ECOLOGY or ANURAN POPULATIONS INHABITING THERMALLY STRESSED AQUATIC ECOSYSTEMS, WITH EMPHASIS ’ 0N LARVAL RANA PiPlE‘NS ” - AND BUFO TERRIESTRIIS" _" ’ . Dissertation for the Degreé‘faf Ph. D. MICHIGAN STATE UNWERSITY DAViD H. NELSON 1 97 4 This is to certify that the thesis entitled ECOLOGY OF ANURAN POPULATIONS INHABITING THERMALLY STRESSED AQUATIC ECOSYSTEMS, WITH EMPHASIS ON RANA PIPIENS AND BUFO TERRES'I‘RIS presented by DAVID H. NELSON has been accepted towards fulfillment of the requirements for m—degree in Wm logy A, l/ZL’fl-"Iocm my 241v»? (Jk Major professor fl Date {/(CLti-X) , q /(/;.7 L/ \ 0-7639 . ‘lllllhfijfl “MB 8' 30W 300K BINDERY INC. ‘9'! LIBRAPY BINDERS ll‘n sputum“ mama“ . mmol "a 8' In“ 5 mg... ABSTRACT ECOLOGY OF ANURAN POPULATIONS INHABITING THERMALLY STRESSED AQUATIC ECOSYSTEMS, WITH EMPHASIS 0N LARVAL RANA PIPIENS AND BUFO TERRESTRIS By David H. Nelson Field and laboratory studies were conducted to determine the reSponses of anuran populations to thermally stressed aquatic ecosystems. Adult and larval amphibians were sampled in and around a cool arm of a 67 ha reservoir that receives high temperature effluent from a nuclear production reactor on the Savannah River Plant (SRP) in South Carolina. Adult amphibians were sampled in a mark-release program over a 13-month period by pitfall traps placed peripheral to the reservoir. Species diversity, relative abundance, seasonal occurrence and activity patterns were compared among dominant anurans inhabiting the area. These patterns for some species were compared with data from nearby unheated areas and analyzed in terms of the thermal gradient (16-45 C) extending the length of the reservoir's cool arm. Larval amphibians were routinely sampled for l5 successive months by dipnets and minnow traps from two stations in the heated reservoir and from five cool seepage ponds flanking the reservoir. All specimens were identified, and larval Rana pipiens and Bufo terrestris were measured for length and staged according to the level of development. David H. Nelson In the laboratory, embryonic 3, pipiens were reared through metamorphosis to compare survivorship, growth and developmental rates at four constant temperature regimes: 20 C, 25 C, 30 C, and 35 C. Pitfall trapping studies revealed that three anuran populations dominated (86% of 5,583 captures) the 13 species encountered. Adults and emergent young of the Southern toad (g, terrestris) and the narrow- mouthed toad (Gastrophryne carolinensis) were trapped during the same months at the heated reservoir as they were elsewhere on the SRP. Breeding patterns and activity of the leopard frog (3, pipiens), how- ever, were more extensive at the reservoir than at other areas. Adult 3, pipiens at the reservoir were active all year, and recently trans- formed specimens were trapped during 10 months of the year. Captures of migrating adults and juveniles confirm that both 3, pipiens and g, terrestris bred and developed within protected areas of the heated reservoir. Recapture frequencies ranged from 2% (g, carolinensis) to 16% (g, terrestris). Although larvae of five anuran species were removed from heated reservoir waters, abundance, species diversity and population density were inversely related to the degree of thermal loading sustained. Having the shortest developmental period, 9, terrestris was most suc- cessful, although restricted to a single breeding migration yearly. Embryonic and larval mortality in some hot areas exceeded 90%. Within the limits of thermal tolerance, however, growth of larval 3, pipiens and g, terrestris were both inversely related to temperature and directly related to periods of development. David H. Nelson Although all embryonic 3. pipiens maintained in the laboratory at 35 C died within nine days, most specimens at 30 C, 25 C and 20 C completed development through transformation. Values for mortality (30-37%) and transformation (63-70%) were similar among the three groups. Corroborating field data, specimens reared at higher temperatures (30 C, 25 C) demonstrated reduced growth rates but increased developmental rates when compared to larvae reared at 20 C. Developmental periods required 6 months (30 C), 8 months (25 C), and >17 months (20 C). Survivorship curves were similar among larvae reared at 20 C, 25 C and 30 C. Specimens demonstrating abnormal development (crooked spine and paralyzed hindlimbs) were significantly greater at 30 C than at 25 C or 20 C. Anomalous development is an apparent manifestation of thermal stress observed only in the laboratory. Localized cool stream seepage creates isolated microenvironments that allow for marginal development of the most common anurans (3, pipiens and D, terrestris) in a reservoir receiving supralethal levels of thermal loading ( >50 C). The adaptation to breeding during noctur- nal rainfall fortuitously confers a double advantage especially to anurans breeding in thermally stressed waters. Both field and labora- tory studies confirm that temperatures maintained in excess of 34 C are lethal to eggs, embryos and larvae of most anurans. Within tolerance limits, increased temperatures reduce growth rates but increase develop- ment rates. Field data presented here are usually not available fbr comparison with laboratory populations. David H. Nelson Although temperature responses must be interpreted at sensitive developmental stages for each species, certain generalities of growth and development apparently hold true within the range of thermal tolerance. It is clear that local physical microhabitats can be altered or effluent temperatures regulated in order to guarantee marginal survival of dominant aquatic populations, and thereby safeguard the stability of the stressed community. ECOLOGY 0F ANURAN POPULATIONS INHABITING THERMALLY STRESSED AQUATIC ECOSYSTEMS, WITH EMPHASIS 0N LARVAL RANA PIPIENS AND BUFO TERRESTRIS By 9"“ David H? Nelson A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Zoology 1974 ACKNOWLEDGMENTS I deeply appreciate the support and suggestions of Dr. J. Whitfield Gibbons at the Savannah River Ecology Laboratory. His continued interest and encouragement throughout the three years of this study contributed much to its success. He critically reviewed early versions of the manuscript, advised me in numerous instances, and gave generously of his time and resources. I wish to thank my guidance committee for helpful suggestions, in addition to critical review of the manuscript. pg! major professor, Dr. Marvin Max Hensley, has contributed time and continued encouragement before and during the study. The other committee members: Dr. T. Wayne Porter, Dr. Eugene W. Roelofs, and Dr. Steve N. Stephenson have all offered suggestions and ideas that focused attention on certain key aspects of the study. The entire staff at the Savannah River Ecology Laboratory: research associates, graduate students, secretaries, and technicians all contributed to this work in some capacity, and made the research more challenging and rewarding. I am especially indebted to Kim Fowler (deceased) and John Coker for technical assistance during some parts of the study, to Leah Rutland for typing, to Jean Turner for securing library material, and to Fran Beverly and Bridgette Purvis for managing numerous office matters. Henry Kania and Dr. Robert Beyers offered many helpful suggestions regarding laboratory apparati and techniques. Dr. Becky Sharitz assisted by identifying plants. Many people aided in various aspects of the field work, and I am especially indebted to Joseph Bourque, Linda Briese, John Coker, Kim Fowler, David Meggins, and Charles Bear. Robert Gardner and John Pinder offered helpful suggestions on statistical procedures. In addition to computer privileges, support to scientific meetings and provisions for manuscript preparation, excellent field and laboratory equipment and laboratory facilities were generously provided by the Savannah River Ecology Laboratory. For all of these benefits I am greatly appreciative. I am indebted to the University of Georgia Libraries and Institute of Ecology for library privileges and other services. I also acknowledge the Department of Zoology, Michigan State University and the former secretary, Mrs. Bernadette Henderson, for many efforts on my behalf. ii Special thanks go to the U.S. Atomic Energy Commission for financial support throughout the study. Work was supported by contract AT 38-1-(310) between the Atomic Energy Commission and the University of Georgia while I was a predoctoral fellow supported by the Division of Nuclear Education Training through Oak Ridge Associated Universities (l97l-1972) and the Savannah River Ecology Laboratory (1973). I am grateful to Dr. Ronn G. Altig for help in identifying larval anurans and to Dr. Frederick R. Gehlbach for reviewing a portion of the manuscript. Finally I acknowledge the patient cooperation and assistance of my wife Nancy, without whose encouragement and understanding this thesis would not have been possible. She aided in typing, coding data, and supplying inspiration throughout the study. iii TABLE OF CONTENTS LIST OF TABLES . ......................... LIST OF FIGURES ......................... INTRODUCTION ........................... MATERIALS AND METHODS ...................... Description of the Study Area ................ Field Studies ........................ Pitfall Trapping Survey ................. Reservoir Sampling with Dipnets and Minnow Traps ..... Reservoir Sampling of Larval Bufo terrestris ....... Laboratory Rearing of Larval Rana pipiens .......... Statistical Treatment .................... Field Study: Pitfall Trapping Survey ............ Field Study: Reservoir Sampling with Dipnets and Minnow Traps ........................ Field Study: Reservoir Sampling of Larval Bufo terrestris . . Laboratory Study: Rearing of Larval Rana pipiens ...... DISCUSSION ............................ Field Study: Pitfall Trapping Survey ............ Rana pipiens--Seasonality ................ Bufo terrestris--Seasonality ............... GastrOphryne carolinensis--Seasonality .......... Acris gry]lus--Seasonality ................ Pseudacris triseriata--Seasonality ............ Rana pipjens--Distribution ................ Bufo terrestris--Distribution .............. Gastrophryne carolinensis--Distribution ......... Dominant Anurans-ifiistribution .............. Acris r llus--Distribution ............... Pseudacr s riseriata--Distribution ........... iv Page vi 45 71 75 81 109 114 115 116 Immature Rana ipiens .................. 124 The Gradient E ect ................... 126 Body Lengths of Pitfall-Trapped Bufo terrestris and Rana pipiens on the Savannah River Plant ........ 129 Field Study: Reservoir Sampling with Dipnets and Minnow Traps ........................ 130 Total Lengths of Larval Rana pipiens ........... 133 Field Study: Reservoir Sampling of Larval Bufo terrestris . . 134 Laboratory Study: Rearing of Larval Rana pipiens ...... 140 Accidental Deaths .................... 140 Mortality ........................ l4l Transformees ....................... 142 Missing Data ....................... 143 Growth and Transformation ................ 146 Transformation and Survivorship ............. 152 Percent Survivorship ................... 154 Growth in Body Size ................... 157 Abnormalities ...................... 158 Crooked Spine ...................... 158 Paralyzed Hindlimbs ................... 162 SUMMARY AND CONCLUSIONS ..................... 164 Pitfall Trapping Survey ................... 164 Reservoir Sampling with Dipnets and Minnow Traps ....... 165 Reservoir Sampling of Larval Bufo terrestris ......... 165 Laboratory Rearing of Larval Rana pipiens .......... 166 LITERATURE CITED ......................... 167 Table LIST OF TABLES Mean maximum (upper) and mean minimum (lower) temperatures (n==14) recorded bimonthly in the six thermal zones of Pond C Reservoir and in cool seepage pond A (Fig. 2) ...................... Numbers of amphibians (anurans above and urodeles below) trapped (1971-1972) in pitfalls along the periphery of Pond C Reservoir on the Savannah River Plant in South Carolina ............... Numbers and body lengths (mm) of Rana pipiens trapped in pitfalls along the periphery of Pond C Reservoir (1971-1972) ........................ Numbers and body lengths (mm) of immature Rana pipiens trapped in pitfalls along the periphery of Pond C Reservoir (1971-1972) ................... Numbers and body lengths (mm) of Bufo terrestris trapped in pitfalls along the periphery of POndl C Reservoir (1971-1972) .................. Numbers and body lengths (mm) of Gastrgphryne carolinensis trapped in pitfalls along the periphery of Pond C Reservoir (1971) ................ Numbers and body lengths (mm) of Acris gryllus trapped in pitfalls along the periphery of Pond C Reservoir (1971-1972) ................... Numbers and body lengths (mm) of Pseudacris trisgriata trapped in pitfalls along the periphery of Pond C Reservoir (1971-1972) ................... Numbers and body lengths (mm) of Rana gigiens captured ,in pitfall traps along the periphery 0 on C Reservoir from 26 February 1971 to 31 March 1972 ........... vi Page 46 47 48 50 51 52 53 55 56 Table Page 10. Numbers and body lengths (mm) of Bufo terrestris captured in pitfall traps along the periphery of Pogg C Reservoir from 26 February 1971 to 31 March 7 9 ........................... 5 11. Numbers and body lengths (mm) of Gastrophryne carolinensis captured in pitfall traps along the periphery 0f Pond C Reservoir from 26 February 1971 to 31 March 1972 ..................... 58 12. Numbers of three species of anurans: Rana pipiens (5:21), Bufo terrestris (8,3,) and Gastroghrxne caro inensis (§,c.) trapped in pitfa s a ong t e periphery 6f Pond C Reservoir from 26 February 1971 to 31 March 1972 ..................... 59 13. Numbers and body lengths (mm) of Acris r llus captured in pitfall traps along the periphery of Pond C Reservoir from 26 February 1971 to 31 March 1972 ........................... 60 14. Numbers and body lengths (mm) of Pseudacris triseriata captured in pitfall traps along the periphery of Pond C Reservoir from 26 February 1971 to 31 March 1972 . . . . 61 15. Numbers of immature (body length <47 mm) Rana pipiens initially captured in pitfall traps ............ 62 16. Mean numbers of Rana pipiens caught per pitfall trap area in each of the thermal zones along the periphery of Pond C Reservoir .................... 66 17. Mean numbers of Bufo terrestris caught per pitfall trap area in each of the thermal zones along the periphery of Pond C Reservoir ............... 67 18. Mean numbers of Gastrophyne carolinensis caught per pitfall trap area in each of the thermal zones along the periphery of Pond C Reservoir ............. 67 19. Mean numbers of Acris gryllus and Pseudacris triseriata caught per pitfall trap area in each of the thermal zones along the periphery of Pond C Reservoir ....... 68 20. Rana pipiens trapped yearly in pitfall traps along the periphery 0f three aquatic habitats on the Savannah River Plant in South Carolina ............... 69 vii Table Page 21. Bufo terrestris trapped yearly in pitfalls along the periphery of three aquatic habitats on the Savannah River Plant in South Carolina ............... 70 22. Larval amphibians present (+) or absent (0) in one or more of 15 monthly dipnet samples (June 1971 to September 1972) in the vicinity of Pond C Reservoir (&==eggs deposited but no larvae found) .......... 72 23. Adult Bufo terrestris trapped in pitfalls along Pond C Reservoir ......................... 76 24. Bufo terrestris larvae sampled from different thermal regimes in the vicinity of Pond C Reservoir (n =20 for each sample except for area K where n =10) ........ 77 25. Comparisons of stages of development and total lengths (mm) of larval toads collected in weekly samples from three thermal regimes (n =20) ............... 80 26. Mean total lengths (mm) of larval toads at comparable stages of development, collected from cool and heated areas; (n) =sample size .................. 80 27. Laboratory reared Rana pipiens; lOO embryos (stage 11 or 18) originally placed’in each aquarium ......... 82 28. Comparison of larval mortality and transformation at three thermal regimes (excluding specimens not accounted for) ...................... 83 29. Body lengths (mm) of 683 transforming Rana pipiens reared at three different temperaturesTih the laboratory ........................ 83 30. Comparisons of body lengths of Rana pipiens reared in the laboratory at three different temperatures ..... 87 31. Body lengths (mm) of 683 transforming Rana pipiens at three different temperatures in the lanratory (1971-1972) ........................ 88 32. Time required for laboratory reared larval R. pipiens to reach comparable levels of survivorship (transformees + "natural“ mortality) at different thermal regimes . . . . 93 viii Table 33. £311. 2355. 23(5. Body lengths (mm) of transforming Rana pipiens reared in the laboratory at three different temperatures. Larvae are compared by stages of development (Gosner, 1960) ...................... Occurrence of developmental abnormalities in larval Rana pipiens laboratory reared at three different thermaTTregimes ...................... Body lengths of 683 transforming Rana pipiens reared in the laboratory at three temperatures. Larvae with normal spines and crooked spines are compared ....... Laboratory reared Rana pipiens with normal and crooked spines. Body lengths (mm) of transforming larvae reared at three thermal regimes are compared by stages of development ................... ix Page 99 104 107 108 I" M- —-__..——-._..-—_-' Figure omvmmbum 1 C). '1 1 . 12. 13. LIST OF FIGURES Pond C Reservoir on the Savannah River Plant, South Carolina ..................... The cool arm of Pond C Reservoir ............ End view of pitfalls .................. Drift fence ...................... Cool seepage pond A .................. Cool seepage pond I .................. Reservoir shoreline V ................. Reservoir shoreline K ................. Sites at the reservoir from which larval populations of Bufo terrestris were sampled ............ Experimental apparatus employed to rear embryonic Rana pipiens through metamorphosis in the laboratory . . View of larval Rana pipiens kept at 20 C ........ Pitfall-drift fence section Y (Fig. 2) ......... Total lengths of larval R, pipiens collected from three reservoir sites receiv ng ifferential levels of thermal loading ................... Mean stage of development (Limbaugh and Volpe, 1957) of larval B, terrestris collected in three weekly samples from three areas along Pond C Reservoir Body lengths of laboratory reared larval R, pipiens transforming at different thermal regimes; 400 embryos (stage 11 or 18) were originally introduced at each temperature ...................... Page 10 12 16 18 21 23 25 27 31 35 38 65 74 79 85 Figure 16. 17. 18. 19. 20. 21. 22. Biweekly survivorship rates, excluding mortality, of laboratory reared larval R. pipiens at three different thermal regimes .................... Biweekly survivorship, including mortality, of laboratory reared larval R. pipiens at three different thermal regimes .................... Percent survivorship of Rana pipiens laboratory reared at three different thermhl regimes .......... Mean body lengths (-+2 SE) of laboratory reared R. pipiens at three different thermal regimes ....... Larvae (reared at 30 C) demonstrating abnormally developed "crooked spines." Some specimens are upside-down due to helical swimming patterns ...... Larval R. 1 1ens demonstrating edemaceous and emaciated Eogy condition. Specimens demonstrating these anomalous body conditions were most common in the cohort reared at 20 C ............... Mean body lengths (-+2 SE) of laboratory reared larval R. pipiens having normal or crooked spines at three different thermal regimes ........... xi Page 90 92 95 98 101 106 INTRODUCTION National power demands have resulted in increased amounts of heated effluents discharged into natural habitats. The numbers of power plants constructed in this country to meet anticipated electrical demands increases each year (Mihursky and Kennedy, 1967; Summers, 1971). As supplies of fossil fuel decline, nuclear reactors assume more prom- inent roles of satisfying these demands (Clark, 1969; Levin gt.gl,, 1972). The dearth of knowledge on the impact of thermal loading (pollution) as a stress imposed upon the natural flora and fauna presents an immediate practical problem in sound resource and waste management (Cairns, 1972; Mihursky, 1969; Nelkin, 1971). Literature dealing with the effects of thermal pollution has been increasing in recent years (Coutant, 1968, 1969, 1970, 1971; Kennedy and Mihursky, 1967; Raney and Menzel, 1969). Symposia conducted to elucidate some: of the major issues (Gibbons and Sharitz, 1974; Krenkel and Parker, 1969), have revealed that little is known about interpreting and pre- dicting responses of stressed populations, communities and ecosystems to thermal loading (Levin gt_al,, 1970). To understand the impact of thermal loading, specific studies are needed to delineate responses of component populations in each affected ecosystem. General concepts based on field and laboratory studies are needed to provide a foundation for maintaining stable community dynamics in such environments. The aquatic breeding habits of most amphibians subject them to thermal changes in both aquatic and terrestrial ecosystems. Their widespread occurrence in both environments makes amphibians useful indicators of thermal stress. Studies of temperature adaptation have been conducted primarily at the subcellular, tissue, and organismal levels (Das and Prosser, 1967; Packard, 1972; Prosser, 1958, 1967; Rose, 1967; Troshin, 1967; Ushakov, 1972; Whittow, 1970). Many workers have described differential sensitivities among tissues, organs and systems, as they are affected by temperature. Thus, studies have been oriented mainly towards biochem- ical, physiological interpretations. Modern terminology and concepts concerning temperature adaptation were presented by Precht (1958) and Prosser (1958), and will not be discussed here. Ecological and physio- logical considerations are reviewed by Prosser, 1973. Other workers (§_g, Brett, 1956; Davenport and Castle, 1895) have reviewed the lit- erature describing rate responses of aquatic forms to temperature. More recent studies delineated maximum thermal tolerances of certain aquatic forms (Bachmann, 1969; Brock, 1970; Moore, 1939; Muto, 1972; Tarzwell, 1970; and Volpe, 1953, 1957a). Some studies involve the determinations of critical thermal maxima (CTM), interpretations of these values, and problems inherent in synergistic agents (Dunlap, 1968; Hutchison, 1961; Seibel, 1970). Reviewing the literature on thermal acclimation of organisms to high temperature, Davenport and Castle (1895) concluded that acclimati- zation to higher temperatures occurred across the plant and animal ' M ‘u up. "I [“1 \ . a?! J '4‘. kingdoms (in organisms subjected to increased temperature) by loss of water from the protoplasm. They recorded heat rigor in the toad Bufo legginosus at 40 C to 44 C. Theories about the physiological mechan- isms responsible for heat death (Fry, 1967; Heilbrunn, 1956; Hoar, 1966; and Vernberg and Vernberg, 1970) are not discussed in this study. Lillie and Knowlton (1897) presented data of others and their own to demonstrate that growth responses of organisms have Optimal levels, and that subminimal and supramaximal temperatures may severely inhibit growth from this optimum. Arrhenius and Van't Hoff devised mathematical formulae to describe the 010 relationship between temperature and chem- ical reactions (Belehradek, 1935). Krogh (1914) showed that temperature influenced embryonic development of a fish, frog, water beetle, and sea urchin in proportion to the temperature increment. Crozier (1926) using a variety of plants and animals, Atlas (1935), Bachmann (1969), Grainger (1959), Hoadley (1938), and Ryan (1941) described growth or developmental phenomena in relation to temperature. Water temperature is generally regarded as the most critical environmental factor to which anurans are exposed during breeding and embryonic development. Development of anuran eggs and larvae have been shown to be directly related to water temperatures (Herreid and Kinney, 1967; Moore, 1939, 1942a; Zweifel, 1968). Thermal tolerances and devel- opmental rates of anuran eggs, embryos and larvae have been correlated with breeding habitat, season, and geographical distribution (Ballinger and McKinney, 1966; Licht, 1971; Moore, 1939, 1949; Ruibal, 1957: Volpe, 1953, 1957a). The effects of thermally stressed aquatic environments on normal growth and developmental processes are difficult to evaluate. Laboratory studies demonstrating thermoregulation in larval amphibians (reviewed by deVlaming and Bury, 1970) are often