Ila-N. (“AVE .5 L7, 5"}: A s- ‘uu “H .5 L. “ ’1, hhbgfi LIBRARY Michigan State University ABSTRACT A COMPARATIVE STUDY OF MONTANE AND LOWLAND LARVAL POPULATIONS OF Leptodactylus albilabris IN PUERTO RICO by William R. Bhajan Significant morphological differences were deters mined between tadpole samples of Leptodactylus allbilabris found at El Yunque, a highland region of Puerto Rico, and comparable samples obtained at Rio Piedras, a lowland area in the same island. These differences might be explained in that the mon— tane population made climatic adjustments with such success that morphological differences resulted. A COMPARATIVE STUDY OF MONTANE AND LOWLAND LARVAL POPULATIONS OF LEPTODACTYLUS ALBILABRIS IN PUERTO RICO By William Rudolph Bhajan A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Zoology 1966 ACKNOWLEDGMENTS This thesis never would have been written but for the fact that the author was fortunate to have met and asso- ciated with Dr. Harold Heatwole of the University of Puerto Rico. Dr. Heatwole was involved in studying Leptodactylus albilabris in Puerto Rico and adjacent areas. The author immediately contracted a debt of interest, enthusiasm, ideas and guidance from Dr. Heatwole. For further guidance and help, acknowledgment is made to Doctors Marvin M. Hensley, Benjamin H. Santa, and John R. Shaver of Michigan State University, who have made help— ful suggestions in analyzing the data and in the writing of this thesis. I would also like to express my appreciation to Dr. T. Wayne Porter and Dr. Stephen N° Stephenson for their participation in the final stages of my program. 11 TABLE OF CONTENTS Page ACKNOWLEDGMENTS . . . . . . . . . . . . . . ii LIST OF TABLES . . . . . . . . . . . . . . iv LIST OF FIGURES . . . . . . . . . . . . . . v INTRODUCTION . . . . . . . . . . . . . . . 1 METHODS AND MATERIALS . . . . . . . . . . . . 9 Description of the Sampling Areas . . . . . 9 Laboratory Analysis of Growth and Temperature Responses . . . . . . . . lO Comparative Larval Morphology of Natural Populations . . . . . . . . . . . . . . ll RESULTS . . . . . . . . . . . . . . . . l3 Larval Culturation . . . . . . . . . . . . 13 Relationship of Temperature and Developmen- tal Rate . . . . . . . . . 13 Relationship of Temperature and Size . . . . . 15 Relationship of Appendage Growth to Body— length . . . . . . . . . . . . . . . 32 Comparison of Developmental Stages . . . . . . 35 Comparison of Tooth Morphology . . . . . . . . 38 DISCUSSION . . . . . . . . . . . . . . . ul SUMMARY AND CONCLUSIONS . . . . . . . . . . . U5 LITERATURE CITED . . . . . . . . . . . . . U8 iii LIST OF TABLES Table Page 1. Leptodactylus albilabris tadpole culture com— parISon of lowland RiofiPiedras and montane El Yunque in relation to temperature . . . . . 1A 2. Measurements of two groups of montane tadpoles cultured at 20°C. . . . . . . . . . . l7 3. Measurements of two groups of lowland tadpoles cultured at 30°C. . . . . . . . . . . 21 A. Measurements of two groups of montane tadpoles cultured at 30°C. . . . . . . . . . . 2A 5. Measurements of one group of lowland tadpoles cultured at 20°C. . . . . . . . . . . 26 6. Measurements of one group of lowland tadpoles cultured at 20°C. . . . . . . . . . . 28 7. A comparison of the maximum weekly growths between body-lengths and appendage—lengths of Leptodactylus albilabris tadpoles from El Yunque and Rio Piedras when reared at 20°C. and 30°C. 33 8. A comparison of measurements at similar larval stages of Leptodactylus albilabris collected from Rio Piedras and El Yunque . . . . . . 37 9. A comparison of the relationship of tooth mor- phology to developmental stages of samples of Rio Piedras and El Yunque populations of Leptodactylus albilabris tadpoles . . . . . 39 iv LIST OF FIGURES Figure Page 1. Distribution of Leptodactylus albilabris in Puerto Rico and location of the study areas . . A 2. Growth rate of two groups of montane El Yunque tadpole sample cultured at 20°C. . . . . . l9 3. Growth rate of montane El Yunque tadpoles and lowland Rio Piedras tadpoles cultured at 30°C. 0 O 0 I O I O O O O O o O 0 23 A. Growth rate of two groups of lowland Rio Piedras tadpoles cultured at 20°C. . . . . . . . 30 5. Tooth morphology of Leptodactylus albilabris tadpole . . . . . . . . . .‘.. . . 31 INTRODUCTION A comprehensive study of the taxonomy and ecology of Leptodactylus albilabris in Puerto Rico was undertaken in 1963 by Dr. Harold Heatwole, then a staff member at the University of Puerto Rico. The project, reported herein, was undertaken at the suggestion of Dr. Heatwole as a contribution to his research on this species of frog. The field and laboratory research was conducted from January, 196A to December, 1964, in Puerto Rico under the direction of Dr. Heatwole. In October, 196“, I was admitted to the Graduate School at Michigan State University where the research program was concluded. Leptodactylus albilabris, a relatively small frog with an average snout-vent measurement approximately 100 mm. exhibits a great variability in coloration and pattern but shows relative uniformity in structural characters throughout its range. Stejneger (1902) described the living adult as having "general color above olive, the dusky marking dark grayish brown, nearly blackish brown below the dorso-lateral fold and on femur; the dorso— lateral fold and narrow edges around the dark markings pale olive gray; the transocular band and cutting edge of lip dark grayish brown; the supra-labial light band pale straw yellow; underside whitish; throat firely sprinkled with dark chocolate brown; iris olive silvery, overlaid with blackish." Morphologically, the species is described as having the vomerine teeth in two curved series behind the choanae; tongue large, slightly indented behind; nostrils nearer the tip of snout than eye; tympanum circular about two— thirds the diameter of the eye; interorbital space equals width of upper eye-lid; first finger much longer than second, which equals fourth; toes slightly webbed at base; third much longer than fifth; subarticular tubercles well develOped and numerous smaller tubercles in series on the sole; two metatarsal tubercles, the inner connected with a slight tarsal fold; heels overlapping when hindlimbs are folded at right angle to axis of body; tarso-metatarsal joint reaching tympanum when