those that had

oviposited but not yet initiated growth of the next group of follicles.






















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The month with the greatest mean reproductive tissue weight was April

1979, when 4 of 9 individuals contained oviducal eggs, including 2 that

had clutches of 10 eggs each. In general, the weight of ovaries

depended upon the stage of vitellogenesis shown by their follicles (see

below). Oviducts exhibited their greatest diameter and wall thickness

during the nesting season (late spring-early summer). Oviducal develop-

ment was somewhat reduced during late summer and reached its minimum in

the fall.


Vitellogenic Cycle

Ovarian follicles were grouped into 5 mm size categories for each

female throughout the year (Table 1). A summary of these data by month

shows the trend toward increasing ovarian follicle size during the late

summer and fall (Table 2). I devised a measure of the stage of vitello-

genesis based on the degree of follicular development within each indi-

vidual. This measure (Vitellogenic Index) provides a means of assigning

a numerical value to the stage of development of each ovary. Based on

the size of her largest follicle, each female was given a number: less

than 5 mm = 1, 5-9 mm = 2, 10-14 mm = 3, 15-19 mm = 4, 20-24 mm = 5, 25-

29 mm = 6, 30 mm or greater = 7. The highest relative value (= 8) was

assigned to those individuals possessing oviducal eggs or corpora lutea.

The values for all females in each month were averaged to give the

overall measure of Vitellogenic Index (Fig. 3). The shape of this curve

graphically depicits the progress of the vitellogenic cycle in my sample

of female tortoises.













Table 1. Summary of data regarding vitellogenesis and egg
production obtained from female Gopherus polyphemus.
The lists of numbers in each column represent the quantity
of oviducal eggs, corpora lutea, and/or ovarian follicles
in each size category found in each female.


FOLLICLES

TORTOISE OVIDUCAL CORPORA 25- 20- 15- 10- 5-
ID DATE EGGS LUTEA e30mm 29mm 24mm 19mm 14mm 9mm <5mm


3 MAR 79
5 APR 79
7 APR 79
10 APR 79
14 APR 79
25 APR 79
25 APR 79
25 APR 79
27 APR 79
28 APR 79
24 MAY 79
13 JUN 79
14 JUN 79
2AUG 79
2 SEP 79
2 SEP 79
LI SEP 79
24 SEP 79
25 SEP 79
30 SEP 79
4 OCT 79
23 OCT 79
24 NOV 79
15 DEC 79
I FEB 80
26 MAR 80
27 MAR 80
8 APR 80
15 APR 80
18 APR 80
31 MAY 80
31 MAY 80
31 MAY 80


3

3
1
4


6
8
7
6 6 I
7
8
9 9

4 4
5


2940
2835
2934
2889
2931
2782b
2810b
2700b
2921
2831
2868
2502
2503
2854
2612
2775
2822
2889
2916
2734
2257
2618
2664
2668
2899
2726
2696
2635
3068
2656
2957b
2958b
2959b


6








2


2
2
2


7 7
7
5
5


10
6
3
3
3
4
I
2

3

4
2
I1



7 2
3


I *

3 *
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* ALL INDIVIDUALS HAD NUMEROUS MINUTE FOLLCLES IN EACH OVARY.
LESS THAN 2mm IN DIAMETER.


MOST OF THESE WERE


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Corpora Lutea

Corpora lutea were found in the ovaries of 12 females, during

April, May, June, and September. The mean corpus luteum diameter for

each individual was plotted against the date on which the female was

examined (Fig. 4). The equation for the resulting least squares

regression was calculated. The identification of small corpora lutea

became very difficult because of their tendency to become embedded in

the surrounding ovarian tissue and the lack of differentiation by color.

The smallest corpora lutea that could be positively identified as such

were about 5 mm in diameter.


Hormonal Fluctuations

Plasma estrogen and progesterone levels were determined for 28

females examined between March 1979 and May 1980. The mean value for

all individuals in each month was calculated (Figs. 5 and 6). Estrogen

levels were elevated in early September and from December through April

(the period of maximum follicular development). Both the spring (March

and April) and fall (September) peaks were significantly greater than

the combined summer (May-August) values (t = 2.32 and 2.94; p < 0.05 for

both). A more brief depression in estrogen levels occurred between the

late September and December peaks (Fig. 5).

Plasma progesterone also exhibited 2 peaks in concentration (Fig.

6). Dramatic increases occurred in April, when all individuals have

either recently ovulated or possessed enlarged follicles that were ready

to be released, and October. Following the April peak, progesterone

levels declined steadily but relatively slowly throughout the summer to

























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minimal values in September (t = 3.11; p < 0.01). The trend following

the October peak was much different. Values declined rapidly to very

low levels in November, and remained there through February (t = 3.20, p

< 0.01, April vs. November-February).

The results from the preliminary hormonal study of 1977 and 1978

provide additional data that do not always agree with those of 1979-

1980. I do not doubt that the 1977 and 1978 determinations accurately

reflected the physiological condition of the tortoises that were

sampled, but I feel that these data may not be representative of wild

populations because 1) the samples were from individuals being kept in

captivity, 2) the reproductive condition of the tortoises was not known,

3) samples were not obtained from throughout the entire year 4) some

individuals were being sampled repeatedly and what effect this had on

the animals was unknown. In spite of these potential problems, the

amount of information on turtle reproductive endocrinology is so limited

that the results of the preliminary studies are of value.

Progesterone determinations were made for females between mid May

and late October 1977, and late February to late August 1978. In 1977,

peaks in concentration occurred in early June and mid July, which does

not conflict with the results of 1979-1980. The few determinations from

August, September, and October 1977 are all consistently low. The sharp

peak shown in October 1979, is conspicuously absent from the earlier

data, although a rapid increase and decrease could have been present but

not observed. The progesterone data for 1978 show an entirely different

picture. Whereas in 1977, concentrations in the 1 to 4 ng/ml range were











common (11 of 35 determinations [31.4%] were greater than 1.0 ng/ml),

the values from 1978 were much lower overall. Of 53 measurements in

1978, only 2 (3.8%) were greater than 1.0 ng/ml. Most samples were

taken in June and July of both years so timing should not be the cause

of this difference. As an extreme example of the low levels of 1978,

one individual was sampled 11 times between 24 February and 27 July, and

not once was her plasma progesterone level above the minimum detectable

concentration of 0.050 mg/ml.

Fewer determinations of plasma estrogen concentration were made

than for progesterone because of the limited amount of plasma in each

sample. Thirty-eight were made for 1978 individuals (vs. 53 for proges-

terone) and none in 1977. The data from the early part of the sampling

period in 1978 agreed with those obtained in 1979-1980, but determina-

tions from the later sampled months differed considerably. Levels in

February were high and approximately equal to those seen in February

1979. Concentrations were declining or low throughout March and April,

similar to the trend a year later. However, by late May and through

July, values were generally rising and high, although some temporary

declines were evident. This trend was quite different from that in

1979, when the lowest levels of the year were seen between May and

August. The possible causes, and the physiological significance, of the

progesterone and estrogen concentrations observed will be discussed

below.












Chin Gland Structure

Each externally visible "gland" is actually a composite of 2 to 4

simple tubular holocrine glands, each of which has a duct leading to a

pore that can be seen at the surface (Figs. 7 and 8). The stratified

squamous epithelium of the epidermis is keratinized but scaleless. The

highly vascular dermis is composed of dense irregular connective tissue

and contains the glands proper. -Beneath the dermis is the somewhat less

dense hypodermis, and the underlying skeletal musculature.