difficult to relate to the more complex natural conditions. Moreover, field data on thermo- regulatory behavior of larval amphibians are scanty (Brattstrom, 1962, 1963). Common problems arise from attempts to formulate generalities which allow comparisons of organisms, populations and ecosystems. Because of its complexity, workers have approached the problem of thermal stress from various perspectives. The conclusions are often confounded by factors such as synergism, acclimation, and variable thermal responses among different age groups. Although some field studies conducted revealed negligible effects of heated effluent (Alabaster, 1964; Merriman, 1970), others (Jones, 1964; Trembley, 1960) substantiate serious damage. Additional studies are needed (Levin gt gl,, 1970) in different habitats in different regions of the country. In the present study, laboratory and field studies were designed to characterize population responses of larval anurans (frogs and toads). To determine how thermally stressed aquatic environments evoke popula- tion responses in field situations, adult and larval amphibians were sampled along the periphery of a reservoir receiving lethal levels of heated effluent from a nuclear production reactor. Cool shoreline seepage in some parts of the reservoir created isolated microenviron- ments marginally habitable to amphibian larvae. Responses to stress were characterized in terms of larval survivorship, growth and development. To compare the success of amphibian populations occurring in the vicinity of the heated reservoir and determine the range of thermal conditions that they can tolerate, the level of thermal loading was related to several variables. The numbers of species occurring around the reservoir, and those breeding, developing, and emerging were related to thermal levels in the different areas. Another goal was to determine the extent to which an aquatic ecosystem can be thermally stressed before it is rendered uninhabitable for naturally occurring amphibians. A basis was sought for predicting the maximum thermal limits that can be safely imposed on the Southeast- ern reservoir studied, yet allow amphibian development. Hazards (g_g, thermal stress, and modifications in food availability, cover and preda- tion) related to the direct thermal influence were also of interest. Prominent population responses should be demonstrable both in the field and laboratory. Consequently, in addition to field studies conducted in the thermally stressed reservoir habitats, larval Rgga_ pipiens Schreber were reared in the laboratory at four constant temper- ature regimes: 20 C, 25 C, 30 C and 35 C. Survivorship, growth, and developmental rates of laboratory reared larvae were compared among the thermal regimes and the laboratory findings compared to those observed in the field. To achieve these goals, four distinct, yet related, programs were pursued (three in the field and one in the laboratory): Post-larval anurans were captured using a drift fence-pitfall trap method throughout the year to monitor seasonal occurrence and movements (localized activity and migrations). The pitfall trapping study was designed to indicate which anurans occur in the vicinity, and thus provide a listing of potential reservoir breeders. The occurrence of recently metamorphosed emergent transformees would also provide evidence of which species successfully completed larval development in the reservoir. Due to placement of pitfalls peripheral to the thermal gradient of the reservoir, both adult breeding migration to and emergence of transformed young from the reservoir would become evident. Seasonal activity and movement patterns were to be related to the thermal gradient and to the location of associated cool seepage ponds flanking the reservoir. After establishing which amphibians were active around the reservoir, a sampling program was undertaken to document which (if any) anurans deposited eggs, and survived through embryonic and larval development there. Samples were systematically removed monthly from both heated and cool areas in the vicinity ' of the reservoir by dipnet. Minnow traps were also maintained in cool and heated aquatic environments for 11 months. Data obtained from these studies were to provide evidence for the presence or absence of larval anurans in different thermal regimes along the thermal gradient. Abundance and species diversity of larval amphibian populations were to be compared among cooled shallows of the heated reservoir and peripheral stream seepage ponds. In the third field study, larvae of the Southern toad (Bufo terrestris Bonnaterre) develOping from eggs naturally deposited in the reservoir were sampled from cool and heated areas. Growth and developmental responses of a single breeding migra- tion of B, terrestris in March 1972, were then compared among several subpopulations in aquatic habitats receiving variable levels of thermal loading. Breeding activity in the thermal waters provided an excellent opportunity to compare growth and developmental rates among larvae at different temperature regimes in a non-laboratory situation. An experimental rearing of Rana pipiens was conducted in the laboratory. Embryos from the field were maintained at four constant temperatures to allow for a comparison of growth and development at different thermal regimes. Manifestations of thermal stress evident among larvae sampled in the reservoir were compared with those observed under laboratory conditions. .1"- if» '1: “‘I MATERIALS AND METHODS Description of the Study Area All field studies were conducted along the periphery of a 67 ha reservoir ("Pond C") formed in 1958 to receive heated effluent from a nucflear production reactor on the Savannah River Plant near Aiken, South Carwalina. Temperatures of the reservoir are classified as "Secret" infkarmation and only those relevant to the ecological studies are reported. Heated effluent is carried to the reservoir through a thermal canal. After entering the reservoir, thermal effluent passes down the main channel (Fig. l) and thereafter into another reservoir: Par Pond. Since the current does not flow directly into the cool arm of Pond C Reservoir, temperatures there consist of a thermal gradient which was diilided into six thermal zones for the present study (Fig. 2). At the "Knxth of the cool arm (zone VI), water temperatures may approach 50 C and exceed the thermal tolerance of most organisms with the exception of thermophilic bacteria and bluegreen algae. Temperatures are considerably I‘Educed at the opposite extreme of the thermal gradient (zone I, Fig. 2). I"current stream flow there allows water temperatures at times to aPproach levels typical for this region of the country. Most common anurans can be collected or heard calling there in season. Fig. l. Pond C Reservoir on the Savannah River Plant, South Carolina. Letters represent locations of drift fence-pitfall trapping areas along the periphery of the reservoir's cool arm. Arrows indicate the direction that heated effluents flow from the thermal canal through the reservoir. C D E w F Pond C GH reservoir X I J r--1--fi Y K o 200 L rneters Z .M S“: / \\\ thermal effluent Figure l 11 Fig. 2. The cool arm of Pond C Reservoir. Pitfall trapping areas are grouped into thermal zones along the thermal gradient. Temperatures represent the mean minimum and mean maximum (N==15) for March 1972. Seasonal temperature data for the six zones are given in Table 1. Seepage ponds are apparent near sites A, B, I, J, and Z. 12 A .................................. - 22—31° u '3 I .......................................... <9. 27-34° V D II ..................................... E.......... 28-35° F In ooooooooooooooooooooooooo G 'x ......... ii ............... 28-36° I. IV 29-36° Y J V ................................... K 30-37° 2‘ L VI ............................ M Figure 2 13 The reservoir was constructed over a natural creek bed. The water table along most of the reservoir, therefore, is very close to the surface of the ground. Stream seepage is evident in low-1 ying areas throughout the length of the cool arm. Several cool seepage ponds occur contiguous to the reservoir (Fig. 2). All five ponds (except the one near site Z) maintain readily apparent and steady flow of the under- ground water seepage into the reservoir, and may on occasion contact the reservoir prOper. The terrestrial habitats surrounding the reser- voir are characterized by mixed hardwood and pine of the "Oak-Pine Forest Region" (Braun, 1964); the soil type is predominately sand. Field Studies ELtfall Trappim Survey Beginning at the terminal pool (upstream end) of the cool arm 0? the reservoir, pitfall-drift fence sections were laid at regular 'UTtervals on both sides (Fig. l). Thirteen sections were placed along the eastern periphery of the reservoir at intervals of 100 m. On the west side, six additional pitfall-drift fence sections were spaced at lntervals of 200 m. Thus, traps were distributed throughout the length 9f the thermal gradient. Habitats ranged from low, marshy areas to hlgrn xeric ones and from open grasslands to dense woods. Traps were situated as close to the water as practical (2-5 m), 31though a high water table or elevated terrain in some areas necessi- tated placement of cans farther away from shore. The traps sampled more than 2.2 km of shoreline along the thermal gradient. Each trap consisted 14 of four pitfalls (cans 42 cm deep and 35 cm in diameter), and 15.2 m of aluminum flashing (51 cm high). Flashing was buried 4-5 cm below the soil level and fastened with wire to 1.3 m metal stakes driven into the ground (Fig. 3). A 10 m section of drift fencing was laid in a straight line with a bucket (pitfall) buried at each end of both sides, flush with the soil surface. Holes were punched in the bottom of pit- fall cans to allow for drainage of rain. A 1.3 m section of flashing extended beyond each bucket on both sides, at each end of the drift fence at 60° angles (Fig. 4). Amphibians, reptiles and other small animals encountered the drift fence and followed it to the pitfalls at either end. Frequency of field visits during the year varied with the catch. In the spring, traps were usually checked daily: in the sumner, every 2-3 days; and in the winter, every 3-4 days. Travel between traps was by boat, as most drift fences were not readily accessible by road. Amphibians were removed from cans and the following data recorded: Species, snout-vent length, sex, and whether the individual wasnew or a recapture. Each specimen was toe clipped and released outside the drift fence lateral to the pitfall from which it was removed. The OUtside toe on the right forelimb was removed to allow discrimination 0f initial captures and subsequent recaptures. Throughout the duration 01’ the study, maximum-minimum thermomenters were maintained along the Shoreline of the thermal gradient. Data were recorded every 2-3 days. 15 Fig. 3. End view of pitfalls. Two cans were buried at each end of the drift fence. 16 Figure 3 17 Fig. 4. Drift fence. Organisms encountering the fence follow it and move towards the terminally placed pitfalls. Figure 4 ’fi uni or.“ flu To his 19 Reservoir Sampling with Dipnets and Minnow‘Traps Since the presence of larvae would confirm successful breeding and development, several areas were selected for a monthly sampling program: cool seepage ponds A, B, I, Z and heated reservoir shorelines V and K (Figs. 2, 5, 6, 7, and 8). During the latter part (25-30 days) in each month (June 1971 through July 1972), the sites selected were routinely sampled using two dipnets: a small mesh Turtox Bueno model and a large 6 mm mesh nylon net. Sampling periods at each site lasted for a minimum of 20 minutes and continued thereafter until no new species were encountered. Specimens were immediately placed in 150 ml of 10% formalin. To insure proper preservation of specimens, these solutions were changed later the same day and one week thereafter. Larvae were brought into the laboratory for identification, using Altig's (1970) key, measurement (total length to nearest 0.2 mm), and staging according to the level of development (Gosner, 1960). Two locations in the reservoir were regularly sampled: near pitfall sites V and K (Figs. 7 and 8). Both received cool stream seep- age that usually reduced the water temperature in the shallows 10-20 C below those of nearby shores. The gradually sloping shoreline at site V contained shallows ranging from a few millimeters to 0.3 m. It con- tained emergent vegetation (mostly Scirpus americanus) for a distance of approximately 40 m. Site K, approximately 600 m closer to the hotter end of the thermal gradient, was the warmest area sampled routinely. Emergent vegetation there was restricted to a small stand of cattail (Iypha latifolia). Other reservoir areas receiving seepage were sampled periodically. 