Freeze Tolerance as an Overwintering Adaptation in Cope's Grey Treefrog (Hyla chrysoscelis)
Jon P. Costanzo; Michael F. Wright; Richard E. Lee, Jr.
Copeia, Vol. 1992, No. 2. (May 1, 1992), pp. 565-569.
Stable URL: http://links.jstor.org/sici?sici=0045-8511%2819920501%293%3A1992%3A2%3C565%3AFTAAOA%3E2.0.CO%3B2-B
Copeia is currently published by American Society of Ichthyologists and Herpetologists.
Your use of the JSTOR archive indicates your acceptance of JSTOR's Terms and Conditions of Use, available at http://www.jstor.org/about/terms.html. JSTOR's Terms and Conditions of Use provides, in part, that unless you have obtained prior permission, you may not download an entire issue of a journal or multiple copies of articles, and you may use content in the JSTOR archive only for your personal, non-commercial use.
Please contact the publisher regarding any further use of this work. Publisher contact information may be obtained at http://www.jstor.org/journals/asih.html.
Each copy of any part of a JSTOR transmission must contain the same copyright notice that appears on the screen or printed page of such transmission.
The JSTOR Archive is a trusted digital repository providing for long-term preservation and access to leading academic journals and scholarly literature from around the world. The Archive is supported by libraries, scholarly societies, publishers, and foundations. It is an initiative of JSTOR, a not-for-profit organization with a mission to help the scholarly community take advantage of advances in technology. For more information regarding JSTOR, please contact [email protected].
http://www.jstor.org Tue Jan 8 16:23:10 2008 SHORTER CONTRIBUTIONS: HERPETOLOGY chromosome homology, such as banding, is essays in honor of M. J. D. White. W. R. Atchley needed to further illuminate chromosome evo- and D. S. Woodruff (eds.). Cambridge Univ. Press, lution within the Xantusiidae. London, England. MACGREGOR,H. C., AND J. VARLEY.1988. Working with animal chromosomes. John Wiley and Sons, Acknowledgments.-This study would not have New York, New York. been possible without the help of the Museo OLMO,E. 1986. A. Reptilia, p. 1-100. In: Animal Nacional de Historia Natural de Cuba and the cytogenetics. Volume 4: Chordata 3. B. John (ed.). Universidad de La Habana, coorganizers (with Gebriider Borntraeger, Berlin, Germany. Pennsylvania State University) of the expedi- , AND G. ODIERNA.1980. Chromosomal evo- tion that obtained Cricosaura tjpica. Special lution and DNA of cordylid lizards. Herpetologica thanks to G. Silva and members of the expe- 36:311-316. dition. We thank A. Wynn for suggestions in PRESCH,W. 1988. Phylogenetic relationships of the karyotype preparation, G. Zug for cataloging Scincomorpha, p. 471-492. In: Phylogenetic rela- tionships of the lizard families. R. Estes and G. K. of specimens, L. R. Maxson for providing re- Pregill (eds.). Stanford Univ. Press, Stanford, Cal- search facilities, and S. W. Schaeffer for use of ifornia. equipment. Supported by a grant from the Na- SCHWENK,K. 1988. Comparative morphology of the tional Science Foundation (BSR-8906325 to lepidosaur tongue and its relevance to squamate SBH). phylogeny, p. 569-598. In: Phylogenetic relation- ships of the lizard families. R. Estesand G. K. Pregill (eds.). Stanford Univ. Press, Stanford, California.