Each of the individual secretary glands is surrounded by a dense

connective tissue capsule that is differentiated from, but grades into,

the connective tissue of the dermis. Septa that are continuous with

this surrounding capsule extend into and partially subdivide each gland.

There are no associated muscle fibers that might aid in secretion. The

cells making up each gland appear similar to those of mammalian seba-

ceous glands. Three structurally and functionally separate layers can

be identified within each gland. The basal or germinative zone is the

peripheral region of the gland adjacent to the surrounding capsule. It

is composed of only a few layers of small cells that have small nuclei

and little cytoplasm. In keeping with the holocrine nature of these

glands, it is assumed that the basal zone is a region of mitosis and the

source of the secretary cells. Inside the basal zone is the much

thicker proliferative zone. The cells in this layer possess nuclei that

are 2 to 3 times as large as those in the basal zone, and amounts of

cytoplasm that are relatively even more abundant. Presumably as these

cells are enlarging they are manufacturing the secretary product of the




























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of a gopher tortoise. Portions of the
basal, proliferative, and defoliative
zones and the duct can be seen (63X).


Figure 8.











gland. The thickness of this zone varies with the activity of the

gland. If the gland is secreting, it may be up to 50 cell layers thick,

whereas in an inactive gland it may be almost unidentifiable. Inside

the proliferative zone is the relatively narrow defoliative zone, where

the secretary cells degenerate and the glandular product is released

into the duct. Before the cells reach the surface of the defoliative

zone they become reduced in size again, presumably as the metabolic

activity of the cell slows down. When the gland is active, the surface

of this layer has a ragged appearance as the cellular components are

cast off with the secretary product.


Female Chin Glands

Female chin gland development was maximal during April and May,

with decreased volumes from June to August (Fig. 9). A secondary

increase in chin gland size occurred in early September, after which the

trend toward reduced size was resumed. Minimal sizes were seen in

December, at which time the calculated volumes were only about 13% of

the May maximum. A trend toward increasing sizes began after December

and led into high values again the following April.

Microscopic examination of chin gland tissue revealed no cellular

differences between enlarged and shrunken glands, other than the

relative thickness of the proliferative zone (Fig. 10 and 11). The

inner and outer zones appear almost the same in all months, except for

the degree of "raggedness" of the defoliative zone. Even within the

proliferative zone, the cells themselves do not vary substantially in

size or shape, only in their number. When stained with the standard
























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Figure 10.


Section through a gopher tortoise chin gland in
the "active" condition. Note especially the
great width of the proliferative zone and the
uneven edge of the defoliative zone bordering
the duct (125X).


Figure 11.


Section through a gopher tortoise chin gland in
the "inactive" condition. Compared to Figure 10
the proliferative zone has almost disappeared
and cells bordering the duct form a smooth intact
boundary (125X).










hematoxylin and eosin technique, no staining differences between active

and inactive glands could be detected.


Maturity, Fecundity, and Young

Values in the literature for size at sexual maturity for north

Florida gopher tortoises are females--220 to 230 mm plastron length

(Iverson 1980); males--230 mm carapace length (Auffenberg and Iverson

1979), which also equals 230 mm plastron length following the conversion

factors of Taylor (1982). It was not the purpose of the present study

to determine precisely size at sexual maturity, but some revealing data

regarding the subject were obtained. Using the presence of sperm in

either the seminiferous tubules or epididymis as the criterion for

maturity, all 23 males examined were mature. The smallest of these had

a plastron length of 182 mm, substantially smaller than the previously

established lower limit. Additionally, 6 other males had plastron

lengths less than 230 mm and were sexually mature. The data for females

do not entirely support the limits set by Iverson (1980). Females were

judged to be mature if they possessed oviducal eggs, corpora lutea, or

ovarian follicles greater than 5 mm in diameter. Of 31 females

examined, 4 were immature. The largest of these had a plastron length of

228 mm. The smallest mature female was 210 mm in plastron length,

although all other mature females were at least 220 mm.

Clutch size varied from 2 to 10 in 24 females that had either

oviducal eggs, corpora lutea, or enlarged follicles. Clutch size when

calculated for all criteria combined was 6.71 + 2.22 (mean + std. dev.).

When determined separately, mean clutch size based on oviducal eggs












(6.86 + 3.07, n = 7) is most similar to the overall mean. The cal-

culated mean based on number of corpora lutea (6.08 + 2.39, n = 12) is

probably lower because of the difficulty in identifying some of the

smaller ones within the ovaries of individuals that ovulated some time

ago. Conversely, the mean of enlarged follicles (7.25 + 1.36, n = 12)

is larger because not all follicles of "mature" size are ovulated. The

mean clutch size of my sample population (6.71) is much closer to the

figure derived by Landers et al. (1980) for G. polyphemus in south

Georgia (7.0) than to Iverson's (1980) estimate for north Florida

gophers (5.18).

Five females were examined in which both oviducal eggs and corpora

lutea were present. In 3 of these the number of oviducal eggs and

corpora lutea in the right and left halves of the reproductive tract

were not equal, indicating transuterine migration. In 2 of these 3, net

migration was away from the side with the greater number of corpora

lutea. Iverson (1980) found 4 females with evidence of transuterine

migration. Net migration was away from the more productive ovary in 2

of those individuals, but Iverson concluded that transuterine migration

probably served to equalize reproductive tract volume. The few data

available on the subject do not constitute a basis for drawing any

conclusions about the adaptive significance of transuterine migration.

I examined the reproductive tracts of 7 females whose oviducts

contained shelled eggs. In 4 of these cases (5, 7, and 28 April 1979,

and 18 April 1980) calcification of the eggs was not very far

progressed. After removal from the females, these eggs were preserved










along with the remainder of the reproductive tracts. The ovaries and

oviducts of one female were obtained from a hunter (25 April 1979). The

6 well-calcified eggs from that individual were removed and eaten by the

hunter. The other 2 clutches of oviducal eggs were removed from indi-

viduals I had dissected (13 June 1979 and 26 March 1980) and looked as

if they were ready for oviposition. I incubated both of these clutches

but only one (13 June 1979) developed. All 4 of the eggs hatched, after

108, 109, 110, 111 days incubation at 28 C. Measurements of the

hatchlings were taken 2, 19, and 282 days after the last one emerged

from its shell (Table 3).



Male Reproductive Cycle

Testicular Changes

Mean monthly weights of male reproductive tissue (testes plus

epididymides) showed significant seasonal variation (Fig. 12). Testes

were lightest during the winter, spring, and early summer. Means of the

values from September and October (n = 7) were significantly greater

(0.278 vs. 0.134 g/100 g body weight) than values from the rest of the

year taken together (n = 16). In Figure 13, which depicts this

relationship, testes weights that have been adjusted for body size are

plotted against total body length. At all body sizes the values from

September and October are consistently greater than those from all other

months. The fall values (late September-October) were significantly














Table 3. Mass and total length measurements of 4 hatchling gopher
tortoises from a clutch of 4 eggs removed from a female
13 June 1979. Hatchlings are listed in order of emergence
from their eggshells. Mass in grams; Total shell length
in mm. Tortoise 122 died in December 1979.


4 October 1979 21 October 1979 10 July 1980
Tortoise ID Mass Total Length Mass Total Length Mass Total Length


L1 28.1 49.2 30.4 51.6 51.7 62.6

R1 28.8 47.7 29.4 49.2 50.5 60.1

L2 26.8 47.5 29.9 50.1 45.6 59.6

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greater than those of July (t = 5.94; p < 0.01) and December (t = 4.19;

p < 0.01).

Changes in the tissue types within each male's testes were shown in

two ways. The relative proportion of each testis that was composed of

the various tissue types showed dramatic variation between months (Fig.