20 Fig. 5. Cool seepage pond A. This cool pond is located near pitfall trapping area A (Fig. 2). The cool arm of the heated reservoir is shown in the background. 21 22 Fig. 6. Cool seepage pond I. This cool pond is located near pit- fall trapping area I (Fig. 2). Shoreline seepage is evident in the foreground and drainage into the reservoir can be seen in the background. 23 Figure 6 24 Fig. 7. Reservoir shoreline V. The shallow shoreline of Pond C Reservoir is near pitfall trapping area V (Fig. 2). Cool seepage here reduces shoreline temperatures to levels habitable to larval amphibians. 25 . I I ‘ _ 'fl’zl“o"fl'.f‘ - a ‘ . . (T.‘/ ‘t ’ s i. Figure 7 26 Fig. 8. Reservoir shore K. This reservoir site (near pitfall trapping area K) was the hottest area sampled routinely for larval amphibians. Shoreline seepage, evident in the foreground, reduces water temperatures to allow for marginal survival of anuran larvae there. Both 3. pipiens and B, terrestris bred there; both incurred high mortality. (A minnow trap is present in the water just behind the log.) 27 3.3.1, ...... 1.... . .u. -... .. 4 L .r. .. Marti . ”pagwflcsw at} :6. 9. M. ...,..... 2.31%.? "RAJ/v ...l. v.1 . Y.“ ’..‘ . ~. .~O. - '\.Ou" I\' . ”£576.... 1 . .. . ,. 2. r.., .1 1cm... . . a . L111 10 Figure 8 28 The seepage ponds chosen for routine sampling were located near pitfall sites A, B, I, and 2 (Fig. 2). Additional sites were sampled periodically for comparison. Pond designations represent the names of pitfall trap sites to which they are nearest. The association of seep- age ponds with the reservoir ranged from a continuously flowing stream connection (8) to total separation (Z). Pond I was connected to the reservoir at high water levels only (mostly spring and winter), and Pond A was rarely ever continuous with the reservoir. To supplement the monthly sampling program, three to four minnow traps were maintained in each of the six locations mentioned (from 3 September 1971 to 2 August 1972). Other areas were also sampled with minnow traps from time to time. During each field visit to the pitfalls, the minnow traps were checked also. The specimens caught were removed from traps and recorded by species. Anuran larvae were staged and measured in the field, or preserved and taken to the laboratory for staging and measuring. Reservoir Samplinggof Larval Bufo terrestris Large numbers of larval Bufo terrestris were encountered on 29 March 1972, in cool seepage ponds flanking Pond C Reservoir and along the shallow shoreline in the cooler parts of the reservoir proper. Swarming masses of larvae were numerous in cooler reservoir shallows (3-10 cm deep) within 0.5 m of the shoreline, venturing into deeper, warmer water only if provoked from the shore. Larval densities were noticeably reduced or absent in shoreline areas with little or no seepage, sparse vegetation, and thus, high temperatures. 29 On 29 March, samples were taken from several locations in the heated reservoir and from cool, peripheral seepage ponds to compare growth rates and develOpment of larvae from different thermal regimes. During the following three weeks, successive samples were removed weekly from sites where p0pulation densities were sufficient. Sampled areas were separated by thermal barriers of open shore without seepage or vegetation. Increased temperatures (5-10 C above ambient) probably precluded movement along most of the shoreline, thus maintaining the integrity of sampled "populations.” Each sample of larval B, terrestris (30 specimens, if available) was removed with a fine mesh dip net and preserved in the field. Sam- ples were taken to the laboratory where they were identified (Altig, 1970), measured (total lengths), and staged (Gosner, 1960) according to the level of development. In an initial analysis, ten larvae were examined from each sample. Since these data indicated trends in growth and development, 20 additional specimens were examined from areas where sample sizes permitted. For the supplemental analyses, the staging system illustrated by Limbaugh and Volpe (1957) was adopted. The thermal ranges (given in the text) represent the daily mean minimum and daily mean maximum temperatures recorded at each study site during the four week period of study. Sufficient numbers of larvae were available for three consecu- tive samples at sampling areas 8, A and V (Fig. 9). Area B (a cool pond, peripheral to the reservoir) receives a constant flow of underground stream seepage and is continuous with the reservoir only at high water 30 Fig. 9. Sites at the reservoir from which larval populations of Bufo terrestris were sampled. Areas A, D, V, and K were along the shore of the heated reservoir; area B was a nearby cool seepage pond. Temperatures represent mean maximum recordings over periods of two to three days (n =15) during March 1972. 31 A — (32.9 0) '\~ B — (24.4 C) (34,4 C)—V D-(34.9 C) ’Pond C reservoir K— (35.7 0) fl 0 200 meters sis rr’ \T‘ 4 thermal effluent \ Figure 9 32 levels. Temperatures there ranged from 8 to 24 C. The pond (B) was completely separated during the four weeks of the sampling period; thus, it received no thermal effluent. Area A was located at the cooler end of the thermal gradient in the reservoir proper where temperatures ranged from 21 to 33 C. Compared to area V, area A received "moderate" thermal loading. Area V (Fig. 7) was the warmest area (27-34 C) from which successive samples were available and was 350 m closer to the thermal inflow than thermally intermediate area A. Extensive cool shallows in the reservoir at area V served as thermal refuge areas for certain aquatic forms (invertebrates, fish and amphibians) not found in the reservoir proper. The shore at area V was very gradually sloped, with a pool (2.2 m in diameter and 0.5 to 1.0 m deep) that always contained water. When reactor flow was temporarily interrupted, the reservoir water level fell 0.3 to 1.3 m, completely exposing 1-8 m of the pre- viously submerged shoreline. Then only the pool area remained, surrounded by dry land. Although successive samples were unavailable, larvae were removed from heated areas 0 (28-35 C) and K (29-36 C; Fig. 9). Area 0 received little seepage, thus larvae did not have access to cool shal- lows as in area V. Area K (Fig. 8) was the hottest site from which larval amphibians were ever collected. It received cool seepage, and was partially protected from lethal reservoir temperatures by a shallow sand bank between the pool and the reservoir. 33 Larval condition (length and stage of development) can be considered as a function of age and temperature. If larvae were the same age (representing a single breeding migration), any significant difference in growth and development could be attributed to temperature. Two major questions were of concern regarding growth and development. First, do they vary as a function of thermal regime? To test whether developmental stages and lengths were the same at three thermal regimes (areas A, B and V), specimens were compared weekly with the Kruskal- Wallis nonparametric analysis of variance (Sokal and Rohlf, 1969). The null hypothesis was that the three populations were at comparable stages of development, and lengths. The second question was whether lengths at a given developmental stage varied among the different thermal regimes. Lengths at comparable stages of development were pooled among sampling areas and tested with the Kruskal-Wallis nonparametric analysis of variance. Laboratory Rearjgg of Larval Rana pipiens Sixteen 20 gallon (84.6 1) aquaria, placed on metal stands, were employed: four replicate aquaria in series at each temperature (Fig. 10). Each tank was originally filled with 84.6 1 of water from Par Pond on the Savannah River Plant. Evaporation was compensated by the addition of (glass) distilled water back to the original level. To reduce tempera- ture fluctuations within the aquaria, the four sides of each were covered with fiberglas insulation. Covers made of 1.2 cm plexiglas rested flush on the top of each tank. The insulation was found to 34 Fig. 10. Experimental apparatus employed to rear embryonic Rana pipiens through metamorphosis in the laboratory. Four replicate aquaria were maintained at each of the four temperatures: 20 C, 25 C, 30 C, and 35 C. 35 36 reduce temperature fluctuations appreciably at the warmer thermal regimes, and the plexiglas covers considerably reduced evaporation and stabilized temperatures in the aquaria. At the beginning of the study, builders sand was soaked and rinsed for 24 hours in running water, then placed to cover the lower 2 cm of each aquarium bottom. On 16 December 1971, the sand in each tank of the remaining thermal regimes was removed and replaced with gravel and undergravel filters weighted down by two large rocks (Fig. 11). Rocks were later replaced with small culture dishes. Water was continuously circulated among the four replicate aquaria at each temperature. Air lift pumps were employed to move water from the bottom of one tank to the surface of the tank next in the series (Fig. 10). Excelon (polyurethane) tubing (1.9 cm in diameter), 18 ga. syringe needles, and bent sections of 8 mm (0.0.) pyrex glass tubing were used. Air pressure was supplied by a 3/4 H.P., two cycle oil- less Bell & Gossett air compressor, set to deliver at a pressure of approximately 5 psi. To maximize efficiency of air lift pumps, the water column was lifted from the floor (95-98 cm). This system was found to circulate water at an average rate of 0.6 l/minute. Inter- connecting, inverted U tubes of 13 mm (0.0.) pyrex glass tubing were also placed as siphons among the four aquaria in each series. These prevented water overflow created by differential transfer rates of water by the four different air lift pumps. 37 Fig. 11. View of larval Rana pipiens kept at 20 C. Rocks weighing down undergravel filters were later replaced with glass culture dishes. Considerable size differences among larvae indicated highly variable growth rates at 20 C. 38 ill : I)HAII* I I.: +11”: " " 11‘ (:21. ' {“1 Figure 11 39 To assess effects of continual thermal loading on larval growth and development, embryonic Rana pipiens were reared at different thermal regimes. 