HABITAT IN KANSASClarke (1958:40-45) reportedT. o. ornatain all terrestrial communities studied in Osage County; he considered the subspecies to be characteristic of the "… cultivated-field community …" and to be of frequent occurrence in (but not characteristic of) the "… Oak-Walnut Hillside Forest …, Buckbrush-Sumac …, and Prairie communities …". Brennan (1937:345) foundT. o. ornatato be equally abundant in mixed prairie and prairie-streamside habitats in Ellis County; the subspecies was much rarer on rocky hillsides and in the habitat surrounding prairie ponds. Carpenter (1940:641) listedT. o. ornataas an inhabitant of "… tall and mixed-grass prairies …" (also in Oklahoma and Nebraska). Fitch (1958:99) found the order of preference for habitats at the Natural History Reservation to be grazed pasture land, woodland, open fields with undisturbedprairie vegetation, and fallow fields with a rank growth of weeds.At the Damm Farm the greatest number of box turtles was collected on the pasture, especially in three areas designated inPlate 15as the "northwest corner," "southern ravine," and "house pond" areas. These three areas had several features in common. All contained ravines and rocky slopes that provided many places of concealment (dens, burrows of larger animals, and suitable substrate for the excavation of earthen forms). All contained water (in ponds and intermittent streams) for most of the year; and, all were frequented daily by cattle that left an abundant supply of dung in which box turtles foraged. In addition, each of the three areas contained at least one mulberry tree, under which fruit was abundant in the months of June and July.The relative numbers of box turtles found in different areas on the Damm Farm were, of course, governed to some extent by my activity in these areas and by the relative ease with which box turtles were seen in different types of vegetational cover. Turtles were more easily seen in the pasture (especially in sparsely vegetated or denuded areas) where much of my field work was done on horseback, than in the wooded areas, where excursions were usually made on foot. It was evident, however, after mapping known ranges and studying patterns of movement in marked turtles, that concentrations in the three above-mentioned areas of pasture were an indication of actual preference by turtles for the more favorable habitat in these areas rather than the result of incomplete sampling.REPRODUCTIONMatingMating takes place throughout the season of activity but is most common in spring—soon after emergence from hibernation—and in autumn. Turtles frequently copulated in the laboratory in spring and autumn. Copulation was observed under natural conditions on several occasions but only once at the Damm Farm.Norris and Zwiefel (1950:4) saw two captive individuals ofT. o. luteolacopulating on 12 August; copulation lasted two hours. Brumwell (1940:391-2) gave the following description of mating inT. o. ornata. A male pursued a female for nearly half an hour, first nudging the margins of her shell and later approaching her rapidly from the rear and hurling himself on her back in an attempt to mount, at the same time emitting a stream of liquid from each nostril. The liquid was presumably water; both sexes had imbibedwater in a pond just before courtship began. Brumwell suggested that pressure on the plastron of the male had forced the water out his nostrils. The pair remained in the coital position for 30 minutes after the male had achieved intromission. In another instance, Brumwell (loc. cit.) saw four males pursuing a single female, the males exhibiting the same behavior (nudging and lunging) outlined above. Males that attempted to mount other males were repelled by defensive snapping of the approached male. The female also snapped at some of the males that tried to mount her. One male was finally successful in mounting and was henceforth unmolested by the other males. Brumwell suggested that shell biting and tapping may be methods of sex-recognition.In the several instances of mating that I observed, the male, after mounting the shell of the female (Pl. 28), gripped her, with the first claws of his hind feet, just beneath her legs or on the skin of the gluteal region and, with the remaining three claws, gripped the posterior edges of her plastron. In most instances the female secured the male's legs by hooking her own legs around them. The coital position ofT. ornataseems to differ from that ofT. carolina, at least in regard to the position of the male's legs. The coital positions ofT. carolinaillustrated by Cahn (1937:94, Fig. 13) are physically impossible forT. ornata.InT. ornatathe pressure exerted on the male's legs by the female probably impairs circulation and probably is painful to the male, especially after coitus, when the male falls backward but is still held by the female. The heavily developed musculature of the legs of males may be an adaptation to strengthen the legs for this temporary period of stress. Evans (1953:191) and Cahn and Conder (1932:87-88) observed the hind legs of males ofT. carolinato be noticeably weakened after copulation, causing the males to remain inactive for several hours.Evans (op. cit.) observed 72 matings ofT. carolinaand divided the process into three phases as follows: 1) circling, pushing and biting by the male; 2) mounting (female with shell closed); and, 3) coition (female with shell open). Penn and Pottharst (1940:26) reported that captiveT. carolinain New Orleans mated chiefly