14). In the spring (the period of courtship and mating and when overall

testis size is minimal) the proportion of the testis that is composed of

seminiferous tubules was at its lowest level (only about 10% in April).

By September and October, however, the tubules had become predominant,

representing almost 60% of the testicular tissue. The combined mean for

the males examined in September and October (58.8 + 4.5%, n = 7) was

49.8% greater than that of individuals from all other months (39.2 +

4.2%, n = 16). The difference between September-October and April was

highly significant (t = 15.8; p < 0.001).

Changes in the diameter of the seminiferous tubules and their

lumens exhibited seasonal variation that corresponded to the propor-

tional changes. Tubules increased slowly in size during the summer, but

enlarged rapidly and reached their greatest diameter in the fall (t =

5.76; p < 0.001, September-October vs. April) (Fig. 15). Mean tubule

and lumen diameters for the September/October group (0.277 + 0.038 and

0.183 + 0.027 mm, n = 7) are 180 and 130% greater than the mean of those

individuals from the rest of the year (0.154 + 0.038 and 0.079 + 0.027

mm, n = 16). During the fall period of tubular enlargement, the lumens

showed a relatively greater increase in size than did the entire tubule

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absolute difference between the tubule and lumen diameters remained

about the same for all months (only one was outside the range of 0.06 to

0.11 mm), the dramatic increase in size by both resulted in a greater

proportional change in the lumen (maximum 3.7 times minimum vs. 2.7

times for tubule).


Spermatogenic Cycle

A series of spermatogenic stages was described to evaluate the

cellular changes taking place within the seminiferous tubules of each

male. The number of cell layers making up the wall of the tubule, the

relative numbers of the meiotic cell types contained within the wall,

and the presence of sperm in the tubule lumen were considered prior to

categorizing each male into one of the 4 stages (Table 4). Stage 1

(inactive) represents the period of minimal spermatogenic activity. The

tubule wall is only 1 or 2 cell layers thick and is comprised only of

spermatogonia and Sertoli cells (Fig. 16). Individuals classified as

Stage 2 (maturing) exhibit proliferation of spermatogonia, differenti-

ation of spermatogonia into primary spermatocytes, and production of

many secondary spermatocytes and some spermatids via meiotic divisions.

No sperm are present yet (Fig. 17). The period of maximal sperm produc-

tion (spermiogenesis) characterizes Stage 3. Large numbers of both

spermatids and sperm may be found within the tubules of males in this

stage, but only one or the other may be abundant, depending upon the

exact time of the cycle (Fig. 18). During Stage 4 sperm leave the

seminiferous tubules and pass out to the epididymis (spermiation).













Table 4. Characteristics of the four stages of spermatogenesis
found in male Gopherus polyphemus.







Stage Condition of Seminiferous Tubules



1 Inactive; only spermatogonia; tubules reduced

in diameter.


2 Maturing; mitosis of spermatogonia; many

spermatocytes, some spermatids, no sperm

present; tubule diameter increasing.


3 Spermiogenesis; few to many spermatids and

sperm; tubule diameter maximum.


4 Spermiation; sperm abundant in lumen or passing

into epididymis; few spermatocytes or spermatids

forming.
































Figure 16.


Figure 17.


Section through a gopher tortoise testis in
the inactive condition (Stage 1). The tubule
wall is thin and contains only spermatogonia
and Sertoli cells (500X).


Section through a maturing gopher tortoise testis
(Stage 2). The enlarging tubule has a wall
composed of several layers of spermatogonia,
spermatocytes, and spermatids (500X).










Therefore, sperm may or may not be abundant in the tubule lumen. Unlike

Stage 3, few new spermatocytes or spermatids are forming (Fig. 19).

The number of males exhibiting each spermatogenic stage was

recorded by month (Table 5). The resulting trend in the timing of each

stage during the annual cycle correlates closely with changes in overall

testis size and detailed changes in seminiferous tubule diameter and

relative tissue composition of the gonad. Individuals exhibiting Stage

1 are found from December to April, when testis size, tubule diameter,

and relative tubule proportion are minimal. During Stage 2 (June and

July) tubules are enlarging and becoming a greater proportion of the

testicular tissue. Stage 3 extends from July to September and corre-

sponds to the period when gonadal weight, tubule diameter, and relative

tubule abundance all increase to their maxima. Throughout most of Stage

4 (September to December) these three variables are declining to their

basal levels.

Changes in the epididymides reflected what was occurring within the

testes (Fig. 20). During the spring and summer (the breeding season and

about 2 months afterward) the mean diameters of both the ductus

epididymis and the included sperm mass decreased slowly. In September,

however, they both increased dramatically in size, reaching levels that

were maintained throughout the winter and until the following breeding

season (t = 5.58 [ductus] and 11.9 [sperm mass]; p < 0.01 and 0.001,

September-October vs. July). The fall increase was a result of the

movement of sperm from the testes during and shortly after the period of

maximum spermatogenic activity (Figs. 21 and 22).





























Figure 18. Section through a gopher tortoise testis undergoing
maximum spermiogenesis (Stage 3). The large diameter
tubule contains many spermatids and sperm (500X).


Figure 19.


Section through a gopher tortoise testis with
abundant sperm in the tubule lumen (Stage 4).
Although sperm are numerous, few spermatocytes
or spermatids remain in the tubule wall. Tubule
diameter remains large (500X).

















Table 5. Monthly number of male Gopherus
polyphemus in each spermatogenic
stage.





Month 1 2 3 4


Mar 3

Apr 3

Jun 3

Jul 2 1

Sep 5 1

Oct 1

Dec 2 1

Feb 1










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Figure 21.


Figure 22.


Section through the epididymis of a gopher tortoise
in late fall. Spermiation is complete, resulting in
the large mass of sperm present within the ductus
epididymis. Only a small portion of the duct may be
seen in the field (125X).


Section through the epididymis of a gopher tortoise
in early fall. Maximum spermatogenesis has not yet
occurred. The number of sperm within the duct
represents the minimum amount seen during the annual
cycle. Compare the diameter of the duct to that in
Figure 21 (125X).










Testosterone Fluctuations

Plasma testosterone exhibited decreasing concentrations just prior

to and throughout the breeding season (February to May), and reached its

lowest level of the year in June (Fig. 23). Despite the large differ-

ence between the February and June means, the difference is not statis-

tically significant, however, due to the large amount of variability in

the data and small sample sizes. After June, the hormone's concentra-

tion rose again, reaching relatively high levels again in September and

October. This secondary peak in the fall preceded another decline

during the early part of the winter period of inactivity. The major

(spring) peak in plasma testosterone occurred at a time when the

tortoises were emerging from their burrows and becoming more active, but

declining values were well underway by the time courtship and copulation

would be expected. The late summer/fall increase corresponded to the

period of active spermatogenesis, with the highest values occurring

during maximum spermatogenesis (September). Chin gland volumes also

showed a secondary increase at the same time.

The preliminary results from 1977 and 1978 generally agreed with

those obtained in 1979-1980. Since data were collected between May and

October 1977, and between February and August 1978, it is not possible

to compare values for all months (Table 6). However, the two greatest

monthly means were both observed in February (1978 and 1979-1980).

Values declined rapidly in March of both years. The three months in

which the mean concentrations were the lowest were May 1977, May 1978,

and June 1979, indicating that late spring and early summer were
















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Table 6. Plasma testosterone levels (ng/ml) in male
gopher tortoises. Means were calculated
from the values of all males sampled each
month. The numbers in parentheses are
sample sizes.


