Chapter II: Part 2

There is a marked alternation of ovarian activity in _T. ornata_, one ovary being more active than its partner in a given season. The less active ovary is more active than its partner in the following season. For example, a specimen killed in July had four corpora lutea on the right ovary and two on the left and there were five enlarged follicles (of the medium size group), representing the next set of eggs to be ovulated, four on the left ovary and one on the right. Similar alternation of ovarian activity was observed, to a greater or lesser extent, in nearly all of the females examined. Many subadult females that were approaching their first breeding season (as evidenced by the presence of large ovarian follicles but no indication of former ovulation) had but one active ovary. This may account in part for the tendency of small, young females to lay clutches smaller than average. One ovary may become senile in old females before its partner does; this may explain the occasional absence or atrophy of one ovary in large females that I have examined.

In all the specimens examined, it was evident that ovulation had occurred or would occur in two successive seasons. Senile or young females might, however, be expected to skip a laying season if only one ovary was functioning.

After ovulation, the collapsed follicle assumes a cuplike shape and becomes a glandular corpus luteum (Pl. 20, Fig. 2). Corpora lutea are approximately eight millimeters in diameter and are easily discernible at least until the eggs are laid; they are somewhat less distinct after preservation. Corpora lutea undergo rapid involution following oviposition and, after two to three weeks, are little more than small puckerings on the ovarian epithelium. At this stage they are properly referred to as corpora albicantia and are discernible only after careful examination of the ovary under low magnification. Corpora albicantia remain on the ovary until April of the year following ovulation but disappear in May and are never present after the new set of eggs is ovulated. Ovaries of some subadults (that would have laid first in the season following capture) contained enlarged follicles and, but for their lack of corpora lutea and corpora albicantia, were indistinguishable from those of older, fully mature females.

Altland (1951:605-610) gave a histological description of the corpus luteum of _Terrapene carolina_. Corpora lutea were glandular and filled with lipoidal material until the eggs were laid. Atresia of corpora lutea began when eggs were laid, was completed by mid-August, and was coincident with atresia of large follicles that did not undergo ovulation. Altland did not describe the gross external appearance of the corpus albicans.

The corpus luteum of oviparous reptiles seems to be closely associated with the intrauterine life of the eggs and, in viviparous reptiles, it may be an important factor in maintaining optimum gestational environment; however, its functions in all reptiles are poorly understood (Miller, 1948:200-201).

Information gleaned from records of gravid females and known dates of nesting suggests that eggs are retained in the oviducts two to three weeks before laying. Once they are ovulated, the eggs are exposed to but few hazards until laid; counts of corpora lutea are an accurate indication of the number of eggs laid. In the gravid females examined by me, number of corpora lutea on the ovaries was equal, in all but one instance, to the number of oviducal eggs. In the single instance in which an extra corpus luteum was found, one egg had probably been laid before the specimen was captured. The high incidence of correspondence between counts of corpora lutea and counts of oviducal eggs indicates also that _T. ornata_ deposits the entire complement of oviducal eggs at one time, not singly or in smaller groups.

Extrauterine migration of ova, whereby eggs from one ovary pass into the oviduct of the opposite side, is of common occurrence in _T. ornata_ and is known to occur also in _T. carolina_, _Chrysemys picta_, _Emydoidea blandingi_, _Pseudemys scripta_, _Cnemidophorus sexlineatus_, and in several mammals (Legler, 1958). This ovular migration may serve to redistribute eggs to the oviducts when the ovaries are functioning at unequal rates.

The eggs acquire shells soon after they enter the oviducts. No shell-less eggs were found in oviducts but several specimens of _T. ornata_ had oviducal eggs, the thin, parchmentlike shells of which lacked the outer calceous layer; in these specimens the corpora lutea were fresh, probably not more than two days old. Eggs that had remained in the oviducts longer had a calceous layer on the outside of the shell. Eggs having incompletely developed shells were successfully incubated in the laboratory. Cagle (1950:38) found shelled but yolkless eggs in the oviducts of several _Pseudemys scripta_ but found no yolkless eggs in nests. No yolkless eggs were found in specimens of _T. ornata_ in the course of the present study.

The uterine portion of the oviducts becomes darkened (pale gray to intense black) in the breeding season. Darkening of oviducts seemed to coincide with the period when eggs were in the oviducts and it persisted for a variable length of time after the eggs were laid. Oviducts of immature females were ordinarily pale.

Nesting

Ornate box turtles nest chiefly in June. Some females nest as early as the first week of May or as late as mid-July but the nesting season reaches its peak in mid-June. Eggs nearly ready to be laid were in oviducts (determined by bimanual palpation in the field or by dissection in the laboratory) of many females captured in June; nearly half of the records so obtained were in the second week of that month. Early records of shelled oviducal eggs were April 25 (specimen from Ottawa County, Oklahoma), May 5, and May 22. The two latest records are for females retaining oviducal eggs on July 2 and 11. Known dates for nesting of free-living females were distributed rather evenly through the month of June. It is worthy of note that all (four) of the nestings known to occur in July were by captive females. Females of _T. ornata_, like those of some other turtles (Cagle and Tihen, 1948; Risley, 1933:694), seem to retain their eggs until conditions are suitable for nesting. Most of the reports in the literature of nesting after mid-July represent records for captive females.

Nests of _T. o. ornata_ were so well-concealed that they were difficult to find even when a gravid female had been followed to the approximate location by means of a trailing thread. Females spend one to several days seeking a site for the nest, usually traveling a circuitous route within a restricted area. Movements of nest-seeking females were more extensive than those of males and non-gravid females observed in the same periods.

