Geographic variation in energy storage and physiological responses to freezing in the gray treefrogs Hyla versicolor and H.

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1 The Journl of Experimentl Biology 26, The Compny of Biologists Ltd doi:1.1242/je Geogrphic vrition in energy storge nd physiologicl responses to freezing in the gry treefrogs Hyl versicolor nd H. chrysoscelis Json T. Irwin 1, * nd Richrd E. Lee, Jr 2 1 Deprtment of Biology, Bucknell University, Lewisurg, PA USA 17837, USA nd 2 Deprtment of Zoology, Mimi University, Oxford, OH USA 4556, USA *Author for correspondence (e-mil: jirwin@ucknell.edu) Accepted 19 My 23 The physiologicl responses supporting freeze tolernce in nurns re well known, ut the evolution of this trit remins little studied. This is the first common-grden study of geogrphic vrition in cryoprotective responses to freezing nd the degree of freeze tolernce. We studied the gry treefrogs (Hyl versicolor nd H. chrysoscelis) from symptric sites in Minnesot, Indin nd Missouri. Ptterns in the literture suggest tht northern frogs produce more cryoprotectnts upon freezing, ut we found no geogrphic vrition in cryoprotective responses or degree of freeze tolernce. The concentrtion of glucose produced upon freezing ws higher thn previously reported for this species (liver: 475 µmol g 1 dry mss). Unfrozen frogs hd high levels of glycerol (liver: pprox. 15 µmol g 1 dry mss), nd did not produce more upon Summry freezing. Liver glycogen content (concentrtion multiplied y liver mss) ws highest in frogs from Minnesot nd Missouri, nd ws stored in preference to lipids in Minnesot frogs, possily to provide energy for the longer northern winters. Minnesot frogs ccumulted more ice (53.4±1.8%) fter freezing to 2.5 C thn Indin frogs (45.5±3.3%). The two species differed in ody size ut not in ny of the physiologicl prmeters mesured. We conclude tht these popultions show no dptive vrition in freeze tolernce nd tht compring pulished studies my e misleding ecuse of different cclimtion nd feeding regimes. Key words: freeze tolernce, gry treefrog, Hyl versicolor, H. chrysoscelis, cryoprotection, liver, glucose, glycogen. Introduction Although freeze tolernce in nurns hs een known since 1982 (Schmid, 1982), little work hs een done on the ecologicl nd evolutionry significnce of this remrkle trit. Most work to dte hs focused on physiologicl chnges tht occur on freezing nd the vrious dpttions promoting freeze tolernce (for reviews, see Costnzo nd Lee, 1994; Storey nd Storey, 1996). Studies tht focused on the evolution of freeze tolernce hve compred the physiologicl responses to freezing of freeze-tolernt nd freeze-intolernt mphiin species (Costnzo et l., 1993; Swnson et l., 1996) nd hve considered the link etween dehydrtion tolernce nd freeze tolernce (for review, see Storey nd Storey, 1996). However, some sic issues in the evolution of freeze tolernce hve not een ddressed. How mny times hs freeze tolernce evolved? Do mphiins in northern regions tolerte greter degree of freezing? Our study is the first to consider these issues using common-grden pproch with individuls collected cross rod geogrphic rnge. In ddition, our model for this study, the gry treefrog species complex (Hyl versicolor nd H. chrysoscelis), llows comprison of the physiologicl responses to freezing etween closely relted diploid nd tetrploid species. The gry-treefrog species complex hs unique ttriutes tht fcilitte the study of evolutionry physiology. First, the tetrploid Hyl versicolor hs evolved independently t lest three times from the diploid H. chrysoscelis (Ptcek et l., 1994). Although these two species re often found in symptry, the phylogenetic lineges within either of these species re generlly lloptric (i.e. evolutionry rnches do not hve overlpping geogrphic rnges), thus tetrploids re often symptric with diploids tht re not of the diploid linege from which the tetrploids evolved. There is evidence tht symptric diploid nd tetrploid frogs undergo prllel selection for protein lleles (Romno et l., 1987) nd tht desicction tolernce vries more mong sites thn etween these two species (Rlin, 1981), so we compred frogs from sites where oth species occur in symptry. Do diploids nd tetrploids living in the sme environment hve the sme physiologicl responses to freezing? Are tetrploids more similr to prentl diploids or symptric diploids? These two species re good model for the study of cold tolernce ecuse they re found cross lrge geogrphic re. Both species re widely distriuted throughout the estern nd southern United Sttes nd west to the Gret Plins nd re symptric in mny plces throughout their rnge. However, in locl res these two species re not necessrily in the sme