hind limbs are carried forward along the body; skin smooth above and below; numerous small, pointed tubercles on the outer surface of the tibia; a narrow dorsolateral glandular fold and another similar one, though less prnounced, on sides from shoulder to groin; a strong glandular fold from posterior angle of eye over tympanum to shoulder; ventral disk plainly marked by two transverse dermal folds, one between the forelimbs, the other across the belly, these joined by a longitudinal dermal fold on each side. Leptodactylus albilabris is found on the Caribbean islands of Puerto Rico, Vieques, Culebra, St. Thomas, Tortola, Jost Van Dyke, Anegador, and St. Croix. In the Antilles, it is restricted to the Virgin Islands, St. Croix, Vieques and Puerto Rico. In Puerto Rico (Figure l) the species occurs where suitable moisture conditions exist from sea level to an altitude of about 2000 feet. In the Northern Coastal Plain, Leptodactylus albilabris is abundant at the lower altitudes and populations extend into the highlands be- hind. The animals are especially abundant in moist meadows and irrigated fields of sugar cane. Stomachs examined contained land snails, spiders, and a variety of insects including beetles, ants, flies, catepillar and cockroaches. The breeding season of this frog seems to be early in the year. Eggs were found in January and March at El Yunque and Rio Piedras respectively. Full grown tadpoles were observed in the Catalina planatation on March 1, and on April 7, eggs and all stages of tadpoles were observed in a little stream begind the town of Utuado (Figure l). The tadpole of Leptodactylus albilabris is distinctive and readily identified by "length of body about one and one- third its width and slightly less than one—half the length of the tail; nostrils nearer the eyes than the end of the snout' distance between eyes one—fifth more than distance between the nostrils and considerably less than width of * l__ Arecibo «w 0 Bayamon Rio Piedras 0 El Yunque *Lares *Utuado *Caguas *Mayaguez *Barranqnitas *Maricao *Adjuntas *Mbonito * Humacao * *Ponce Coamo ”u Figure 1. Localities known for Leptodactylus albilabris in Puerto Rico. The circles (0) indicate the locations of the study areas. mouth; distance between nostrils equals their distance from eyes, as well as the diameter of the eyes; spiraculum on left side, directed backward and upward, situated above a line drawn between the base of the muscular part of the tail and the mouth and nearer to the posterior extremity of the body, being about halfway between anterior border of eye and insertion of hind legs; anus a long tube, median and larger than the spiraculum, tail about four times as long as deep, ending in an obtuse point; both upper and lower crests confined to the tail and nearly equal in depth, their edges being nearly parallel until the terminal third; the depth of the muscular part of the tail at its base about two-thirds the greatest total depth" (Stejneger, 1902). Objectives of the Study The tadpoles of Leptodactylus albilabris in Puerto Rico differ morphologically in accordance with habitat. In the lowlands, at a temperature of approximately 30° C. the tadpoles are smaller in body than the montane population. The montane population also exhibits longer appendages. The question therefore, arises as to whether the two populations represented distinct subspecies of Leptodactylus albilabris, or merely express clinal responses to their habitats. The findings of the data are examined in the light of Allen's and Bergmann's Rules. I will present a morphological comparison of the larvae from highland and lowland populations to further delineate the problem. Next, experimental rearing of larvae under controlled conditions was conducted. The objective was to ascertain if morphological and/or behavior responses of the two populations that would develop. A montane pOpulation group was cultured in mon— tane conditions and another in lowland conditions. Securing these conditions in the laboratory was effected through temperature control. The same procedure was followed for a lowland population sample. Furthermore, the possibility that acclimatization might be responsible for morphological differences between populations was of interest. Acclimatization is a process in which organisms adjust to new climatic conditions and embraces the whole complex of adaptation to the varying factors in the new environment. Adaptation to environmental temperatures is important for the survival of a species. Animals must be able to function under available temperatures and these temperature relations are of paramount importance to the complete ecolo- gical understanding of any animal species. The nature of these adjustments is of great interest. Oxygen consumption in cold adapted fishes is several times as great as in the warm adapted fish (Wells, 1935; Peiss and Field, 1950; Scholander, et al., 1953). Stuart (1951) found that only an increase in haemoglobin could balance the oxygen carrying capacity of the blood in cold adapted fish. Fishes acclimated to cold water are less tolerant to higher temperatures than those acclimated to warm water (Hathway, 1927; Brett, 1944; Fry, 19A7). That such phenomena are characteristic of most aquatic poikilotherms is well established (Prosser, et al., 1950). Considerable literature involving temperature acclima— tization is avialable for adult amphibians. Among the more notable are those Bullock (1955), Ruibal (1955), Prosser (1955) and Highton (1962). These authors have shown that the compensatory metabolic rates exhibited by northern or cold adapted anurans are in keeping with the general concept that activity rates are greater at a given tempera- ture in cold blooded animals from northern latitudes when compared with the same or closely related forms from southern latitudes. Moore (1939, 1940, 19u2, 19u6, 19u9), Volpe (1953) and Goin (1962) have investigated the adaptation of frog embryos to temperature. These authors demonstrated that warm adapted species have a more rapid rate of development. They also found that the time of tadpole transformation into the adult is progressively greater to the north. Most of the above work was done in the United States and very little in the tropics. Part of the data to be presented in this paper areconcerned with