Activities of one gravid female, typical in most respects of the activities of several other gravid females observed (for periods of one to 23 days) at the Damm Farm, illustrate pre-nesting behavior (Fig. 29). A trailer was attached to the female on the morning of June 7. She was recovered early on the following afternoon; her movements in the elapsed period had been restricted to a small, deep, ravine 150 feet long and 20 to 30 feet wide. She had traversed each edge of the ravine at least once and had crossed it six or seven times, keeping mostly to areas on the upper parts of south--or west--facing slopes where vegetation was sparse or lacking. In six places she had dug into the ground, probably to test the suitability of the soil for nesting. In three places she dug beneath rocks that jutted out from the bank, and in two places merely scratched away the upper crust of soil. Her most recent attempt at digging (probably late the previous evening or in early morning on the day of her capture) consisted of a flask-shaped cavity that, but for the lack of eggs and a covering of earth, was like a completed nest (Pl. 21, Fig. 1). The cavity was 55 millimeters deep, 80 millimeters wide at the bottom, and 60 millimeters wide at the opening. For several inches about the opening the earth was slightly damp. That piled on the rim of the opening was of the consistency of thick mud, indicating that the female had voided fluid first on the surface of the earth and again inside the cavity to soften the soil. Subsequently during eight days her activities were similar but not so extensive as on the day described above. It was determined by daily palpation that she laid her eggs somewhere in the general area of the ravine on June 15 but the nest could not be found.

No completed nests containing eggs were discovered at the Damm Farm but the locations of several robbed nests and partly completed nests provided some information on preferred sites. The nests found were on bare, well-drained, sloping areas and were protected from erosion by upslope clumps of sod or rocks. The nest cavity illustrated in Plate 21 was at the edge of the sod-line on the upper lip of the west-facing bank of a ravine. One nest had been excavated in a shallow den beneath an overhanging limestone rock. Three nests were on west- or south-facing slopes and one was on the north-facing bank of a ravine. Box turtles presumably select bare areas for nesting because of the greater ease of digging. One female at the Damm Farm was thought to have laid her eggs in a cultivated field and William R. Brecheisen told me he discovered two nests in a wheat field being plowed in July, 1955.

The repeated excavation of trial nest cavities presumably exhausts the supply of liquid in the female's bladder. Frequent imbibing of water is probably necessary if the search for a nesting site is continued for more than a day or two. Standing water was usually available in ponds, ravines, ditches, and other low areas at the Damm Farm in June. Nesting in June, therefore, is advantageous not only because of the greater length of time provided for incubation and hatching but also because of the amount of water available for drinking. Females can probably be more selective in the choice of a nesting site if their explorations are not limited by lack of water.

Females of _T. ornata_, in all instances known to me, began excavation of their nests in early evening and laid their eggs after dark; Allard (1935:328) reported the same behavior for _T. carolina_.

William R. Brecheisen, on July 22, 1955, at his farm, two miles south and one mile west of Welda, Anderson County, Kansas, observed that a large female began digging a nest in an earth-filled stock tank at 6:00 P. M. At first she moved her body about on the surface of the earth, loosening it and pushing it aside with all four legs, making a depression approximately two inches deep and large enough to accommodate her body. At 7:30 P. M. she began digging alternately with her hind feet at the bottom of the depression. Digging continued until 10:00 P. M., at which time the nest cavity was three inches deep, and three inches in diameter, with a smaller opening at the top. Six eggs were laid in the next half-hour. Covering of the nest probably took more than one hour but observations were terminated after the final egg was laid. By the following morning the nest-site had been completely covered and was no different in appearance from the rest of the earthen floor of the tank. (Brecheisen observed more of the nesting than anyone else has recorded and I am obliged to him for permission to abstract, as per the above paragraph, the notes that he wrote on the matter.)

A nest made by a captive female at the Reservation was of normal proportions except for an accessory cavity that opened from the neck of the nest, immediately below the surface of the ground. This smaller cavity contained a single egg. This peculiar nest may have resulted from the efforts of two different females since several were kept in the same outdoor pen.

Ten adult females were kept in an outdoor cage in the summer of 1955. The cage was raised off the ground on stilts and its floor was covered with 12 inches of black, loamy soil. A small pan of water was always available in the cage and the turtles were fed greens, fruit, and table scraps each evening. Nesting activity was first noted on June 21, when one of the females was digging a hole in a corner of the enclosure. She dug with alternate strokes of her fully-extended hind legs in the manner described (Legler, 1954:141) for painted turtles (_Chrysemys picta bellii_). Nevertheless, digging was much less efficient than in _Chrysemys_, because of the narrow hind foot of the female _T. ornata_; approximately half of the earth removed by any one stroke rolled back into the nest or was pulled back when she reinserted her leg. The female stopped digging when I made sudden movements or held my hand in front of her. Digging continued for approximately 45 minutes; then the female moved away and burrowed elsewhere in the cage. The nest cavity that she left was little more than a shallow depression. Three other females were digging nests early in the evening on July 3, 5, and 8; in each of these instances the female stopped digging to eat when food was placed in the cage and completed the nesting process, unobserved, later in the evening. In each instance where nest-digging by captive females was observed, the hind quarters of the female rested in a preliminary, shallow depression, and the anterior end of the body was tilted upward at an angle of 20 to 30 degrees. In late June and early July several eggs were found, unburied, on the floor of the cage and in the pan of water.

The excavation of a preliminary cavity by captive females may not represent a natural phenomenon. Allard (1935) made no mention of it in his meticulous description of the nesting process in _T. carolina_. It is worthy of mention, however, that Booth (1958:261) reported the digging of a preliminary cavity by a captive individual of _Gopherus agassizi_.