BEZY,R. L. 1972. Karyotypic variation and evolution CARLAANN HASSAND S. BLAIRHEDGES, De- of the lizards in the family Xantusiidae. Contr. Sci. partment of Biology, 208 Mueller Lab, Pennsyl- Nat. Hist. Mus. Los Angeles County 227: 1-29. vania State University, University Park, Pennsyl- -. 1984. Systematics of xantusiid lizards of the vania 16802. Accepted 1 April 1991. genusLepidophyma in northeastern Mexico. Ibid. 349: 1-16. BICKHAM,J. W. 1984. Patterns and modes of chro- mosomal evolution in reptiles, p. 13-40. In: Chro- mosomes in evolution of eukaryotic groups. Vol- ume 11. A. K. Sharma and A. Sharma (eds.). CRC Press, Boca Raton, Florida. CAPRIGLIONE,T. 1987. New data on karyotypes of some Scincidae. Caryologia 40: 109-1 14. Copria, 1992(2), pp. 565-569 O 1992 by the American Soclety of CROTHER,B. I.,M. M. MIYAMOTO, AND W. F. PRESCH. Ichthyologists and Herpetologists 1986. Phylogeny and biogeography of the lizard family Xantusiidae. Syst. Zool. 35:37-45. FREEZE TOLERANCE AS AN OVERWIN- DEWEESE,J. E., AND J. W. WRIGHT.1970. A prelim- TERING ADAPTATION IN COPE'S GREY inary karyological analysis of scincid lizards. Mam- TREEFROG (HYLA CHRYSOSCELZS).-Many malian Chromos. Newsl. 11:95-96. temperate zone ectotherms are confronted with ESTES,R., K. DE QUERIOZ,AND J. GAUTHIER.1988. severe environmental challenges during winter. Phylogenetic relationships within Squamata, p. 119- Aquatic forms usually are protected from freez- 281. In: Phylogenetic relationships of the lizard families. R. Estes and G. K. Pregill (eds.). Stanford ing temperatures owing to the high thermal Univ. Press, Stanford, California. buffering capacity of water. Terrestrial verte- GORMAN,G. C. 1970. Chromosomes and the system- brate ectotherms ugenerallv avoid extreme win- atics of the family Teiidae (Sauria, Reptilia). Copeia ter temperatures by hibernating within insulat-
1970:230-245. ed refugesu below the frostline: however, some -. 1973. The chromosomes of the Reptilia, a may be exposed to potentially lethal environ- t to taxonomic interpretation, p. 349-424. In: Cy- mental temperatures. Those overwintering totaxonomy and vertebrate evolution. A. B. Chi- above the frostline must survive either by ex- arelli and E. Capanna (eds.). Academic Press, Lon- tensive supercooling or by tolerating the for- don, England. mation of ice within body tissues. HEDGES,S. B., R. L. BEZY,AND L. R. MAXSON.1991. Phylogenetic relationships and biogeography of Deep and prolonged supercooling is a major xantusiid lizards inferred from mitochondria1 DNA overwintering adaptation of many terrestrial in- sequences. Mol. Biol. Evol. 8:767-780. vertebrates (Lee, 1989). However, the biophys- KING,M. 198 1. Chromosome change and speciation ical constraints of large body mass (i.e., water in lizards, p. 262-285. In: Evolution and speciation: volume) preclude this strategy for most verte- COPEIA, 1992, NO. 2 brate ectotherms (Costanzo et al., 1988; Cos- crickets and flesh fly larvae ad libitum. On 15 tanzo and Claussen, 1990). Manv, .~olar fishes Oct., the frogs were habituated to 15 C, 12:12 avoid freezing by supercooling, but these pro- (L:D) for 2 wk during which time food was with- duce specific antifreeze proteins that inhibit the held. Subsequently, the frogs were exposed to growth of ice crystals (DeVries, 1983). hibernative conditions [4 C, 0:24 (L:D)] until At least several species of amphibians and they were tested for freeze tolerance (3-8 Jan. reptiles survive the repeated freezing and thaw- 1990). ing of extracellular fluids. Natural freeze tol- Eight frogs, held individually within plastic erance has been conclusively demonstrated in (50 ml) centrifuge tubes, were cooled inside an four species of frogs: Rana syluatica (Lotshaw, insulatedjar submerged in a cold (-6.5 C) bath. 1977),Hjla crucifer, H. uersicolor (Schmid, 1982), The airspace in each tube was insulated with Pseudacris triseriata (Storey and Storey, 1986); plastic foam to reduce the rate of ice formation. two species of turtles: Chrysemys picta (Storey et A 30-gauge thermocouple positioned against al., 1988), Terrapene carolina (Costanzo and the abdomen provided a continuous recording Claussen, 1990);and one snake: Thamnophis sir- of body temperature on a data logger (OM 50 1- talis (Costanzo et al., 1988). Laboratory