the adaptation of the embryonic characters of Leptodactylus albilabris for the lower temperatures which occur at the montane rain forest of El Yunque, Puerto Rico. METHODS AND MATERIALS Descriptionfiof the Sampling Areas Schmidt (1930) provides the following pertinent and precise account: Puerto Rico includes a wide range of habitat condi— tions from the extremely wet mountain rain forest of Luquillo, where mountain palms and hardwoods are hung with lianas and draped with moss that never dries out, to the opposite extreme of aridity on the south—west corner of the island, where a cactus flora predominates. Puerto Rico, though only 100 miles long and about 35 miles wide, is mountainous and therefore lends itself to habitats with significant temperature differences. The island is traversed by several mountain ranges with a broad northern plain. Rio Piedras is situated on this coastal plain while El Yunque is situated on the eastern end of the Luquillo mountain range. These two sites typically represent a grassland region and a tropical rain forest region and were selected as sampling areas. Rio Piedras, at latitude 18° 25'Noand longitude 66° OA'W-,has an elevation of about 75 feet above sea level. It has an annual rainfall of 77.1 inches and it is also traversed by numerous streams. The average temperature is 82°F. El Verde, the second study area, is located 1500 feet above sea level at latitude 18° 21'N. and longitude 65° A9' 10 on the lower montane rain forest up the side of the El Yunque mountain in the Luquillo Experimental Forest of eastern Puerto Rico. Here the average temperature is 68°F. Laboratory Analysis of Growth and Temperature Responses Broods of Leptodactylus albilabris eggs covered with foam were found near the running streams of El Yunque. Two clutches numbering about 100 eggs each, were collected on January 25, 196A, and kept in separate containers until the tadpoles emerged. Eggs of the Rio Piedras population were found in shallow stagnant pools, under stones and near streams on March 2A, 1964. Two clutches, also of approximately 100 eggs each, were obtained from this area. The lowland clutches were treated similarly as highland clutches by using separate containers. The tadpoles of the montane pOpulations emerged on January 26, 196A. Forty tadpoles from each clutch were measured and placed in individual open containers. These tadpoles were arranged in groups as follows: Group 1 (a): 25 tadpoles each in its individual container were cultured at 20°C. Group 2 (a): also 25 in number from Group (a) was cultured at 30°C. Group 1 (b): 15 in number was cultured at 20°C. Group 2 (b): 15 in number from group (b) was cultured at 30°C. 11 The purpose of the grouping was to culture the mon— tane population in montane temperature conditions (group la and 1b) and also to culture montane population in lowland temperature conditions (group 2a and 2b). The lowland population sample was similarly cultured. No feeding was necessary during the first week due to food nutrients present in the yolk sac. Tap water, aerated for at least 2“ hours, was used for culturing tadpoles. The water was changed daily. The individual tadpoles were fed daily with canned spinach. During the process of metamor— phosis, the water level was reduced gradually. When forelegs appeared, small pebbles were inserted in the cheese cloth covered jars. Culturing was stopped when the entire tail had been absorbed. Since data on geographic variation is usually based upon morphological characteristics, attempts were made to obtain morphometric data. The specimens were measured weekly and replaced in their containers. Measurements were made by removing the specimens to a petri dish which was placed over graph paper that was calibrated in millimeters. The animals were then examined through a binocular dissecting sc0pe. Comparative Larval Morphology of Natural Populations Samples from the field were needed for comparison with live tadpoles being reared in the laboratory. Tadpoles were caught by a net in various pools and running streams in both 12 sampling areas. The montane tadpoles were collected at El Yunque in July and August, 196“, and samples from the lowland Rio Piedras were collected in May and June, 1965. These samples were fixed in 10% formalin and after fixation were transferred to 70% alcohol. Two hundred and fifty tadpoles, from a pOpulation estimated roughly at 1,000, were collected from El Yunque and 175, from a population of approximately 800, from Rio Piedras were randomly chosen and examined. The larvae were measured, tooth morphology diagrammed, and developmental stages determined, based on Gosner's (1960) simplified table for staging anuran embryos and larvae. Those tadpoles showing variations from normal tooth morphology were separated in bottles and labelled for future reference. RESULTS Larval Culturation Relationship offiTemperaturejand Developmental Rate: The rearing of larvae under controlled laboratory temperature produced the following observations: (1) (2) The montane population cultured at the temperature approximating its environment (20°C.) metamorphosed within llldays whereas the lowland population cultured at the temperature approximating its environment (30°C.) metamorphosed within 25 days (Table l). The montane pOpulation at 30°C. takes 30.5 days to transform from the tadpole stage whereas the lowland population at 20°C. required 252 days for this process (Table 1). The results of this phase of the study indicate that a differential response to temperature was expressed as follows: (1) (2) The larval period of the montane population at its environmental temperature (20°C.) is A.A times longer than the lowland population in the latters environmental temperature (30°C.). The metamorphosis rate of samples from the montane population is increased when reared at the lowland temperature (30°C.) by 3.6M times its normal larval period. 