1977 1978 1979-1980


February


March

April


May


June


July

August


September

October

November

December


6.51 (7)

38.37 (10)

27.96 (6)

14.11 (2)

31.62 (2)

23.82 (3)


89.87 (3)

9.26 (3)

10.86 (11)

4.83 (16)

19.54 (18)

9.27 (5)

39.91 (10)


103.99 (3)

38.50 (3)

35.28 (3)


9.42 (3)

37.69 (3)


73.36 (5)

41.73 (1)


43.14 (3)











consistently the seasons when testosterone levels were minimal. The

secondary fall peak present in 1979-1980 was seen to a lesser degree in

1978, but was not observed in 1977. An increasing trend was indicated

between July and August 1978, so that values in September could have

approached the level of those from September 1979, but since data col-

lection in 1978 did not continue past August, this possibility could not

be confirmed. Since the animals sampled in 1977 had all been recently

captured and relocated, the hormone levels seen during the summer and

fall could have been affected by the stress of those activities.


Male Chin Glands

Maximum male chin gland volumes were observed in March and April

(Fig. 24). Following this portion of the breeding period volumes

declined until mid-summer (t = 5.15; p < 0.01, April vs. July), then

exhibited secondary increases in September and October (t = 4.46 and

6.31; p < 0.02 and p < 0.01, vs. July and December). Subsequently, they

declined again during late fall and reached their smallest size of the

year in mid-winter (December-January). Glands enlarged again in late

winter/early spring, prior to the next breeding season. The values from

the spring (April) and fall (late September-October) peaks were not

significantly different (t = 2.15; p < 0.1).

Microscopic changes in male chin gland structure were similar to

those found for females. There was no cellular or structural difference

between enlarged and reduced glands, except for a change in the thick-

ness of the proliferative zone. Once again, the factor causing the

overall change in chin gland volume was the number of cell layers







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present in that region, rather than a difference in cell type or size

anywhere within the gland.















DISCUSSION


Female gopher tortoises exhibit a distinct temporal separation of

the vitellogenic and ovulatory portions of the reproductive cycle.

There has been no evidence of the production of more than one clutch

annually by females of this species. All follicles ovulated each

nesting season (April through June) have undergone enlargement during

the previous fall and reached ovulatory size by December. A comparison

of maximum ovarian follicle sizes and the diameters of ova removed from

eggs by Iverson (1980) also indicates that no vitellogenic enlargement

of follicles occurs during the mating or nesting seasons. This scheme

differs from those of Chrysemys picta (Gibbons 1968), Trionyx muticus

(Plummer 1977), and Kinosternon subrubrum (Iverson 1979), all of which

can produce more than one clutch per nesting season. These species have

a period of vitellogenesis in the fall, but they also can form at least

one more clutch that undergoes rapid vitellogenesis shortly after the

first nesting of the year. The most extreme example of multiple nest-

ings among turtles involves the sea turtles. Female Chelonia mydas may

nest several times in a season and lay 100 eggs per nest, although they

do not nest every year (Carr 1967). A female of any species that

produces several clutches of eggs during one nesting season is precluded

by size constraints from possessing within her body more than a rela-

tively small percentage of the enlarged follicles at any one time.












By necessity, vitellogenesis must be progressing in a series of stages

during the nesting season. Domantay (1968) has reported that female

Chelonia mydas contain a graded series of follicles, with each set

successively moving up in size category as each successive clutch is

laid. The giant Aldabran tortoise (Geochelone gigantea) exhibits a

condition similar to that of the gopher tortoise with respect to vitel-

logenesis (Bourn 1977). This species undergoes vitellogenesis between

November/December and June/July, with ovulation of a single clutch

following soon after. Callard et al. (1978) reported that in Chrysemys

picta 3 distinct sets of ovarian follicles could be identified based on

their grouping into size categories. Callard concluded that only one set

of follicles was ovulated each year in females from the population that

was studied, and that that set had been undergoing vitellogenic growth

for 3 years.

The selective advantage of increasing clutch size and/or number of

clutches per season is clear, and many turtle species produce large

numbers of eggs per year by doing one or the other, or both (Moll 1979).

The factors placing limitations on these variables are not as easily

evaluated. Climate, diet, energetic efficiency of egg production, and

metabolic capabilities could all affect the ability of individuals of a

species to produce multiple clutches. The gopher tortoise possesses one

of the lowest annual reproductive potentials of any turtle, producing

only 1 clutch of 5-7 eggs per year (Moll 1979; Iverson 1980; Landers et

al. 1980). Additionally, the proportion of animals that survives to

hatch, grow, and become sexually mature is extremely small (Auffenberg











and Iverson 1979; Alford 1980). The ultimate causes of this low repro-

ductive potential and the question of whether the species can survive

under the conditions it faces today need immediate study (Auffenberg

1969; Auffenberg and Franz 1982).

Female reproductive tissue weights show the expected trends during

the annual cycle based upon the observed periods of vitellogenesis and

ovulation. Ovarian recrudescence follows oviposition by about 3 months,

after a summer-long period of minimal development. The occurrence of

mating and oviposition in the spring followed by vitellogenesis in the

fall is typical of other temperate turtle species (Gibbons 1968; Lewis

et al. 1979; Plummer 1977; Iverson 1979). Ovarian follicles grow at a

rate of about 5 mm per month between August and December. In August,

follicles grow out of the "less than 5 mm" category, and by December

have reached the maximum ovulatoryy) size of slightly over 30 mm.

The role played by plasma steroids in the control of the reproduc-

tive cycles of female turtles has not always been clear. The results of

the few studies that have been completed have not consistently indicated

the same relationships between ovarian and behavioral events and fluctu-

ations in measured hormone levels. Possible causes of these differences

include 1) the taxonomic diversity of the species examined, 2) the

range in geographic sources of animals, 3) various ecological positions

filled by the species examined, 4) different treatments and experimental

designs.

Klicka and Mahmoud (1977) gave exogenous progesterone injections to

mature preovulatory painted turtles (Chrysemys picta) to test for the












effect of this steroid on ovarian weight, follicle size, and oviduct

weight. While ovarian weight was not significantly changed in response

to this hormonal treatment, both follicle diameter and oviduct weight

were reduced. In experiments testing for the effects of progesterone on

oviposition in females bearing oviducal eggs, Klicka and Mahmoud found

that the hormone prevented oviposition to a great extent. Progesterone

was also shown to be very effective in preventing ovulation. No preovu-

latory turtle that received exogenous progesterone subsequently ovu-

lated. The authors concluded that progesterone acted as an antigonadal

agent in C. picta by affecting the pituitary and reducing production of

gonadotropin from that organ, rather than disrupting estrogen levels as

had been proposed for lizards (Callard et al. 1972). In addition to

affecting the reproductive tissues, the hormone caused a significant

reduction in pituitary weight compared to controls, but its antigonadal

effects were overcome when it was administered in conjunction with

pregnant mare serum gonadotropin. The physiological mechanism of pro-

gesterone control over gonadal events was not clearly elucidated by

Klicka and Mahmoud, but some insight into its action was gained. High

progesterone levels might inhibit ovulation as long as a previous clutch

of eggs remains in the oviducts but upon oviposition the level of the

hormone falls and ovulation of mature follicles could again occur. A

high progesterone concentration could also prevent oviposition by

blocking contraction of the smooth muscles of the oviduct, in a manner

similar to that in which progesterone from the placenta maintains preg-

nancy in mammals (Turner and Bagnara 1976).