Eggs

The number of eggs in 23 clutches ranged from two to eight (mean, 4.7 A+- 1.37 [sigma]); clutches of four, five, and six eggs were most common, occurring in 18 (78 per cent) instances. The tendency for large females to lay more eggs than small females (Fig. 6) was not so pronounced as that reported by Cagle (1950:38) for _Pseudemys scripta_. The small size of _T. ornata_, in comparison with other emyid turtles, seemingly limits the number of eggs that can be accommodated internally. The number of eggs per clutch in T. carolina [2 to 7, average 4.2, Allard (1935:331)], is nearly the same as that of _T. ornata_.

Shells of the eggs are translucent and pinkish or yellowish when the eggs are in the oviducts. After several days outside the oviducts the shells become chalky-white and nearly opaque. Eggs incubated in the laboratory retained the pinkish color somewhat longer than elsewhere on their under-surfaces, which were in contact with moist cotton, but eventually even this part of the shell became white. Infertile eggs remained translucent and eventually became dark yellow, never becoming white; they could be distinguished from fertile eggs on the basis of color alone. Shells of infertile eggs became brittle and slimy after several weeks.

The outer layer of the shell of a freshly laid egg is brittle and cracks when the egg is dented. After a few days, when the eggs begin to expand, the shell becomes flexible and has a leathery texture. The shell is finely granulated but appears smooth to the unaided eye. The granulations are approximately the same as those illustrated by Agassiz (1857:Pl. 7, Fig. 18) for _T. carolina_.

Eggs are ellipsoidal. Data concerning size and weight (consisting of mean, one standard deviation, and extremes, respectively) taken from 42 eggs (representing 9 clutches) within 24 hours after they were laid, or dissected from oviducts, are as follows: length, 36.06 A+- 2.77 (31.3-40.9); width, 21.72 A+- 1.04 (20.0-26.3); and weight, 10.09 A+- 1.31 (8.0-14.3). There was a general tendency for smaller clutches to have larger eggs; the largest and heaviest were in the smallest clutch (two eggs) and the smallest were in the largest clutch (eight eggs). Risley (1933:697) reported such a correlation in _Sternotherus odoratus_, as did Allard (1935:331) in _T. carolina_. Measurements in the literature of the size of eggs of _T. ornata_ suggest a width greater than that stated above, probably because some eggs already had begun to expand when measured.

Eggs of _T. ornata_ expand in the course of incubation, as do other reptilian eggs with flexible shells, owing to absorption of water. In the laboratory, 48 eggs increased by an average of approximately three grams in weight and three millimeters in width over the entire period of incubation; increase in width coincided with decrease in length. Cotton in incubation dishes was kept moist enough so that some water could be squeezed from it. When the cotton was constantly moist, eggs showed a fairly steady expansion from the first week of incubation until hatching. The process could be reversed by allowing the cotton to dry. Eggs that were allowed to dry for a day or more became grossly dented or collapsed. Eggs at the periphery of the incubation dish were ordinarily more seriously affected by drying than were those at the center or in the bottom of the dish. A generous re-wetting of desiccated eggs and cotton caused the eggs to swell to their original proportions within 24 hours. Recessions occurred, however, even in the clutches that received the most nearly even amount of moisture. Increases in weight and size seemed to reach a peak in the middle of the incubation period and again immediately before hatching. Infertile eggs expanded in the same manner as fertile eggs in the first week or two of incubation, but thereafter gradually regressed in bulk or failed to re-expand after temporary periods of dryness. Fertile eggs that were in good condition had a characteristically turgid, springy feel and could be bounced off a hard surface.

Temporary lack of moisture usually did not kill embryos; prolonged dryness, combined with high temperatures, probably could not be tolerated. Lynn and Ullrich (1950), by desiccating the eggs of _Chrysemys picta_ and _Chelydra serpentina_, produced abnormalities in the young ranging from slight irregularities of the shell to eyeless monstrosities; eggs desiccated in the latter half of incubation produced a higher percentage of abnormal young than eggs that were desiccated earlier.

In 1956, three fertile eggs, from clutches that were at different stages of incubation, were immersed in water for 48 hours. The eggs rested on the bottom of the bowl in the same position in which they had been placed in the incubation dishes; when turned, they returned invariably to the original position. The embryos in two of the eggs (one and 27 days old at the time of immersion) were still living ten days after the eggs were removed from the water; the embryo in the remaining egg (21 days old at the time of immersion) was dead. Eggs immersed in water increased in size and weight at the same rate as eggs in incubation dishes, indicating that absorption of water probably operates on a threshold principle, the amount absorbed being no more than normal even under wet conditions.

Natural nests usually are in well-drained areas, but water probably stands in some nests for short periods after heavy rains. Provided the nest cavity itself is not damaged, water in the nest is probably more beneficial than harmful to the eggs; however, nests that are inundated during floods probably have little chance of survival.

Embryonic Development

Eggs were examined by transmitted light in the course of incubation. At the time of laying (or removal from oviducts) no embryonic structures were discernible even in eggs that had been retained in the oviducts of captive females some weeks past the normal time of laying; a colorless blastodisc could be seen if eggs were opened. Embryonic structures first became visible at eight to ten days of incubation; at this time vascularization of the blastodisc was evident and the eyes appeared as dark spots. Heart beats were observed in most embryos by the fifteenth day but were evident in a few as early as the tenth day. The pulse of a fifteen-day-old embryo averaged 72 beats per minute at a temperature of 30 degrees. Embryos at fifteen days, measured in a straight line from cephalic flexure to posteriormost portion of body, were approximately nine to ten millimeters long and at 22 days were 14 millimeters long. At approximately 35 days the eggs became dark red; embryonic structures were discernible thereafter only in eggs that had embryos situated at one end, close to the shell.