2 286 J. T. Irwin nd R. E. Lee, Jr Tle 1. Summry of pulished ccounts of plsm cryoprotectnt concentrtion during freezing in dult gry treefrogs, Hyl versicolor nd H. chrysoscelis [Glucose] [Glycerol] Rering/collection Popultion (mmol l 1 ) (mmol l 1 ) conditions Reference Hyl versicolor Hncock Co., IL L rered Lyne, 1999 Hncock Co., IL L rered Lyne nd Jones, 21 Fyette Co., IN 22.7 <1 L rered Lyne nd Lee, 1989 Hennepin Co., MN Nil ~3* L rered Schmid, 1982 Ontrio L rered Storey nd Storey, 1985 Ontrio Spring collected Storey nd Storey, 1987 Hyl chrysoscelis Butler Co., OH 24.9 <.1 L rered Costnzo et l., 1992 Minnesot No mention Yes* Fll collected Schmid, 1986 *Mesured in muscle extrcts nd ldder urine. Plsm concentrtion reported in ll other cses. hitts: t lest in Wisconsin, H. versicolor is widely distriuted ut H. chrysoscelis is generlly limited to regions of grsslnd nd svnnh (Jslow nd Vogt, 1997). These species lso differ in the northern extent of their rnge: H. versicolor extends frther north into Mnito, Ontrio nd New Brunswick (Preston, 1982; McAlpine et l., 1991). Given tht H. versicolor reches so much frther north, it is possile tht this species is etter le to survive northern winters thn its diploid prentl species. The mount of cryoprotectnt produced is directly relted to the degree of freeze tolernce, t lest in nother freeze-tolernt frog, Rn sylvtic (Costnzo et l., 1993). Therefore, we expect frogs from northern popultions to produce more cryoprotectnt upon freezing. Indeed, pulished ccounts suggest tht oth gry treefrogs (Tle 1) nd wood frogs (Storey nd Storey, 1988; Costnzo nd Lee, 1994) in colder regions produce more cryoprotectnt thn those from southern portions of the rnge. However, differences mong studies in methodology, especilly cclimtion regimes, mke comprisons cross studies difficult nd inconclusive (Lyne, 1999). Also, no single study hs directly compred the physiologicl responses to freezing of the tetrploid Hyl versicolor to its diploid ncestor H. chrysoscelis. Our study is the first to use common-grden pproch to descrie geogrphic vrition of freeze tolernce in n mphiin species. This pproch llows us to identify differences due to genetic dpttion to the locl environment. In ddition, our pproch llows comprison etween species nd mong phylogenetic lineges in well-studied species complex. Mterils nd methods Mle gry treefrogs were collected from three popultions where Hyl chrysoscelis Cope (Hc) nd H. versicolor Le Conte (Hv) occur in symptry: Clerwter Co. nd djcent Becker Co. in Minnesot (MN), Phelps Co. in Missouri (MO) nd Union Co. in Indin (IN). All frogs were collected during the reeding seson nd identified to species y chrcteristics of the reeding cll. They were trnsported ck to Ohio where they were housed in outdoor enclosures t the Mimi University Ecology Reserch Center nd fed crickets (size 2.5, Topht Frms, Portge, MI, USA) twice per week until the frogs stopped feeding with the onset of cold wether. On Novemer 15, the frogs were moved to smller cges with soil floor covered y lef litter nd these were held in drkness in wlk-in cold room t 4 C. To test their physiologicl responses to freezing, ech frog ws plced in 5 ml test tue with thermocouple djcent to the frog s ventrl surfce. After locking the opening of the tue with fom nd connecting the thermocouple to multichnnel chrt recorder, the tues were sumerged into cold th (RTE-14, Nesl, Portsmouth, NH, USA) t.8 C. Once cooled to temperture of.5 to.8 C, the frogs were stimulted to freeze y ppliction of erosol coolnt to the outside of the tue. The frogs were held t.8 C for 1 h, then cooled to 2.5 t rte of.4 C h 1 ( rte oserved in wood frogs freezing in nture; J. T. Irwin nd J. P. Costnzo, unpulished dt) thus reching the trget temperture fter 42.5 h. Once t 2.5 C, they were held t this temperture for n dditionl 4 h to llow equilirium ice content to e reched. Upon removl from the