under conditions of optimum temperature (21 to 27° C.) and high humidity; some matings took place in a pool of water. Males pushed females about after mating, often rolling them over several times.Because ornate box turtles observed by me were able easily to right themselves from an inverted position on substrata of allkinds, males left lying on their backs after copulation are probably in no danger of perishing in this position, as was suggested by Allard (1939) forT. carolina.InseminationOviducts of several females were flushed by means of a pipette to determine whether they contained sperm. Approximately half of the females captured in May, 1956, had sperm in their oviducts, but females captured in June and July did not. Sperm flushed from the oviducts were in clumps of several hundred and showed no sign of motility a few minutes after the female was anesthetized with chloroform. No sperm were found in the oviducts of immature females but one female of nearly adult size was observed in copulation with a mature male.Thorough examination of microscopic sections of oviduct (taken at various times in the season of activity) usually revealed a few sperm lodged in the folds (Pl. 19, Fig. 8) of the cephalic as well as the caudal portion of the tube, but no specialized seminal receptacles such as occur in snakes (Fox, 1956) were present. Fertilization without reinsemination probably occurs inT. ornata. Ewing (1943) and Finneran (1948:126) reported that females ofT. carolinaproduced fertile eggs for periods of four and two years, respectively, after being removed from all contact with males.Sexual Cycle of MalesTestes were preserved in each month from April to October. The following description of spermatogenesis is based chiefly on material collected in 1955, although testes were preserved also in 1954. Comparison of material obtained in 1954 and 1955 revealed that spermatogenesis began earlier and was more advanced on any given date in 1955 than in 1954.Testes of mature individuals are pale yellow and slightly oblong. The epididymis is ordinarily dark brown or black and contrasts sharply with the color of the testes. Size of testes was expressed as the average length (greatest diameter) of both testes. Testes are smallest in April, immediately after emergence from hibernation, and largest in early September (Pl. 20, Figs. 3-4). They are nearly spherical when of maximum size; increase in bulk, therefore, is relatively greater than the increase in size shown inFigure 3. They increase in size from April until early June, recede during most of June, and again increase in size in July and August. They remainlarge from early September until hibernation is begun, becoming only slightly smaller in late September and October.Increase in size following emergence from hibernation may be due in part to proliferation of the sustentacular cytoplasm. Decrease in size in early June is correlated with the end of the period of most active mating; maximal size is coincident with the peak of the spermatogenic cycle in early September.Fig. 3.Seasonal fluctuations in size (average greatest diameter) of testes inT. o. ornataas determined by examination of 40 specimens from eastern Kansas.Spermatogenesis (refer toPl. 19, Figs. 1-5) begins in early May when a few spermatogonia appear in the seminiferous tubules. The histological appearance of testes preserved in April and May is much the same. Nuclei of Sertoli cells, which outnumber the spermatogonia, are evident at the periphery of the tubules and the clear cytoplasm of the cells extends into and nearly fills the lumina. The few darkly stained spermatids that are present in April are cells that probably were produced in the previous summer. Sperm are present in small groups within the sustentacular cytoplasm, but ordinarily are absent in the lumina.Primary spermatocytes appear in the tubules from mid-May to early June. By mid-May there are practically no sperm at any place in the tubules. The sustentacular cytoplasm has a less compact arrangement in late May than in April.Spermatogenesis is well under way by mid-June; at this time, two or three distinct layers of primary and secondary spermatocytes are present and these cells outnumber the Sertoli cells. The lumina are filled with cellular detritus and are no longer bordered by a clear ring of sustentacular cytoplasm. No sperm are present.Spermatids appear in late June and a few of them undergo metamorphosis in early July; by mid-July, spermatids and secondary spermatocytes are the dominant cells in the seminiferous tubules, although spermatogonia are still active.By late August, clusters of sperm and metamorphosing spermatids surround the Sertoli cells; large numbers of sperm as well as sloughed cells representing various spermatogenic stages are present in the lumina. Secondary spermatocytes are still evident near the periphery of the tubules but they are much less numerous than spermatids. The germinal epithelium is still semiactive and small groups of primary spermatocytes are present in nearly all of the tubules.The spermatogenic cycle is completed in the latter half of October when most of the spermatozoa pass into the epididymides. A few spermatozoa and spermatids remain in the seminiferous tubules during hibernation. Although no testicular material was obtained from hibernating turtles, comparisons of sections made in October and April show that the germinal epithelium remains inactive from autumn until spring. Possibly some spermiogenesis takes place in the