Part 6

Further evidence for multiple clutches was the absence of enlarged ovarian follicles in some females obtained in September. Atretic follicles, ordinarily orange, brown, or purplish, were observed on the ovaries of many of the females examined; in most instances, not more than two follicles of the small or medium size groups were atretic. Atresia was in no instance great enough to account for the complete loss of enlarged follicles.

Further study probably will show that many of the females laying in May and early June lay again before the end of July, and that eggs in the oviducts of females captured in the latter month frequently represent second clutches. Under favorable conditions, eggs laid by the end of July would have a good chance of hatching before the advent of cold weather in autumn; turtles hatching too late to escape from the nest could burrow into its sides and probably escape freezing temperatures.

Cagle's findings concerning Pseudemys scripta (1950:38) and Chrysemys picta (1954:228-9) suggest that these species lay more than one clutch per season, at least in the southern parts of their ranges. Carr (1952) indicated that multiple layings were known in most species of marine turtles (families Dermochelydae and Chelonidae) and strongly suspected in other species. Other turtles recorded to have produced multiple clutches in a single season (based chiefly on captive specimens or cultured populations) include: the starred tortoise, Geochelone elegans (Deraniyagala, 1939:287); the Asiatic trionychid, Lissemys punctata (op. cit.:304); the diamond-backed terrapin, Malaclemys terrapin (Hildebrand and Prytherch, 1947:2); and the Japanese soft-shelled turtle, Trionyx japonicus (Mitsukuri, 1895, cited by Cagle, 1950:38).

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.