cold th frogs were doule-pithed nd rpidly dissected in wlk-in cold room t 4 C. Ech frog s entire liver ws removed, weighed nd, fter tking 15 mg susmple for determintion of wter content, frozen in liquid nitrogen. The right thigh musculture ws similrly removed nd susmpled, then lso frozen in liquid nitrogen. Tissue susmples were weighed, dried to constnt mss t 6 C nd reweighed to determine wter content. The hert nd surrounding lood vessels (sinus venosus, right nd left truncus rteriosus) were removed nd centrifuged to collect lood from within these structures into heprinized cpillry tues. The hert nd the remining crcss were frozen in liquid nitrogen. Blood retrieved from the hert ws preserved in 37% uffered formldehyde. Mesurements of red lood cells (length nd

3 Freeze tolernce in gry treefrogs 2861 width, N=1 cells per frog) under 4 mgnifiction were performed to confirm the species identifiction originlly mde through reeding cll chrcteristics (Mtson, 199). Control frogs (held unfrozen t C) were similrly treted except tht lood ws smpled directly into hemtocrit tues from the severed truncus rteriosus. To mesure cryoprotectnt (glucose nd glycerol) concentrtions, frog tissues were homogenized in ice-cold.6 mol l 1 perchloric cid, then neutrlized with hlf volume of 1 mol l 1 KHCO 3. Mesurements of glycogen required n dditionl step: 1 µl smple of the perchloric cid homogente ws incuted (37 C for 3 h) with myloglucosidse (Sigm Chemicl Co., St Louis, MO, USA) in 1 ml of sodium cette uffer (119 mmol l 1 sodium cette, 77 mmol l 1 cetic cid, ph 4.8) to convert ll glycogen to glucose. To stop this rection, the enzyme ws destroyed y ddition of more.6 mol l 1 perchloric cid nd the solution ws gin neutrlized with 1 mol l 1 KHCO 3. Mesurements of free glucose oth in the originl extrct nd fter digestion with myloglucosidse were performed using the glucose oxidse procedure (No. 51, Sigm Chemicl Co.). Glycogen ws expressed in glucose units nd ws clculted y sutrcting the free tissue glucose from the totl glucose fter myloglucosidse digestion. Tissue glycerol ws mesured using the glycerol phosphte oxidse procedure (No A, Sigm Chemicl Co.) nd lctte using the lctte oxidse procedure (No. 735, Sigm Chemicl Co.). After dissection of the tissue smples, ech frog crcss ws immeditely frozen in liquid nitrogen nd stored t 8 C. The crcsses were lter weighed, dried to constnt mss nd reweighed. Dried crcsses were pulverized in coffee grinder, nd the lipids were extrcted nd quntified using chloroform/methnol procedure (Teitz, 197). An dditionl susmple of frogs ws frozen y the ove protocol, then mesured for ice content. These frogs were rpidly trnsferred from the cold th using pre-chilled forceps to 1 ml of distilled wter in n insulted clorimeter t room temperture. The temperture chnge of the wter ws monitored with thermocouple connected to McL (AD Instruments, Colordo Springs, CO, USA) dt cquisition system nd ws used to clculte the frog s ice content following the methods of Lee nd Lewis (1985) nd Lyne nd Lee (1989). These frogs were dried t 6 C to constnt mss to estimte wter content of ech individul, requirement for clcultion of ice content. We used nlysis of vrince (ANOVA; PROC GLM, SAS) to identify which fctors (specificlly the species, geogrphic origin nd freezing tretment) significntly ffected the physiologicl chrcteristics including tissue concentrtions of metolites nd ice content. The model included ll interctions nd the dt in the figures re presented s lestsqure mens (SAS, LSMEANS). Percent dt were rcsine, squre-root trnsformed efore nlysis. In compring totl liver glycogen content, we performed n nlysis of covrince (ANCOVA; PROC GLM, SAS) with ody mss (logtrnsformed) s the covrite. Agin, we present lest-squre mens, these eing djusted for ody size. An experiment-wise error rte of.5 ws used in ll nlyses. Smple size for ech group used in the physiologicl ssys ws 12, except for the Missouri H. chrysoscelis controls (N=5) nd frozen smples (N=6), Missouri H. chrysoscelis controls (N=3) nd frozen smples (N=5), nd ll Minnesot groups (N=11 for ech species/tretment comintion). Ice content ws sed on N=5 for ech popultion/species comintion tested. Lipid concentrtions were mesured on 16 Minnesot frogs, 5 Missouri frogs nd 12 Indin frogs. ANCOVA ws not used ecuse mss (log-trnsformed) ws not significnt fctor in the nlysis of lipid concentrtion. The degree of freeze tolernce ws ssessed s survivl of freezing to vrious tempertures. The freezing protocol for these ssessments mtched tht of the physiologicl tests, ut longer tests were used to rech lower tempertures. Frogs were thwed t C for 24 h, nd then llowed to recover on wet filter pper in drkness t 4 C. The frogs were checked throughout recovery for two sic responses: lim retrction (ility to pull in the hindlim when retrcted mnully) nd righting response (ility to right itself when turned on its dorsum). The time when these responses were first oserved ws recorded. Frogs were judged to hve survived only if they exhiited norml posture nd ehvior. All of the experiments presented here were conducted using identicl methods during either the winter of or the winter of These two yers were compred y including yer s vrile in the ANOVA model used for the physiologicl comprisons (PROC GLM, SAS). These two yers were never significntly different, thus the dt were comined, nd this fctor ws dropped from the regression model. Metolite concentrtions re given in µmol g 1 dry tissue mss ecuse tissue wter content chnged gretly with freezing. All dt re presented s lest-squre mens ± S.E.M., except when the dt were not corrected for Stndrd mss (g) c Fig. 1. Differences in stndrd ody mss mong the popultions nd species (oth control nd frozen frogs re included in ech men). Mens not shring letter were significntly different (Bonferroni multiple comprison, α=.5). IN, Indin; MN, Minnesot; MO, Missouri; Hc, H. chrysoscelis; Hv, H. versicolor. c

4 2862 J. T. Irwin nd R. E. Lee, Jr ody mss, in which cse they re mens ± stndrd error of the men (S.E.M.). The discussion elow considers the sttisticl significnce of the min effects in the ANOVA model (popultion, species, freezing tretment). Results As previously shown (Mtson, 199), H. versicolor ws generlly lrger thn H. chrysoscelis (F 1,15 =17.1, P<.1) nd northern frogs were significntly smller thn their Glucose (µmol g 1 dry mss) Glycerol (µmol g 1 dry mss) Lctte (µmol g 1 dry mss) A Liver glucose C Liver glycerol E Liver lctte Control Frozen B Thigh muscle glucose southern counterprts (F 2,15 =51.2, P<.1) (Fig. 1). Interestingly, there ws lso species-popultion interction (F 2,15 =4.7, P=.11) ecuse there ws no significnt difference in ody size etween the Minnesot H. chrysoscelis nd H. versicolor (Fig. 1). The popultions nd species smpled for this study exhiit very little vrition in their physiologicl responses to freezing. Liver glucose incresed significntly with freezing (F 1,99 =719.1, P<.1) from seline levels of pprox. 23 µmol g 1 dry mss to pprox. 46 µmol g 1 dry mss (Fig. 2A). In the liver there were no D Thigh muscle glycerol F Thigh muscle lctte Fig. 2. Glucose (A,B), glycerol (C,D), nd lctte (E,F) concentrtions in the liver (A,C,E) nd thigh muscle (B,D,F) of control (lck rs) nd frozen (white rs) frogs. Freezing significntly incresed tissue glucose concentrtion (liver: F=719, P<.1; thigh muscle: F=81.1, P<.1) nd lctte concentrtion (liver: F=17.9, P>.1; thigh muscle: F=19.8, P<.1). When letters re present, mens not shring letter were significntly different. Arevitions s in Fig. 1. significnt differences in glucose concentrtions mong the popultions (F 2,99 =1.1, P=.325) or the species (F 1,99 =3.7, P=.59). In the thigh muscle glucose lso incresed with freezing (F 1,99 =63.1, P<.1) from pprox. 13 µmol g 1 dry mss up to pprox. 32 µmol g 1 dry mss (Fig. 2B). As in the liver, there were no significnt differences etween the species (F 1,99 =.1, P=.777) ut the popultions were significntly different (F 2,99 =4.5, P=.14) ecuse the Missouri H. chrysoscelis controls hd slightly higher glucose concentrtions thn the other groups (Fig. 2B). No differences mong the popultions were present in the frozen frogs. Glycerol followed different pttern. Glycerol ws t very high concentrtions in oth the frozen frogs nd the control frogs. In fct, the overll ANOVA ws not significnt for glycerol either in liver (F 11,99 =1.3, P=.24) or thigh muscle (F 11,99 =.91, P=.532). Thus, there were no significnt differences etween the species or mong the popultions, nor were there ny differences induced y freezing. Glycerol levels were typiclly 13 µmol g 1 dry mss in the liver (Fig. 2C) ut out 19 µmol g 1 dry mss in the thigh muscle (Fig. 2D). A significnt mount of lctte, yproduct of neroic metolism, ws ccumulted during freezing in oth the liver (F 1,99 =16.9, P<.1; Fig. 2E) nd thigh muscle (F 1,99 =19.8, P<.1; Fig. 2F). The liver lctte concentrtions rose from typiclly 5 6 µmol g 1 dry mss up to 18 µmol g 1 dry mss (ut were higher in the Missouri nimls, see elow). Concentrtions in the thigh muscle incresed from 21 to 38 µmol g 1 dry mss. There ws lso significnt effect of popultion on the ccumultion of lctte in liver (F 2,99 =7.5,