early phases of hibernation or in the period in late autumn when turtles are intermittently active. It is uncertain whether the reorganization of the sustentacular cytoplasm occurs in autumn, in spring, or in the course of hibernation.The seminiferous tubules of immature males are small, lack lumina, and contain a few large but inactive spermatogonia (Pl. 19, Fig. 6). The testes of specimens that were nearly mature contained primary and secondary spermatocytes but lacked lumina; it was thought that such individuals would have matured in the following summer and bred in the following autumn.Mature sperm were found in epididymides at all times of the year but were most numerous in spring and autumn, the period betweenspermatogenic cycles (Pl. 19, Fig. 7). Sperm expelled from the epididymides in autumn matings are seemingly replaced by others from the seminiferous tubules; the epididymides become much smaller when their supply of sperm is nearly exhausted after spring mating.Risley (1938:304) found the testes of the common musk turtle,Sternotherus odoratus, to be largest in August and smallest in early May. Recession of testes in spring was coincident with the period of active breeding; increase in size, later in the season, corresponded to increasing spermatogenic activity and enlargement of seminiferous tubules. Altland (1951:600-603) found the spermatogenic cycle ofTerrapene carolinato be nearly like that ofSternotherus odoratus. Fox (1952) found that testes of garter snakes (Thamnophis sirtalisandT. elegans) in California reached a peak of spermatogenic activity in midsummer, regressed in the latter half of the summer, and were inactive in winter.The spermatogenic cycle ofT. ornataas here reported, differs in no important respect from those ofThamnophis,Sternotherus odoratus, orTerrapene carolina, except that inT. ornatathe cycle begins and ends somewhat later in the season of activity. In most of the lizards that have been studied (Fox, 1952:492-3), spermatogenesis reaches a peak in spring (more or less coincident with the mating period and with ovulation) and the germinal epithelium remains active in winter.Sternotherus,Terrapene, andThamnophisare alike in completing spermatogenesis late in the season and storing spermatozoa, in the seminiferous tubules or in the epididymides, during hibernation.It is noteworthy that, in the turtles and snakes mentioned above, sperm produced in autumn are used to fertilize eggs laid in the following year, and mating [with the exception ofThamnophis elegans, (Fox, 1956)] occurs in both spring and autumn. It is not definitely known in any of these instances, whether sperm resulting from autumn or spring inseminations (or both) fertilize the eggs. Risley (1933:693) found motile sperm in the oviducts of femaleSternotherus odoratusthat had recently emerged from hibernation; he believed that spring mating, although it commonly occurred, was not necessary to fertilize eggs. Disadvantages, if any, of completing spermatogenesis well in advance of ovulation seem to be at least partly counteracted by two annual mating periods or by mating throughout the season of activity.Sexual Cycle of FemalesThe following account of oögenesis is based on examination of preserved ovaries from 68 mature specimens. The ages of most specimens were known, inasmuch as the specimens were used in studies of growth as well as gametogenesis. Other data were obtained from adult females that were dissected but not preserved, and from immature females.Fig. 4.Seasonal fluctuations in ovarian weight inT. o. ornata, as determined by examination of 60 specimens from eastern Kansas.Size of ovarian follicles was determined by means of a clear plastic gauge containing notches 5, 10, 15, 20, and 25 millimeters wide. The number of follicles within a given size range could be quickly determined by finding the smallest notch into which the follicles fit. It was necessary to weigh all ovaries after preservation since some of them had not been weighed when fresh. Since all ovarian samples were preserved in the same manner, weightsremained relatively the same. Preserved material was lighter than fresh by an average of 13 per cent. Follicles less than one millimeter in diameter were not counted. Corpora lutea and corpora albicantia were studied under a binocular dissecting microscope. No histological studies were made of the female reproductive system.Ovarian follicles and oviducal eggs were recorded separately for the right and left sides. Each ovary was always kept associated with the oviduct of the same side, but in some instances it was not recorded whether the organs were left or right.Ovaries ordinarily weighed most in October, March, and April, when most females contained enlarged follicles, and least in August and September when the supply of enlarged follicles was usually exhausted (Figs.4and5).Fig. 5. The seasonal occurrence of enlarged ovarian follicles in femalesFig. 