A “circannian” rhythm in hibernating species of the genus Citellus with observations on their physiological evolution

Comp. Biochem. Physiol., 1966, Vol. 19, pp. 603 to 617. Pergamon Press Ltd. Printed in Great Britain

A "CIRCANNIAN" RHYTHM IN HIBERNATING SPECIES OF THE GENUS C I T E L L U S W I T H OBSERVATIONS ON T H E I R PHYSIOLOGICAL EVOLUTION E. T . P E N G E L L E Y and K. H. K E L L Y Department of Life Sciences, University of California, Riverside, California, U.S.A. (Received 31 M a y 1966)

A l ~ t r a c t 1 1 . Continuous hibernation or estivation in Citellus lateralis, C. mohavensis, C. tereticaudus, C. variegatus and C. beecheyi never exceeds about 2 weeks. The frequency of arousal is a function of the ambient temperature, though in the latter four species not necessarily a direct one. A theory is proposed to explain this phenomenon. 2. An endogenous rhythm, termed "circannian", of about a year's duration is demonstrated in all five species. The physiological evolution of this rhythm and hibernation are discussed, and Zeitgebers speculated upon. The adaptive value of such a rhythm in the ecology of the various species is demonstrated. 3. Neither the length of the hibernation period nor the active period seems to be affected by castration, thus arguing against gonadal atrophy or hypertrophy being a causative factor in the onset or termination of hibernation. 4. There is a marked cyclic rise and fall in body weight of at least three species, and the whole hibernation periods are closely related to this. It is thought that this cycle is a reflection of a deeper endogenous rhythm probably functioning in the central nervous system. 5. Subspecies of C. lateralis from the most northern and most southern part of its range show no difference in their physiological behavior with respect to hibernation. INTRODUCTION THE factors, both internal and external, which influence the onset and termination of hibernation in small mammals have been of increasing concern to physiologists, in their attempt to understand this remarkable phenomenon. L y m a n (1954), using the hamster (Mesocricetus auratus), uncovered much useful information, but the main problems remain unanswered, and still do. See also the recent work of Hoffman & Reiter (1965) on the hamster. In a thorough study of the goldenmantled ground squirrel (Citellus lateralis tescorum), Pengelley & Fisher (1957, 1961, 1963) demonstrated that continuous hibernation was never of long duration, but that overall periods, termed whole hibernation periods, alternated with long periods of activity, apparently on a seasonal basis. T h i s rhythmic behavior took place under year-round constant environmental conditions of temperature and photoperiod, as well as food and water. T h i s led the authors to conclude that in this species the basic factor underlying hibernation was an endogenous r h y t h m 603

604

E. T. PEN(IELLEYAND K. H. KELLY

of about a year's duration. Pengelley (1966a) extended this theory, terming it a "circannian" (L. circa = about, annum = year) rhythm, in keeping with the terminology of Halberg et al. (1959) who described the now well-established daily rhythms as "circadian" (L. circa ~ about, dies -~ day). Citellus lateralis tescorum is only one species of a large genus which in North America ranges from the rigorous environment of Alaska (Citellus undulatus) to the hot deserts of the south-western United States (Citellus tereticaudus, Citellus leucurus and others) (Hall & Kelson, 1959), and thus inevitably exhibit a vast range of physiological and behavioral adaptations. Even the single species Citellus lateralis ranges from the Rocky Mountains of northern British Columbia (subspecies tescorum) to the San Bernardino Mountains in southern California (subspecies bernardinus). It therefore seemed profitable to compare the physiological behavior of various species within this genus when exposed to controlled and constant environmental conditions for long periods of time. It was hoped to uncover some of the factors determining the onset and termination of hibernation within the genus, also something about the evolution of hibernation and estivation, and how these adapt the various species to their greatly different environmental niches. No distinction will be made here between these two phenomena of hibernation and estivation, since this problem has been discussed by Bartholomew & Hudson (1960), who argue that physiologically they are the same. It was also intended to see if there were any major physiological differences, so far as hibernation was concerned, between the two subspecies of Citellus lateralis, namely tescorum from the most northern part of its range and bernardinus from the most southern part. The species chosen were C. lateralis bernardinus (the subspecies will only be referred to when pertinent) which was known to hibernate (Mullally, 1953), C. mohavensis which according to Bartholomew & Hudson (1960) hibernated and estivated, C. tereticaudus which Hudson (1964) considers to be an estivator, C. variegatus which hibernated in response to food deprivation (Pengelley, 1964), and C. beecheyi which hibernates sporadically (Strumwasser, 1960). MATERIALS AND METHODS All animals were trapped live in the wild during the summer and early fall. The C. lateralis bernardinus were taken near Big Bear in the San Bernardino Mountains of southern California; C. mohavensis in the Mohave Desert near Lancaster, California; C. tereticaudus in the Colorado Desert near Palm Springs, California; C. variegatus just north of Las Vegas, Nevada; and C. beecheyi in Riverside, California. Where castration was performed, this was done shortly after capture. The animals were all housed in separate cages with bedding, food (Purina Chow) and water ad libitum. Initially they were in a room at 23°C and a 12-hr artificial photoperiod, but during the latter part of September were all transferred to the appropriate experimental conditions. Thereafter all disturbances were kept to a minimum, but observations were made every day. Hibernation was determined by using the "sawdust technique" of Lyman (1948) and Pengelley & Fisher (1961) which does not disturb the animal in any observable way.