5 Freeze tolernce in gry treefrogs A 4 A Tissue wter content (%) B Control Frozen Fig. 3. Liver (A) nd thigh muscle (B) tissue wter content of control (lck rs) nd frozen (white rs) frogs. Liver wter content lwys fell significntly with freezing, ut there were no differences etween species or mong the popultions within the control or frozen groups. Arevitions s in Fig. 1. Glycogen (µmol g 1 dry mss) B Fig. 4. Liver (A) nd thigh muscle (B) glycogen concentrtion (in glucose units) for control frogs of the vrious species nd popultions smpled. There were no significnt differences mong the control frogs in liver concentrtion ut MN frogs hd higher muscle concentrtion. Arevitions s in Fig. 1. P=.1), proly ecuse the Missouri nimls ccumulted more lctte during freezing, especilly the Missouri H. versicolor. No popultion differences were oserved in the thigh muscle, which ws proly due to the high vriility of lctte concentrtion in this tissue. Tissue wter content ws significntly reduced y freezing. This ws true for liver (F 1,99 =148.5, P<.1; Fig. 3A) nd thigh muscle (F 1,99 =156.6, P<.1; Fig. 3B) s wter ws drwn from the tissues into growing ice crystls (Lee et l., 1992). In liver, wter content fell from 71% to 61% (lestsqure mens) nd in thigh muscle it fell from 74% to 63% (lest-squre mens). The liver wter content did not differ etween the species (F 1,99 =.8, P=.378) or mong the popultions (F 2,99 =2.8, P=.69). The sme ws true of thigh muscle (F 1,99 =.2, P=.653 for species; F 2,99 =.7, P=.516 for popultion). Liver glycogen, the proposed source for glucose nd glycerol (Storey nd Storey, 1985), ws mesurly reduced y freezing (F 1,99 =12.2, P<.1) from 281 to 2283 µmol g 1 dry mss (lest-squre men, ll frogs included) (not shown). There were no significnt differences mong the popultions of control frogs (Fig. 4A). In the thigh muscle there ws significnt difference mong the popultions (F 2,99 =69., P<.1; Fig. 4B). The Minnesot frogs hd nerly twice the muscle glycogen concentrtion (219 µmol g 1 dry mss) of their Indin (131 µmol g 1 dry mss) nd Missouri (125 µmol g 1 dry mss) counterprts. Liver glycogen concentrtion, expressed on per grm sis, is not the est indictor of glycogen vilility. We clculted totl liver glycogen content, rther thn concentrtion, y multiplying liver glycogen concentrtion y intct liver mss. Body mss (log-trnsformed) ws included s covrite in this nlysis ecuse it strongly influenced liver glycogen content (F 1,5 =9.8, P=.9) through effects on liver size. Totl liver glycogen ws significntly reduced with freezing from 389 to 26 µmol (P<.1; lest-squre men, ll frogs included) s it ws moilized to produce glucose. The popultions differed significntly in totl liver glycogen content (F 2,5 =9.1, P<.1) with control Minnesot nd Missouri frogs hving significntly higher liver glycogen contents thn control Indin frogs (Fig. 5A). Accumultion of high levels of glycogen in control northern frogs cme t the expense of lipid storge. The popultions differed significntly in crcss lipid content (F 2,3 =7.2, P<.1) with the Minnesot frogs hving the lowest concentrtion (Fig. 5B). To demonstrte tht this is not simply ecuse Minnesot frogs were smller, we clculted rtio of