5.The seasonal occurrence of enlarged ovarian follicles in females ofT. o. ornata, expressed, for each month, as the percentage of total females that contained two or more follicles having diameters greater than 15 mm. Total number of females in each of the samples is shown in parentheses at the top of each bar.The ovarian cycle begins in July or August, after ovulation has occurred. At that time many minute follicles form on the germinal ridges of the ovaries. On the basis of the material that I examined, it seems that ovarian follicles either grow to nearly mature size in the season preceding ovulation and remain quiescent over winter or grow rapidly in the period of approximately six weeks between spring emergence and ovulation. Altland (1951:603-5) reportedthat the former condition was the usual one inT. carolina; he suggested that possibly some of the enlarged follicles were absorbed during hibernation.Examination of yolks of oviducal eggs revealed that follicles mature when they reach a diameter of 16 to 20 millimeters and a weight of two to two and one-half grams (Pl. 20, Fig. 1).The enlarged follicles remaining on the ovaries after ovulation (excluding those smaller than six mm.) can be grouped according to diameter as: large (greater than 15 mm.), medium (11 to 15 mm.), and small (six to 10 mm.). Ten females collected in the period from June 2 to 8, after they had ovulated, all had follicles falling in at least one of these size groups, and eight had follicles falling in two or more of the groups. In females having enlarged follicles of more than one of the size groups, there were several follicles in each of two groups and no follicles, or only one follicle, in the remaining group. Enlarged follicles represent future clutches but whether the enlarged follicles will be ovulated in the same season or in a later season is questionable.Evidence found in the present study suggested that at least a few females lay more than one clutch of eggs per year. Among 34 specimens obtained in June and July, eight (24 per cent) had corpora lutea (or easily discernible corpora albicantia) and at least two follicles more than 15 millimeters in diameter; in three specimens (9 per cent) the ovaries bore fresh corpora lutea (representing recent ovulations) and a set of older corpora lutea (representing ovulations that had occurred several weeks previously). It was thought that each of these eleven females (33 per cent of sample) had produced or would have produced two clutches of eggs in the season of its capture. The number of large follicles present after the first set of ovulations (mean, 3.5) was fewer in most instances than the average clutch-size (see below), indicating that second clutches are smaller than first clutches. Smaller second clutches were found also inT. carolina(Legler, 1958).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 layingin 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 concerningPseudemys scripta(1950:38) andChrysemys 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 inT. 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 shapeand 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 sub-adults (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 ofTerrapene 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 thatT. ornatadeposits 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 inT. ornataand is known to occur also inT. 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 ofT. ornatahad 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 severalPseudemys scriptabut found no yolkless eggs in nests. No yolkless eggs were found in specimens ofT. ornatain 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.NestingOrnate 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 ofT. 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 ofT. o. ornatawere 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 inPlate 21was 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 ofT. 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 forT. 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 elsehas 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 inChrysemys, because of the narrow hind foot of the femaleT. 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 inT. carolina. It is worthy of mention, however, that Booth (1958:261) reported the digging of a preliminary cavity by a captive individual ofGopherus agassizi.EggsThe number of eggs in 23 clutches ranged from two to eight (mean, 4.7 ± 1.37 σs]); 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) forPseudemys scripta. The small size ofT. ornata, in comparison with other emyid turtles, seemingly limits the number of eggs that can be accommodated internally. The number of eggs per clutch inT. carolina[2 to 7, average 4.2, Allard (1935:331)], is nearly the same as that ofT. ornata.Fig. 6. The relation of plastral length to number of eggs laidFig. 6.The relation of plastral length to number of eggs laid by 21 females ofT. o. ornatafrom eastern Kansas.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) forT. 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 tendencyfor 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 inSternotherus odoratus, as did Allard (1935:331) inT. carolina. Measurements in the literature of the size of eggs ofT. ornatasuggest a width greater than that stated above, probably because some eggs already had begun to expand when measured.Eggs ofT. ornataexpand 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 ofChrysemys pictaandChelydra 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 DevelopmentEggs 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, eggswere 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 ofSternotherus odoratusto be longer at lower temperatures; corresponding observations were made by Allard (1935:332) and Driver (1946:173) on the eggs ofTerrapene carolina. Cagle (1950:40) and Cunningham (1939) found no distinct differences in length of incubation period for eggs ofPseudemys scriptaandMalaclemys 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.