0n 2 September 1971, two egg masses were collected among dense shoreline vegetation of Dick's Pond, a 0.97 ha farm pond on the Savannah River Plant, 6.6 km from Pond C Reservoir. The embryos were carefully separated in the laboratory, staged, divided into groups of 100, and placed in culture dishes half-filled with pond water at 18 C. Each group was counted twice: only normal embryos were included. Only nine dead or distorted embryos were found among the approximately 2,000 eggs examined. A group of 100 embryos was introduced into each of the four series of aquaria at each constant temperature regime (20 C, 25 C, 30 C, 35 C). Groups of the more advanced cohort (stage 18: "muscular re- sponse," Gosner, 1960) were placed in the last two tanks (C and D) at each temperature. The less advanced group (stage 11: "mid-gastrula") was placed in the first two tanks (A and B). To prevent movement of embryos by circulating water currents, the eggs were temporarily placed inside 26 ga., stainless steel screen cylinders (approximately 21 cm in diameter), placed upright in the center of each tank. Embryos remained inside the screen cylinders from five to seven days, until reaching the first larval, feeding stage (26). At that point the screen cylinders were removed and larvae became dis-_ tributed throughout the aquaria. Barriers of stainless steel screen were placed around the heaters and interconnecting siphons to reduce experimental mortality of larvae. To keep larval amphibians from being transferred from one aquarium to the next by the air lift pumps, or 40 interconnecting U tube siphons, the entrances to both were surrounded by cylindrical siphon covers. Each of these cylinders was made from 36 ga. stainless steel screen, and measured 4-6 cm in diameter. Fiberglas insect screening was used to bind the bottom of the siphon cover. It was attached to the stainless steel with nichrome wire. Water was continuously circulated through outside filters, driven by air lift pumps. Originally four liter jars containing charcoal and glass wool were used. As the growing larvae began to eat more, and deposit greater amounts of fecal debris (on 16 December 1971), more efficient metaframe outside filters were adopted. At the same time, undergravel filters were also installed. The sand was removed from each aquarium, and gravel was placed over the undergravel filters. First rocks, then later culture dishes were placed over each side of the undergravel filters to prevent their rising. Since tadpoles demonstrate a preference for "close quarters," they tended to collect in corners along the air stands of undergravel filters, behind siphon covers, at the end of the aquarium, and under leaves of spinach. For this reason, precautions had to be taken to prevent larvae from working their way below the undergravel filters. Mortality of several larvae resulted from such an occurrence. They died below the undergravel filters, apparently unable to get out. Constant temperatures were maintained by "Jumo" (Preiser) mercury thermoregulators, relay boxes (Precision Temperature Regulator, Eastern Industries), and heaters prepared in the laboratory (Fig. 10). The relay boxes automatically turned on heaters as the temperature fell 41 below that for which the thermoregulator had been set. They automatically switched the heaters off when the thermoregulator was restored to the temperature for which it was set. Temperature fluctuations rarely exceeded 1 C in 24 hours. Since each temperature series was supplied with a single thermoregulator, any difference in heaters would perpetuate a warmer or cooler temperature in certain tanks. Whereas an identical heater was required for each aquarium to reduce temperature variation, they were constructed from glass tubing, nickel-chromium wire, sand, and insulated wire, and connected in parallel. An efficient heater, in order to maintain temperature constancy, should at best be working half of the time for maximum efficiency. This heating rate reduces the time lags that result from on-off switched systems and the accompanying thermal fluctuation. In order to increase the heating surface of the heaters, 13 mm (0.0.) pyrex glass tubing was bent into a U-shape, with dimensions (28 cm wide x 33 cm high) appro- priate to fit into the end of each aquarium. Twenty feet (6.1 m) of nichrome wire was coiled and placed into the lower 17.8 cm of the U, and strained sand placed around the heating coil as insulation. Water circulated from one aquarium to the next; it was withdrawn from the bottom of the cool and and deposited at the surface of the end in which the heater was positioned. This system of circulation (in addition to the water agitation created by undergravel filters) prevented thermal stratification within tanks. 42 To insure that the room temperature did not exceed the minimal temperature regime of 20 C, an air conditioner was constantly run and generally maintained a temperature of 17 to 19 C. A cool room tempera- ture obviated the need for other mechanisms to reduce temperatures. Temperatures were checked daily at 0900 and 2100 hours for the first two weeks; observations of embryonic conditions and counts were recorded each time. Thereafter, temperatures and accounts of larvae in each tank were recorded once at each daily check. The upper and lower diel tem- perature fluctuations were recorded for each temperature treatment by maximum-minimum thermometers. They provide a profile of minor fluctua- tions. Except for two major power failures, totalling less than 29 hours, daily temperatures did not fluctuate more than :1 C. In order to provide a unifbrm source of lighting, the windows in the laboratory were covered with aluminum foil. All illumination came from the eight fluorescent ceiling lamps. A uniform periodicity, a 12 hour light-12 hour dark diel cycle, was employed throughout the study with the aid of an electric timing switch. The plexiglas covers on aquaria allowed uniform illumination to each. Frozen spinach was fed ad libitum. For the first 10 months of the experiment, freshly thawed spinach was added daily. Thereafter (when a few larvae remained at 20 C only) spinach was added every other day. Decomposition of spinach at 20 C was very slow. Food was not allowed to remain inside an aquarium for more than two days. At each feeding, old spinach was removed with a hand net. Any spinach or fecal debris was thoroughly removed with fine mesh nets, and the new spinach 43 placed on top of the undergravel filters, covered with pieces of gravel to keep it weighted down. Daily procedures involved recording of temperatures, feeding, cleaning, measuring and staging specimens, and recording data on dead or transfbrming larvae. Charcoal and glass wool in outside filters were changed one to three times a week as necessary. At each checking, temperatures at the bottom, cool end were recorded for each tank. Data provided by a maximum-minimum thermometer in each thermal regime pro- vided a constant record of temperature variation. Dead specimens were recorded, measured, staged and preserved when discovered. Any abnormalities were noted. All larvae removed were preserved in a 7.6 X 2.5 cm vial in 10% formalin and stored for a double checking of data. The formalin was replaced within two weeks with a fresh solution to assure proper preservation. Statistical Treatment Certain statistical conventions are consistently employed in the text, figures and tables. The total sample sizes are represented by "N"; the numbers of observations in subsamples are represented by "n.” The standard error of the mean (SE) is the measure of variance most commonly employed although the range is occasionally given. The designation "CV" represents the coefficient of variation. The original experimental design called for a comparison of larval growth and development as a function of the major covarients temperature (thermal regime) and age. The same analysis was chosen 44 for larval R, pipiens reared in the laboratory and for larval B, terrestris sampled weekly from the reservoir. In preliminary exam- ination, data sets were analyzed for variance homogeneity and normality (Bartletts' test and Kolmogrof-Smirnoff test: after Sokal and Rohlf (1969). All data sets were heteroscedastic--uncorrected by transfor- mations (log, square root, square, and reciprocal). About half of the data sets were normally distributed. Since the assumptions of par- ametric statistics could not be satisfied, data were compared with the Kruskal-Wallis nonparametric analysis of variance and chi square tests. RESULTS Field Study: Pitfall Trapping Survey A bimonthly thermal profile of mean minimum and mean maximum temperatures was recorded in arbitrarily assigned zones along the cool arm of the reservoir, and attests to the seasonal perpetuation of a thermal gradient there (Table 1). Nine species of urodeles (salaman- ders) and 13 species of anurans (frogs and toads) were collected in pitfall traps over the 13 month period (Table 2). The purpose of the study was to focus on the major amphibian populations breeding in the reservoir, hence further consideration will be limited to the five dominant anuran species for which sample sizes were larger. The numbers of salamanders trapped were low in comparison to frogs and toads. Of 1,784 Rana pipiens trapped, 301 (17%) were recaptured (Table 3). The majority of specimens (63%) were caught from May through July. To compare size classes, the ranges of body lengths are presented for monthly total captures. Adult specimens exceed 49 mm and emergent young range from 20 to 33 mm (Wright and Wright, 1949). Although adults were found to be active throughout the year, emergent transformees were trapped every month but February, 1972. The low mean body lengths of initially captured specimens from March to July (smaller than those for total captures or recaptures), 45 46 Table 1. Mean maximum (upper) and mean minimum (lower) temperatures (n==14) recorded bimonthly in the six thermal zones of Pond C Reservoir and in cool seepage pond A (Fig. 2). The zones represent the thermal gradient that occurs in the cool arm of Pond C Reservoir on the Savannah River Plant in South Carolina Cool Thermal Zone Pond A I II III IV V VI February 16.2 27.8 32.3 32.7 33.4 34.1 35.8 5.8 17.3 25.2 26.1 26.4 27.1 28.5 April 29.0 34.1 38.0 37.9 38.9 39.6 41.1 12.4 26.8 31.0 32.9 32.2 33.1 35.0 June 33.3 39.7 42.6 43.0 43.3 44.4 45.2 21.3 33.1 35.8 38.0 37.9 39.1 40.1 August 38.9 37.7 40.6 40.5 40.9 41.5 42.3 24.3 33.7 34.6 36.3 36.3 37.4 38.1 October 23.5 35.3 39.8 40.4 41.0 42.1 43.2 17.7 29.4 34.4 36.3 36.3 37.5 38.7 December 18.8 29.4 32.5 33.0 33.7 34.0 36.2 9.2 20.8 25.0 27.7 28.1 27.5 30.3 47 .xgmzenmm NNuNN Noe NFco anaFFm>m mecca .copFeuouzmmm use .moozuzm .Nmozgzu .mazumcmoEmmo «esopmxns< .cowwxpovammo .sz3 cm: .m35Fmgusnouoz .cocozmem "gouge cF agocmm mFouoLN «an»: .msaoFcnwom .mFLomuzmma .mFeo< .m:NNSNOmemN .onN .mcmm ”mgocmm cmgzc n2 NeF e N N NF N N o N N Nm NN NN o F mamocFaaflm .a NNN.N Nos em om NN NN NNN Noe FNN Nan Neo.F .wmm .NMN .hww .mm: .1 F o o o o o .o o o F o o o o l o nm N F o o o o o F o o o N o o F mm NF o o o o F o e N N o N F o o mm «N o o N N F N o F e N F o F o «emcee mm Ne o o o o o o o o NF mm o o o o mFmFomem mm eN e o N N N N N N N N o o o o mcmstmFo mm em F o N N F o e FF NF FN F F o o Fxoosnro; mm NNF F o F o F N NF NN N N oN e o o mchonmmFmo um NNF N NF mm N F N N e N Ne mm NF N N mumFemegp up NNN NF e N NF NN Ne Nu NN NF F o NF NF o szFxgm MK NNN.F o o o F NF NoF NFN NNF NNN NNN Ne FF o o mchocFFoemo mm FeN.F NNN N N N F NF «N NN NN NoN FN FeN NNN NF mFepmmggmu mm NNN.F NF FF Fe NN ¢N FN oe NN FeN Noe NON NNF NeF NN mcmFmFm .m quoF on: no; emu owe >oz poo mom mz< Fae and am: La< gm: anon mmmFooam mcFFoLNu spzom cF pcmFa Lo>Fm zmccm>mm mFFNFuFa cF FNNNFIFFNFV tongue» AzoFon mmFouoL: one on» co LFo>memm u use; Fo NewcaFgma one meoFm m>onm memeacov mcanFsa5m mo Namesaz .N anmF 48 .zgmzenmu NNiNN Low NFco oFaNFFm>m mamas Fom . Mme.F eNN.F N: 33 ml 26 N. 8 «F11. NNAN :N 93 NFII 6:: NF.F N.Nm _F -- -- o NN-om NF.F N.NN FF Newsanma mo.e e.NN N NN.F N.Fm mm NN-NN NN.F o.Ne oe Ngazcaa NF.m N.me e om.e o.Nm eN NN-NN «N.N N.Ne NN Langmuao NF.N o.me N NN.N N.em NF oe-om NN.F ¢.Ne 3N Lansa>oz Fm.e N.m¢ N mN.m N.NN me 4N-ON om.F N.oe NN Canopus NN.F o.Ne a eN.m o.em NN FN-NN NN.F N.ee Nm amasauawm Ne.o o.e¢ oF 4N.m N.NN Ne NN-NN «o.F N.Ne mm bm=m=< NN.o N.NN Nm NN.F N.Nm NON NN-NN Ne.o o.Nm FeN NFae NN.o o.mm FN NN.F o.Nm New NN-oN NN.o N.Nm NNN «:35 ee.o N.Nm NoF Nm.o m.Nm NNN NN-FN NN.o o.mm Nae Na: Ne.o e.Nm mN NN.o e.mm as. ON-NN mm.o o.em NNF FFLa< NN.F N.Nm NF No.F N.FN mNF NN-NN oe.o N.NN NeF saga: -- o.Nm N em.o F.em em NN-mN 4N.o N.Nm NN asgazanae mm m 2 mm NF z 2.3.5. mm w z moezuamoom mogzuamu FmeFcF moezuamo FmpoF FNanuFuva LFo>memm u econ mo Ngozngoa may meoFm mFFmeFQ :F vegans» mcmFawm,m:mm.Fo FEEV mgpmcmF Neon New mean=z .m anNh 49 indicate that emergent young comprise the majority of specimens trapped then. The percentage of specimens recaptured increased during the first four months to 22% of the total specimens captured in May. Except for February 1972, when all 11 specimens trapped were recaptures, the high- est monthly recapture rate was 29% (November). Mean body lengths of immature (‘<47 mm) B, pipiens (Table 4), verify that emergent trans- formees dominate monthly captures for most of the year. A biweekly analysis of trapping data reveals a highly significant correlation between the mean body length and number of R. pipiens trapped biweekly (Spearman r = -0.813, P <0.0001). Bufo terrestris showed a distinctive bimodal seasonal activity pattern. The periods of greatest capture were March-April and June (Table 5). Emerging transformees (5-10 mm) were encountered only in May, June and July. No B, terrestris were caught from November to January. A total of 272 (17%) of the individuals were recaptured. Gastrophryne carolinensis (the narrow-mouthed toad) was trapped from April to December (Table 6) demonstrating a bimodal seasonal activ- ity pattern. Most of the specimens (87%) were trapped from June through September, and only 3% were recaptured. Emergent young (10-13 mm) were trapped from July to October. The numbers of the southern cricket frog (Acris gryllus) trapped monthly also represent bimodal seasonality (Table 7). Emergent young (10-15 mm) were trapped from July to December. Numbers of specimens trapped were much fewer than those of R, pipiens, B, terrestris or G, carolinensis. Only eight (3% of 271) were recaptured. 