6 2864 J. T. Irwin nd R. E. Lee, Jr Crcss lipid content (g) Rtio of liver glycogen content to crcss lipid content (µmol g 1 ) Glycogen content (µmol) 6 A B C IN IN IN Fig. 5. (A) Totl liver glycogen in control frogs estimted y multiplying liver glycogen concentrtion ( µmol g 1 dry mss) y intct liver mss. (B) Totl crcss lipid content. (C) Rtio of liver glycogen to crcss lipid content. In ll cses mens not shring letter were significntly different (Bonferroni multiple comprison, α=.5). IN, Indin; MN, Minnesot; MO, Missouri. totl liver glycogen to totl crcss lipid content (Fig. 5C). Also, significnt negtive correltion exists etween liver glycogen concentrtion nd crcss lipid content (F 1,32 =6., P=.2, r 2 =16%) in control frogs. Thus, there is n pprent trde-off etween glycogen nd lipid storge. The mount of ice tht ccumulted during freeze to 2.5 C ws mesured on Minnesot H. chrysoscelis nd H. versicolor, nd Indin H. chrysoscelis (Fig. 6). The only significnt effect in this comprison ws popultion (F 1,14 =7.8, P=.16), with Minnesot frogs ccumulting more ice thn the Indin frogs. There were no mjor differences mong the groups in the MN MN MN MO MO MO Body wter frozen (%) Fig. 6. The ice content of frogs from severl popultions/species fter freezing to 2.5 C. Minnesot frogs ccumulted more ice thn the Indin frogs. IN, Indin; MN, Minnesot; MO, Missouri; Hc, H. chrysoscelis; Hv, H. versicolor. Tle 2. Survivl of gry treefrogs of oth species nd from severl popultions to vrious freezing tempertures Temperture ( C) Popultion/Species Indin H. chrysoscelis 3/3 6/6 6/12 /9 H. versicolor 3/3 6/6 2/1 3/9 /1 Minnesot H. chrysoscelis 4/5 1/4 H. versicolor 5/5 2/7 Missouri H. chrysoscelis 1/3 H. versicolor 2/3 Frogs were not used in more thn one experiment. minimum temperture survived. There my hve een slightly higher survivl t 5.5 nd 6.5 C in the Minnesot frogs ut smple sizes were too low to chieve sttisticl significnce (Tle 2). Mesurements of recovery prmeters were sed only on survivors, thus smple size ws low nd sttisticl nlysis not possile. On verge, frogs frozen to 5.5 nd 6.5 C took twice s long to recover lim-retrction ility (pprox. 11 h) thn those frozen to 3.5 nd 4.5 C (pprox. 5 h), nd similr pttern ws present in recovery of the righting response. There were no consistent differences etween the species or mong the popultions in the time to recover lim retrction or the righting response. Discussion This study provided new insights into the use of cryoprotectnts in the gry treefrog species group. Becuse this study ws the first to compre the two species of gry treefrogs nd lso the first to include severl geogrphic loctions in single study, we hve een le to dispel some misconceptions

7 Freeze tolernce in gry treefrogs 2865 regrding nurn freeze tolernce tht hve risen from the comprison of pulished studies. Lyne (1999) pointed out tht interpopultionl comprisons hve een hmpered y different methods for cold-conditioning of frogs, n oservtion sed on his finding tht Illinois H. versicolor hd higher degree of freeze tolernce thn reported in previous studies of the sme species from similr climte in Indin. To eliminte this prolem, we rised gry treefrogs from severl popultions together, thus llowing comprisons of geogrphic nd interspecific vrition in freeze tolernce. The following discussion compres our experimentl groups in terms of cryoprotective responses, liver glycogen concentrtion (the source for cryoprotectnts), nd their degree of freeze tolernce nd recovery from freezing. In contrst to other studies, we found tht gry treefrogs produce s much glucose s other freeze-tolernt mphiins. Indeed, glucose concentrtions were s high s those reported in Ontrio wood frogs R. sylvtic (Storey nd Storey, 1984; Storey, 1987). Given its high concentrtion, glucose is likely to ply cryoprotective role, s it does in the wood frog (Costnzo et l., 1993). Why hve other studies not reported high glucose concentrtions upon freezing? First, most studies of gry treefrogs hve only mesured glucose concentrtions in the plsm, not the liver. The liver is likely the site of glucose synthesis (Storey nd Storey, 1985), nd we found very high concentrtions of glucose in this orgn. The only other study to mesure liver glucose in frozen gry treefrogs (Storey nd Storey, 1985) found only 16.6 µmol g 1 dry mss in the single dult mle smpled wheres we typiclly sw 18 µmol g 1 dry mss. We lso found more glucose in the thigh muscle: 12.2 µmol g 1 fresh mss in our study versus pprox. 