HABITAT IN KANSAS
Clarke (1958:40-45) reportedT. o. ornatain all terrestrial communities studied in Osage County; he considered the subspecies to be characteristic of the "… cultivated-field community …" and to be of frequent occurrence in (but not characteristic of) the "… Oak-Walnut Hillside Forest …, Buckbrush-Sumac …, and Prairie communities …". Brennan (1937:345) foundT. o. ornatato be equally abundant in mixed prairie and prairie-streamside habitats in Ellis County; the subspecies was much rarer on rocky hillsides and in the habitat surrounding prairie ponds. Carpenter (1940:641) listedT. o. ornataas an inhabitant of "… tall and mixed-grass prairies …" (also in Oklahoma and Nebraska). Fitch (1958:99) found the order of preference for habitats at the Natural History Reservation to be grazed pasture land, woodland, open fields with undisturbedprairie vegetation, and fallow fields with a rank growth of weeds.
At the Damm Farm the greatest number of box turtles was collected on the pasture, especially in three areas designated inPlate 15as the "northwest corner," "southern ravine," and "house pond" areas. These three areas had several features in common. All contained ravines and rocky slopes that provided many places of concealment (dens, burrows of larger animals, and suitable substrate for the excavation of earthen forms). All contained water (in ponds and intermittent streams) for most of the year; and, all were frequented daily by cattle that left an abundant supply of dung in which box turtles foraged. In addition, each of the three areas contained at least one mulberry tree, under which fruit was abundant in the months of June and July.
The relative numbers of box turtles found in different areas on the Damm Farm were, of course, governed to some extent by my activity in these areas and by the relative ease with which box turtles were seen in different types of vegetational cover. Turtles were more easily seen in the pasture (especially in sparsely vegetated or denuded areas) where much of my field work was done on horseback, than in the wooded areas, where excursions were usually made on foot. It was evident, however, after mapping known ranges and studying patterns of movement in marked turtles, that concentrations in the three above-mentioned areas of pasture were an indication of actual preference by turtles for the more favorable habitat in these areas rather than the result of incomplete sampling.
REPRODUCTION
Mating
Mating takes place throughout the season of activity but is most common in spring—soon after emergence from hibernation—and in autumn. Turtles frequently copulated in the laboratory in spring and autumn. Copulation was observed under natural conditions on several occasions but only once at the Damm Farm.
Norris and Zwiefel (1950:4) saw two captive individuals ofT. o. luteolacopulating on 12 August; copulation lasted two hours. Brumwell (1940:391-2) gave the following description of mating inT. o. ornata. A male pursued a female for nearly half an hour, first nudging the margins of her shell and later approaching her rapidly from the rear and hurling himself on her back in an attempt to mount, at the same time emitting a stream of liquid from each nostril. The liquid was presumably water; both sexes had imbibedwater in a pond just before courtship began. Brumwell suggested that pressure on the plastron of the male had forced the water out his nostrils. The pair remained in the coital position for 30 minutes after the male had achieved intromission. In another instance, Brumwell (loc. cit.) saw four males pursuing a single female, the males exhibiting the same behavior (nudging and lunging) outlined above. Males that attempted to mount other males were repelled by defensive snapping of the approached male. The female also snapped at some of the males that tried to mount her. One male was finally successful in mounting and was henceforth unmolested by the other males. Brumwell suggested that shell biting and tapping may be methods of sex-recognition.
In the several instances of mating that I observed, the male, after mounting the shell of the female (Pl. 28), gripped her, with the first claws of his hind feet, just beneath her legs or on the skin of the gluteal region and, with the remaining three claws, gripped the posterior edges of her plastron. In most instances the female secured the male's legs by hooking her own legs around them. The coital position ofT. ornataseems to differ from that ofT. carolina, at least in regard to the position of the male's legs. The coital positions ofT. carolinaillustrated by Cahn (1937:94, Fig. 13) are physically impossible forT. ornata.
InT. ornatathe pressure exerted on the male's legs by the female probably impairs circulation and probably is painful to the male, especially after coitus, when the male falls backward but is still held by the female. The heavily developed musculature of the legs of males may be an adaptation to strengthen the legs for this temporary period of stress. Evans (1953:191) and Cahn and Conder (1932:87-88) observed the hind legs of males ofT. carolinato be noticeably weakened after copulation, causing the males to remain inactive for several hours.
Evans (op. cit.) observed 72 matings ofT. carolinaand divided the process into three phases as follows: 1) circling, pushing and biting by the male; 2) mounting (female with shell closed); and, 3) coition (female with shell open). Penn and Pottharst (1940:26) reported that captiveT. carolinain New Orleans mated chiefly under conditions of optimum temperature (21 to 27° C.) and high humidity; some matings took place in a pool of water. Males pushed females about after mating, often rolling them over several times.