13 1n. m.om HHH mm mmm coasts Hessaa co newcmfl awasmsa om H sons: m assesses ma ADV am am mamapomm om SLMSCMh mm Amv m mOH ma mm: mm Suwanee ma ADV :HH mm mm: mm ssaseme mm Amy H mm om Hfipa< mm cones ma ADV mm om Hasa< mm none: mm Aav m mmm m nmnsmomm mm noses ma ADV mmm m sensoomo mm coca: mm Amy a .000m .ooom .000m .000m mamonasosMpmz wefieopam Ammmpv mwmpm Ammwcv mwmum IIIIIIII amass: access Hm>pmH ho npwcmq Hm>nwa ao cpwcmq pcmEooam>mp Hm>pmq HmpcmEHmexm msvcsw Hm mmncmfim cam Hw>nwa no cednmm .mQSanmQEmp on endpmamn 2H ozvcsw Hm endpcoe one manpmam OHm ccmasoa mo comfismasoo oQSpHSO maoacwp mannwafinam msHmpoMUOmeqll.H mqm¢9 15 (3) The rate of metamorphosis of samples from the lowland population is 10 times slower when cul- tured at montane temperatures. The results of the experiment would seem to indicate that a rise in temperature is followed by hastened metamor— phosis whereas a decrease in temperature has a retarding effect on metamorphosis. The montane pOpulation at 30°C. does not duplicate the larval time span of the lowland popula- tion but takes 1.2 times longer. Similarly the lowland pOpulation at 20°C. takes 2.3 times longer than the montane population at 20°C. ' The montane population appears to be more adaptable to temperature change than does the lowland population. Relationship of Temperature and Size.—-The growth rates of the montane and lowland tadpoles were observed by comparing the maximum weekly mean lengths, utilizing the formulag. % increase/decreasezsize at low temp.-size at high temp.X100 size at high temp. The following conclusions were made as a result of such analysis: (1) The montane population (Table 2, Figure 2) when cultured at the temperature approximating its environment (20°C.) has an increase in body length of 8.86%; an increase in tail length of 25.12%; an increase in hind leg length cf 7.7A%; and an 16 TABLE 2.—-Measurements of two groups (1a and lb) of montane El Yunque tadpoles cultured at 20°C. x refers to the mean length and 28x denotes plus and minus twice the standard error of the mean. 17 El Yunque 20°C.(la) El Yunque 20°C.(lb) Date 23; ,Range No. E 23; Range No. Body 5.77 0.11 5.25--6.50 25 Jan. 26 Tail 11.81 0.17 10.50-13.00 25 Body 6.88 0.23 5.50— 8.00 21 5.12 0.12 “.50- 5.50 15 Feb. 2 Tail l“.20 0.5“ 10.00-16.00 21 10.53 0.38 9.00-11.50 15 - Body 8.11 0.3“ 6.50-10.00 21 6.68 0.27 6.00- 7.50 1“ Feb. 10 Tail. 17.78 0.63 13.00-21.00 21 13.89 0.“1 12.50-15.00 1“ Body 9.70 0.50 8.00-11.00 21 8.5“ 0.““ 7.00- 9.50 1“ Feb. 17 Tail 21 93 0.65 18.00-2“.00 21 17.89 0.“5 16.00-19.00 1“ Body 10 63 0.26 9.00-11.50 21 10.28 0.3“ 9.00-11.00 1“ Feb. 2“ Tail 26.25 0.75 22.00—27.00 21 20.85 0.75 18.00-23.00 1“ Body 12.89 0.““ 10.50-1“.00 20 12.23 0.28 11.50-13.00 1“ Mar. 1 Tail 29.“5 1.03 2“.00-35.00 20 27.50 0.96 2“.00-30.00 1“ Body ‘ 1“ 2“ 0.5“ 11.50—16.00 19 13.77 0.36 13.00-1“.50 1“ Mar. 8 Tail 32.76 1.16 27.00937.00 19 30.30 1.0“ 27.00-33.00 1“ Body .15.03 0.“8 12.50-16.50 19 1“.69 0.38 13.50-15.50 1“ Tail 35.26 1.16 29.00-39.00 19 33.1“ 0.95 29.00—35.00 1“ Mar. 15 Hindleg 0.16 0.22 0.00- 2.00 19 0.“8 _0.36 0.00- 1.50 1“ Body 15.6“ 0.53 12.75-17.00 19 15.19 0.37 13.75-16.00 1“ Tail 37.57 1.26 31.00-“2.00 19 35.25 1.11 30.00-38.00 1“ Mar. 21 Hindleg 1.63 0.70 0.00- 5.00 19 2.71 0.98 0.00- 6.00 1“ Body 15.92 0.“8 13.00-1700 19 15.5“ 0.35 1“.00-16.25 1“ Tail 39 26 1.3“ 32.00-“3.00 19 36.25 1.29 30.00-“0.00 1“ Mar. 29 Hindleg 3.90 1.60 0.00-12.00 19 5.61 1.92 1.00-12.00 1“ Body 1“ 57 0.“3 12.50—16.00 19 1“.28 1.03 13.00-15.00 1“ Tail 38 10 2.02 30.00-““.00 19 3“.92 1.37 30.00-39.00 l“ Hindleg 6.13 2:26 0.00-15.00 19 7.85 2.28 3.00-15.00 1“ Apr. 6 Foreleg 0.31 0.62 0.00- 6.00 19 Body 15.03 0.“0 12.50-16.00 19 1“.60 0.33 13.00-15.00 1“ Tail 37.21 2.20 25.00-“5.00 19 36.1“ 1.13 31.00-38.00 1“ Hindleg 10 “2 3.20 0.00-22.00 19 12.78 3.16 “.00-23.00 1“ Apr. 13 Foreleg 1.63 1.50 0.00— 9.00 19 0.93 1.26 0.00- 7.00 1“ Body 1“.88 0.32 1“.00—16.00 18 1“.70 0.“0 13.00-16.00 1“ Tail 31 11 “.78 1.00~“3.00 18 31.00 1.69 5.00-38.00 l“ Hindleg 1“.66 3.“8 0.00-25.00 18 16.96 3.“1 “.50-25.00 1“ Apr. 20 Foreleg 3.78 2.12 0.00-11.00 18 “.89 2.uo 0.00-11.00 1“ Body 1“.61 0.36 l“.OO—16.50 18 l“.61 0.69 12.00-18.00 1“ Tail 22.77 7.30 0.00—“2.00 18 21.1“ 6.“0 0.00—35.00 1“ Hindleg 18.6“ 3.78 0.00-27.00 18 19.30 3.““ “.50-2“.00 1“ Apr. 27 Foreleg 5.53 1.98 0.00-11.00 18 6.66 2.08 0.00-10.50 1“ Body l“.36 0.38 13.00-15.50 1“ 1“.50 0.58 12.00-16.00 12 Tail 18.86 7.86 0.00-“1.00 1“ 1“.“2 8.20 0.00—3“.00 12 Hindleg 18.75 “.32 0.00-27.00 1“ 20.58 3.17 10.00-26.00 12 May “ Foreleg 7.1“ 1.7“ 0.00—10.00 1“ 7.70 2.1“ 0.00-11.00 ’12 Body 1“.9l 0.“9 13.00-16.00 12 l“.66 0.62 12.50-16.00 9 Tail 6.92 7.00 0.00-32.00 12 3-11 “.0“ 0.00-18.00 9 Hindleg 20.13 5.3“ 0.00-28.00 12 22.20 3.02 15.00-28.00 9 May 11 Foreleg 8.29 2.30 0.00-11.50 12 9.17 0.90 7.00-10.50 9 Body l“.87 1.31 13.00-16.00 “ 1“.33 1.“5 13.00-15.50 3 Tail 7.50 15.00 0.00-30.00 “ 0.00 0.00 0.00- 0.00 3 Hindleg 16.13 8.63 3.50-23.00 “ 20.66 2.96 18.50-23.50 3 May 18 Foreleg 7.00 “.66 0.00— 9.50 “ 9.00 1.00 8.50-10.00 '3 Body 13.00 1 Tail 30.00 1 Hindleg 3.75 1 May 25 Foreleg 0.00 1 18 Figure 2.--Growth rate of two groups of montane El Yunque tadpoles (la and lb) cultured at 20°C. The horizontal line represents the mean of the sample; the vertical line, the range; and the box, plus and minus twice the standard error of the mean. Growth rate in mm. “0-- l9 E1 Yun ue 20°C. (la) ———— Body _.._.. Taié - ~~~-~-H1n leg 1 ~- ---- Foreleg .L In 11 i f 90 100 110 120 Days “0 El Yunque 20°C. (lb) 35 -> - '%U % - ' \ .o -. .11 II. % \' _- 25 - - - '\ 5’ " .1 ”,1 20 -- 1%: :;.-- ' ' u .. \I 15 _ :jI : . I ,8} f . - 10 __ _. " .\ ‘ ' xii-B 0 31' - an. i i I : i I i i I i I 10 '20 3O “0 50 60 7O 80 90 100 110 120 20 increase in fore leg of 9% as compared to the lowland population (Table 3) cultured at the temperature approximating its environment (30°C.). (2) The montane population (Table “, Figure 3) cultured at 30°C. has an increase in body length of “.2%; a decrease in tail length of 17.7%; an increase in hind leg length of l9.“6%; and an increase in fore leg of 53.7% as compared when raised at the temperature approximating its evnironment (20°C.). (3) The lowland population cultured at 20°C. (Tables 5 and 6, Figures “ and 5) has a decrease in body length of l“.“%; a decrease in tail length of 17.8%; a decrease in hind leg length of 132%; and a decrease in fore leg length of 33.3% as compared when raised at the temperature approximating its environment (30°C.). When both population samples are cultured at their native temperatures, the El Yunque population develops bigger body, tail, hind and fore legs. At reversed temperatures, the montane population continues to develop bigger body, hind and fore legs, whereas the lowland population develops much smaller body, tail, hind and fore legs. It would seem that the montane population is more adaptable at both native and experimental temperature con— 21 ma 00.0 -00.a 0m.0 m0.a am 00.0HI00.