The first workers to measure changes in circulating hormone levels

coincident with the reproductive cycle of a female turtle were Callard

et al. (1978). They examined the relationship between the plasma

steroids estrogen, progesterone, and testosterone and the reproductive

cycle of Chrysemys picta. Their study revealed dramatic increases in

the levels of all 3 hormones just prior to ovulation (July) and equally

rapid returns to low levels shortly thereafter. Levels of estrogen and

testosterone also rose significantly again during fall ovarian recru-

descence. Callard et al. concluded that estrogen facilitated the

production of vitellogenin in the spring (final maturation of preovula-

tory follicles) and fall (ovarian recrudescence), but questioned the

fact that the period of greatest follicular growth (fall OR) was charac-

terized by the lesser of the two peaks of estrogen. They also

questioned the absence of a significant progesterone peak in the fall,

when it might be expected that progesterone would exhibit a rise con-

comitant with that of estrogen, as was the case in the periovulatory

phase. Callard et al. hypothesized that the role(s) of progesterone in

the spring is/are to 1) play a role in follicular maturation by slowing

yolk uptake into the follicle just prior to ovulation, 2) facilitate

ovulation once follicles are mature, then cause a reduction in gonado-

tropic hormone level, 3) decrease oviduct motility after ovulation until

eggs can be shelled and readied for oviposition.

Licht et al. (1979) made repeated measurements of plasma steroid

levels in female green sea turtles (Chelonia mydas) throughout the

nesting season. Their results were similar to those of Callard et al.











(1978) in that for each ovulation there was a distinct peak in proges-

terone. Since these females nested repeatedly approximately every two

weeks, a series of periovulatory peaks resulted, separated from each

other by periods characterized by much lower levels. These data

supported the hypothesis of Callard et al. that progesterone is involved

in the final stages of follicular maturation. Licht et al. found,

however, that estrogen did not also show periovulatory peaks in the

green sea turtle. Since it was believed that estrogen played a role in

the accumulation of vitellogenin for each clutch of eggs, the observed

levels could not be explained. After having shown a moderate rise

during the mating (pre-nesting) period, the levels of the hormone

remained low throughout the nesting season. The cause for the observed

differences in the hormonal fluctuations between C. picta and C. mydas

is unknown, but Licht et al. hypothesized that it may be a result of the

different environments to which the two species are adapted (temperate

lakes vs. tropical oceans) or the striking differences between their

periods of ovarian growth and reproductive seasons (relatively short,

producing one or two clutches vs. prolonged, with several clutches).

Concentrations of progesterone and estrogen were determined in the

female snapping turtle (Chelydra serpentina) by Lewis et al. (1979).

Measurements were made in December (hibernation), May (prior to ovula-

tion), June (following ovulation), and September (ovarian recrudescence

and vitellogenesis). The possession of data from only 4 times during

the annual cycle makes evaluation of the results of this study somewhat

difficult, because of the possibility of missing significant











fluctuations during other periods. The conclusions to be drawn, how-

ever, are not substantially different from the general trends seen in

other studies. Estrogen concentrations were highest in May and

September, the periods of follicular maturation and ovarian recrudes-

cence. Progesterone was high in June, when oviducal eggs and corpora

lutea were present, but consistently low at the other 3 samplings.

In light of previous studies, the roles played by the steroid

hormones estrogen and progesterone in the control of ovarian and ovi-

ducal events in female Gopherus polyphemus may be evaluated (Table 7).

Near the time of ovulation, estrogen level is high, facilitating final

growth of follicles prior to their release. Rising and high levels of

progesterone prevent ovulation and end follicular growth. After

estrogen and progesterone concentrations fall, vitellogenesis ends (E)

and ovulation can occur (P), but oviposition is still prevented until

the levels of progesterone become low enough, allowing sufficient time

for albumin and eggshell deposition within the oviducts. During the

summer, estrogen is basal and no ovarian growth occurs. Progesterone

continues to decline, removing any inhibition to ovulation or oviposi-

tion that might not yet have occurred. In August and September the

estrogen level rises dramatically, resulting in substantial follicular

yolk accumulation and ovarian weight increases. Progesterone continues

to decline during this period. In October, progesterone rises and

estrogen falls sharply. The increased antigonadal effect of

progesterone and the removal of the stimulus for vitellogenin accumula-

tion become evident. The result is that ovarian growth is halted and











Table 7. The role of estrogen and progesterone in the
control of ovarian and oviducal events in
female Gopherus polyphemus.


Hormonal
Months) Fluctuation Events


March-April




April-May




May-August

May-September


August-September


October


E high
P rises



E falls
P falls



E low


P falls slowly


E rises
P low


--follicles in final maturation
- ends follicular maturation
prevents ovulation
reduces excitability of oviduct

- vitellogenesis ends
- ovulation facilitated by sharp decline
oviposition facilitated when moderate
levels are reached

- vitellogenesis prevented

+ ovulation and oviposition may occur
antigonadal effect removed
ovaries prepared for fall vitellogenesis

-ovarian growth and vitellogenesis begins
+antigonadal effect not expressed


E drops sharply *vitellogenesis not
facilitated

P rises sharply--antigonadal effect


November-December E rises again -+ follicular growth
stimulated

P falls again -antigonadal effect
removed .


no ovarian growth
between September
and November


1) follicles reach
near ovulatory
size
2) reproductive tissue
weight attains
maximum and levels
off


December-February E high

P low


+ follicles maintained in near-ovulatory
stage
+ ovarian growth and maintenance not
inhibited











follicles enlarge only slightly. A reversal of the hormonal condition

takes place in November, with estrogen returning toward maximal levels

and progesterone nearing zero. In response, yolk deposition resumes

again shortly thereafter. From December until nearly the next nesting

season estrogen remains high, progesterone remains low, and ovarian

follicle diameters remain near nesting season levels. As the period of

ovulation approaches in the spring, the concentrations of both hormones

begin their cyclic fluctuations once again.

These hypotheses regarding the relationship between the plasma

steroids and the female reproductive cycle generally agree with those

proposed by Klicka and Mahmoud (1977), Callard et al. (1978), and Licht

et al. (1979). My data differed substantially only from those of Licht

et al. who found no periovulatory or vitellogenic estrogen peaks in

Chelonia mydas. The facilitation of yolk accumulation by estrogen in

Gopherus polyphemus seems clear, as it does in Chrysemys picta (Callard

et al. 1978). The hormone or other factor that influences vitello-

genesis in the absence of elevated estrogen levels in the green sea

turtle is unknown.

The dramatic change in the relative concentrations of G. polyphemus

estrogen and progesterone during October was quite interesting.

Although Callard et al. (1978) did report a minor increase in progeste-

rone during September for C. picta, a sharp rise and decline during the

fall had not previously been reported for any turtle. This temporary

increase seemed to be the cause of striking effects on the concentration

of plasma estrogen and the rate of follicular growth. The estrogen











level plunged while progesterone was elevated, and the previously steady

increase in ovarian weight was reduced to zero at the same time.

Whether progesterone exerted this effect by disrupting the estrogen

concentration directly (Callard et al. 1972) or by reducing gonadotropin

secretion via some influence on the hypothalamo-hypophyseal pathway

(Klicka and Mahmoud 1977) is not known. Whatever the mechanism, the

evidence for a strong antigonadal effect by progesterone seems clear.

The significance of the pause in fall ovarian growth may also be

questioned. Why would a long-term physiological process be temporarily

suspended, only to be resumed later at about the same rate? The most

likely explanation is that it in some way saves energy. The production

and maintenance of a clutch of large follicles presumably involves a

great energetic cost. Perhaps under favorable conditions (food, water,

temperature), when follicular growth is proceeding rapidly, a mechanism

to slow ovarian enlargement would be advantageous by reducing the period

of time that the clutch of mature (large) follicles would have to be

maintained. An alternative hypothesis is that harsh environmental con-

ditions that occurred between September and November necessitated sus-

pension of ovarian development, and that this was mediated through the

endocrine system.