Because ornate box turtles observed by me were able easily to right themselves from an inverted position on substrata of allkinds, males left lying on their backs after copulation are probably in no danger of perishing in this position, as was suggested by Allard (1939) forT. carolina.
Insemination
Oviducts of several females were flushed by means of a pipette to determine whether they contained sperm. Approximately half of the females captured in May, 1956, had sperm in their oviducts, but females captured in June and July did not. Sperm flushed from the oviducts were in clumps of several hundred and showed no sign of motility a few minutes after the female was anesthetized with chloroform. No sperm were found in the oviducts of immature females but one female of nearly adult size was observed in copulation with a mature male.
Thorough examination of microscopic sections of oviduct (taken at various times in the season of activity) usually revealed a few sperm lodged in the folds (Pl. 19, Fig. 8) of the cephalic as well as the caudal portion of the tube, but no specialized seminal receptacles such as occur in snakes (Fox, 1956) were present. Fertilization without reinsemination probably occurs inT. ornata. Ewing (1943) and Finneran (1948:126) reported that females ofT. carolinaproduced fertile eggs for periods of four and two years, respectively, after being removed from all contact with males.
Sexual Cycle of Males
Testes were preserved in each month from April to October. The following description of spermatogenesis is based chiefly on material collected in 1955, although testes were preserved also in 1954. Comparison of material obtained in 1954 and 1955 revealed that spermatogenesis began earlier and was more advanced on any given date in 1955 than in 1954.
Testes of mature individuals are pale yellow and slightly oblong. The epididymis is ordinarily dark brown or black and contrasts sharply with the color of the testes. Size of testes was expressed as the average length (greatest diameter) of both testes. Testes are smallest in April, immediately after emergence from hibernation, and largest in early September (Pl. 20, Figs. 3-4). They are nearly spherical when of maximum size; increase in bulk, therefore, is relatively greater than the increase in size shown inFigure 3. They increase in size from April until early June, recede during most of June, and again increase in size in July and August. They remainlarge from early September until hibernation is begun, becoming only slightly smaller in late September and October.
Increase in size following emergence from hibernation may be due in part to proliferation of the sustentacular cytoplasm. Decrease in size in early June is correlated with the end of the period of most active mating; maximal size is coincident with the peak of the spermatogenic cycle in early September.
Fig. 3.Seasonal fluctuations in size (average greatest diameter) of testes inT. o. ornataas determined by examination of 40 specimens from eastern Kansas.
Fig. 3.Seasonal fluctuations in size (average greatest diameter) of testes inT. o. ornataas determined by examination of 40 specimens from eastern Kansas.
Spermatogenesis (refer toPl. 19, Figs. 1-5) begins in early May when a few spermatogonia appear in the seminiferous tubules. The histological appearance of testes preserved in April and May is much the same. Nuclei of Sertoli cells, which outnumber the spermatogonia, are evident at the periphery of the tubules and the clear cytoplasm of the cells extends into and nearly fills the lumina. The few darkly stained spermatids that are present in April are cells that probably were produced in the previous summer. Sperm are present in small groups within the sustentacular cytoplasm, but ordinarily are absent in the lumina.
Primary spermatocytes appear in the tubules from mid-May to early June. By mid-May there are practically no sperm at any place in the tubules. The sustentacular cytoplasm has a less compact arrangement in late May than in April.
Spermatogenesis is well under way by mid-June; at this time, two or three distinct layers of primary and secondary spermatocytes are present and these cells outnumber the Sertoli cells. The lumina are filled with cellular detritus and are no longer bordered by a clear ring of sustentacular cytoplasm. No sperm are present.
Spermatids appear in late June and a few of them undergo metamorphosis in early July; by mid-July, spermatids and secondary spermatocytes are the dominant cells in the seminiferous tubules, although spermatogonia are still active.
By late August, clusters of sperm and metamorphosing spermatids surround the Sertoli cells; large numbers of sperm as well as sloughed cells representing various spermatogenic stages are present in the lumina. Secondary spermatocytes are still evident near the periphery of the tubules but they are much less numerous than spermatids. The germinal epithelium is still semiactive and small groups of primary spermatocytes are present in nearly all of the tubules.
The spermatogenic cycle is completed in the latter half of October when most of the spermatozoa pass into the epididymides. A few spermatozoa and spermatids remain in the seminiferous tubules during hibernation. Although no testicular material was obtained from hibernating turtles, comparisons of sections made in October and April show that the germinal epithelium remains inactive from autumn until spring. Possibly some spermiogenesis takes place in the early phases of hibernation or in the period in late autumn when turtles are intermittently active. It is uncertain whether the reorganization of the sustentacular cytoplasm occurs in autumn, in spring, or in the course of hibernation.