~ am.0 00.0 woaooom 00 .000 ma 00.00-00.5H 00.0 00.0H am 00.00-00.0H m0.0 00.00 woaooam ma 00.0 100.0 00.0 00.0 am 00.0 :00.0 00.0 00.0 Hams ma 00.0HI0m.mH mm.0 0m.0a am 00.0an0m.mfi am.0 mm.0H 000m ma 00.0HI00.HH 00.0 00.mH am 0m.mfiu00.a 00.0 00.0H woaoofim ma .000 ma 00.mmu00.0m as.0 0a.0m am 00.0mu00.0m 00.0 00.0m Hams ma 00.0Hu0m.HH mm.0 m0.mH 00 00.0HI00.NH 00.0 0m.mH soom ma 00.00-00.00 Hm.0 00.Hm mm 00.mm:00.aa 00.0 0H.Hm Hams 0 .900 ma 00.HHI00.0H NH.0 mm.0fi mm 00.0HI00.00 mm.0 mm.0H zoom ma 00.0 Imm.s mH.0 0a.s mm 00.0 Imm.a0 HH.0 00.5 Hams mm .002 ma 00.0 uom.m HH.0 m0.m mm 00.0 I00.m0 0s.0 00.m zoom .02 owcmm mmm m .02 owcmm mmm m open Aomv.000m mmooofim on Ammv.0°0m monooam oam .Ooom PM UoQSszo moHoQUMp mmpcofim cam UCMHSOH we now use wmv mdzopw 03p mo memEonzwmozll.m mqm I' '~\ " “\~.‘._'_" 1.1:;1 I I Figure 5. Tooth morphology of Eggtodactzlus albilabris tadpole. 32 ditions and the lowland population is affected at the reduced temperaturec Relationship of Appendage Growth to Bodyflengthe——Table 7 expresses the differential changes that develOped between the body and its appendages; namely, the tail, hind legs and fore legs. These data were derived by dividing the maximum weekly mean body length into the maximum weekly mean length of the appendage being measured, in an effort to ascertain any differences thatmay have developed between the popula- tions during the developmental periode The data, thusly treated, revealed the following information° (1) In their natural environmental temperature con— ditions, the lowland population indicated a ratio of tail-length to body~length 0°32 lower than that of the montane pOpulation. (2) The lowland pOpulation maintained a more or less constant prOportion between body and tail-lengths at both temperatures, (3) The montane pOpulation decreased its proportion of tail to body—lengths by O.Hu when cultured at 3000, (4) The length of the hind leg to that of the body- length of both the montane population and the low— land pOpulation at their native temperature con— ditions share a common ratio of 1,35. (5) The ratio of hind leg and body-lengths of the lowland population, at reversed temperature (20°C,), is reduced by one—half. 33 TABLE 7.--A comparison of the maximum weekly growths between body-lengths and appendage—lengths of Leptodactylus albilabris tadpoles from El Yunque and Rio Piedras when reared at 20°C. and 30°C. Maximum Appendage/ weekly ; Maximum Body Location . mean 23x Range No. length ratio Char. El Yunque 20°C. (1a) 15.92 0.98 13.00-17.00 19 17.80 El Yunque 20°C. (lb) 15.5“ 0.35 19.00—16.25 1a 18.00 Rio Piedras 30°C. (2a) 1h.53 0.27 13.50—16.00 29 16.00 R10 Piedras 30°C. (2b) 14.36 0.33 13.50—15.00 15 15.00 . Bodv El Yunque '30°C. (2a) 16.88 0.39 16.00-18.00 18 18.00 El Yunque 30°C. (2b) 15.87 0.33 15.00-16.50 13 16.50 Rio Piedras 20°C. (1a) 12.u3' 0.9M 11.00-1u.oo 7 lu.00 Rio Piedras 20°C. (lb) 12.82 0.58 12.00-1H.25 7 19.25 El Yunque 20°C. (la) 39.26 1.3a 32.00-93.00 19 “5.00 El Yunque 20°C. (1b) 36.25 1.29 30.00-90.00 1a “0.00 .hO Rio Piedras 30°C. (2a) 30.80 0.6u 28.00-3u.oo 2n 3u.oo Rio Piedras 30°C. (2b) ' 29.70 0.77 28.00-33.00 15 33.00 .08 . Tail El Yunque 30°C. (2a) 32.87 1.09 28.00-36.00 20 36.00 El Yunque 30°C. (2b) 31.30 1.53 27.00—35.00 13 35.00 .96 Rio Piedras 20°C. (la) 25.98 0.55 23.00-28.00 28 32.00 Rio Piedras 20°C. (lb) 26.00 0.97 29.00-30.00 15 30.00 .09 El Yunque 20°C. (1a) 20.13 5.3“ 0.00-28.00 12 28.00 El Yunque 20°C. (lb) 22.20 3.02 15.00-28.00 9 28.00 .35 Rio Piedras 30°C. (2a) 20.92 0.83 l”.00-2fl.00 2” 29.00 Rio Piedras 30°C. (2b) 18.86 0.60 17.00-20.00' 15 20.00 .35 Hixwileég El Yunque 30°C. (2a) 25.80 0.70 23.00-29.00 18 29.00 El Yunque 30°C. (2b) 29.73 0.99 21.50-27.50 13 27.50 .56 Rio Piedras 20°C. (la) 8.56 2.53 1.50-18.00 21 18.00 Rio Piedras 20°C. (lb) 8.38 5.15 2.00-16.00 6 17.00 .67 El Yunque 20°C. (1a) 8.29 2.30 0.00-11.50 12 11.50 El Yunque 20°C. (1b) 9.17 0.90 7.00-10.50 9 11.00 .55 Rio Piedras 30°C. (2a) 8.20 0.37 7.00-10.00 29 10.00 Rio Piedras 30°C. (2b) 7.82 0.36 7.00- 9.00 15 9.00 .55 Foreleg El Yunque 30°C. (2a) 13.9“ 0.67 11.00-15.00 18 15.00 El Yunque 30°C. (2b) 13.40 0.56 11.50-15.50 13 15.50 .82 'Rio Piedras 20°C. (1a) 6.00 0.00 6.00- 6.00 u 8.00 R10 Piedraw 20°C. (1b) . 6.00 0.00 5.00- 7.00 4 7.00 .98 (7) (8) (9) (10) 34 At reversed temperature (3000.), the El Yunque population increases its proportion of hind leg- length to body-length by 0.21. When these two populations are cultured at reverse temperature conditions, the ratios of hind leg to body—lengths and foreleg to body-lengths of the lowland population is approximately one—half that of the montane pOpulation. The length of the foreleg to that of the body— length of both the montane population and the lowland pOpulation share a common ratio of 0.55. At reversed temperature (30°C.), the El Yunque population increases its proportion of foreleg to body—length by 0.27. At reversed temperature (20°C.), the lowland popu- lation decreases its prOportion of foreleg to body—length by 0.07. Allen's Rule states that protruding body parts, such as tail, ears, bill extremities, and so forth, are relatively shorter in the cooler parts of the range of a species than in the warmer parts. The data, when subjected to this interpre_ tation, indicates that the Rule is maintained by four of the ratios measured. Specifically, they are: (l) (2) Increased hindleg to body proportion of the mon- tane population cultured at 30°C. Reduced hindleg to body proportion of the low— land pOpulatiOn cultured at 20°C. 35 (3) Increased foreleg to body proportion of the mon— tane population cultured at 3030. (U) Reduced foreleg to body proportion of the lowland population cultured at 2000. On the other hand, Allen's Rule is not maintained with respect to the larger tail to body proportion of the montane population as compared to that of the lowland population. Futhermore, the Rule appeared to be inconsequential when comparing the hindleg to body proportion and foreleg to body proportion when both populations are cultured at their native temperature conditions. Comparison of Developmental Stages Gosner (1960) charted the developmental stages of larval development on the basis of the presence or absence of con— spicuous features in varying degrees of noticeable changes. The criteria utilized by Gosner (op. cit.) were employed during this study to compare the growth patterns of the lowland and highland populations of Leptodactylus albilabris larvae. The stages, used herein, are listed below with their associated distinguishing features. Stages 17-21: based on the development of the oral suckers° Stage 22: tail fins become transparent and circula— tion begins. Stage 23: oral disc and labial tooth-rows begin to differentiate. 