The earliest that Iverson (1980) found oviducal eggs in a north

Florida gopher tortoise was 26 April. Based on the literature, the

females he examined, and personal observations of others, Iverson con-

cluded that most oviposition occurs between mid May and mid June. I

believe that the main portion of the nesting season begins earlier than











this in north Florida. Of the 12 females I examined in April, 5

possessed oviducal eggs. If eggs are retained in the oviducts about 2

weeks (Powell 1967; Gibbons 1968; Moll 1973; Tinkle and Gibbons 1977),

42% of my April sample would have oviposited no later than 12 May. An

additional 4 females with follicles 30 mm or greater in diameter were

examined before 15 April. If only 2 of these individuals had ovulated

within the next 2 weeks, 50% of the April females would have nested

before 15 May.

The discovery during this study of a female with well-calcified

oviducal eggs on 26 March was quite surprising, and would seem to add

even more support to the idea that the nesting season is well underway

by mid May. The exact reproductive condition of this female, however,

must be questioned. Primarily because of the presence of an additional

5 ovarian follicles greater than or equal to 30 mm in diameter, I wonder

whether the two well-calcified oviducal eggs were produced from the same

group of enlarged follicles. Also, these eggs failed to develop when

incubated, suggesting that they had either not been fertilized or were

adversely affected by oviducal retention. Although none of the other 12

individuals examined between August 1979 and March 1980 contained ovi-

ducal eggs, the possibility exists that the 2 eggs found on 26 March

represent a clutch (or part of a clutch) that was produced but not laid

the previous year. Potentially, these eggs could have been laid with

the 5 for 1980, assuming all 5 of those were to be ovulated. Although

the production of viable eggs by 26 March in north Florida cannot be

ruled out, all of the evidence from this and other studies indicates










that the nesting season of the gopher tortoise is not yet underway by

that date. I conclude that in north Florida the major period of ovi-

position begins between mid and late April rather than mid May.

It seems that most females have nested by mid June. Of four indi-

viduals examined in May, one (on the 24th) possessed no oviducal eggs,

corpora lutea, or follicles greater than 14 mm in diameter, indicating

ovulation and oviposition had occurred some time ago. The other three

(all on the 31st) all had corpora lutea, but had been obtained from a

hunter and the presence of oviducal eggs could not be determined. Of

two tortoises from 13 and 14 June, one had corpora lutea but no oviducal

eggs, so had nested. The other had oviducal eggs that later hatched,

indicating that they were ready to be laid. I have no evidence that any

of the other females examined would have nested after mid June.

In male gopher tortoises the period of maximum testicular size is

coincident with the period of spermatogenesis but temporally distinct

from the mating period. This condition is typical of several other

temperate turtle species including Chrysemys picta (Gibbons 1968;

Callard et al. 1976), Sternotherus odoratus (McPherson and Marion 1981;

Mahmoud and Klicka 1972), Trionyx muticus (Plummer 1977), Trionyx

spiniferus (Robinson and Murphy 1978), Trionyx sinensis (Lofts and Tsui

1977), and Testudo hermanni (Kuchling et al. 1981). Singh (1977)

reported, however, that largest testis size and greatest spermatogenic

activity were concurrent with the breeding period in the Indian pond

turtle Lissemys punctata granosa.











Environmental factors that are the ultimate causes of changes in

testicular size and spermatogenic stages are less well understood for

turtles than for lizards (Licht 1967, 1969, 1971, 1972; Licht et al.

1969; Noeske and Meier 1977). McPherson and Marion (1981) hypothesized

that in Sternotherus odoratus temperature and photoperiod interacted to

initiate the production and maturation of sperm and to bring about the

end of spermatogenesis. Singh (1977) felt that in addition to those two

factors, changes in precipitation were important in controlling the

testicular cycle in populations of Lissemys living in areas with dis-

tinct wet and dry seasons. One or more of these environmental variables

are also suspected of influencing the timing of spermatogenesis in G.

polyphemus.

Changes in the development of the epididymides of the gopher

tortoise seem to be in response to testicular events and mating

activity. Hormonal or other factors may also be of importance in

influencing epididymal diameter, but the major determinant seems to be

the number of sperm contained within the duct. The very close

association seen between the diameters of the ductus epididymis and the

mass of sperm contained within it indicates that the duct expands in the

fall to accommodate the sperm that are leaving the testis and contracts

in the spring following ejaculation.

Few studies examining the role of the endocrine system in the

control of the male reproductive cycle in turtles have been completed.

Other than G. polyphemus, only Chrysemys picta (Callard et al. 1976),

Chelonia mydas (Licht et al. 1979), and Testudo hermanni (Kuchling et











al. 1981) have been examined. The geographic ranges and ecological

positions of these species differ, and the timing of specific spermato-

genic stages varies somewhat between them. The relationship between

fluctuating levels of plasma testosterone and testicular events within

each, however, is surprisingly similar (Fig. 25).

In G. polyphemus the plasma testosterone level declines during the

prebreeding and breeding periods, rebounds during the summer and peaks

during fall spermatogenesis, and then declines again during the winter

before peaking again prior to next year's mating season. Of the other

three studies that examined plasma testosterone fluctuations, only that

of Kuchling et al. spanned an entire year. Their results from T.

hermanni were similar to those of the gopher tortoise. Testosterone

declined during the mating season, rebounded during fall

spermatogenesis, and returned to low levels for the winter. The levels

of circulating testosterone Kuchling et al. found at the "breeding peak"

were relatively low compared to those in the fall. These results seem

not to conform to mine, since values increased between January and

April, a period that includes part of the mating season. I propose,

however, that because no determinations were made in February or March,

the main part of the "spring peak" was not observed. Substantially

higher concentrations of plasma testosterone were probably present in

February and/or March, so that the value in April was part of a declin-

ing trend rather than a peak as it seemed due to a lack of data points

in the preceding two months. Licht et al. (1979) made testosterone

determinations between March and June, months that spanned the















































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prebreeding and mating periods of the C. mydas population they studied.

For this portion of the annual cycle the trend is the same as that of G.

polyphemus. Values peak during the prebreeding period and decline just

prior to and during the mating season. Callard et al. (1976) studied C.

picta from April until December. The mating period included April and

May, and was a time when plasma testosterone concentrations declined

precipitously. Levels were basal during the summer, but rose dramati-

cally in the fall during spermatogenesis. If endocrine control of the

spermatogenic cycle of C. picta is similar to that of G. polyphemus, I

suspect that the increasing testosterone concentration Callard et al.

found in December would not lead directly to the high levels present in

April. A mid winter period of lowered levels probably separates two

peaks in the annual cycle.

In male mammals, both spermatogenesis and the behaviors associated

with courtship and mating are dependent on the presence of androgens.

Testosterone (and its "active form," the metabolite dihydrotestosterone)

is one of the main circulating androgens of testicular origin (Lofts

1968; Turner and Bagnara 1976). In seasonally-breeding mammals elevated

plasma testosterone concentrations are correlated with concurrent semi-

niferous tubule development and reproductive behavior. Spermatogenesis

in most turtles that have been studied, however, occurs months after the

breeding season at a time when the testicular interstitium is atrophied,

but when there is a second peak in plasma testosterone.











When considering the role played by the endocrine system in the

control of reproductive events in turtles, some questions come to mind.