The seminiferous tubules of immature males are small, lack lumina, and contain a few large but inactive spermatogonia (Pl. 19, Fig. 6). The testes of specimens that were nearly mature contained primary and secondary spermatocytes but lacked lumina; it was thought that such individuals would have matured in the following summer and bred in the following autumn.
Mature sperm were found in epididymides at all times of the year but were most numerous in spring and autumn, the period betweenspermatogenic cycles (Pl. 19, Fig. 7). Sperm expelled from the epididymides in autumn matings are seemingly replaced by others from the seminiferous tubules; the epididymides become much smaller when their supply of sperm is nearly exhausted after spring mating.
Risley (1938:304) found the testes of the common musk turtle,Sternotherus odoratus, to be largest in August and smallest in early May. Recession of testes in spring was coincident with the period of active breeding; increase in size, later in the season, corresponded to increasing spermatogenic activity and enlargement of seminiferous tubules. Altland (1951:600-603) found the spermatogenic cycle ofTerrapene carolinato be nearly like that ofSternotherus odoratus. Fox (1952) found that testes of garter snakes (Thamnophis sirtalisandT. elegans) in California reached a peak of spermatogenic activity in midsummer, regressed in the latter half of the summer, and were inactive in winter.
The spermatogenic cycle ofT. ornataas here reported, differs in no important respect from those ofThamnophis,Sternotherus odoratus, orTerrapene carolina, except that inT. ornatathe cycle begins and ends somewhat later in the season of activity. In most of the lizards that have been studied (Fox, 1952:492-3), spermatogenesis reaches a peak in spring (more or less coincident with the mating period and with ovulation) and the germinal epithelium remains active in winter.Sternotherus,Terrapene, andThamnophisare alike in completing spermatogenesis late in the season and storing spermatozoa, in the seminiferous tubules or in the epididymides, during hibernation.
It is noteworthy that, in the turtles and snakes mentioned above, sperm produced in autumn are used to fertilize eggs laid in the following year, and mating [with the exception ofThamnophis elegans, (Fox, 1956)] occurs in both spring and autumn. It is not definitely known in any of these instances, whether sperm resulting from autumn or spring inseminations (or both) fertilize the eggs. Risley (1933:693) found motile sperm in the oviducts of femaleSternotherus odoratusthat had recently emerged from hibernation; he believed that spring mating, although it commonly occurred, was not necessary to fertilize eggs. Disadvantages, if any, of completing spermatogenesis well in advance of ovulation seem to be at least partly counteracted by two annual mating periods or by mating throughout the season of activity.
Sexual Cycle of Females
The following account of oögenesis is based on examination of preserved ovaries from 68 mature specimens. The ages of most specimens were known, inasmuch as the specimens were used in studies of growth as well as gametogenesis. Other data were obtained from adult females that were dissected but not preserved, and from immature females.
Fig. 4.Seasonal fluctuations in ovarian weight inT. o. ornata, as determined by examination of 60 specimens from eastern Kansas.
Fig. 4.Seasonal fluctuations in ovarian weight inT. o. ornata, as determined by examination of 60 specimens from eastern Kansas.
Size of ovarian follicles was determined by means of a clear plastic gauge containing notches 5, 10, 15, 20, and 25 millimeters wide. The number of follicles within a given size range could be quickly determined by finding the smallest notch into which the follicles fit. It was necessary to weigh all ovaries after preservation since some of them had not been weighed when fresh. Since all ovarian samples were preserved in the same manner, weightsremained relatively the same. Preserved material was lighter than fresh by an average of 13 per cent. Follicles less than one millimeter in diameter were not counted. Corpora lutea and corpora albicantia were studied under a binocular dissecting microscope. No histological studies were made of the female reproductive system.
Ovarian follicles and oviducal eggs were recorded separately for the right and left sides. Each ovary was always kept associated with the oviduct of the same side, but in some instances it was not recorded whether the organs were left or right.
Ovaries ordinarily weighed most in October, March, and April, when most females contained enlarged follicles, and least in August and September when the supply of enlarged follicles was usually exhausted (Figs.4and5).
Fig. 5. The seasonal occurrence of enlarged ovarian follicles in femalesFig. 5.The seasonal occurrence of enlarged ovarian follicles in females ofT. o. ornata, expressed, for each month, as the percentage of total females that contained two or more follicles having diameters greater than 15 mm. Total number of females in each of the samples is shown in parentheses at the top of each bar.
