After ovulation, the collapsed follicle a.s.sumes 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 a.s.sociated 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 pa.s.s 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 s.e.xlineatus_, 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 sh.e.l.ls soon after they enter the oviducts. No sh.e.l.l-less eggs were found in oviducts but several specimens of _T.

ornata_ had oviducal eggs, the thin, parchmentlike sh.e.l.ls 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 sh.e.l.l. Eggs having incompletely developed sh.e.l.ls were successfully incubated in the laboratory. Cagle (1950:38) found sh.e.l.led 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 sh.e.l.led 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, ill.u.s.trate 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 spa.r.s.e 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 ill.u.s.trated 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 sc.r.a.ps 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, un.o.bserved, 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 aga.s.sizi_.

Eggs

The number of eggs in 23 clutches ranged from two to eight (mean, 4.7 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 p.r.o.nounced 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_.

[Ill.u.s.tration: FIG. 6. The relation of plastral length to number of eggs laid by 21 females of _T. o. ornata_ from eastern Kansas.]

Sh.e.l.ls of the eggs are translucent and pinkish or yellowish when the eggs are in the oviducts. After several days outside the oviducts the sh.e.l.ls 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 sh.e.l.l 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. Sh.e.l.ls of infertile eggs became brittle and slimy after several weeks.

The outer layer of the sh.e.l.l 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 sh.e.l.l becomes flexible and has a leathery texture. The sh.e.l.l is finely granulated but appears smooth to the unaided eye. The granulations are approximately the same as those ill.u.s.trated by Aga.s.siz (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 2.77 (31.3-40.9); width, 21.72 1.04 (20.0-26.3); and weight, 10.09 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 sh.e.l.ls, 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 sh.e.l.l 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 sh.e.l.l.

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 r.e.t.a.r.d 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 gra.s.sland 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 sh.e.l.l 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 sh.e.l.l and cause the outer layer to crumble away, especially where the head and forequarters of the embryo lie against the sh.e.l.l. Some embryos could be seen spasmodically thrusting the head and neck dorsally against the sh.e.l.l.