50 Table 4. Numbers and body lengths (mm) of immature Rana i iens trapped in pitfalls along the periphery of Pond C Reservoir (l97l-l972) Month N Y'Length SE Range Februarya 59 34.02 0.273 29-39 March 132 31.77 ‘ 0.291 25-46 April 159 33.23 0.216 27-42 May 473 32.38 0.156 21-43 June 383 32.10 0.274 20-44 July 223 33.97 0.331 23-46 August 40 38.82 _ 0.831 28-46 September 18 34.78 1.708 25-44 October 33 30.12 1.157 20-44 November 8 34.12 1.597 30-43 December 10 32.90 1.703 25-41 January 9 31.56 0.973 26-36 February 0 -- -- -- March ____jz 33.11 1.540 23-40 1,556 aData available only for 26-28 February 51 .xgmacmn so..gmnsmooo .Lmaeo>oz mchzu unmamo mcoz n .Ngmagnmu NN-NN Noe NFco anmFFm>m apnea NNN NNN.F oNN.F NN.o N.NN .Nm- NN.o N.NN .mNm- FN-ee NN.o N.NN .Nmm- Note: - - o FN.F N.NN N NN1Ne FN.F N.NN N Ngngnmu -1 - o - - o 1- 1- 11 o nxgmacma -Lonem>oz NN.F N.Ne N NN.N F.oN oF FN-NN NN.N N.NN NF L8238 Ne.N N.Ne FF NN.N N.Ne NF NN1om NN.F N.Ne «N Loneopqmm NF.N N.Ne NF NN.N N.Ne NN NN-NN NN.N N.Ne oN umzma< NF.N F.N¢ NF Fe.F N.NN NN FN-oF NN.F N.NN NN ana NN.F N.NN NN NN.F N.NN NNF NN-N NN.F N.NN FNN mesa NN.F N.NN NF FN.F F.NN NN oN1N NN.F F.NN FN Na: eN.o N.NN em NN.N N.NN «NF NN-NN NF.N N.NN NeN FFea< NN.N N.NN ow NN.N N.NN NFN NN-Ne NN.N N.NN NNN nose: 1- - o NN.N N.NN oF NN-Ne NN.N N.NN oF mxgmagnou mm .x 2 mm .x z omega mm .M 2 moespamoom mmeaaamu FNFchH mmezunmu quoF ANNNF1FNNF on» NcoFm mFFaFuFa :F tongue» mFgomoLeou omzN Fo FEEV New :mF Neon new memasaz LFo>gmmmm N econ mo xgmganoa .N anmh Table 6. Numbers and body lengths (mm) of Gastrophryne carolinensis trapped in pitfalls along the periphery of Pond C Reserv01r No specimens were caught February-March __ Number of N X Length SE Range Recaptures April 10 26.4 0.91 22-32 0 May 42 27.8 0.39 23-32 0 June 321 28.3 0.12 22-34 10 July 395 29.9 0.13 13-36 15 August 159 29.6 0.30 12-37 9 September 318 13.7 0.22 10-33 2 October 105 18.4 0.30 10-29 4 November 13 18.6 1.85 15-40 0 December ____jL 16.0 -- 16 __J; 1,369 40 Table 7. Numbers and body lengths (mm) of Acris r llus trapped in pitfalls along the periphery of Pond C ReseFVBTF'1157131972) __ Number of N X Length SE Range Recaptures Februarya O -- -- -- -- March 13 22.0 0.47 19-24 2 April 16 22.8 0.71 17-29 0 May 0 -- -- -- 0 June 1 16.0 -- l6 0 July 15 14.9 0.70 10-21 0 August 33 16.5 0.44 11-22 0 September 75 17.2 0.32 11-23 3 October 42 18.1 0.46 14-25 2 November 35 19.0 0.46 14-25 1 December 14 21.1 0.82 15-28 0 January 5 22.4 0.75 20-24 0 February 4 21.2 1.44 19-25 0 March _l_8_ 21 .6 0.51 17-25 __g 271 8 aData available only for 26-28 February. 54 The last species for which seasonal data are presented is Pseudacris triseriata feriarum (the upland chorus frog). Seven (4%) of the 173 specimens trapped and marked were recaptured (Table 8). Adult frogs were at the reservoir throughout the year and emergent transformees (7-11 mm) were caught in May and June. Body lengths of R. pipiens, B, terrestris, and G, carolinensis caught in each trap area and each thermal zone were similar among total captures, initial captures and recaptures for each species (Tables 9, 10 and 11). Dispersal of the three species among traps, however, differs considerably. To allow for a comparison of trap affinities among the three dominant species, the numbers of specimens and trap rankings are presented together (Table 12). Although some trap sites were ranked consistently high for each species (A, B and C) and some consistently low (F, G and L), others exhibited considerable disparity among the three dominant species (V, W and Z). Data for Acris gryllus (Table 13) and Pseudacris triseriata (Table 14) both differ considerably among body lengths and numbers trapped. In addition to determining which adult amphibians occur and breed in the vicinity of the heated reservoir, a major goal of the pitfall trapping study was to reveal the reservoir areas in which larval development occurred. Emergence patterns of immature R, pipiens along the periphery of Pond C Reservoir can be seen by comparing initial captures from each trap area (Table 15). Of 1,306 initially captured immature g, pipiens, 49% were caught in five (26%) of the 19 trap sites. All of these areas were associated with nearby cool seepage ponds or Table 8. Numbers and body lengths (mm) of Pseudacris triseriata trapped in pitfalls along the periphery of Pond C Reservoir (197l-1972) __ Number of N X Length SE Range Recaptures Februarya 2 29.5 -- 27—32 0 March 6 26.0 0.86 24—29 1 April 15 19.4 0.83 15-27 0 May 33 16.8 0.90 11-30 0 June 43 15.1 0.55 7-25 2 July 2 15.0 -- l5 0 August 4 22.0 2.48 15-26 0 September 2 25.0 -- 25 0 October 5 27.4 1.03 24-29 0 November 1 28.0 -- 28 0 December 5 30.0 1.14 26-33 0 January 33 29.3 0.39 25-33 1 February 13 28.7 0.58 24-32 2 March __8_ 27.8 0.56 26-30 __1_ 173 7 3Data available only for 26-28 February. 56 .mnm mNe.F NNN.F - -1 1- o NN-NN NN.N N.NN NF NN.N N.NN NF 2 FN-NN NN.N F.FN N NN-NN NN.N N.NN NN NN.N N.NN NN .N H> NN-om NN.N N.Ne e FN-NN NN.F N.NN NN NN.F N.NN Ne N NN-FN NN.N N.NN N NN1NN NN.N N.NN Ne NN.N N.NN NN x oe-FN NF.N o.FN N NN-oN NN.N N.FN NN NN.N N.FN eN > > oN-NN NN.F N.NN om eN-NN NN.N N.NN NNF NN.N N.NN NNF .N NN-NN NN.F N.NN NN NN-FN NN.N N.NN NNF NN.N N.NN NNF .F NN 1- N.NN F NN-NN NN.F N.NN mm NN.F N.NN cm x >H NN-NN - N.NN N NN-NN NN.F N.NN NF NN.F N.NN ON I NN-NN NN.N N.NN N Ne-NN NN.N N.NN NN NN.N N.NN NN N NN-NN NN.F N.Ne NN NN-NN NN.N N.Fe NFF NN.N N.NN FNF 3 FFN Fe-NN - N.NN N oe-NN NN.N N.NN NN NN.N N.NN NN N NN-NN oF.F N.NN NF NN-NN NN.N N.NN NN NN.N N.NN NNF N eN-NN NN.N N.NN NN NN-NN NN.N N.NN NNF NN.N N.NN FNN > NH Ne-NN NN.N N.NN NF NN-NN NN.N N.NN «N NN.N F.NN NN N oN-NN NN.N N.NN eF NN-NN NN.N N.NN NN NN.N N.NN No N Ne-NN NN.N N.NN e NN-NN NN.N N.NN NN NN.N N.NN NN a H NN-NN NN.N N.FN Ne mN-FN FN.o N.NN NNF NN.N N.NN NNF .N NN-om NN.F N.NN NF NN-NN NN.N N.NN NNF NN.N N.NN NNF .< magma NN gamcm4.w z magma NN camco4.m 2 mm numcm4.w z QNLF mcoN FaggozF mmeauamomm mmeauamN FNFchH mmezuamN quoF AN .NFN mmmv anew on» Law: econ omaaomm Fooo a mo mucwmmga on» mwumoFucF N.V Fonsxm sz .Ncm Fooo one an mchcFmon .ucmFumgm FNELmzu mg» mcoFa mucoN an umazogm «gm mqmgF .NNN_ muse: _m op FNNF Nemaegom NN sage LFo>Lommm N econ No Neonanma esp meoFm mane» FFmFuFa :F umgzuamo mcmF F mama No NEEV mgumcmF Neon New msoasaz .N anmF 57 .whw NNN.F NmN.F NN-NN NF.N N.FN NF NN-NN NN.F N.Ne NN FF.F N.Ne NN z - 1- N.NN N NN-NN NN.F N.NN NN NN.F N.NN NN .N N> NN1Fe NN.N F.NN N NN-NN NN.F N.NN NN NN.F F.NN NN N NN1NN NN.N N.NN NF NN-NN NN.N N.NN NN NN.N N.NN NN x NN-NN NN.F F.N< FF NN-eF NN.F N.NN NN NN.F N.Ne me > > NN-ee NN.N F.FN NN NN1NN FN.F N.NN NN NN.N N.NN NN .N NN-Ne NN.F N.NN NF NN-eN NN.F N.NN Ne NN.F N.NN NN .N eN-Ne NN.N N.NN N NN-NN NN.N N.NN NF NN.N N.NN NN x >H NN-Fe NN.N N.NN N NN-NN NN.F N.Ne NN FN.F F.N¢ we : NN1N¢ NN.N N.NN e FN-NN NN.F N.Ne NN NN.F N.Ne NN N FN-Ne NN.F N.NN N NN-NN NN.F N.NN FN NN.F N.NN Ne 3 NNN NN-NN NN.F N.NN NF FN-NN NN.F N.NN NN NN.F F.NN Ne N NN-NN NN.F N.NN NF NN-NN FF.F N.Ne NN NN.N N.NN em N - 1- N.NN F FN-NF FF.¢ N.NN NF NF.¢ N.Ne NF > HF NN-Ne NN.N F.FN N . NN-NN NF.F N.NN NN NN.F . N.NN NN N NN-Ne NN.F N.NN NF NN-NN NN.N N.NN NNF NN.N N.NN «NF N 1- - N.NN F NN-NF NN.N N.NN eF NF.N N.Ne NF N H NN-FN NN.N N.FN NN FN-N NN.N N.NN NNN NN.N N.NN NNN .N NN-NN NF.F N.FN NN NN-N FN.F N.NN NNN NF.F N.Ne NNN .< magma mm sumcmN.M z omcmm NN spmcmN.w 2 mm cumcm4.m z NNLF mcoN FNELNNN mmezuamomm maespnmN FmFchH mmezpamN quoN awe» on» New: econ mmmqmom Fooo a No wocomoga ecu mmumoncF F.v Fonezm ocF .ucm Fooo esp an Nchszwn .ucoFNNLN FNELNNN as» mcoFN moco~ Na NoazoLN NNNLF .NNNF cogmz _N on _NN_ Newsgnou NN sage gFo>gomo¢ N econ No NNNNNFLNN mg» NcoFm mane» FFNFNFN :F cogapgmu mFepmogemu omzN No NEEV mnemcoF Neon new mg¢a§=z .NF anmF 58 N.N- ERN. N.NN-u. 11 - N.FN F NN1NN NN.N N.NN NF NN.N N.NN NF 2 eN-NN FN.N N.NN NF NN-NF NN.N N.NF NNN NN.N N.NF NNN .N N> - - N.NN F NN-NN NN.N N.FN NF NN.N N.NN NF N - - N.NN F NN1NF NF.F N.NN NN NF.F N.NN NN x NN-NN - N.FN N NN-eF NN.N N.NN eN NN.N N.NN NN > > NN1NN - N.NN N NN1NF NN.F N.NN Ne NN.F N.NN NN .N - - N.NF F NN-eF NN.N N.NN NN NN.F F.NN NN .H NN-NN NN.N N.NN e Ne-eF NN.N N.NN eN NN.N N.NN NN x >H NN-NF NN.N N.NN N NN-NF NN.F N.NN NN NN.F N.NN NN I - - - N eN-NF NN.N F.NN NN NN.N F.NN NN N - 1- - N NN-NF NN.N N.NN Ne NN.N N.NN me 3 FNF - - - N NN-NN - N.NN N 11 N.NN N N NN-NN NN.N N.NN N NN1NF NN.N N.NN Ne NN.N N.NN Ne N NN-NN - N.FN N eN-NF NN.N N.NN NN FN.N N.NN NN > . NF - - N.NN F NN1NF FN.N N.NN Ne NN.N N.NN Ne N - - N.NF F NN-NF NN.N N.NN NNF NN.N N.NN «NF N 1- 1- N.NN F eN-NF NN.N N.NN NN NN.N N.NN NN N F NN-NN - N.NN N NN-NF NN.N N.NN NNF NN.N N.NN NNF .N FN-NN NN.N N.NN N NN-NF NN.N N.NN NNF FN.N N.NN NNF .< mNcmm NN schmN.M z oNcmm NN cpmcmN.M z NN NNNNNN.M z NNLN mcoN FNELNNN megapamumm moezpamN FNFNFNN moeaunmN quoN NNN» on» New: Neon mNmwam Fooo a No mocmmmea asp NmpouFNcF N.v Foaezm mcF .ucm Fooo an» an NchcFNon .NcoFumgN FNELNNN on“ NcoFm mmcoN an umasoeN men NNNLF .NNNF,:oLmz,FN op N NgmagnmN NN sage eFo>gomoN N ucoN No NumcaFeoN NNN NcoFm mane» FFNFNFN :F noeapamo mchmcFFoemo m: g; oepmmN No NEEV mgamcoF Neon New mg¢n5=z .FF anmN 59 NNN.” NNN.F NNN.F N N.F N.NF NF N NF NN NF NN NF 2 N F NF N NN« N«N NN NN .N H> NF NF NF «F NN NF NN N« N NF N.NF N NF NNF NN NN NN x N.N N NF NF NFN NN N« «N > > « N N N «NN NN NN NNF .N N NF N N NNN NN NN NNF .H «F N NF NF «FF NN NN «N x >N N.NF «F NF NF NN NN «« NN 1 NF N.NF «F N.NF NN NN NN NN N NF N.FF NF « «NN N« N« FNF 3 FNN NF . NF FF N.NF NN N N« NN N N NF « N FNN N« «N NNF N N.N N NF F «NN NN NF FNN > FH FF N.FF N N.FF NNF N« NN NN N N N N N NNN «NF ««F NN N NF N NF N.FF NNF NN NF NN N F N « F N NNN NNF NN« NNF .N F N N N NNN NNF NNN NNF .< 3:5. N.N. N.N N.N z N.N N.N N.N 8.: 2.3 wngw>< quoN FNELNNN mNchcmN mocmuczn< moFomaN emu z amen mg» Lam: Neon mNmammm Fooo N we mocmmmga mg» moumoFocF N.N Foaeam NNN mucoFngN FNELNNN use NcoFm monoN Na NoazosN men NNNLN .Nth Nogmz FN op FNNF NnggnmN NN scum grm>mmmwm N mean we Ngoganwm msw,N=oFm mFFmNuFN NF Negqmgu N.Nnmq mchmcFFoemu chugnomemN New N.N.Nv mFepmmsemu NNNN .Aam.mv mcmFan new: "memescm No moFuwNm mossy No mLmNENz .NF aanN Table 13. Numbers and body lengths (mm) of Agris gryllus captured in pitfall traps along the periphery of Pond C Reservoir from 26 February 1971 to 31 March 1972. Eight specimens were recaptured. indicates the presence of a cool seepage pond near the trap The symbol (') Thermal __ Zone Trap N X Length SE Range A' 17 16.9 0.62 21-21 B' 18 19.2 0.71 14-24 I u 16 16.2 0.47 14-20 C 9 19.5 1.15 11-22 D 9 21.6 0.74 19-26 II V 3 18.6 1.45 16-21 E 21 19.7 0.75 15-25 F 5 22.2 1.02 19-25 III W 22 18.5 0.67 13-25 G 6 18. 1.30 15-23 H 5 16.6 1.36 15-22 IV X 1 17.0 -- 17 I‘ 60 18.1 0.48 ll-28 J' 33 16.9 0.55 10-25 V Y 3 20.3 1.45 18-23 K 14 18.2 0.88 14-23 L 11 20.5 0.90 15-25 VI 2' 2 21.0 -- 17-25 M 16 21.2 0.70 17-29 61 Table 14. Numbers and body lengths (mm) of Pseudacris triseriata captured in pitfall traps along the periphery of Pohd'C Reservoir from 26 February 1971 to 31 March 1972. Seven specimens were recaptured. The symbol (') indicates the presence of a cool seepage pond near the rap Thermal __ Zone Trap N X Length SE Range A' 6 19.0 3.32 7-26 B' 7 27.3 1.70 19-32 I u 2 25.5 -- 22-29 D 3 28.0 1.52 26-31 0 -- -- -- II V 13 20.2 1.61 15-30 E 6 30 8 1.40 24-33 F 1 30.0 -- 30 III 21.7 3.66 18-29 G 2 29.5 -- 29-30 H 1 32.0 -- 32 IV 24 4 1.27 19-30 I’ 24 3 3.38 20-31 J' 0 -- -- -- V Y 9 24.3 1.52 16-32 32.0 -- -- L 2 30.0 -- 28-32 VI 2' 106 20.1 0 68 11-32 M 0 -- -- -- 62 .NNNNLNNN NN-NN Low NFco «FNNFFN>N NNNNN F NF N N N N «N N F N F N z N N.N NN N N N «NF «NN «N «« «N N .N H> N NF NN N N N N «N «N « N F N N NF «« «N N N N «NF «N N N N x N N NN N N «« «FF ««F «N «N ««F «NF > > N « NN «N N «« «N «NN «FN «NF «N F .N N N NNF «FF «N N «NF «NN «NN «NF ««F N .N N «F NN N N N «N «FF «N N N N x >H N NF «F N N N N N F « N N N F N.NF NN N N N N « «N F N N N N N NN N N « N «NN «NF N F N 3 FFH F N.NF NN N N F «N N N « N F N - « N1 NN N N N «N- -«FF -«Nm - mm--«Nm---m--111m-1- - - - N NF NN N F N N «NN «NN «NN F N > NH « NF NN N N F «N «N «NN F «N F N m N.N Q o o N ««F N «FF « a FF N .. « FF «N N N «F «N «NF «FN N N N N . F N N NFF N N N N «NN «NN N «NN N .N N F NNF «N «N N NN «NN «FN « «N F .