Fig. 5.The seasonal occurrence of enlarged ovarian follicles in females ofT. o. ornata, expressed, for each month, as the percentage of total females that contained two or more follicles having diameters greater than 15 mm. Total number of females in each of the samples is shown in parentheses at the top of each bar.
The ovarian cycle begins in July or August, after ovulation has occurred. At that time many minute follicles form on the germinal ridges of the ovaries. On the basis of the material that I examined, it seems that ovarian follicles either grow to nearly mature size in the season preceding ovulation and remain quiescent over winter or grow rapidly in the period of approximately six weeks between spring emergence and ovulation. Altland (1951:603-5) reportedthat the former condition was the usual one inT. carolina; he suggested that possibly some of the enlarged follicles were absorbed during hibernation.
Examination of yolks of oviducal eggs revealed that follicles mature when they reach a diameter of 16 to 20 millimeters and a weight of two to two and one-half grams (Pl. 20, Fig. 1).
The enlarged follicles remaining on the ovaries after ovulation (excluding those smaller than six mm.) can be grouped according to diameter as: large (greater than 15 mm.), medium (11 to 15 mm.), and small (six to 10 mm.). Ten females collected in the period from June 2 to 8, after they had ovulated, all had follicles falling in at least one of these size groups, and eight had follicles falling in two or more of the groups. In females having enlarged follicles of more than one of the size groups, there were several follicles in each of two groups and no follicles, or only one follicle, in the remaining group. Enlarged follicles represent future clutches but whether the enlarged follicles will be ovulated in the same season or in a later season is questionable.
Evidence found in the present study suggested that at least a few females lay more than one clutch of eggs per year. Among 34 specimens obtained in June and July, eight (24 per cent) had corpora lutea (or easily discernible corpora albicantia) and at least two follicles more than 15 millimeters in diameter; in three specimens (9 per cent) the ovaries bore fresh corpora lutea (representing recent ovulations) and a set of older corpora lutea (representing ovulations that had occurred several weeks previously). It was thought that each of these eleven females (33 per cent of sample) had produced or would have produced two clutches of eggs in the season of its capture. The number of large follicles present after the first set of ovulations (mean, 3.5) was fewer in most instances than the average clutch-size (see below), indicating that second clutches are smaller than first clutches. Smaller second clutches were found also inT. carolina(Legler, 1958).
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 layingin 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 concerningPseudemys scripta(1950:38) andChrysemys 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 inT. 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 shapeand 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 sub-adults (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 ofTerrapene 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 thatT. ornatadeposits 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 inT. ornataand is known to occur also inT. 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 ofT. ornatahad 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 severalPseudemys scriptabut found no yolkless eggs in nests. No yolkless eggs were found in specimens ofT. ornatain 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 ofT. 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 ofT. o. ornatawere 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 inPlate 21was 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 ofT. 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 forT. 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 elsehas 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 inChrysemys, because of the narrow hind foot of the femaleT. 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 inT. carolina. It is worthy of mention, however, that Booth (1958:261) reported the digging of a preliminary cavity by a captive individual ofGopherus agassizi.
Eggs
The number of eggs in 23 clutches ranged from two to eight (mean, 4.7 ± 1.37 σs]); 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) forPseudemys scripta. The small size ofT. ornata, in comparison with other emyid turtles, seemingly limits the number of eggs that can be accommodated internally. The number of eggs per clutch inT. carolina[2 to 7, average 4.2, Allard (1935:331)], is nearly the same as that ofT. ornata.
Fig. 6. The relation of plastral length to number of eggs laidFig. 6.The relation of plastral length to number of eggs laid by 21 females ofT. o. ornatafrom eastern Kansas.
Fig. 6.The relation of plastral length to number of eggs laid by 21 females ofT. o. ornatafrom eastern Kansas.
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) forT. 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 tendencyfor 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 inSternotherus odoratus, as did Allard (1935:331) inT. carolina. Measurements in the literature of the size of eggs ofT. ornatasuggest a width greater than that stated above, probably because some eggs already had begun to expand when measured.
Eggs ofT. ornataexpand 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 ofChrysemys pictaandChelydra 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, eggswere 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 ofSternotherus odoratusto be longer at lower temperatures; corresponding observations were made by Allard (1935:332) and Driver (1946:173) on the eggs ofTerrapene carolina. Cagle (1950:40) and Cunningham (1939) found no distinct differences in length of incubation period for eggs ofPseudemys scriptaandMalaclemys 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.