exper- C, Omega Electronics). iments involving freezing in other species, in- Ice formation in supercooled frogs was in- cluding toads (Storey and Storey, 1986), aquatic duced by briefly applying aerosol coolant to the frogs (Weigmann, 1929; Lotshaw, 1977; exterior of each tube. The onset of ice forma- Schmid, 1982), plethodontid and ambystomid tion was clearly indicated by the release of the salamanders (Storey and Storey, 1986; our un- latent heat of fusion. Freezing proceeded until publ. data), and lizards (Weigmann, 1929; Spel- the following criteria were met: (1) a minimum lerberg, 1972; Claussen et al., 1990), have re- of 24 hr had elapsed, and (2) body temperature sulted in death, irreparable injury, or had declined to -2.5 C or below. The frogs ecologically negligible survival rates. were then transferred to a cold room (4 C) and We hypothesized that Cope's gray treefrog allowed to thaw. Following a 3 d habituation (H. chr~soscelis)is tolerant of body freezing be- period they were transferred to 22 C and tested cause this species is similar to H. versicolor, a for recovery criteria. We judged that the frogs known freeze-tolerant form, in morphological, had recovered fully if they fed (caught and in- genetic, and ecophysiological characteristics gested live crickets), maintained normal body (Ralin, 1977). The two species differ primarily postures, and locomoted spontaneously. in chromosome compliment (H. chr~soscelisis The blood of nine additional frogs (collected diploid, whereas H. versicolor is tetraploid) and in Butler County, Ohio, during June 1990 and in the pulse frequency of mating calls (Ralin, exposed to hibernative conditions for 30 d) was 1977. 198 1). The H. chrvsoscelis-versicolor com- analyzed to determine whether glucose or glyc- plex is distributed widely over eastern North erol, cryoprotectants utilized by freeze-tolerant America and ranges from the Gulf Coast of the frogs (Storey, 1990),are mobilized by H. chrysos- United States to Manitoba and Nova Scotia, celis. Blood was obtained from six rapidly thawed Canada (Conant, 1975). Geographically these frogs previously frozen to -2.9 C (f0.1 C, SEM) species may be widely separated although they for 53.5 h and from three unfrozen (control) are sympatric over some parts of their ranges frogs; it was collected directly from the ventricle (Ralin, 1977). We investigated freeze tolerance and centrifuged to separate the plasma. The in a population of H. chr~soscelisfrom southwest plasma was assayed for glucose using a colori- Ohio, located only 32 km from a breeding pop- metric procedure (no. 5 10, Sigma Chemical ulation ofH. versicolor, individuals that are known Company, St. Louis, Missouri) and for glycerol to be freeze tolerant (Layne and Lee, 1989). using established methods (Baust et al., 1983) for high-performance liquid chromatography Materials and methods.-Cope's gray treefrogs (HPLC). The osmotic concentration of plasma (H. chrysoscelis) were collected as they called from was measured using a Wescor 5500 vapor pres- a small breeding pond in Butler County, Ohio, sure osmometer. during June 1989. Eight adult males [n mass f Although H. chr~soscelisand H. versicolor are standard error of the mean (SEM) = 11.2 f 0.4 morphologically indistinguishable, each species g] were kept on damp moss inside an environ- can be identified on the basis of its karyotype. mental chamber. Frogs were exposed to 23 C One drop of blood from two unfrozen frogs was under a 12: 12 (L:D) photoregime and were fed separately mixed with 0.5 ml buffered formalin SHORTER CONTRIBUTIONS: HERPETOLOGY 567
(pH 6.8) and examined at 100 x on a phase TABLE1. FREEZING-INDUCEDCHANGES IN BLOOD contrast microscope. Cell dimensions (length GLUCOSEAND OSMOLALITYIN COPE'SGRAY TREE- and width) were measured for random samples FROG,Hyla chrysoscelis, TESTEDDURING SUMMER.Means of erythrocytes using an ocular reticule. Eryth- are shown t 1 SEM. rocyte measurements of our frogs (19.5 x 13.4 Plasma pm) were much smaller than those (22.3 x 16.1 Plasma glucose osmolality pm) from sympatric H. versicolor U. Vaughn, un- Group (pmol/ml) (mOsm) Mass (g) n publ.). Thus, the frogs used in our experiments Unfrozen 1.0 + 0.2 249 + 2 9.7 t 0.6 3 were undoubtedly the diploid form, H. chrjsos- Frozen 24.9 + 3.3 274 t 4 8.2 + 0.5 6 celis.