Incubation periods for 49 eggs (representing 12 clutches) kept in the laboratory ranged from 56 to 127 days, depending on the temperature of the air during the incubation period. In 1955, eggs were kept at my home in Lawrence where air temperatures were uncomfortably hot in summer and fluctuations of 20 degrees (Fahrenheit) or more in a 24-hour period were common. The following summer eggs were kept in my office at the Museum where temperatures were but slightly cooler than in my home and subject also to wide variation. In 1957 this part of the Museum was air-conditioned and kept at approximately 75 degrees. The greater lengths of incubation periods at lower temperatures are shown in Table 1. Risley (1933:698) found the incubation period of _Sternotherus odoratus_ to be longer at lower temperatures; corresponding observations were made by Allard (1935:332) and Driver (1946:173) on the eggs of _Terrapene carolina_. Cagle (1950:40) and Cunningham (1939) found no distinct differences in length of incubation period for eggs of _Pseudemys scripta_ and _Malaclemys terrapin_, respectively, at different temperatures within the range tolerated by the eggs.

Most nests observed in the field were in open situations where they would receive the direct rays of the sun for at least part of the day; the shorter average incubation periods (59 and 70 days, respectively), observed in 1955 and 1956, therefore, more nearly reflect the time of incubation under natural conditions than does the excessively long period (125 days at 75 degrees) observed in 1957 under cooler, more nearly even temperatures.

TABLE 1.--The Relationship of Temperature and Duration of Incubation
Period as Determined from Laboratory Studies of 49 Eggs of
_T. ornata_.
=============+===================+=========+========+=================
Average | Period of | Number | Number |
daily | incubation (Days) | of | of | Remarks
temperature |--------+----------|clutches | eggs |
(Fahrenheit) | Mean | Range | | |
-------------+--------+----------+---------+--------+-----------------
| | | | | Wide daily
91 | 59 | 56-64 | 6 | 24 | fluctuations in
| | | | | temperature
-------------+--------+----------+---------+--------+-----------------
| | | | | Wide daily
82 | 70 | 67-73 | 4 | 21 | fluctuations in
| | | | | temperature
-------------+--------+----------+---------+--------+-----------------
| | | | | Temperature
75 | 125 | 124-127 | 2 | 4 | thermostatically
| | | | | controlled
-------------+--------+----------+---------+--------+-----------------

Sixty-five days seems to be a realistic estimate of a typical incubation period under natural conditions; eggs laid in mid-June would hatch by mid-August. Even in years when summer temperatures are much cooler than normal, eggs probably hatch by the end of October. Hatchlings or eggs would have a poor chance of surviving a winter in nests on exposed cut-banks or in other unprotected situations. Overwintering in the nest, hatchlings might survive more often than eggs, since hatchlings could burrow into the walls and floor of the nest cavity. Unsuitable environmental conditions that delay the nesting season and retard the rate of embryonic development may, in some years, be important limiting factors on populations of ornate box turtles.

In areas where _T. ornata_ and _T. carolina_ are sympatric (for example, in Illinois, Kansas, and Missouri) the two species occupy different habitats, _ornata_ preferring open grassland and _carolina_ wooded situations. Under natural conditions, the average incubation periods of these two species can be expected to differ, _T. carolina_ having a somewhat longer period due to lower temperatures in nests that are shaded. In the light of these speculations, the remark of Cahn (1937:102)--that _T. ornata_ nested later in the season (in Illinois) and compensated for this by having a shorter incubation period--is understandable.

The range of temperatures tolerated by developing eggs probably varies with the stage of embryonic development. When temperatures in the laboratory were 102 to 107 degrees Fahrenheit for approximately eight hours, due to a defect in a thermostat, the young in two eggs of _T. ornata_, that had begun to hatch on the previous day, were killed, as were the nearly full-term embryos in a number of eggs of _T. carolina_ (southern Mississippi) kept in the same container. A five-day-old hatchling of _T. ornata_, kept in the same container, survived the high temperatures with no apparent ill effects. Cagle (1950:41) found that eggs of _Pseudemys scripta_ could not withstand temperatures of 10 degrees for two weeks nor would they survive if incubated at 40 degrees. Cunningham (1939) reported that eggs of _Malaclemys terrapin_ could not survive prolonged exposure to temperatures of 35 to 40.6 degrees but tolerated temporary exposure to temperatures as high as 46 degrees.

In the summer of 1955, a clutch of three eggs, all of which contained nearly full-term embryos, was placed in a refrigerator for 48 hours. The temperature in the refrigerator was maintained at approximately 4.5 degrees; maximum and minimum temperatures for the 48 hour period were 2.8 and 9.5 degrees, respectively. When the eggs were removed from the refrigerator they showed gains in weight and increases in size comparable to eggs, containing embryos of the same age, used as controls. The experimental eggs began to hatch two days after they were removed to normal temperatures--approximately 24 hours later than the controls.

In the late stages of incubation, the outer layer of the shell becomes brittle and is covered with a mosaic of fine cracks or is raised into small welts. Several days before hatching, movements of the embryo disturb the surface of the shell and cause the outer layer to crumble away, especially where the head and forequarters of the embryo lie against the shell. Some embryos could be seen spasmodically thrusting the head and neck dorsally against the shell.

The role of the caruncle in opening the shell seems to vary among different species of turtles. Cagle (1950:41) reported that it was used only occasionally by _Pseudemys scripta_; Allard (1935:332) thought that it was not used by _Terrapene carolina_; and, the observations of Booth (1958:262) and Grant (1936:228) indicate that embryos of _Gopherus agassizi_ use the caruncle at least in the initial rupturing of the shell.