A "CIRCANNIAN ~ RHYTHM IN HIBERNATING SPECIES OF CITELLUS

605

RESULTS

Daily behavioral response of hibernating animals Figure 1 shows the typical daily behavioral response to two different environmental temperatures of five species of Citellus during a period when the frequency of hibernation was at its maximum for each animal. The total time span is 34 days.

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DAYS FIG. 1. D a i l y p a t t e r n of h i b e r n a t i o n for five species of Citellus at 12°C a n d 3°C a m b i e n t t e m p e r a t u r e s . Solid bars indicate c o n t i n u o u s h i b e r n a t i o n , vertical lines arousals.

The black horizontal bars represent periods of continuous hibernation and vertical lines an arousal with subsequent re-entry into hibernation. The data are for individual animals only, but are based on not less than ten animals for each species, none of which varied appreciably from the patterns shown here. It is quite clear that in no case does continuous hibernation exceed a period of 10 days (i.e.C. lateralis at 3°C), but a period of about 5 days is more typical. There are, however, obvious differences between the species, and on the basis of their tendency to hibernate regularly with fewer interruptions of an active state they may be rated as follows: C. lateralis, C. mohavensis, C. beecheyi, C. variegatus and C. tereticaudus. Thus C. lateralis may be rated a relatively "good" hibernator, and C. tereticaudus a relatively "poor" one. In C. lateralis the periods of continuous hibernation are on the average longer at an ambient temperature of 3°C than at 12°C. This phenomenon has been reported previously (Pengelley & Fisher, 1961), and a theory proposed to account for it. However, in all the other species the situation is either apparently the reverse (C. variegatus) or there is no significant difference between the two ambient temperatures (C. mohavensis, C. beecheyi and C. tereticaudus). A theory to account for

606

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this will be discussed later, but it is pertinent to note here the body temperatures to which the various species fall under the two different ambient temperatures. At both 3°C and 12°C, the body temperature of hibernating C. lateralis falls to the respective ambient temperatures and remains there until the next arousal. This is true also for all the other species at 12°C ambient, but is not so at 3°C ambient. At this latter ambient temperature the four species in question tend to hold their body temperatures 5-7°C above the ambient, that is at about 8-10°C (Pengelley, 1964). It seems as if these four species differ from C. Iateralis in having some form of "thermostat" which holds their body temperature several degrees above ambient when this falls below about 10°C. By careful observation it has been determined that each arousal from hibernation is complete, in the sense that the active homothermic temperature of 37-38°C is regained before re-entry into hibernation. In all species urination invariably takes place before the animal enters hibernation again. Long-term response to constant environment

In Figs. 2, 3 and 4 data are plotted which represent the response to different ambient temperatures and a photoperiod of 12 hr of the five species for a period in excess of a year. The whole hibernation period for each animal is represented by solid horizontal bars and comprises the entire period between onset and termination of hibernation, with the exception that the periodic arousals (Fig. 1) are not indicated (Pengelley & Fisher, 1961, 1963) Where no clear overall period of

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hibernation was apparent, shorter intermittent periods are represented by vertical lines. Animals which were castrated are marked with a C (ordinate), while those that died during the experiment are marked with an X at the appropriate time. I I