Results.-The eight frogs tested in winter cooled to a mean (+SEM) temperature of -1.8 f 0.2 durations. In the present study, all H. chrysoscelis C before body freezing was induced. Following recovered fully from rigorous tests of their abil- ice nucleation they cooled slowly (f = 0.13 + ity to survive ice formation in body tissues. 0.01 C/h), remained frozen for 24.0-40.0 h (f Freeze tolerance in this species is likely an im- = 28.3 f 1.7 h), and reached final body tem- portant adaptation promoting overwinter sur- peratures of -2.5 to -2.9 C (2 = -2.7 + 0.1 vival. W. D. Schmid (1986) reported that H. C). Upon their removal from the cooling cham- chrjsoscelis survived freezing in the laboratory, ber, the limbs and skin of the frogs were rigid but did not provide details of the freezing pro- indicating that much ice had formed within their tocol (e.g., cooling rate, equilibrium tempera- bodies. Respiratory activity was not detected. ture, duration), cryoprotectant concentrations, Most frogs regained the righting response and or survival criteria. the ability to retract their limbs within 48 h of The ice contents of treefrogs were not mea- thawing. one individual appeared dead follow- sured because a limited number of s~ecimens ing thawing; however, by 72 h, it resumed a were available for experimentation. However, normal posture and eventually recovered fully. the dynamics of ice formation in H. chrysoscelis Feeding did not commence until >7 d post- probably are similar to those in sympatric H. thawing, but all frogs eventually accepted food uersicolor (Layne and Lee, 1989). Presumably, and remained healthy for at least 6 wk. about 40-50% of the total body water in H. The marked elevation in plasma glucose con- chrysoscelis would have frozen under our exper- centration in frozen vs unfrozen frogs tested in imental conditions; this amount is less than-the summer (Table 1) was highly significant (t = lethal ice content (52-62%) for H. uersicolor dur- 4.9, df = 7, P = 0.002; t-test). The osmotic ing winter (Layne and Lee, 1989). Subsequent concentration of plasma from frozen frogs was research on H. chrysoscelis should address the significantly (t = 4.4, df = 7, P = 0.003; t-test) physiological limits to, and the effect of season greater than that from control frogs (Table 1); on, its capacity for freeze tolerance. Several in- correlation analysis showed that plasma osmo- vestigators (Schmid, 1982; Layne and Lee, 1989) lality was directly related (r2 = 0.85, df = 5, P have reported a diminished freeze tolerance in = 0.009) to plasma glucose concentration. Glyc- treefrogs during late spring relative to autumn erol concentrations in all plasma samples were and winter. low (
SPELLERBERG,I. F. 1972. Temperature tolerances of defend their calling sites against intruders southeast Australian reptiles examined in relation (Ovaska, 1992). Therefore, I predicted that to reptile thermoregulatory behavior and distri- movements of adult males are confined to the bution. Oecologia 9:23-46. vicinity of their calling sites and that males ex- STOREY,J. M., AND K. B. STOREY.1985. Adaptations hibit site-tenacity and move shorter distances of metabolism for freeze tolerance in the gray tree than females. I also predicted that movements frog, Hyla versicolor. Can. J. Zool. 63:49-54. STOREY,K. B. 1990. Life in a frozen state: adaptive of both adults and juveniles are shorter at high strategies for natural freeze tolerance in amphibi- than low population densities. Woolbright's ans and reptiles. Am. J. Physiol. 258:R59-R568. (1985) study on movement patterns of E. coqui -, andJ. M. Storey. 