In the three instances in which hatching was closely observed in _T. ornata_, the caruncle made the initial opening in the shell; claws of the forefeet may have torn shells in other hatchings that were not so closely observed. In all observed instances, the shell was first opened at a point opposite the anterior end of the embryo. The initial opening had the appearance of a three-cornered tear. A quantity of albuminous fluid oozed from eggs as soon as the shells were punctured.

The initial tear is enlarged by lateral movements of the front feet, and later the hind feet reach forward and lengthen the tear farther posteriorly. In many instances a tear develops on each side and the egg has the appearance of being cleft longitudinally. The young turtle emerges from the anterior end of the shell or backs out of the shell through a lateral tear.

The process of hatching, from rupture of shell to completion of emergence, extended over three to four days in the laboratory. Many hatchlings from time to time crawled back into the shell over a period of several days after hatching was completed. In a clutch of eggs kept in a pail of earth, by William R. Brecheisen, eight days elapsed between onset of hatching and appearance of the first hatchling at the surface.

A nest in an outdoor pen at the Reservation was discovered in early October. The cap had been recently perforated and the hatchlings had escaped. One of them, judged to be approximately two weeks old, was found in a burrow nearby. The cavity of the nest appeared to have been enlarged by the young. The eggs were probably laid in early July. Emergence of young from the nest had been delayed for a time after hatching, until rain softened the ground in late September and early October.

Fertility and Prenatal Mortality

Eggs were incubated in the laboratory at more nearly optimum temperature and humidity than were eggs in natural nests. Percentage of prenatal mortality probably was lower in laboratory-incubated eggs than in those under natural conditions.

Of sixty eggs studied in the laboratory, 45 (75 per cent) were fertile; 36 (80 per cent) of the fertile eggs (those in which the blastodisc was at some time discernible by transmitted light) hatched successfully. In six clutches all the eggs were fertile and five of these clutches hatched with 100 per cent success. One clutch contained eggs that were all infertile and another clutch had four infertile eggs and two fertile eggs that failed to hatch. Among nine fertile eggs that failed to survive, four casualties occurred in the late stages of incubation or after hatching had begun, indicating that these are probably critical periods.

Fertility of eggs was not correlated with size or age of female, with size of clutch, or with size of egg. Eggs laid in the laboratory had higher rates of infertility and prenatal mortality than did eggs dissected from oviducts. Handling of eggs in removing them from nests to incubation dishes, after embryonic development had begun, might have been responsible for reduced viability (Table 2).

TABLE 2.--Comparative Rates of Fertility and Prenatal Mortality for Eggs Dissected from Oviducts and for Eggs That Were Laid in the Laboratory and Subsequently Removed to Incubation Dishes. ===========================+==============+=============== | Eggs removed | Eggs dissected NUMBER OR PERCENTAGE | from nests | from oviducts ---------------------------+--------------+--------------- Number of eggs examined | 22 | 38 ---------------------------+--------------+--------------- Percentage of fertile eggs | 64 | 82 ---------------------------+--------------+--------------- Percentage of fertile | | eggs hatched | 50 | 94 ---------------------------+--------------+--------------- Percentage of eggs hatched | 32 | 76 ---------------------------+--------------+---------------

Reproductive Potential

Assuming that 4.7 eggs are laid per season, that all eggs are fertile and all hatch, that all young survive to maturity, that half the hatchlings are females, and that females first lay eggs in the eleventh year, the progeny of a single mature female would number 699 after twenty years. Considering that infertility and prenatal mortality eliminate approximately 40 per cent of eggs laid (according to laboratory findings) the average number of surviving young per clutch would be 2.8 and the total progeny, after 20 years, would be 270, provided that only one clutch of eggs was laid per year. But it is thought that, on the average, one third of the female population produces two clutches of eggs in a single season. If the second clutch contains 3.5 eggs (resulting in 2.1 surviving young when factors of infertility and prenatal mortality are considered), the progeny of a single female, after 20 years, would number approximately 380. Postnatal mortality reduces the progeny to a still smaller number.

The small number of eggs laid each year and the long period required to reach sexual maturity make the reproductive potential of _T. ornata_ smaller than that of the other turtles of the Great Plains, and much smaller than nearly any of the non-chelonian reptiles of the same region.

Number of Reproductive Years

The total span of reproductive years is difficult to determine; I am unable to ascertain the age of a turtle that has stopped growing. No clearly defined external characteristics of senility were discovered in the populations studied. A male that I examined had one atrophied testis. In another male both testes were shrunken and discolored and appeared to be encased by fibrous tissue. Both males were large, well past the age of regular growth, and had smoothly worn shells. Several old females had seemingly inactive ovaries. Reproductive processes probably continue throughout life in most members of the population, although possibly at a somewhat reduced rate in later life.

GROWTH AND DEVELOPMENT

Initiation of Growth

Young box turtles became active and alert as soon as they hatched, and remained so until low temperatures induced quiescence. If sand or soil was available, hatchlings soon burrowed into it and became inactive. Covering containers with damp cotton also induced inactivity; the hatchlings usually made no attempt to burrow through the confining layer. Desire to feed varied in hatchlings of the same brood and seemed not to be correlated with retraction of the yolk sac or retention of the caruncle. Some hatchlings actively pursued mealworms; on subsequent feedings they learned to associate my presence with food and eagerly took mealworms from forceps or from my hand. Meat, vegetables, and most other motionless but edible objects were ignored by hatchlings but some individuals learned to eat meat after several forced feedings. Hatchlings that regularly took food in the first month of life ordinarily grew faster than hatchlings that did not eat. Many of the hatchlings in the laboratory showed no areas of new epidermal growth on the shell in the time between hatching and first (induced) hibernation.