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FIG. 3. Whole hibernation periods (solid bars and lines) for C. mohavensis and C. tereticaudus at 12°C and 3°C ambient temperatures. C indicates castrated animal. X indicates death. Photoperiod 12 hr. C. lateralis bernardinus (Fig. 2) followed closely the predicted pattern of response on the basis of previous work (Pengelley & Fisher, 1963). Thus there is a distinct eireannian rhythm during which the animals alternate between a long period of hibernation and a long period of the homothermie active state. The period of hibernation corresponds approximately to what would be fall and winter in the wild state, while the active homothermie period corresponds to spring and summer. In other words, an annual rhythm is maintained under constant environmental conditions. This situation is most obvious in C. lateraIis and C. mohavensis and while the same phenomenon seems to be present in the other three species, its clear demonstration is complicated by several factors. As can be seen C. tereticaudus does not survive well under the relatively cool ambient temperatures of 3°C and 12°C, nevertheless what data are available from Fig. 3 show

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that in no case did an animal hibernate during the summer, while those that survived into the second winter period did in fact hibernate sporadically during this time. The pattern of hibernation periods in C. variegatus and C. beecheyi (Fig. 4) although less precise than C. lateralis is nevertheless clearly one of an annual rhythm, there being no case in which an animal hibernated during the summer. I

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FzG. 4. Whole hibernation periods (solid bars and lines) for C. variegatus and C. beecheyi at 12°C and 3°C ambient temperatures. C indicates castrated animal. X indicates death. Photoperiod 12 hr. That C. tereticaudus, C. variegatus and C. beecheyi might not show such a pronounced hibernating pattern as C. lateralis is to be expected, since it has been demonstrated in C. variegatus (Pengelley, 1964) and in C. tereticaudus (Hudson, 1964) that lack of food is a stimulus to hibernation in these species. However, in the present experiments food and water were available at all times, and thus would be inhibitors to hibernation. Table 1 summarizes the data in days for C. lateralis and C. mohavensis over complete periods of hibernation and activity, as well as the total period "onset to onset". Upon analysis by White's (1952) modification of the Mann & Whitney rank test for significance it is found that at the 5 per cent level in C. lateralis there is no significant difference in the periods of hibernation, activity or in the total period onset to onset between normal and castrated animals at 12°C or 3°C. Or, in other words, castration does not affect the hibernating behavior. There is also no significant difference between males and females at 12°C, but at 3°C there

A "CIRCANNIAN"RHYTHM IN HIBERNATINGSPECIES OF CITELLUS

609

TABLE 1--SUMMARY OF WHOLE HIBERNATION PERIODS~ ACTIVE PERIODS AND TOTAL PERIODS (ONSET TO ONSET) IN MALE, FEMALE AND CASTRATED ANIMALS OF C. lateralis AND C. mohavensis

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is a statistically significant difference in the total period (onset to onset) between males and females. This phenomenon was also found in C. lateralis tescorum (Pengelley & Fisher, 1963). This difference is due to the considerably longer active period of the males, normal or castrated, for there is no significant difference in the corresponding hibernation period between the two sexes. There is also no significant difference in the hibernating, active or total periods of normal or castrated animals when compared at the two ambient temperatures of 12°C and 3°C. Unfortunately the death rate of C. mohavensis was rather high and consequently there are insu~cient animals for statistical analysis. Nevertheless from inspection of Fig. 3 and Table 1 it does not seem that castration affected the hibernating behavior of this species, and so far as it is possible to judge this is true also for the other three species.

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FIG. 5. Cyclic body-weight pattern in relation to hibernation period (solid bars) for C. tereticaudus and C. mohavensis at 12°C ambient temperature, and C. lateralis 12°C and 3°C ambient temperatures. Photoperiod 12 hr.

A " C I R C A N N I A N " RHYTHM IN HIBERNATING SPECIES OF C 1 T E L L U S

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Body weight patterns Figure 5 shows the variations in body weight for representative animals of three species, C. tereticaudus, C. mohavensis and C. lateralis, with the latter at two different ambient temperatures. The hibernation periods (black bars) are also plotted relative to the body weights. This data clearly indicate for all three species that the body weight shows an approximate annual cycle, which is not dependent upon the food supply since this was available ad libitum at all times. In C. lateralis and C. mohavensis the onset of hibernation is clearly associated with a maximum or declining body weight, while its termination occurs at the minimum weight in the overall cycle. In C. tereticaudus, on the other hand, there is no clear association between body weight and the onset or termination of hibernation. It should also be pointed out that the weight peaks of C. lateralis closely coincide at the two ambient temperatures of the experiment, while those of C. mohavensis and C. tereticaudus occur much earlier in the cycle and consequently are "out of phase" with C. lateralis both so far as weight and hibernation periods are concerned. This is well demonstrated by comparing the second hibernation onset time of C. lateralis (Fig. 2) with that of C. mohavensis (Fig. 3). The remarkable coincidence of this onset in the animals of the latter species is highly suggestive of an accurate timing mechanism. DISCUSSION