1986. Freeze tolerance and in Puerto Rico provided a basis for interspecific intolerance as strategies of winter survival in ter- comparisons. restrially-hibernating amphibians. Comp. Bio- chem. Physiol. 83A:613-617. Methods.-Two study sites that differed in veg- -. -. 1988. Freeze tolerance in animals. Physiol. etation type and structure were approx. 1.5 km Rev. 68:27-84. --, S. P. J. BROOKS,T. A. CHURCHILL,apart in St. James, Barbados, West Indies. The Bellairs site, located near the grounds of the AND R.J. BROOKS.1988. Hatchling turtles survive freezing during winter hibernation. Proc. Natl. Bellairs Research Institute of McGill Universi- Acad. Sci. U.S. 85:8350-8354. ty, was an untended flower bed (25 m long, 3- WEIGMANN,R. 1929. Die wirkung starker abkuhlung 7 m wide) separated by lawns and paved areas auf amphibien und reptilien. Zeitschrift fiir Wis- from other suitable habitats for frogs. Dense senschaffliche Zoologie 13454 1-692. ground vegetation included bromeliads (Bill- bergia spp.), which provided retreats for the JON P. COSTANZO,MICHAEL F. WRIGHT,AND frogs. The Greenwich site was in a forested gul- RICHARDE. LEE,JR. Departmentof Zoology, Mi- ly along a seasonal stream near the village of ami University, Oxford, Ohio 45056. Accepted Greenwich. Ground vegetation was sparse, and 27 Nov. 1990. frogs sheltered under rocks rather than in veg- etation. To document nightly movements of adult males and females, I followed frogs dusted with fluorescent pigments (Woolbright, 1985) and located their positions hourly all night using a copno, 1992(2), pp 569-573 portable ultraviolet light source. I caught frogs O 1992 by the Amertcan Society of (calling males and adult females) soon after dark ichthyologists and Herpetologists and, for each frog, recorded sex and snout-vent SHORT- AND LONG-TERM MOVEMENTS length (SVL) and sprinkled its dorsum with pig- OF THE FROG ELEUTHERODACTYLUS ments (red, orange, or green) from a small vial. JOHNSTONE1 IN BARBADOS, WEST IN- I released each frog at its original location min- DIES.-Movements of individuals influence so- utes after capture. At each subsequent sighting cial organization and genetic relationships with- of a marked frog, I placed a small numbered in and between populations. Following daily tag within 5 cm of the frog to aid in mapping movements of individuals can elucidate differ- its positions the following day. I measured the ences between the sexes and age/size classes in distance (a) between subsequent sightings, (b) foraging and mating behavior. Movements of between two farthest sightings, and (c) total dis- adults and juveniles over longer time spans in- tance moved by each individual. I marked 10- fluence genetic structure of populations. Sev- 18 frogs on seven nights during the rainy season eral studies have addressed movements of pond- between 16 July and 20 Sept. 1988 at Green- breeding anurans during the reproductive wich site. On several occasions, however, heavy season (e.g., Fellers, 1979; Given, 1988; Perrill rains after midnight washed the pigments off. and Shephard, 1989), but little information ex- Data for 20 females and 19 males resighted all ists on terrestrial frogs without breeding mi- night and at least five times were obtained from grations.- four nights (16, 22 July; 8 Aug.; 20 Sept.). I was I examined movements by the terrestrially less successful in relocating marked frogs at Bel- breeding frog Eleutherodactylusjohnstonei night- lairs site due to dense vegetation and obtained ly and over several months at two sites in Bar- comparable data for only nine males and three bados, West Indies. Males are territorial and females (22,24,29,June 1988). I used the Mann-