Size and Appearance at Hatching

The proportions of the shell change somewhat in the first few weeks of life. At hatching the shell may be misshapen as a result of confinement in the egg. Early changes in proportions of the shell result from expansion--widening and, to a lesser degree, lengthening of the carapace--immediately after hatching. Subsequent retraction or rupture of the yolk sac and closure of the navel are accompanied by a decrease in height of shell and slight, further widening of the carapace.

The yolk sac retracts mainly between the time when the egg shell is first punctured and the time when the turtle actually emerges from the shell. When hatching is completed, the yolk sac usually protrudes no more than two millimeters, but in some individuals it is large and retracts slowly over a period of several days.

One individual began hatching on November 11 and was completely out of the egg shell next day; the yolk sac was 15 millimeters in diameter, protruded six millimeters from the umbilical opening, and hindered the hatchling's movements. The sac broke two days later, smearing the bottom of the turtle's dish with semifluid yolk. The hatchling then became more active. Twenty-six days later the turtle was still in good condition and its navel was nearly closed. A turtle that hatched with a large yolk sac in a natural nest possibly would benefit, through increased ease of mobility, if the yolk sac ruptured.

A recently hatched turtle was found at the Reservation in October, 1954, and was kept in a moist terrarium in the laboratory where it died the following May. The turtle was sluggish and ate only five or six mealworms while in captivity; no growth was detectable on the laminae of the shell. Autopsy revealed a vestige of the retracted yolk sac, approximately one millimeter in diameter, on the small intestine.

The navel ("umbilical scar") of captive hatchlings ordinarily closed by the end of the second month but in three instances remained open more than 99 days. The position of the navel is marked by a crescent-shaped crease, on the abdominal lamina, that persists until the plastron is worn down in later years (Pl. 24, Fig. 1).

The caruncle ("egg tooth") (Fig. 7) remains attached to the horny maxillary beak for a variable length of time; 93 per cent of the live hatchlings kept in the laboratory retained the caruncle on the tenth day, 71 per cent on the twentieth day, and only 10 per cent on the thirtieth day of life. Few individuals retained the caruncle when they entered hibernation late in November, and none retained it upon emergence from hibernation. Activities in the first few days or weeks of life influence the length of time that the caruncle is retained; turtles that begin feeding soon after hatching probably lose the caruncle more quickly than do those that remain quiescent. The caruncles of some laboratory specimens became worn before finally dropping off. Almost every caruncle present after 50 days could be flicked off easily with a probe or fingernail. The initiation of growth of the horny maxillary beak probably causes some loosening of the caruncle. The caruncle may aid hatchlings in escaping from the nest.

After the caruncle falls off, a distinct boss remains, marking its former place on the horny beak (Pl. 25, Fig. 1); this boss is gradually obliterated over a period of weeks by wear and by differential growth, and is seldom visible in turtles that have begun their first full year of growth. The "first full year of growth" is here considered to be the period of growth beginning in the spring after hatching.

Growth of Epidermal Laminae

Growth of ornate box turtles was studied by measuring recaptured turtles in the field, by periodically measuring captive hatchlings and juveniles, and by measuring growth-rings on the epidermal laminae of preserved specimens. Studies of growth-rings provided by far the greatest volume of information on growth, not only for the years in which field work was done, but for the entire life of each specimen examined.

It was necessary to determine the physical nature of growth-rings and the manner in which they were formed before growth could be analyzed. Examination of epidermal laminae on the shell of a box turtle reveals that each has a series of grooves--growth-rings--on its surface. The deeper grooves are major growth-rings; they occur at varying distances from one another and run parallel to the growing borders of the lamina. Major growth-rings vary in number from one to 14 or more, depending on the age of the turtle (Pl. 22). In juvenal turtles and in young adults, major growth-rings are distinct and deep. Other grooves on the shell--minor growth-rings--have the same relationship to the borders of the laminae but are shallower and less distinct than major growth-rings. One to several minor growth-rings usually occur on each smooth area of epidermis between major growth-rings. As the shell of an adult turtle becomes worn, the minor growth-rings disappear and the major rings become less distinct. Both sets of rings may be completely obliterated in old turtles but the major rings usually remain visible until several years after puberty.

In cross section, major growth-rings are V- or U-shaped. The inner wall of each groove is the peripheral edge of the part of the scute last formed whereas the outer wall represents the inner edge of the next new area of epidermal growth. The gap produced on the surface of the lamina (the open part of the groove) results from cessation of growth at the onset of hibernation. Minor growth-rings are shallow and barely discernible in cross-section (Fig. 8). It may therefore be understood that growth-rings are compound in origin; each ring is formed in part at the beginning of hibernation and in part at the beginning of the following growing season.

The few publications discussing growth in turtles express conflicting views as to the exact mode of growth of epidermal laminae. Carr (1952:22) briefly discussed growth of turtle scutes in general and stated that eccentric growth results from an entirely new laminal layer forming beneath, and projecting past the edges of the existing lamina. Ewing (1939) found the scutes of _T. carolina_ to be the thickest at the areola and successively thinner in the following eight annual zones of growth; parts of scutes formed subsequent to the ninth year varied irregularly in thickness. He stated that epidermal growth took place at the margins of the laminae rather than over their entire under-surfaces.