36 Stages 23-25; develOpment of Operculum and consequent Stage disappearance of external gills. 25: spiracle present on left side. Stages 26-30: length/diameter relationship of the developing limb—bud. Stages 31-37: development of toes. Stages 38-90: proportional changes in the length of in— Stage Stage dividual toes and the appearance of meta— tarsal and subarticular tubercles. Ml: disappearance of cloacal tail piece. 82: appearance of forelimbs. Stages u3-A6: identified by metamorphosis of the head indicated by changes in the mouth. Samples selected at random from each population were examined and their stages ascertained (Table 8). The follow— ing are some observations that were made: (1) (3) (Li) At stage 26, the montane pOpulation is 1.97 times larger in body and 2.3 times larger in tail length than the lowland. At stage 27, the montane population is 1.75 times larger in body and 2.07 times larger in tail length than the lowland. At stage 31, the montane population is 1.50 times larger in body and 1.75 times larger in tail length than the lowland. At stage 36, the montane population is 1.95 times larger in body and 1.79 times larger in tail length than the lowland. 37 TABLE 8.——A comparison of measurements at similar larval stages of Leptodactylus albrilabris collected from Rio Piedras and El Yunque. El Yunque Body Length Tail Length Total Length Stage x range i range 2 range NO 29 6.31 5.50- 8.00 10.99 9.75-1l.50 16.75 15.25-19.50 9 25 7.81 6.50- 9.50 12.25 9.00-20.00 20.06 16.00-19.50 12 26 11.10 8.25—13.25 17.67 11.00—22.00 28.77 20.00-35.00 35 27 11.59 '8.50-19.50 18.90 12.50-23.00 29.99 21.00—99.75 60 28 12.35 7.50-16.00 19.96 19.00—28.50 32.31 23.00-99.50 51 29 13.27 11.50-19.75 21.30 15.00-25.00 39.57 25.50-37.50 10 20 13.12 12.50-19.00 22.08 21.00-23.00 35.20 39.00-37.00 6 31 13.17 12.50-15.00 21.33 20.00-23.00 39.50 32.50—37.00 9 32 13.37 13.00-13.75 21.50 20.00—23.00 39.87 33.00-36.75 2 33 13.19 12.50—19.00 21.17 18.50-23.50 39.36 31.00-37.25 9 39 13.25 - 13.25 21.50 21.50 39.75 39.75 1 35 13.75 13.75 29.25 29.25 38.00 38.00 1 36 13.75 10.00-15.00‘ 23.66 22.00-25.00 37.91 33.00-90.00 6 37 19.50 19.00—15.00 29.87 29.75-25.00 39.37 39.00-39.75 2 38 15.12 19.75-15.50 29.75 29.50-25.00 39.87 39.75-90.00 2 39 19.87 19.00-16.00 23.75 17.50—29.00 38.62 33.00-99.50 16 90 19.10 13.00-15.00 29.00 22.00-27.00 38.10 35.00-91.00 13 91 13.80 11.50-16.00 29.20 21.50526.00 38.00 39.00-92.00 5 92 13.62 13.00-19.25 27.00 27.00-27.00 90.62 90.00-91.25 2 93 13.87 13.00-15.00 29.87 22.00-28.50 38.79 35.00-92.00 9 Rio Piedras 26 5.63 5.00- 5.50 7.61 7.00-10.00 13.29 12.00-16.25 11 27 6.60 5.75- 8.25 8.88 7.00—11.50 15.98 12.50-21.25 99 28 7.95 6.00- 8.25 10.20 8.00—12.50 17.65 19.50-20.25 23 29 8.90 7.00- 9.00 11.58 9.50-13.00 19.98 16.50-22.00 20 30 8.50 8.00— 9.50 11.60 10.00-13.50 20.10 17.50-22.50 12 31 8.70 7.50— 9.50 12.19 10.00-19.00 20.89 17.50-23.50 8 32 8.80 8.00-10.00 11.90 10.00-15.00 20.70 18.00—25.00 8 33 8.85 8.00- 9.50 12.20 10.50-13.50 21.05 19.50-23.00 10 39 9.21 8.25-10.25 12.96 11.50-15.25 22.17 20.00—25.25 6 35 9.30 8.75-10.75 12.00 10.00-19.25 21.30 18.00-25.00 5 36 9.96 8.00-11.50 13.20 11.00-16.50 22.66 19.00—28.00 13 37 10.00 10.00 16.00 16.00 26.00 26.00 1 38 10.99 9.25—12.00 15.00 13.00-17.00 25.99 29.00-29.00 9 39 ' 11.60 11.00—12.00 17.20 19.50-19.00 28.80 26.00—30.75 9 90 13.00 13.00 19.00 ' 19.00 27.00 27.00 1 38 (5) At stage 39, the montane population is 1.28 times larger in body and 1.37 times larger in tail length than the lowland. (6) At stage 90, total length diminishes through re— sorption of the tail, and larval mouth parts begin to break down. On the basis of these comparisons, at comparable stages, the montane tadpoles are longer in both body and tail measurements than those from the lowland habitat. Comparispn of Tooth Morphology A tadpole acquires rows of teeth during its period of early development and loses them during the period of meta— morphosis° The standard tooth morphology of the Leptodactylus albilabris tadpole (Figure 6) exhibits two upper tooth rows (1 and 2) with a median space on the second upper row, and three tooth rows (3, 9, and 5) below the mandibles with a smaller median space on the first lower row. Table 9 summarizes the conditions of tooth row numbers at the various developmental stages within the two pOpula- tions of larvae studied. A "typical" tooth row is one with a continuous series of teeth present; "poorly developed" refers to the presence of a ridge or part of it with labial teeth discontinuous; and "absent" denotes no ridge or labial teeth. L78 - 1‘ .T:f... TABLE 9.-A comparison of the relationship of tooth morphology to developmental stages of samples of Rio Piedras and El Yunque populations of Leptodactylus albilabris tadpoles. Stages 33 39 35 Tooth row no. Loc. Condition u2 13 91 .36 37 38 39 -90 32 28 29 30 31 26 27 29 25 l7 17 10 59 98 10 6 92 b»: 3 IKO l Typical Pobrly dev. E.Y. Hm \Or-l J'r-i Absent [\N 11 6 20 I7 10 9 92 35 58 51 Typical R.P. E.Y. Poorly dev.. Absent 39 3 l 10 9 16 2 l2 6 19 - - 10 99 23 1 l2 35 59 51 10 04>! O 0 mm Typical R.P. E.Y. Poorly dev. Absent 2 9 0 7 93 2O 19 10 8 35 56 50 Typical E.Y. Poorly dev. Absent \OH 12 6 6 91 21 18 u 3a 5a us 9 Typical - -— 1 0I 2 R.P. E.Y. Poorly dev. Absent \DH 6 10 o 11 99 23 2o. 12 '35 60 51 ‘12 0 9 04M ,CISIIJ Total no. examined 90 The pattern of tooth rows and mandibles in the mon— tane population is essentially the same as those in the lowland. However, abnormalities of labial teeth as illustrated by "poorly developed" and "absent" develop more frequently by the lowland sample in the earlier stages of development. For instance, at stage 