The results of these experiments clearly indicate four distinct phenomena. Firstly, that continuous hibernation in most, probably all, members of this genus never exceeds a period of about 2 weeks, and that the lengths of these periods are a function of the ambient temperature, though not necessarily a direct one. Secondly, that there is clear evidence in this genus of an endogenous circannian rhythm alternating between overall periods of hibernation and activity. Thirdly, that the lengths of the overall periods are not the result of sexual activity in the form of atrophy or hypertrophy of the gonads, since they are unaffected by castration. Fourthly, that the cyclic rise and fall of body weight over a period of about a year is a reflection of the endogenous rhythm, and is closely related to the onset and termination of hibernation. In addition there does not appear to be any difference in the behavior of the subspecies bernardinus (southern form) as compared to the northern form, tescorum (Pengelley & Fisher, 1961, 1963). These four phenomena will be discussed in turn. Pengelley & Fisher (1961) pointed out that in C. lateralis, C. tridecemlineatus and C. columbianus periodic arousals from hibernation were a function of the ambient temperature. The higher the latter, the more frequent the arousals. They also demonstrated that at each of these arousals the animals urinated, though they rarely ate or drank. Since it is thermodynamically impossible for a metanephric kidney to produce anything but the most minute amounts of urine at body temperatures characteristic of hibernation (Hong, 1957; Winton & Bayliss, 1955), and since Pengelley & Fisher (1961) proved there was no increase in the volume of bladder urine between 1 and 12 days of continuous hibernation at 2°C, the

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E. T. PI~NGELLEY$d~lDK. H. KELLY

latter authors proposed that the cause of these periodic arousals was a gradually increasing blood concentration of the end-products of metabolism, which after a period of approximately 12 days reached a critical level triggering the arousal mechanism which caused the animal to regain the homothermic temperature of 37°C. At this temperature the kidneys again functioned normally, removing the end-products of metabolism in the form of urine, the bladder filled causing the animal to urinate, after which it re-entered hibernation for another 12 days or so. If this theory is correct, it should follow that at higher ambient temperatures the arousals should be more frequent since the rate of production of metabolic endproducts would be greater. This is generally true (Pengelley & Fisher, 1961; Fisher, 1964). Furthermore, it should follow that the concentration of endproducts in the blood would gradually increase over the 12 day period. Taking non-protein nitrogen as a metabolic end-product, Fisher (1964) was unable to demonstrate that this was the case. However, further experiments using refined techniques to elucidate this problem are under way in our laboratory. Unlike C. lateralis the four other species in this investigation do not show a decreased frequency of periodic arousals at the lower ambient temperature of 3°C. The reason for this is probably that these four species are apparently unable to safely permit their body temperatures to fall below 8-10°C, and thus at an ambient of 3°C they have to produce more heat than C. lateralis in order to maintain the higher body temperature. This would necessarily give rise to an increased production of metabolic end-products and consequently an increased frequency of arousals during which these are eliminated. The higher critical body temperature of the four species under discussion is in all probability an adaptation to their respective habitats which are arid deserts and plateaux, comprising the upper and lower sonoran life zones, and in their winter burrows it is unlikely that the ambient temperatures ever fall below 8-10°C. C. lateralis, on the other hand, is indigenous to the boreal life zone where its winter burrow temperatures fall closer to 0°C, and thus there is a great advantage for this animal to be able to louver its body temperature to about I°C which indeed it does. Halberg et al. (1959) and Aschoff (1963, 1965) have pointed out that for the widespread and well-studied 24 hr endogenous rhythms, commonly called circadian rhythms, three criteria are necessary for their establishment. Firstly, the rhythm must not be synchronous with any environmental periodic stimulus; secondly, the period of the rhythm may deviate from the time being measured, i.e. 24 hr for circadian rhythms; and thirdly, it must be relatively temperatureindependent. All these criteria are satisfied in our data for a circannian rhythm of hibernation of about a year's duration. Certainly this seems to be true for C. lateralis and C'. mohavensis. The reasons for the less precise data in the case of the other three species have already been speculated upon, but there is yet another possibility upon which we wish to theorize. Cade (1964) has presented evidence to show that in the Marmotini, which includes the genus Citellus, the evolutionary changes since the Oligocene seem to have been from an ancestral hibernator undergoing a period of fattening towards more recent physiological organizations in