If testosterone levels are high in both spring and fall, why would

courtship occur only in the spring and spermatogenesis only in the fall?

Part of the answer hinges on the fact that the few data gathered so far

for turtles do not clearly indicate the relative magnitudes of the

spring and fall peaks. By relying on the information from the gopher

tortoise as a basis for hypothesizing, it could be concluded that the

levels of testosterone circulating in the fall are not high enough to

elicit reproductive behavior, or at least to the degree seen in the

spring. Although the spring and fall peaks in testosterone concentra-

tion are not statistically different, the higher levels seen in the

spring may nonetheless be of biological importance. If, in the gopher

tortoise, a behavioral response to hormone concentration is dose-

dependent, as has been proposed for the lizard Anolis carolinensis

(Tokarz and Crews 1980), then the overall greater values seen in the

spring should correspond to the period of more intense courtship.

Courtship in captive animals has been repeatedly observed as late in the

year as November (Douglass 1976), and it would not be surprising that

reproductive behavior and elevated plasma testosterone are positively

correlated in the fall as well as the spring. The significant increases

in chin gland volume occurring in September and October lend credence to

the idea that some level of fall mating might occur. When considering

possible explanations for a lack of androgen-dependent spermatogenesis

in the spring, it must be remembered that the data gathered so far in











the study of turtle reproductive endocrinology have only been for tes-

tosterone levels in the peripheral circulation. Levels of testosterone

within the seminiferous tubules have not been determined, even though it

is at that location where the concentration is important when consider-

ing spermatogenesis. Harris and Barthke (1974) showed that in the rat,

testosterone concentration could be 20 times greater within the tubules

than in the general circulation. In the fall, levels within the semi-

niferous tubules of the gopher tortoise may be much higher than those

measured, whereas the cirulating concentration in the spring may not be

great enough to stimulate tubular development. If the conclusion is

that spermatogenesis occurs in response to testosterone from a source

other than the testicular interstitium, then another possible origin

must be identified. A likely candidate is the Sertoli cell, present

within each seminiferous tubule and shown in at least one study (Lofts

and Bern 1972) to exhibit a secretary appearance during the production

of sperm. Lacy (1960, 1973) and Lofts (1972) have for some time

advanced the idea that the Sertoli cell might play a role in regulating

spermatogenesis through the production of hormones.

The genus Gopherus is unique among the testudinids in that its 4

species possess integumentary glands on each mandibular ramus

(Auffenberg 1969). Agassiz (1857) first reported these structures, and

pointed out that those of G. berlandieri were more developed than those

of G. polyphemus. The glands were subsequently reported in G. agassizii

(Grant 1936) and G. flavomarginatus (Legler and Webb 1966). Smith and

Brown (1948) incorrectly stated that the only species to possess these











glands was G. berlandieri, and that only in males of that species were

they functional.

Despite the fact that their existence has been known for some time,

these subdentary glands have received little attention. In only a very

few studies have attempts been made to examine them in detail or to

investigate their function. In his initial description of the courtship

of G. polyphemus, Auffenberg (1966) reported that the glands (and pre-

sumably their exudate) were employed in the behavioral sequence. When

courting, male gophers vigorously bob their heads in a vertical arc

directly in front of females. Auffenberg concluded that this served to

waft the scent from the glands toward the female. Auffenberg (1969)

also reported that females rubbed their chins with their forelegs during

the courtship sequence, presumably causing the secretary product of the

glands to be expressed. The legs were then extended anteriorly and

"presented" to the male facing her. Auffenberg concluded that these

actions served to facilitate olfactory communication between the court-

ing tortoises and indicated a readiness to mate. The results of the

histological portion of the present study support Auffenberg's

hypothesis. Since there is no muscular tissue incorporated into the

structure of the gland, accumulated secretary product can be forced out

of the duct during courtship encounters only by vigorous contraction of

the deep underlying skeletal muscle or by pressing on the gland

externally.

Rose et al. (1969) studied the chin glands of both sexes of all

four Gopherus species and described electrophoretic differences between










the glandular products of each. Rose et al. stated that chin glands are

largest in male G. berlandieri and G. agassizii, and weakly developed in

all females. They found that the glandular exudate contained esterases,

phospholipids, triglycerides, free fatty acids and cholesterol. Rose et

al. concluded that chin glands probably served as olfactory as well as

visual cues during courtship and/or combat. They also speculated that

the lipid component of the chin gland exudate might be a factor that

elicits the behavioral response in gophers since that fraction has a

characteristic odor to humans.

Rose (1970) found nine fatty acids in the chin gland secretary

product of male G. berlandieri, and tested the effect of painting the

head of a model tortoise with a solution containing those compounds in

the correct concentrations. Rose found a highly significant increase in

the number of positive responses of both sexes to the "painted" model as

compared to those directed toward the model before the fatty acid

solution was added. The behavioral responses in these tests were

combat-like rather than courtship-like (for example, no mounting

attempts by males). Rose speculated that this might have been because

the test solution was patterned after the chin gland exudate of males.

The small size of G. berlandieri females' chin glands prevented the

collection of sufficient secretary product to allow completion of a

complimentary analysis and test.

Weaver (1970) tested the ability of G. berlandieri to differentiate

between male and female conspecifics. Weaver found that the tortoises

used a variety of cues for sex recognition, including chemical signals











originating from the chin glands. He also found that behavioral

responses following encounters occurring when the chin glands were

secreting differed from those seen when the glands were inactive.

Weaver reported that male G. polyphemus rubbed their forelimbs against

the jaw in a manner previously described for females.

Literature references to chin glands have usually included the

terms "active," "secreting," "enlarged," or evertedd" and their antonyms

to refer to the physiological state of the structures. It had become

accepted that the glands were "active" during the breeding season and

"inactive" throughout the rest of the year, although I am not aware of

any efforts to try to quantify the changes in size. I wanted to examine

this secondary sexual characteristic and to correlate changes in gland

size with other reproductive variables including hormonal fluctuations.

Several points can be drawn from the chin gland volume data, some

of which are unexpected. Males and females exhibited almost exactly the

same trends in chin gland size fluctuations, with glands becoming

enlarged and reduced almost simultaneously at most times of the year.

Male glands are only slightly larger than those of females. Female
3.
glands are usually about 20 mm; smaller than the males', or only about

10% less when both are swollen. Most surprising is that the glands of

both sexes show significant enlargement in the fall as well as during

the spring breeding period. There are no reports of this in the litera-

ture and I am not aware of anyone who has observed this previously.

It is assumed that, because they are secondary sexual characteris-

tics, gopher tortoise chin glands would be under the influence of the










endocrine system. This hypothesis is supported by the increases in

estrogen and testosterone (for females and males respectively) that

occur in association with greater activity of the glands. Hormonal

levels are elevated in the spring and fall, and chin glands enlarge in

the same seasons. However, close examination reveals that the spring

size increase follows the spring hormone peak by about 2 months (April

vs. February for males; May vs. March for females), whereas the fall

glands peak up to one month after the hormones. Possible explanations

for these phenomena include 1) other hormones are involved--but the data

for the only other hormone for which there is sufficient information do

not support this, 2) the endocrine system is not involved in control of

chin gland size, 3) other factors are acting synergistically with

hormone levels to influence chin gland size--including climatic factors,

physiological condition, reproductive state, or refractoriness. At this

time the last explanation seems most likely.