26 the low— land tadpoles have 27% abnormal conditions as compared to the 3% exhibited by the montane group, whereas, at stage 39 the montane tadpoles have a slightly larger tooth abnormality (19%) as compared to the lowland group with 19%. The early abnormality of the lowland tadpoles may be due to a combination of rapid growth and ease of injury to the smaller teeth as compared to the montane adaptation of larger teeth for swift running streams and a longer tadpole phase. DISCUSSION An important phase of adaptation is the development of morphological or physiological adjustments of animals to meet new environments. These adaptations have a hereditary basis and are subject to selection. The constancy of some obviously important features are maintained by these adjustments of the organism. More generally, however, the adaptations increase the likelihood of survival for the individual in question; the adaptive adjustments necessary for survival may bring about marked altera~ tions in the appearance of the individual or in physiological processes vital for its continued survival. The genetic changes that comprise the fundamental adaptive responses of a population are not always easily visualized. The anatomical adaptations and growth rates of the two populations of Leptodactylus albilabris illustrate the fact that adaptation to a cooler temperature and higher altitude is accomplished by the modification of existing structures and difference in growth rate. The montane tadpoles developed larger and less abnormal teeth, possibly an adjustment for feeding in swift streams. Futhermore, they expressed a more rapid rate of development at experimental temperatures approximating that of their environment that did the lowland population at the same low 91 92 experimental temperature. The montane population sample developed very rapidly and transformed a few days later than the lowland population sample at the temperature approximating that of the lowland. Burgess (1950) studied the development of Spade—foot toad larvae (Scaphiopus hammondii) under laboratory conditions and found an acceleration of growth due to higher temperature. Volpe (1958) proposed a hypothesis that the relatively rapid rates of embryonic develOpment in toads are correlated with the limited period of time available for larval growth in temporary pools. However, temperature apparently plays an important role in affecting growth rates as indicated by the data obtained in this study. Temperature has a fundamental effect on size and growth as suggested by the fact that most poikilotherms grow larger when raised at low temperatures than at higher temperatures and pOpulations of the same species are larger from colder environments. MacArthur and Baillie (1929), Imai (1937), and Ryan (1991) studied the response of animals to temperature and suggested that there is a shift in the anabolic-catabolic balance at different temperatures. Bullock (1955) and Prosser (1955) found that northern species maintain a higher metabolic rate at low temperatures than do related southern species. Wimpenny (1990), Newell (1999), and Dunbar (1953) described this size-temperature relationship as "delayed ontogeny." 93 Bergmann's and Allen's Rules are closely related to size and temperature. Accafiiing to Mayr (1992), Bergmann's Rule states "the smaller-sized geographic races of a species are found in the warmer parts of the range, the larger sized races in the cooler districts." Bergmann's principle is expressed by the lowland and highland populations cultured at their respective temperatures approximating their environment (Table 7). The montane population showed an increase in body length of 8.86% over the lowland population. At reversed temperature, the montane population body—length is increased by 9.2% and the body- 1ength of the lowland group is reduced by 19.9%. Carter (1961) has suggested that, probably, the most important general advantage of large over small size is the greater independence from the external environment which larger size allows. Protoplasm characteristically requires uniform maintenance of internal conditions unlike those of the exterior. The surface—volume ratio is less in large bodies than in similar smaller ones; hence, the proportion of living tissues in contact with the external environment is less in larger than in smaller animals. The montane population showed superior survival character— istics as compared with the lowland population when the two groups were reared under reversed temperature conditons. The upland animals acquired a larger body in disregard to Bergmann's Rule and also developed longer appendages, contrary to expecta- tion in keeping with Allen's Rule. The single apparent exception 99 to this phenomenon was expressed by the tail reduction of the montane population. The data indicate, therefore, that the battle for survival in the more hazardous montane ecological location was advantageous to developing a population better equipped than the lowland population. The ratios of appendages to body—lengths were constant for both populations at their natural environmental temperatures. The total larval develOpmental period, when compared at normal and experimental temperatures, revealed additional substantiary data. The montane pOpulation approximated favourably the length of the developmental period of the low— land pOpulation when the culture temperatures were reversed. On the other hand, the lowland population at 20°C. (the mon— tane temperature) did not respond in a similar fashion. The survival inferiority of this population, perhaps, did not make for easy adaptation. (l) (2) (3) (9) (5) (6) SUMMARY AND CONCLUSIONS Larvae from both lowland Rio Piedras and montane El Yunque populations of Leptodactylus albilabris were subjected to normal and experimental tempera— tures. Field collections of both population larvae supplement the study of tooth morphology and meas— urements at similar stages. Comparison of the normal developmental rate of larvae from the lowland at 30°C. and montane at 20°C. reveals that the latter develOped much slower and transformation took place in about 111 days as compared to the lowland at 25 days. At reverse temperatures, the montane population takes 30.5 days, whereas the lowland population takes 252 days to transform into adult. At normal temperatures, the montane population develops longer body, tail, hind and forelegs than the lowland pOpulation. At reverse temperatures, the montane population continues to develop longer body, hind and forelegs, whereas there is a decrease in body, tail, hind and forelegs of the lowland pOpulation. 