It is evident that the mode of scutular growth described by Carr (_loc. cit._) applies to emyid turtles that shed the epidermal laminae more or less regularly (for example, _Chrysemys_ and _Pseudemys_). In these aquatic emyids a layer of the scute, the older portion, periodically becomes loose and exfoliates usually in one thin, micalike piece; since the loosened portion of the scute corresponds in size to the scute below, it must be concluded that a layer of epidermis is shed from the entire upper surface of the scute, including the area of new epidermal growth. Box turtles ordinarily do not shed the older parts of their scutes; the areola and successively younger portions of the lamina remain attached to the shell until worn off. The appearance of a single unworn scute, especially one of the centrals or the posterior laterals, closely resembles a low, lopsided pyramid.

Examination of parasagittal sections of scutes revealed that they were composed of layers, the number of layers varying with the age of the scute. A scute from a hatchling consists of one layer. A scute that shows a single season of growth has two layers; a new layer is added in each subsequent season of growth. Stratification is most evident in the part of the scute that was formed in the first three or four seasons and becomes increasingly less distinct in newer parts of the scute. It may further be understood that scutes grow in the manner described by Carr (_loc. cit._).

When the epidermal laminae are removed, a sheet of tough, pale grayish tissue remains firmly attached to the bones of the shell beneath. This layer probably includes, or consists of, germinal epithelium. Contrasting pale and dark areas of the germinal layer correspond to the pattern of markings on the scute removed.

Growth of epidermal laminae is presumably stimulated by growth of the bony shell. As the bone grows, the germinal layer of the epidermis grows with it. When growth ceases at the beginning of hibernation, the thin edges of the scutes are slightly down-turned where they enter the interlaminal seams (Fig. 8). When growth is resumed in spring, the germinal layer of the epidermis, rather than continuing to add to the edge of the existing scute, forms an entirely new layer of epidermis. The new layer is thin and indistinct under the oldest part of the scute but becomes more distinct toward its periphery. Immediately proximal to the edge of the scute, the new layer becomes greatly thickened, and, where it passes under the edge, it bulges upward, recurving the free edge of the scute above. At this time the formation of a major growth-ring is completed. The newly-formed epidermis, projecting from under the edges of the scute, is paler and softer than the older parts of the scute; the presence or absence of areas of newly formed epidermis enables one to determine quickly whether a turtle is growing in the season in which it is captured. There is little actual increase in thickness of the scute after the first three or four years of growth. The epidermal laminae are therefore like low pyramids only in appearance. This appearance of thickness is enhanced by the contours of bony shell which correspond to the contours of the scutes.

Minor growth-rings differ from major growth-rings in appearance and in origin. Ewing (_op. cit._: 91) recognized the difference in appearance and referred to minor growth-rings as "pseudoannual growth zones." Minor growth-rings result from temporary cessations of growth that occur in the course of the growing season, not at the onset of hibernation. They are mere dips or depressions in the surface of the scute. The occurrence of minor growth-rings indicates that interruptions in growth of short duration do not result in the formation of a new layer of epidermis. Slowing of growth or its temporary cessation may be caused by injuries, periods of quiescence due to dry, hot, or cold weather, lack of food, and possibly by physiological stress, especially in females, in the season of reproduction. Minor growth-rings that lie immediately proximal to major growth-rings (Pl. 22, Fig. 2), are the result of temporary dormancy in a period of cold weather at the end of a growing season, followed by nearly normal activity in a warmer period before winter-long hibernation is begun. Cagle (1946:699) stated that sliders (_Pseudemys scripta elegans_) remaining several weeks in a pond that had become barren of food would stop growing and develop a growth-ring on the epidermal laminae; he did not indicate, however, whether these growth-rings differ from those formed during hibernation.

In species that periodically shed scutes a zone of fracture develops between the old and new layers of the scute as each new layer of epidermis is formed, and the old layer is shed. Considering reptiles as a group, skin shedding is of general occurrence; the process in _Pseudemys_ and _Chrysemys_ differs in no basic respect from that in most reptiles. Retention of scutes in terrestrial emyids and in testudinids is one of many specializations for existence on land. Retention of scutes protects the shell of terrestrial chelonians against wear. Some box turtles were observed to have several scutes of the carapace in the process of exfoliation but no exfoliation was observed on the plastron. Exfoliation ordinarily occurred on the scutes of the carapace that were the least worn; the exfoliating portion included the areola and the three or four oldest (first formed) layers of the scute. The layer of scute exposed was smooth and had yellow markings that were only slightly less distinct than those on the portion that was exfoliating.

Wear on the shell of a box turtle reduces the thickness of scutes, as does the shedding of scutes in the aquatic emyids mentioned. It is noteworthy that any of the layers in the scute of a box turtle can form the cornified surface of the scute when the layers above it wear away or are shed.

It is uncertain whether turtles that have ceased to grow at a measurable rate continue to elaborate a new layer of epidermis at the beginning of each season. Greatly worn shells of ornate box turtles, particularly those of the subspecies _luteola_, have only a thin layer of epidermis through which the bones of the shell and the sutures between the bones are visible. I suspect that, in these old individuals, the germinal layer of the epidermis does not become active each year but retains the capacity to elaborate new epidermis if the shell becomes worn thin enough to expose and endanger the bone beneath it. The germinal layer of old turtles loses the capacity to produce color.

Major growth-rings constitute a valuable and accurate history of growth that can be studied at any time in the life of the turtle if they have not been obliterated. They are accurate indicators of age only as long as regular growth continues but may be used to study early years of growth even in turtles that are no longer growing. Minor growth-rings, if properly interpreted, provide additional information on growing conditions in the course of each growing season.