5.6 µmol g 1 fresh mss in Storey nd Storey (1985). Liver glycogen concentrtion nd lortory cclimtion regimes my ccount for this difference (see elow). In our study, glycerol ws present in high concentrtions efore freezing, nd not further elevted y freezing. This is in contrst to other studies where glycerol ws very low initilly nd production stimulted only upon freezing (e.g. Storey nd Storey, 1985). The hypothesis of Lyne nd Jones (21) tht longer, cooler cclimtion periods my stimulte glycerol production is consistent with our dt, since our frogs were cclimted nturlly outdoors until moved to 4 C on Novemer 15. The environmentl conditions during this period such s drought stress or nturl chnges in photoperiod my hve stimulted glycerol production (s hppens in some insects; Rojs et l., 1986). The cues inititing glycerol production in the gry treefrogs require more study. Until now, there hve een no reports of H. chrysoscelis using glycerol s cryoprotectnt (other thn rief mention without ny supporting dt y Schmid (1986). Only Costnzo et l. (1992) mesured glycerol in H. chrysoscelis nd they found no detectle mounts, ut these mesurements were mde on summer nimls following short-term cold cclimtion. Given our results using nimls from popultion in Indin close to tht studied y Costnzo et l. (1992), s well popultions from Minnesot nd Missouri, it is cler tht H. chrysoscelis cn produce sustntil quntities of glycerol s cryoprotectnt, just s H. versicolor does. In fct, the two species did not differ in glycerol production or, indeed, in ny of their physiologicl responses to freezing. The concentrtions of glycerol tht we mesured were sustntilly higher thn those reported previously from Indin nd Illinois (Lyne nd Lee, 1989; Lyne, 1999; Lyne nd Jones, 21). Our results re more similr to those of treefrogs studied in Ontrio nd Minnesot (Schmid, 1982; Storey nd Storey, 1985). This strengthens the rgument y Lyne (1999) tht interpopultion comprisons re plgued y methodologicl differences. All of the species nd popultions we studied responded to freezing in essentilly the sme wy, thus there re no geneticlly sed differences in freeze tolernce due to ploidy or geogrphic loction. Wht ccounts for the differences seen etween our work nd the previous studies? Why did we see higher glucose nd glycerol production? These differences re likely to stem from differences in glycogen vilility. Unfortuntely, only one previous study of gry treefrogs included mesurements of glycogen in the liver nd muscle. This work focused mostly on juveniles, which typiclly hve low glycogen concentrtions (Storey nd Storey, 1985). The one dult mesured ( mle collected in the fll nd housed in the lortory for one month) hd 342 µmol glycogen g 1 dry mss, less thn the 6 1 µmol g 1 dry mss we mesured here. Our dt re more similr to the more extensive smples mde on wood frogs from Ontrio, which typiclly hve 7 1 µmol g 1 dry mss. These wood frogs lso produce levels of glucose similr to those tht we found in the gry treefrogs (Storey nd Storey, 1985; Storey, 1987). Thus, gry treefrogs with lrge heptic glycogen reserves produce glucose upon freezing much like the wood frog does. The differences in glycogen concentrtion proly stem from differences in the cclimtion regime. Frogs ccumulte glycogen with the onset of cold wether (Psnen nd Koskel, 1974; Smith, 195). However, this requires tht the pproprite cue, low temperture, is present nd tht food is still ville from which glycogen reserves cn e creted (Blier nd Guderley, 1986). In the previous studies of gry treefrogs, ll of the nimls were step-cclimted. Tht is, the frogs were moved through one or more rupt steps of progressively colder tempertures nd shorter photoperiods. However, upon the first drop in temperture, food ws withheld (e.g. Storey nd Storey, 1985; Lyne nd Lee, 1989; Costnzo et l., 1992; Lyne, 1999). Thus, lthough the cue for glycogen ccumultion ws present, the frogs no longer hd food source ville from which to produce glycogen. The result ws lower tissue glycogen content. In contrst, the frogs used in our experiments were rised outdoors where they experienced the nturl chnges in sesonl temperture, precipittion nd dy length. During this time, we continued to feed the frogs nd they te redily on wrm dys, even in lte Octoer. Thus, they hd greter opportunity to ccumulte glycogen. The mount of glycogen ccumulted ws most similr to those of wood frogs collected in Ontrio during the