95 (7) (8) The the basis (1) (2) (3) (9) (5) 96 Field collections reveal that at similar stages, the montane population is bigger in body, tail, hind and forelegs. The pattern of tooth rows and mandible in the montane population is essentially the same as those of the lowland population. analysis of the data, presented herein, provides for the following conclusions: At normal temperatures, the lowland pOpulation develops more rapidly due to the higher temperature which occurs there as compared to the lower temperature condition of the montane population. A rise in temperature is followed by hastened metamorphosis, whereas a decrease in temperature has a retarding effect on metamorphosis. The rate of development of tadpoles from the mon- tane region is less affected by a change in temperature than that of the lowland population. Selection for increasing size may be inferred to be the result of the statistical advantage that slightly superior bulk gives the montane pOpulation a wider range of temperature conditions. The conspicuous difference in growth rate is highly interesting in that is suggests an adaptation of the montane population for the lower temperature which occurs there. (6) (7) (8) (9) 97 From the standpoint of evolution, this adaptive change is accumulative in the sense that it may not appear until the montane popula— tion has been subjected to the existing lower temperature for several generations. The pattern of tooth rows and mandible is essen— tially the same for both pOpulations.- Experimentation has provided evidence which does not point convincingly towards recognizing the two populations as distinct subspecies. Both populations are members of the same species. Some time ago, rather recently in terms of evolutionary chronology, migration occurred and the montane population developed peculiar morphological characteristics in the process of adaption to the new habitat. Should isolation continue it is feasible that speciation would occur. LITERATURE CITED Brett, J. R. 1999. Some lethal temperature relations of Algonquin fishes. Univ. Toronto Stud. Biol., Ser. 52; PUbl. onto FiSh Res. Lab. 6451-499 Bullock, T. H. 1955. Compensation for temperature in the metabolism and activity of poikilotherms. Biol. Rev. 30:311-392. Burgess, R. C. 1950. Development of Spade—foot toad larvae under laboratory conditions. COpeia 1950:99—51. Carter, G. S. 1961. A general zoology of the invertebrates. Sidgwick and Jackson, London, 921 pp. Dunbar, M. J. 1953. Arctic and subarctic marine ecology: immediate problems. Arctic 6:75—90. Fry, F. E. J. 1997. Effects of the environment on animal activity. Univ. Toronto Stud. Biol., Ser. 59; Publ. Ont. Fish Res. Lab. 68:5-62. Goin, O. B. and C. J. 1962. Amphibian eggs and the montane environment. Evolution 16:362—371. Gosner, K. L. 1960. A simplified table for staging anuran embryos and larvae with note on identification. Herpetologica 16:183-190. Hathaway, E. S. 1927. Quantitative study of the changes pro— duced by acclimatization in the tolerance of high tem— peratures by fishes and amphibians. Bull. U.S. Bur. Fish. 93:169—192. Highton, R. 1962. Geographical variation in the life history of the slimy salamander. COpeia 1962:597-613. Imai, T. 1937. Influence of temperature on the growth of Drosophila melanogaster. Sci. Rep. Tohuku Imp. Univ. 11:903—917. MacArthur, J. W. and W. H. T. Baillie. 1929. Metabolic activity and duration of life. 1. Influence of temperature on longevity in Daphnia magna. J. Exp. 2001. 53:221—292. 98 99 Mayr, E. 1992. Systematics and origin of the species. Columbia, New York, 339 pp. Moore, J. A. 1939. Temperature tolerance and rates of development in the eggs of amphibia. Ecology 20:959-978. 1990. Adaptive differentiation in the egg membranes of frogs. Amer. Nat. 79:89—93. . 1992a. The role of temperature in speciation of frogs. Biol. Symp. 6:189-213. 1992c. Embryonic temperature tolerance and rate of development in Rana catesbeiana. Biol. Bull. 31:309-326. . 1996. Incipient intraspecific insolating mechanisms in Rana pipiens Schreber. Genetics 31:309-326. . 1999. Geographic variations of adaptive characters in Rana pipiens. Evolution 3:1—29. Newell, N. D. 1999. Phylectic size increase-—an important trend illustrated by fossil invertebrates. Evolution 3:103—129. Peiss, C. N. and J. Field. 1950. The respiratory metabolism of excised tissues of warm and cold adapted fishes. Biol. Bull. 99 (2):213-229. Prosser, C. L., D. W. Bishop, F. A. Brown, T. L. Jahn, and V. J. Wulff. 1950. Comparative animal physiology. W. B. Saunders, Philadelphia, 888 pp. Prosser, C. L. 1955. Physiological variation in animals. Biol. Rev. 30:229-262. Ruibal, R. 1955. A study of altitudinal races in Rana pipiens Evolution 9:322-338. Ryan, F. J. 1991. Temperature change and the subsequent rate of development. J. Exp. Zool. 88:25—59. Schmidt, K. P. 1930. Amphibians and land reptiles of Puerto Rico, with a list of those reported from the Virgin Islands. New York Acad. Sci. 10:1-160. . 1938. A geographical variation gradient in frogs. Z001. Ser. Field Mus. Nat. Hist. 20:377—382. 50 Scholander, P. F., W. Flagg, V. Walter and L. Irving. 1953. Climatic adaptation in arctic and tropical poikilotherms. Physiol. Zool. 26:67—92. Stejneger, L. 1902. The herpethology of Puerto Rico. Report U.S. Nat. Mus. 1902:599—729. Stuart, L. C. 1951. The distributional implication of temperature tolerances and haemoglobin values in the toad Bufo marinus and Bufo bocourti. COpeia 1951: 220-229. Volpe, E. P. 1953. Embryonic temperature adaptation and relations in toads. Physiol. Zool. 26(9):399—355. . 1958. Embryonic temperature tolerance and rate of develOpment in Bufo valliceps. Physiol. 2001. 30(2):l65-l76. Wells, N. A. 1935. Change in rate of respiratory metabolism in a teleost fish induced by accimatization to high and low temperatures. Biol. Bull. 69:361—367. Wimpenny, R. S. 1991. Organic polarity. Some ecological and physiological aspects. Quart. Rev. Biol. 16:389—925. TA ”lllifiil‘aifllllji(illfllflfliiliiiiflfiiifil“