Regardless of the exact relationship between hormonal fluctuations

and changes in chin gland size, the tissues certainly seem to play a

role in the social interactions of gopher tortoises. This is true of

the behaviors seen in the spring breeding season, as has been reported

in the literature previously, but also during the fall. Courtship has

been observed in the field in April (Kenefick 1954) and May (Layne in

Douglass 1976), and the breeding period has generally been considered to

be the spring and early summer (Carr 1952; Ernst and Barbour 1972). In

captivity, however, tortoises have exhibited reproductive behavior in

August, September, and November (Douglass 1976). The occurrence of











enlarged chin glands in the fall suggests that courtship observed in

that season may not be an artifact of captivity. The significance of

possible fall mating in the wild and the number of offspring resulting

from it (if it occurs) are unknown, but the evidence of a second or

extended breeding period is increasing. Since females do not possess

fully enlarged ovarian follicles until December, any sperm transferred

by the males during fall matings could not immediately fertilize ova,

and would therefore have to be stored by the female. There has been

speculation (Iverson 1980) but no evidence that female gopher tortoises

can store sperm.

Combat between tortoises is also commonly observed in the fall

months. Douglass (1976) noted many instances of agonistic behavior

during his study of the mating system of G. polyphemus, and also

reported related observations of other persons. Although these records

were not systematically gathered, Douglass stated that combat between

males was observed in March, August, September, and October. A compila-

tion of Douglass' reports of others' observations reveals that most of

them occurred in the same months. Weaver (1970) reported combat in the

spring and summer. Taken together, these data suggest that there may be

2 periods when combat is most common. In the spring such behavior is

presumed to be associated with dominance hierarchies, maintenance of

territories, and access to females by males (Douglass 1976). If mating

does occur in the fall, enlarged chin glands would presumably serve the

same purpose then that they do in the spring. If fall courtship only

occurs as a result of captivity, then an alternate explanation for the











significance of swollen glands and combat during August, September, and

October is needed. Such behavior could serve to reaffirm the social

system that was established in the spring and to reassert ownership of

burrows. It is possible that this "system" occurs in the fall because

of a general reduction in away-from-burrow activity and social contacts

during the midsummer months. Although tortoises may occasionally be

found away from burrows on hot summer days, even at midday (pers. obs.),

the animals suffer thermal and water stress if subjected to such

conditions for extended periods (Bogert and Cowles 1947; pers. obs.). A

reduction in the opportunity to communicate may occur on the longest

days of the year because of the necessity of spending part of the day

down in the burrow, whereas the more tolerable period in the fall may

allow dominant individuals to reinforce their position through increased

social interaction.

The question of whether or not the gopher tortoise warrants pro-

tection from hunting is a current topic of debate (Matthews 1979;

Anonymous 1979). Dramatic reductions in the number of tortoises have

been documented and further declines have been predicted (Auffenberg and

Franz 1982). There are two primary causes for the smaller population

sizes seen today--habitat destruction due to urbanization and agricul-

ture, and hunting of the animals for food by man. Disagreement occurs

among persons who attempt to determine the relative effects of these two

factors in contributing to the plight of G. polyphemus. In light of

predicted population size declines this point is moot, however, since it

is the cumulative effect of all the factors that determines whether or











not the species will remain viable. Steps need to be taken now to

reduce the loss both of the tortoises and their preferred habitats,

regardless of the exact relationship between the contributing factors.

The results of the present study do not support the contention that

the gopher tortoise can sustain continued human predation at the level

currently being experienced. A previous study (Taylor 1982) showed that

on the average, 20% of mature individuals were removed from a population

during each episode of human predation. Because of the large body size

(i.e., high age) required to attain sexual maturity and the small clutch

size of mature females, replacement of mature individuals through repro-

duction is extremely slow. The results of the attempted behavioral

analyses in the present study draw attention to another potential

problem in current gopher tortoise management policy. Groups of tor-

toises are sometimes relocated from an area that is scheduled to be

developed or disturbed, in an attempt to reestablish the population in

another suitable area. While these efforts are certainly more benefi-

cial to the animals than doing nothing to save them, the positive

effects of such actions may not be as dramatic as anticipated. The

disruption of an established social system (Douglass 1976) and the

relocation of a portion of a colony into an unfamiliar locality may have

severe enough effects that a period of years may be required before the

mating system of the group becomes reestablished to the extent that

significant numbers of young are produced.











The current game management regulations for Florida (Florida Game

and Fresh Water Fish Commission 1981) specify a possession limit of five

gopher tortoises. This rule, however, is based on no biological

evidence whatsoever, and tortoise populations have declined precipi-

tously while it has been in effect. The relationship between the

species' reproductive potential and its status as an exploited game

animal needs thorough investigation. Such studies have recently been

initiated by the Florida Game and Fresh Water Fish Commission. The

management policy to be followed while these important data are being

gathered is still being debated, however. Should the gopher tortoise be

protected immediately and throughout the period that data are being

collected, with the final decision concerning protection to be made at

some time in the future? Or should exploitation be allowed to continue

under current guidelines while studies are being conducted to determine

if the species can withstand such pressure? If the latter course is

followed, and if it is determined that the level of exploitation has

been excessive, then the ability of G. polyphemus to recover, even under

strict future controls, may be seriously affected. In light of

ecological problems and faunal extinctions that have resulted from the

ignorance and disregard of biological principles, the prudent course of

action is to conserve this resource until the determination is made that

it can withstand exploitation. Since human predation of the gopher

tortoise is for the most part recreationally and economically motivated

(Taylor 1982), the protection of the species and the enhanced survival








81



of this unique and biologically important animal would more than

outweigh the cost to hunters.














SUMMARY AND CONCLUSIONS


1. Ovarian follicles that are ovulated in April and May have undergone

enlargement between September and December of the previous year.

The six to seven eggs produced by a typical female are laid between

mid April and mid June.

2. In females, plasma estrogen and progesterone concentrations exhibit

periovulatory peaks, and secondary surges during vitellogenesis.

The antigonadal effect of progesterone is indicated by the

cessation of ovarian growth and declines in estrogen levels

following increases in its concentration.

3. Maximum spermatogenesis is coincident with greatest testicular

weight and seminiferous tubule diameter in September and October.

Testis and seminiferous tubule development are minimal during the

spring breeding period.

4. In males, plasma testosterone peaks just prior to the mating

season, and again during fall spermatogenesis. Both courtship and

sperm production seem to be under endocrine control, but

differences in peripheral and intratubular testosterone

concentrations may be the cause of the temporal separation between

the two.

5. Both males and females exhibit spring and fall increases in chin

gland size that are associated with peaks in steroid hormone








83


concentrations. Fall mating may be of greater significance than

previously thought.

6. Additional protection for the gopher tortoise is warranted.

Overexploitation of this slowly-reproducing resource must be

prevented in order to ensure its future survival.














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BIOGRAPHICAL SKETCH


Robert Wayne Taylor, Jr. was born January 12, 1950, in Leesburg,

Florida. He attended public school in Leesburg and graduated from

Leesburg High School in 1967. He attended Auburn University from 1967

until 1971, graduating with the Bachelor of Science degree in Biological

Sciences.

Mr. Taylor served as a Supply Corps Officer in the United States

Navy from 1971 until 1976, and attained the rank of Lieutenant.

In 1976, he entered the University of Florida as a graduate student

in the Department of Zoology. He was allowed to by-pass the master's

program and to enter the Ph.D. program in June 1978. While at the

University of Florida, he was employed as a Graduate Teaching Assistant

in the Department of Zoology and as a Graduate Research Assistant in the

Florida State Museum. He is a member of the American Society of

Ichthyologists and Herpetologists, the Herpetologists' League, and the

Society for the Study of Amphibians and Reptiles.

Mr. Taylor is married to the former Gloria Gene Cays. They have a

daughter, Meredith Leigh, born February 22, 1980.