< ngucoz Nchcmm quoF NNN gum NN< FNN NNN Ne: La< gm: NNNN NNNF ocoN No .ozv mocmucsn< FNELNNF momesoemcmgF Nae» New: Neon mNmammm Fooo No mocmmmen on» moumoFNcF N.N FoNst NNF .Ncm Fooo mg» pm NchcFNoN NNNFNNLN FNELNN» NNN NcoFm mmcoN NN NmazogN mew mamLF .NNNF Nose: on NNNF LmNEm>oz seem NNNNNLN one: mcosFumam FN choFuFNNm c< .NEE NNV.N moseowmcmgg chNgmeo mco NmmmF um mmNNFocF mFaEmm on» page mwpmoFNcF A«V Fonezm sz .mameb FFaFNF a :F Nosauamo NFFNFNFNF mcanNm menu NEE N«V. NNNNNF NNoNV mgsmeEF mo muonszz .NF NFNNF 63 cool reservoir shallows. .Traps associated with dry wooded areas generally caught the fewest numbers of 3, pipiens. 0f the immatures, 73% occurred in nine (47%) of the trap sites; none of which were surrounded by woody habitat. Of 230 initially captured 8, terrestris, 50% occurred in four (21%) of the pitfall trap sites. Of the initially captured immatures, 74% occurred in eight (42%) of the trap areas. Unlike the more aquatic .B- pipiens, the terrestrial toads were encountered more commonly in trap areas near woods and were less restricted to sites near cool ponds or reservoir seepage. High numbers of initially trapped immature R, pipiens ( <30 mm) usually represented some emergent transformees (Table 15). Although transformed young were encountered other months of the year (except February 1973), only 35 (2.7%) were trapped from November to March. The monthly occurrence of emergent young was not greatest in traps with the highest total capture. When the 19 trap areas are ranked according to total catch of initially captured immature R, pipiens, 49% of the specimens are found to occur in five (26%) of the traps, and 90% of the specimens occurred in 13 (68%) of the traps. The temperature of Pond C Reservoir during the breeding season undoubtedly determines whether or not larvae can develop successfully. As indicated by Table 15, emergent transformees were trapped near pitfall site Y (Figs. 2 and 12) from February to July 1972. No transforming larvae were trapped there in January, February or March of the next year, however. 64 Fig. 12. Pitfall-drift fence section Y (Fig. 2). Partial separation of the seepage basin by land may keep water temperature at sub-lethal levels. Emergent young of Rana pipiens were trapped here from February to August 1971. 65 U C J...- C I! I 3' "'Qranv Q . E O .‘l e 38"“) 1.1) "V"- f 14‘001 l a l 4 ‘44: ,4- . P. 4.1!: I .:E ‘3). iv ‘11" p Ff . - 55:4 " Figure 12 66 A grouping of data into the six major temperature regimes along the thermal gradient (Table 1, Fig. 2) shows that the numbers of R, pipiens and B, terrestris trapped generally decreased at the heated extreme (Tables 16 and 17). Numbers of total captures, initial captures, and initially captured immatures all demonstrate the same phenomenon in generally conforming to the thermal gradient along the reservoir's cool arm. Gastrophryne carolinensis, A, gryllus and E, triseriata, however, do not clearly reflect this gradient in the numbers trapped (Tables 18 and 19). Table 16. Mean numbers of Rana pipiens caught per pitfall trap area in each of the thermal zones along the periphery of Pond C Reservoir Total Initial Innitial Captures Thermal Captures Captures (Immatures) Recaptures Zone (N=1,779) (N=l,484) (N =1,306) (N=295) I 133.5 113.0 95.5 20.5 II 132.7 108.7 96.7 24.0 111 73.7 54.3 42.0 19.3 IV 71.3 63.0 57.3 8.3 V 91.7 76.3 71.3 15.3 VI 45.7 41.7 37.7 4.0 67 Table 17. Mean numbers of Bufo terrestris caught per pitfall trap area in each of the thermal zones along the periphery of Pond C Reservoir Total Initial Initial Captures Thermal Captures Captures (Immatures) Recaptures Zone (N =1,636) (N = 1,364) (N = 230) (N = 272) I 214.8 184.0 22.0 30.0 II 62.7 54.0 14.0 8.7 III 40.3 32.0 9.0 8.3 IV 41.0 32.7 9.3 8.3 V 72.3 55.3 7.0 17.0 VI 42.3 33.3 8.0 8.3 Table 18. Mean numbers of Gastrgphyne carolinensis caught per pitfall trap area in each of the thermal zones along the periphery of Pond C Reservoir Thermal Total Captures Initial Captures Recaptures Zone (N=l,356) (N=l,3l6) (N= 40) I 111.5 109.2 2.2 II 59.0 57.0 2.0 III 24.0 24.0 0 IV 41.0 38.3 2.7 V 54.3 52.7 1.7 v1 125.0' 121.0 4.0 68 Table 19. Mean numbers of Acris gryllus and Pseudacris triseriata caught per pitfall trap area in each of the thermal zones along the periphery of Pond C Reservoir TH§5251 AiNgr%;lus E,(§r:s;g;gta I 15.0 4.5 II 11.0 6.3 III 11.0 2.0 IV 22.0 3.7 V 16.7 3.3 VI - 9.7 36.0 aEight recaptures. bSeven recaptures. Not only were 3. pipiens trapped every month of the year at Pond C Reservoir (Table 3), but young frogs emerged for 11 months of the year there, compared with two or three months elsewhere on the S. R. P. at Risher Pond (1969-1970) and Karen's Pond (1970). Extended breeding at Pond C Reservoir was not accompanied by an apparent size differential among transformed young (Table 20). Bufo terrestris at Pond C Reservoir demonstrated a seasonal activity pattern similar to that at Karen's Pond in 1970 (Table 21). Most larvae from Pond C Reservoir emerged at about the same time of year as they did at Karen's Pond (1970), but may have emerged at a smaller size (cf. Tables 20 and 21). No transformed R, pipiens emigrated from Risher Pond either year. 69 .NcoFngmnga NF New mmmgq NF .«NNFV meoNNFN .3 .N sage NNNNN NNNNLNNN N amnem>oz FNNF ugmoxm LFo>gmmmN NF.N N.NN Nom NN-NN mgpcoe FFa NNNNN NF=N-Naz NNN.F N N=ON NNNNV NF=N-m==N ONNF NN.N N.NN FN NN-NN Nm=N=<-mc=a ANONN sueez-Nga=anN NNN ae=ON LmNmFN NNNN Ner 1‘ NNNF NN.N N.NN N« NN-NN bm=N=<-m==e ANNFN FFLN<-Nga=eNmN «NN mecoe Langm ANONN NF=N-N==N ONNF NN.N N.NN NF FN-NN NFaa-weaa FNFFN gage: NNF mecca m.=meax Nm. x z Nagy emeapaao NNF>FFu< z ame< 1. NNNNNNN New: Nc=o> xmmN No anacoz NNNNNN NNNN NF NNoN No NcmNLmEN 25 NN v NNNEFNNNN «Nana NNN: mgucoz NoumoFch NoFemN NNFLNN :mxmu mogzunmo quop No mNmpcmogoN we» mumoFNNF mwmmgpcmsma :F msmneNz .mcFFogmN NNNoN :F pcmFN eo>Fm smccm>mm NNN co NNNNFNNN NFNNNNN owes» mo NgmNNFgma NNN NcoFm mamep FFNFNFN NF NFsmmN Nunamsu mcmFNFn mama .NN mFNNN 70 .Nconeaeamaa cF ece mmmaa :F .«NNFV mcoNNFN .3 .N EocN eNeNe FNcF NNNNV NNNN aFo>ammwm NF.c N.cF Fm FN-N NFce-ch NNNNN FFca<-ccccz NNN N ecca chF -- -- c NN-NN cccc NNNFN FFca<-acccz NNN cecca cccmFN NNNF -- -- c NN-NN cccc NNNNN Neg-FFcac NFN cecca ccchc NNNNN cmccc<-NFce chF «F.c c.cF cm cN-N cmccc<-ccce NNNNN Nee-ccccz «NF.F cecca c.ccce¥ Nm .N z Ncsv eccccacc NcF>Fcc< z ccc< NNNNNNN New: NNNNF xemN No mcpcoz NNNNNN NeoN :F NeoN No NNNNLNEN 55 NN v mcoEFooaN mNcem cog: msucoz NNF mpeoFecF mmmmgpcoaea :F mg¢nENz empeoFecF eoFaoN NNN NcFeNe cmxeu mocepaeo Feuop No mNepcmucma .ecFFoceN NFNNN :F NNNFN am>Fm Necce>em exp co mpepFneN oneNae owes» me NgNNNFama one NcoFe mFFeFNFN cF NFaemN emaaeap mFaNmmagmp oNNN .FN NFNeF 71 Field Study: Reservoir Sampling_with Dipnets and Mihnow Traps Nine species of amphibians (seven anurans and two urodeles) were removed from aquatic habitats during the 15 month sampling period (Table 22). Very few salamanders were encountered in heated areas of the reservoir. Two larval Manculus quadridigitatus (dwarf salamander) and only one adult Siren intermedia (lesser siren) were removed from a heated area (V). Four anuran species occurred in all areas sampled to some extent. Species diversity was highest in a cool pond not contig- uous with the reservoir (cool pond Z) and lowest in the hotter reservoir location, area K (Figs. 2 and 5-8; Table l). Larval amphibians of only one species (Rana pipiens) were encountered in numbers high enough to allow for a comparison among areas. Data from routine monthly dipnet samples and from the minnow trapping survey were pooled to compare growth among thermally distinct areas (Fig. 13). Plotting of the data for all cool ponds showed that they were virtually the same. Data from Pond 2 were chosen to represent growth at specific stages of development in a cool (control) habitat. Reservoir areas V and K were both heated; data from the two areas are shown to be smaller at virtually every stage of development than larvae at cool Pond Z (Fig. 13). 72 F.NN N.«F N.«F N N N mm>amF o: NNF: moFNEmm N cc F.F NF c.F «N Fa 58.3883 .8 N N« N N N N N mmFoonm .o: FmNoF N N N N N + aoFoocho> mFNm N + N N N + mapmNFNFchemmm NNFNocmz N N N + + + memommeFaF> NNEFmNNNNNuoz N N + + + + mcmuFEmmm mama + + + N + + NchmcFFocmo chuNmoeummN + + + + + + mogmcwu mm»: + + + + + + NNFFNNN mFao< + + + + + + mFammmuamu NFNN + + + + + + memFNFN mcmm aFo>cmmmm cFo>ammmm aFo>cmmmm LFo>cmmmN cFo>commm aFo>cmmmN NNF: mmFuoNN NNNF No NNF; NNF: NNF: NNNNNFucoN uoz onammN mzoFFmNN NNNNNFucoN NNNNNFucoN NNNNNFFcoN .N ecoN FooN .x mma< .> mma< .N ecoa FooN .H ecoa FooN .< ecoa FooN Neceom om>cmF a: pen emaFmoame NNNm."NV aFo>awmmN N ecoa No NchFoF> NNN cF FNNNF conempamm op FNNF oceev mmFNEmm uocaFe NFgucoe NF No «cos co mco cF NNN “comnm co N+V “Newman mcmFNFNasm Fm>amN .NN mFNmF 73 Fig. 13. Total lengths of larval Rana pipiens collected from three reservoir sites receiving differential levels of thermal loading. Pond Z (Fig. 2) is a cool seepage area unaffected by thermal effluent. Shore V (Fig. 7) and Shore K (Fig. 8) are two sites in the heated reservoir where larval development occurred. Few specimens were collected from the hottest sampling station (K). Lengths are compared at specific stages of development (Gosner, 1960). Length (mm ) Total Mean 74 y r 1 vw—f r wF‘ 701- .. A ‘ 0 1A 65- - C) A 46()'- 1. a o 9 ‘ o 9 55- o 0‘ - )2 CD 50" ‘ . . . " C3 (3 4‘ " 451- - A O O . 40- - A 35. . Pond Z O n=165 . A . Shore V A n=183 30. Share K 0 n= 27 . C A 25- 0 4 (W1, .FF—W 26 28 3O 32 34 36 Stage Of Development Figurel3 38 75 Field Study: Reservoir Samplingof Larval Bufo terrestris Larval B, terrestris were sampled weekly at five locations in the vicinity of Pond C Reservoir (Fig. 9). The majority of specimens examined in this study is assumed to be the result of egg masses deposited in the study area on or just prior to 22 March (Table 23). A consistent progression of advancing stages and increasing sizes was observed weekly in all areas except area K (Table 24; Fig. 14). Larvae were more advanced and larger in heated water than cool Pond B. Data from the supplemental analysis only (n =20) were used for statistical tests. Larvae from area K, however, consisted of data from the initial analysis (n==10). After the first sampling period, total lengths and stages of development were positively related to the level of thermal loading in all areas except K (Table 24). Following the breeding migration, developing larvae and three separate egg masses were found at area K, but more than 95% of the eggs were dead. Fewer than 20 living embryos were found among the three egg masses present; all of these were located in the clutch closest to shoreline seepage. Several toad larvae, however, were removed from area K in the small (n =10) samples of 29 March and 5 April. Stages of development and lengths of larvae taken in weekly samples reveal a reversal from the initial relationship among the three areas. Statistical analysis of successive samples indicates that growth and development were significantly different among areas in the first and third weekly sample (Table 25). At a given stage, however, larvae develOping in cool water were larger than those reared in heated areas (Table 26). 76 Table 23. Adult Bufo terrestris trapped in pitfalls along Pond C ReserVBir. ’Traps were checked at three day intervals or after precipitation during the preceding night Date Rainfall (1972) (cm) No. Trapped March 2 0 0 4 0.10 2 8 0.15 0 ll 0 0 15 0.05 0 18 2.26 30 22 2.31 562 25 0 11 27 O O 29 1.47 5 April 1 1.22 31 77 NNF-5 NS 5cm: 9.2% mace N-N .Fc meoFaoa ao>o mcouoeoctonu ENEFch-ENEFme co eoenooon moaeumaoosop :55me .3 :55. .aoaoao aFo>aomoc eopmon one cF ocoz g ecm N .> .< mmoam mocoNFFFo eoomon oc eo>Foooa N econ Foon eceoF ecNoF eceoF oNFm oFNEmm NN.N“ N.NN onpN om>cmF on om>amF on om>cmF on ecoFomeemcF NN.N“ N.NF nchoF FmpoF NN-NF-« eceom eceoe NN.N N N.NN NN.N e. N.«N «N.N n N.NN onpN om>amF on om>.amF oc NN.N“ F.NN NN.N“ N.NF N«.NN N.NF nchoF quoF NN-NF-« NF.NNNNN NN.N.ANNN NN.N.AN.«N NN.N.ANNN NN.N“ F.NN onpN NN.NNNFF FN.NN-NNN NN.NNNNF «N.NNNNF NN.NNNNF nuNcoF quo... NN-N-« NN.NANNN eoFNEmm NN.NNNNN NF.NNNFN NN.NNNFN onNN «N.NN «.«F No: moam FN.N“ N.«F NN.N“ N.«F N«.NN N.NF nuNcoF quoF NN-NN-N NN N.NN NN NM NN NM NN N.N. NN NNF. nonopmaooeoF N N.NN N N.«N N «.«N N N.NN N «.«N x moa< N moc< > moc< < moa< N ecoa FooN mmmog< NeNNN NNF u c oaonz v. moam com 33.8 233 nomo .co-F NN ab N.Foiomoa N ecoa No NchFoF> one :F mosFNoa FmELonu ucoaoNNFe cone eoFoEmm om>amF anumoLaou oFNN .