Nichols (1939a: 16-17) found that the number of growth-rings formed in marked individuals of _T. carolina_ did not correspond to the number of growing seasons elapsed; he concluded that growth-rings were unreliable as indicators of age and that box turtles frequently skipped seasons of growth. Woodbury and Hardy (1948:166-167) and Miller (1955:114) came to approximately the same conclusion concerning _Gopherus agassizi_. It is significant that these workers were studying turtles of all sizes and ages, some of which were past the age of regular, annual growth. Cagle's review of the literature concerning growth-rings in turtles (1946) suggests that, in most of the species studied, growth-rings are formed regularly in individuals that have not attained sexual maturity but are formed irregularly after puberty.

Cagle's (_op. cit._) careful studies of free-living populations of _Pseudemys scripta_ showed that growth-rings, once formed, did not change in size, that the area between any two major growth-rings represented one season of growth, and that growth-rings were reliable indicators of age as long as the impression of the areola remained on the scutes studied. Cagle noted decreasing distinctness of growth-rings after each molt.

The relative lengths of the abdominal lamina and the plastron remain approximately the same throughout life in _T. ornata_. Measurements were made of the plastron, carapace, and abdominal lamina in 103 specimens of _T. o. ornata_ from Kansas and neighboring states. The series of specimens was divided into five nearly equal groups according to length of carapace. Table 3 summarizes the relationship of abdominal length to plastral length, and of carapace length to plastral length. The mathematical mean of the ratio, abdominal length/plastral length, in each of the four groups of larger-sized turtles, was not significantly different from the same ratio in the hatchling group. The relative lengths of carapace and plastron are not so constant; the carapace is usually longer than the plastron in hatchlings and juveniles, but shorter than the plastron in adults, especially adult females.

TABLE 3.--The Relationship of Length of Abdominal Scute to
Plastral Length, and of Plastral Length to Length of Carapace,
in 103 Specimens of _T. o. ornata_ Arranged in Five Groups
According to Length of Carapace. The Relative Lengths of
Abdominal Scute and Plastron are not Significantly Different in
the Five Groups. The Plastron Tends to be Longer than the
Carapace in Specimens of Adult or Nearly Adult Size.
===============+=========+===========================+==================
| | Length of abdominal |Individuals having
| | as a percentage of | plastron longer
LENGTH | Number | length of plastron | than carapace
OF CARAPACE | of | |
|specimens|----------------+----------+------+-----------
| |Mean A+-[sigma]m | Extremes |Number|Percentage
---------------+---------+----------------+----------+------+-----------
Less than | | | | |
50 mm. | 23 | 18.3A+-.498 |13.7-20.3 | 7 | 38.5
(Juveniles) | | | | |
---------------+---------+----------------+----------+------+-----------
50 to 69 mm. | 20 | 17.8A+-.303 |15.2-20.2 | 8 | 40.0
(Juveniles) | | | | |
---------------+---------+----------------+----------+------+-----------
70 to 100 mm. | 20 | 17.9A+-.445 |14.3-20.6 | 15 | 75.0
(Subadults) | | | | |
---------------+---------+----------------+----------+------+-----------
More than | | | | |
100 mm. | 20 | 17.8A+-.236 |16.4-20.6 | 13 | 65.0
(Adult males) | | | | |
---------------+---------+----------------+----------+------+-----------
More than | | | | |
100 mm. | 20 | 18.8A+-.510 |15.1-25.7 | 19 | 95.0
(Adult females)| | | | |
---------------+---------+----------------+----------+------+-----------

The length of any growth-ring on the abdominal lamina can be used to determine the approximate length of the plastron at the time the growth-ring was formed. Actual and relative increases in length of the plastron can be determined in a like manner. For example, a seven-year-old juvenile (KU 3283) with a plastron 74.0 millimeters long had abdominal growth-rings (beginning with areola and ending with the actual length of the abdominal) 5.9, 7.8, 9.5, 10.7, 12.0, 12.5, 14.3, and 14.9 millimeters long. Using the

[AB ABA╣]
proportion, [-- = -- ], where AB is the abdominal length, PL the
[PL X ]

plastral length, ABA╣ the length of any given growth-ring, and X the plastral length at the time growth-ring ABA╣ was formed, the plastral length of this individual was 29.3 millimeters at hatching, 38.8 at the end of the first full season of growth, and 47.2, 53.2, 59.6, 62.1, and 71.0 millimeters at the end of the first, second, third, fourth, fifth, and sixth seasons of growth, respectively. The present length of the abdominal (14.9 mm.) indicates an increment of three millimeters in plastral length in the seventh season, up to the time the turtle was killed (June 25). This method of studying growth in turtles was first used by Sergeev (1937) and later more extensively used by Cagle (1946 and 1948) in his researches on _Pseudemys scripta_. Because the plastron is curved, no straight-line measurement of it or its parts can express true length. Cagle (1946 and 1948) minimized error by expressing plastral length as the sum of the laminal (or growth-ring) lengths. This method was not possible in the present study because growth-rings on parts of one or more laminae (chiefly the gulars and anals) were usually obliterated by wear, even in young specimens. It was necessary to express plastral length as the sum of the lengths of forelobe and hind lobe.

The abdominal lamina was selected for study because of its length (second longest lamina of plastron), greater symmetry, and flattened form. Although the abdominal is probably subject to greater, over-all wear than any other lamina of the shell, wear is even, not localized as it is on the gulars and anals.

In instances where some of the growth-rings on an abdominal lamina were worn but other rings remained distinct, reference to other, less worn lamina permitted a correct interpretation of indistinct rings.

Abdominal laminae were measured at the interlaminal seam; since the laminae frequently did not meet perfectly along the midline (and were of unequal length), the right abdominal was measured in all specimens. Growth-rings on the abdominal laminae were measured in the manner shown in Plate 22.