A ' t C I R C A N N I K N ~ R H Y T H M I N H I B E R N A T I N G SPECIES OF C I T E L L U S

613

which the dependence on hibernation or torpidity is less and less. His conclusion was that there has been in the course of the evolution of the Sciuridae a tendency towards strict homothermy and away from hibernation and/or estivation. This theory is supported by the work of Lyman (1964), who has shown that the perfused isolated heart of C. leucurus, which does not hibernate or estivate (Bartholomew & Hudson, 1960; Hudson, 1962), nevertheless retains the characteristic of all hearts of hibernators so far studied, namely that it is able to beat rhythmically at between 0-2°C, and that its temperature-rate curve is typical of the hearts of hibernators. Thus Lyman concludes that the ancestors of C. leucurus were hibernators. Pengelley (1966b) has also presented evidence from growth rates in Citellus to support this view. It is also pertinent to note that there is good paleontological evidence (Black, 1963) to support the theory that since the Miocene there has been a general trend towards increasing dryness in the western United States, leading up to the present arid deserts. It is therefore reasonable to assume that as the various species of Citellus evolved from the parent stock, Protospermophilus, in the Miocene, they would have had to adapt to more and more arid conditions. This they have done in a variety of ways exemplified by C. mohavensis and C. leucurus which are sympatric in the Mohave Desert (Bartholomew & Hudson, 1960). C. raohavensis deposits large amounts of fat which it can use in hibernation probably in response to a food shortage. C. leucurus, on the other hand, does not appreciably store fat, is strictly homothermic, with a high critical and lethal temperature, and an efficient water economy (Hudson, 1962). Now our data presented here on the physiological behavior of the five species fit well into the scheme of events just discussed. Thus within these species we find a gradation in the rigidity of the circannian rhythm. We start with C. lateralis, in which fattening precedes long periods of obligatory hibernation, which alternate with active periods in a marked rhythm relatively uninfluenced by environmental factors. This is probably the ancestral physiological and behavioral pattern of the genus which adapted it to marked and precise seasonal changes in a boreal life zone, or, in other words, the endogenous circannian rhythm is an excellent adaptation for a "programmed" environment, where each event in the animal's life cycle must occur at an exact time in order for it to survive. For example, if the animal waited for the stimulus of an onset of cold weather in the winter to start storing fat, it would be too late. In fact it is able to "prepare" for winter long in advance by the deposition of fat as the result of the endogenous rhythm. C. raohavensis is confined to the Mohave Desert where the seasonal environmental changes are not as marked and precise as in the boreal life zone, but nevertheless there is a winter period when food must be scarce, with intermittent drought, Thus we find that this animal has a short period of obligatory hibernation, alternating with a long period of the homothermic active state, but that the obligatory hibernation period can be extended in response, probably, to food deprivation (Bartholomew & Hudson, 1960). The large amounts of fat that this animal stores are used not only to tide it over obligatory hibernation, but also to supply metabolic water in periods of drought. The endogenous circannian rhythm of the ancestral