8 2866 J. T. Irwin nd R. E. Lee, Jr lte fll nd used shortly fterwrd for experiments (Storey nd Storey, 1986), n cclimtion regime very similr to the one we used here. Thus, cclimtion nd feeding regimes hve produced pprent geogrphic vrition tht is not sed on locl dpttion. While the mount of glycogen influences cryoprotective responses when compring etween studies, once there is dequte glycogen for mximl cryoprotective response, the ddition of more glycogen does not improve glucose or glycerol production. This is illustrted y the Minnesot frogs in this study. Although they hd lrger glycogen reserves ville (Fig. 5A), these frogs did not produce more glucose (Fig. 2A,B) or glycerol (Fig. 2C,D) thn the other popultions. Thus, the higher glycogen reserves in northern frogs (nd the corresponding drop in lipid storge; Fig. 5) my e n dpttion to provide energy for the extended northern winter (see review in Psnen nd Koskel, 1974) nd/or to enhnce survivl of repeted freeze/thw cycles (Storey, 1987), rther thn to enhnce cryoprotective responses to freezing. The similrity in glycogen content in the two species (rther thn similrity within genetic linege), provides dditionl support tht locl selection drives prllel evolution for physiologicl trits in these two species (Romno et l., 1987). The degree of freeze tolernce did not vry gretly mong the popultions nd species when the frogs were rised in common environment (Tle 1). There ws significntly greter ccumultion of lctte in the liver of Missouri frogs (Fig. 2C), ut even this difference ws slight nd the highest lctte concentrtions were still on pr with those oserved in the study y Storey nd Storey (1985) of Ontrio gry treefrogs. The minimum tempertures survived y gry treefrogs in this study were closer to those of Ontrio frogs (s were the physiologicl responses to freezing discussed ove) thn previous studies of gry treefrogs in the Midwest (Lyne nd Lee, 1989; Costnzo et l., 1992; Lyne, 1999). The lck of vrition nd overll high degree of freeze tolernce my e relted to the high concentrtions of cryoprotectnts produced y these frogs. There do not seem to e ny genetic differences mong the popultions tht limit or enhnce freeze tolernce. This my suggest tht (1) gry treefrogs rised under these conditions re t the physiologicl limit of freeze tolernce nd/or (2) gry treefrogs from different geogrphic res do not experience gret differences in freezing tempertures in nture nd thus do not require higher degree of freeze tolernce in northern locles s originlly predicted. Both of these hypotheses require further investigtion. In summry, there is little dptive vrition in the cryoprotective responses nd survivl of freezing etween H. chrysoscelis nd H. versicolor nd mong gry treefrogs collected from Indin, Minnesot nd Missouri. We must consider, however, tht the common-grden pproch tken in our experiments my hve eliminted vrition in temperture, photoperiod, food vilility, or other fctors tht frogs from these popultions my hve experienced in nture. How these fctors contriute to the degree of nturl freeze tolernce exhiited y gry treefrogs in nture, nd lso the extent nd durtion of freezing conditions tht frogs experience in nture, remin to e explored. The uthors thnk Crl Gerhrdt, Willim Schmid, nd Jon Ross for help in locting collecting sites in Missouri nd Minnesot. Jckie Litzgus, Csey Tucker, Sen Wlker nd Leh Irwin helped collect frogs. Jckie Litzgus nd Shl Hnkison mde sustntil contriutions to feeding nd cring for the frogs. Jon Costnzo ssisted with the iossy protocols, except for the glycogen protocol, which ws kindly provided y Ctherine Bevier. This work ws supported y NSF IBN-924 to R.E.L., Sigm Xi Grnt-in-id to J.T.I., nd Mimi University s summer workshop progrm. J.T.I. ws supported y P.D.F. from NSERC Cnd (held t McGill University) nd the Dvid Burpee Endowment (Bucknell University) during preprtion of the mnuscript. This reserch ws pproved y Mimi University s Animl Cre nd Use Committee. References Blier, P. nd Guderley, H. (1986). The enzymtic nd metolic effects of extended food deprivtion in Rn pipiens. Physiol. Zool. 59, Costnzo, J. P. nd Lee, R. E. (1994). Biophysicl nd physiologicl responses promoting freeze tolernce in vertertes. News Physiol. Sci. 9, Costnzo, J. P., Lee, R. E. nd Lortz, P. L. (1993). Physiologicl responses of freeze-tolernt nd -intolernt frogs: clues to evolution of freeze tolernce. Am. J. Physiol. 265, R721-R725. Costnzo, J. P., Lee, R. E. nd Lortz, P. L. (1993). 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