614

E.T. PENGELLEYAND K. H. KELLY

form has been retained but adapted to a different set of environmental factors. Our data indicate that the overall period of the circannian rhythm in this species is considerably less than a year, i.e. mean 250 days. This may be an artifact of the experiment, since it was not started until after the animals normally enter hibernation in the wild (Bartholomew & Hudson, 1960); on the other hand, the period may in fact be considerably shorter than a year, but under natural conditions the endogenous rhythm may be adjusted back in phase with the seasons by various environmental factors. C. tereticaudus is an animal of the lower sonoran life zone in the Colorado desert, and has apparently gone one step further than C. rnohavensis, in having almost abandoned a period of obligatory hibernation, and retains only traces of an endogenous circannian rhythm, at least so far as hibernation is concerned. Regretfully our data on C. variegatus are sparse, the animal being difficult to trap, but nevertheless what is available fits into the general pattern of the theory being presented here. Its habitat is rather varied, but seems to be mainly in the upper sonoran life zone. It has no marked and precise period of obligatory hibernation as C. lateralis, but nevertheless shows distinct traces of a circannian rhythm in this regard. Furthermore it has been demonstrated by Pengelley (1964) that food deprivation is a marked stimulus to hibernation in this species, and in the wild this is no doubt an important factor in its extension. The last species, C. beecheyi, spans the range, so to speak, of physiological behavior in Citellus in that it may undergo a period of obligatory hibernation as a result of an endogenous circannian rhythm, or may remain strictly homothermic. It is therefore most interesting and pertinent to note that this species also spans the physical range of all the others discussed here with the exception of C. variegatus to which it is closely taxonomically related (Hall & Kelson, 1959) and probably replaces in its particular ecological niche. Thus C. beecheyi is sympatric with C. lateralis in much, if not all, of the boreal life zone, and is also sympatric with C. tereticaudus in the lower sonoran life zone. Some of this sympatry may be due to man's alteration of the environment, but it nevertheless demonstrates the extraordinary adaptability of C. beecheyi which, we suggest, is in part the result of its ability to make use of an endogenous circannian rhythm in the form of hibernation where necessary, or to abandon it and remain homothermic where advantageous. Finally, though not part of our experiments, Hudson's (1962) work on C. leucurus demonstrated that this species is strictly homothermic throughout the year and our observations on this species confirm his data. Thus we feel that our physiological and behavioral data support Cade's (1964) view that the ancestral stock of Citellus was a heterothermic food-hoarding hibernator, and that the direction of evolution has been towards homothermy. Also that the ancestral stock was probably an obligatory hibernator whose behavior was governed by a rather precise circannian rhythm, but that this physiological rhythm has tended to exert less control over behavioral responses as the genus evolved in a homothermic direction as a result of adapting to generally more arid conditions.

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It is now pertinent to ask what is the Zdtgeber or that which entrains the rhythm, thus correcting the difference in its period with that of the annual environmental period. There is at present no satisfactory answer to this problem. Pengelley & Fisher (1963) presented evidence that ambient temperature was the Zeitgeber for the circannian rhythm of C. lateralis. Hock (1955, 1966), however, has disputed this and argued in favor of the photopcriod as a Zeitgeber. Furthermore, Richtcr's (1965) activity data, using blinded ground squirrels (species not given), tend to suggest light as an entraining agent. Despite this the findings of Morris & Morrison (1964) on C. tridecemlineatus do not indicate photoperiod as a Zeitgeber. Satisfactory data on the Zeitgeber for this circannian rhythm have yet to be elucidated. The third major phenomenon presented here concerns the comparison of castrated and control animals. It is not necessary to discuss this in any detail, since it confirms the data of Johnson et al. (1933) on C. tricedemlineatus, namely that hibernation is unaffected by castration. However, by inference such a conclusion argues in favor of an endogenous rhythm rather than exogenous factors directly controlling endogenous ones, as in birds (Rowan, 1938; Farrier, 1961) and other mammals (Bissonette, 1935). Finally, the fourth major phenomenon, that of the body-weight cycle, is very evident in the three species for which we have satisfactory data (Fig. 5), and there can be no doubt that in the obligatory hibernators the onset of hibernation closely follows on the attainment of maximum weight and its termination on the attainment of minimum weight. In the non-obligatory hibernator, C. tereticaudus, the situation is not so clear. This weight cycle seems to us to be a reflection of a deeper endogenous rhythm which controls the former and thus guarantees that a period of obligatory hibernation will in fact be safe for the animal. On the other hand, Mrosovsky (1966) has argued that the situation might be more like an "hour-glass" in which fat deposition could be substituted for the sand, when it runs out hibernation ceases, and when it builds up hibernation starts. This is possible, but seems unlikely for as Pengelley (1966a) has said "someone has to tip the hour-glass". It seems to us far more probable that some other primary site, such as a nucleus of the central nervous system, supplies an oscillation of which the weight cycle is merely a reflection. In general conclusion and in keeping with Richter's (1965) extensive data on short- and long-term "clocks" in many species of animals, including man, we suggest that long-term endogenous rhythms are indeed a biological reality which warrant extensive investigation. Our own experiments with these species of Citellus are being continued indefinitely. Ackno~oledgements---Tlfis study was supported by Grant No. GB-2155 from the National Science Foundation, and secondarily by grants from the Riverside County (California)

Heart Association, the Kaiser Foundation, and from the University of California. We wish to thank Mrs. C. M. Asmundson for her able technical assistance. REFERENCES ASCHO~J. (1963) Comparative physiology: diurnal rhythms..4. Rev. Physiol. 25, 581-600. ASCHOFFJ. (1965) Circadian rhythms in man. Science, N.Y. 148, 1427-14-32.

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