«N anmF 78 Fig. 14. Mean stage of development (Limbaugh and Volpe, 1957) of larval B, terrestris collected in three weekly samples from three areas along Pond C Reservoir. Vertical lines represent:t2 SE. 79 __NQ< NF __..Q._o._ Fe. 36013 ruewdo'aAaq 50 80 Table 25. Comparisons of stages of development and total lengths (mm) gf larval toads collected in weekly samples from three thermal regimes n=20 Stage of Development X'Stage by Study Area B A V Kruskal-Wallis Anova Date 24.4 C 32.9 C 34.4 C H P 3-29-72 31.7 31.5 30.8 6.06* < 0.05 4-5-72 32.1 33.5 34.2 4.53 < 0.50 4-12-72 32.7 34.2 37.6 13.39* < 0.005 Total Length X Total Length by Study Area Kruskal-Wallis Anova B A V Date . 24.4 C 32.9 C 34.4 C H P 3-29-72 15.7 14.8 14.0 7.46* < 0.025 4-5-72 16.6 17.2 18.2 2.48 < 0.50 4-12-72 16.8 17.5 20.1 12.74* < 0.005 Table 26. Mean total lengths (mm) of larval toads at comparable stages of develOpment, collected from cool and heated areas; (n) =sample size. Data for heated areas A and V are pooled for anova comparison with cool area B Kruskal-Wallis Study Areas Anova Stage B--24.4 C A--32.9 C V--34.4 C H P 31 14.7 (20) 14.2 (21) 14.2 (16) 4.11* < 0.05 32 16.0 (16) 15.5 (14) 15.8 (12) 0.44 < 0.50 33 18.0 (24) 16.9 (16) 16.7 (6) 5.04* < 0.025 1'QStatistically significant. 81 Laboratory Study: Rearing of Larval Rana_pipiens Larval development through metamorphosis occurred at all experimental temperatures except 35 C (Table 27). All embryos main- tained at that temperature died within nine days. Hence, data from this temperature will be excluded in comparisons of the thermal regimes. Of the 1,200 specimens introduced at the three other temperatures, 57% transformed into late metamorphic stages (41-46). Accidental deaths represented less than 5%, and mortality accounted for 30% of the orig- inal experimental animals. Data were not available for approximately 9% of the 1,200. Mortality was greatest at 30 C, and transformation greatest at 25 C. An expression of comparable mortality (survivorship) can be made by using a ratio of the two parameters. Values of the transformation rates/mortality rates are 1.81 at 20 C, 2.25 at 25 C and 1.72 at 30 C. These figures suggest that the 25 C regime was most favorable and 30 C regime least favorable to larval development. Approximately 70% of the larvae at the three temperatures transformed; approximately 30% were lost due to mortality (Table 28). Marked differences occurred in both transformation rates and sizes at the three temperatures having normal development. Completion of development by the larvae required more than five (30 C), seven (25 C) and 17 (20 C) months (Fig. 15). Developmental rates were positively realted to temperatures, and transformation apparently required an inordinate amount of time at 20 C. Numbers of specimens transforming were comparable among replicate aquaria at the same temperatures (Table 29), and did not differ significantly from one 82 Table 27. Laboratory reared Rana pipiens; 100 embryos (stage 11 or 18) originally placed in each aquarium. Percentages are based on a total of 400 specimens at each temperature Temper- Replicate Accidental Transfor- Specimens Not atures Aquaria Deaths Mortality notions Accounted For A 29 47 21 20 C B 23 49 26 C 10 27 51 12 D __5_ _3.3__ _§_6__ _7__ 4 8% 28 0% 50.8% 16 5% A 27 64 8 B 13 39 43 5 25 C c 28 67 3 D __ _l_7_ _76__ _7__ 4 0% 27.8% 62 5% 5 8% A 7 44 47 2 B 3 32 60 5 3° C c 5 30 63 1 D __§_ _ZL fl. __7_ 5 2% 33 5% 57.5% 3 8% A 0 92 0 8 B 2 70 0 28 35 C c o 78 o 22 D _9__ _z_3__ __0___ 27 58 570 683 187a aThree transformed specimens escaped. Table 28. 83 Comparison of larval mortality and transformation at three thermal regimes (excluding specimens not accounted for) Temperature N Mortality Transformation 20 C 315 36% 25 C 361 30% 30 C 364 37% Table 29. Body lengths (mm) of 683 transforming Rana pipiens reared at three different temperatures in the laboratory Aquarium N 7' SE Range CV 20A 47 28.6 0.45 22-35 10.8 208. 49 29.3 0.48 22-36 11.4 20C 51 30.8 0.48 23-40 11.2 200 56 31.3 0.46 24-40 10.9 25A 64 26.6 0.29 22-34 8.7 258 43 25.5 0.39 19-32 9.9 25C 67 26.0 0.28 20-32 8.8 250 76 27.0 0.26 23-32 8.4 30A 47 24.7 0.40 20-33 11.0 308 60 24.2 0.34 20-30 10.8 30C 63 24.6 0.32 20-30 10.3 300 60 23.6 0.35 15-29 11.3 683 84 Fig. 15. Mean body lengths (:‘1 SE) of laboratory reared larval B, pipiens transforming monthly at different thermal regimes; 400 embryos (stage 11 or 18) were originally introduced at each temperature. An asterisk indicates that the series was terminated (20 C and 30 C) before all larvae had transformed. 85 mmwo ”"75 _1—5 2 Amp mgzmwgv gfi uoom .m. J], \ .AuN s s s . .m \ 4~“N ~ U on u ... / man i W\\ ON on— . .. x .H «o. \wHu..... .... an . ON on... * no L ”N I o. H mm NHIIJWWY\WMI/Jmfl UH .Aun \ o— .5 .m a . an . vm - r P b on (mm) q45ue1 Apog uoew 86 another (X2==18.6, P:>0.05). Body lengths of transformed larvae among replicate aquaria were significantly different at 20‘C and 25 C but not at 30 C (Table 30). Differences in body lengths of transformees reared at the three temperatures were also highly significant (Table 30). Final transformees at each temperature were largeriin size than larvae developing earlier (Table 31). . Biweekly transformation curves (survivorship excluding "natural" mortality) of larvae were similar for those reared at 25 C and 30 C. The transformation rate at 20 C, however, differed considerably from the other temperatures (Fig. 16). The transformation curves for the 25 C and 30 C series resemble a geometric progression, and the curve for larvae maintained at 20 C more closely approximates an arithmetic (additive) progression, being more linear in appearance. Larval sur- vivorship (Fig. 17) was calculated by adding "natural" mortality and transformation. The proportions of the experimental populations remain- ing in each thermal regime over time, therefore, follow trends similar to those of the transformation curves (survivorship excluding mortality). Larvae reared at 25 C and 30 C demonstrated rapid declines, whereas those at 20 C declined gradually. Since the series at 20 C and 30 C were both terminated, data were plotted with the assumption that all of those living at the time would eventually complete metamorphosis.’ Extrapolating data from the survivorship curves (Fig. 17), zero survivorship should have occurred after 24 weeks at 30 C, 28 weeks at 25 C and 88 weeks at 20 C. The amount of time required to Peach specified levels of survivorship are compared among the three 87 Table 30. Comparisons of body lengths of Rana pipiens reared in the laboratory at three different temperatures. (Kruska1¥Wallis nonpar- ametric analysis of variance) Kruskal-Wallis __ Statistic Temperature N X (H) P 47 28.6 17.02* 0.005 49 29.3 20 C 51 30.8 56 31.3 64 26.6 10.06* 0.025 43 25.5 25 C 67 26.0 76 27.0 47 24.7 2.78 0.05 60 24.2 30 C 63 24.6 60 23.6 20 c 203 30.1 277.57** 0.001 25 c 250 26 4 30 c 230 24 2 *Statistically significant. Table 31. introduced into aquaria on 1 September 1971 Body lengths (mm) of 683 transforming Rana different temperatures in the laboratory (1971-1972). i iens at three pec1mens were Temper- ._ ature Month N X SE Range CV October. 1971 4 19.2 2.17 15-23 22.6 November 133 24.1 0.20 19-33 9.4 30 C December 70 24.2 0.35 16-30 12.2 January, 1972 13 25.5 0.59 22-29 8.4 February 10 26.5 0.78 23-30 9.3 250' November, 1971 78 26.5 0.21 20-32 7.1 December 95 27.1 0.24 19-32 8.7 25 C January, 1972 40 26.8 0.42 23-34 9.9 February 25 25.7 0.42 23-32 8.1 March 10 25.4 0.82 20-28 10.2 April 2 29.5 2.50 27-32 12.0 250' January, 1972 9 30.3 1.08 25-36 10.7 February 34 30.7 0.37 26-35 7.1 March 33 29.2 0.38 24-33 7.4 April 10 29.9 0.74 27-35 7.8 May 17 28.9 0.69 25-33 9.9 June 27 28.7 0.64 23-38 11.5 20 C July 16 28.3 0.78 23-35 11.0 August 9 27.7 1.62 22-37 17.6 September 5 28.8 1.77 22-32 13.8 October 7 29.7 1.04 26-33 9.3 November 4 30.0 1.92 25-33 12.8 December 4 29.8 2.14 25-35 14.4 January, 1973 7 33.7 1.06 30-38 8.3 February 2%%_ 34.8 0.77 28-40 10.2 89 Fig. 16. Biweekly survivorship rates, excluding mortality, of laboratory reared larval B, pipiens at three different thermal regimes. The double line at 20 C and 30 C indicates that these series were terminated before all specimens had transformed. 90 “— nDZOm<_._.<< <2 q 8215 u com u 33": u on... 6 Anon": o co“ 6 1 Amp mezmwmv o o o o o o O O O O m 00 I\ 0 m S" m N H 02 Aulpuow Bugpnpxg dgqsmAgMns % 91 Fig. 17. Biweekly survivorship, including mortality, of laboratoruV' reared larval 3, pipiens at three different thermal regimes. An asterisk indicates that the series was terminated before all larvae had transformed. 92 dqozo.m.<.hm§.<. lie . o.z.o.w. . Div 0 “V 523.5: E «d 00000 a % 00 S . .00.. E 4 .ON m 00 a m. 0. W. .. 00 a4 .OVM 0 w: ... m. . o 4 .00 mm 6 86m ”5 uoom . . a 4 w o . AVVW up; UOWN E 0. .O@ W." Amfim n5 Uoom 4 00003 A “3% . . . . . . . . . lllOOF 93 thermal regimes (Table 32). Although the time required to progress from one level to the next is quite similar at 25 C and 30 C, corresponding time intervals at 20 C are not comparable. To compare overall survival in a constant time reference, percent survival was plotted as a percent of the total time required for each group to terminate (Fig. 18). Total time represents the actual duration of eXperiments. Since all embryos at 35 C died within nine days, the curve presented represents mortality (rather than survivor- ship), and must be cautiously related to the three other groups in which larvae did develop through transformation. After 20% of the total time had passed, all groups (except 35 C) dropped rather abruptly in survival. The actual time intervals represented are quite different in duration, varying from nine days (35 C) to more than 17 months (20 C). Corrected to a uniform time comparison, survivorship curves for larvae reared at 20 C, 25 C and 30 C closely resemble one another. Table 32. Time required for laboratory reared larval R, i iens to reach com arable levels of survivorship (transformees + "natural" mortality) at different thermal regimes. Numbers in parentheses indicate the number of weeks between successive survivorship intervals Weeks Required Percent Survivorship 20 C 25 C 30 C 90 ll 10 7 '75 22 (11) 11 (1) 8 (1) 50 30 (8) 14 (3) 11 (3) 25 44 (14) 16 (2) 13 (2) 10 66 (22) 20 (4) 16 (3) 94 Fig. 18. Percent survivorship of Rana pipiens laboratory reared at three different thermal regimes. Survivorship (excluding accidental deaths) is expressed as a percentage of the total time required for development or death. A11 larvae at 35 C died within nine days; thus, data for this group represent mortality only. 95 as: .22 2: z :32: oo. oo 8 ok 8 on 9. on ca 2 o 3.1 7 m/m . . o I I .. 471/ «I- la/o :2 2:35 . O— 00. I. I! / I / _I o. ” .I’V U 0 “a I .. 404' a \ ON .. .W/ o I . o . J/ .8 . /t // o r ’ // 1 .. U oomr/ /. O? . u.3..w.:: o /. / , .om . . . / (,0 0 on .. o.. ,/ /\ . 00 .0.) ” .l o. I 1 OK .../. o x, r 9’4”: 6.”, . .0” «on. u z / fun”... . ./ Jr; . oo - 4.11 . Ilium... oo— s 1uaalad Bummn 96 Comparison of body lengths at the later stages of development among the experimental thermal regimes demonstrates that size is negatively related to temperature (Fig. 19). Animals reared at warmer temperatures were smaller almost without exception, at any given stage of the final eight metamorphic stages (Table 33). Subjected to the Kruskal-Nallis nonparametric analysis of variance, differences in body: lengths are shown to be statistically significant among the three thermal regimes (stage 42: H=33.7, P<0.005: stage 43: H=50.l, P<0.005: stage 44: H=155.6, P<<0.005). Two major abnormalities encountered among the three thermal regimes were the manifestation of crooked spines (Fig. 20, vertebral columns bent just behind the pelvic girdle) and paralyzed hindlimbs. Larvae with edemaceous and emaciated body conditions (Fig. 21) were also observed. The occurrence of larvae with crooked spines, as well as paralyzed hindlimbs, was significantly greater in aquaria at the higher thermal regimes (Table 34). Overall data suggest that larvae with crooked spines were shorter than those with normal spines (Fig. 22, Table 35). The numbers of specimens affected by crooked spine in each thermal regimes are significantly nonrandom (X2==138.4, P «<0.005). Comparison, however, (Table 36) shows that the mean body lengths at the three temperatures did not differ uniformly with regards to spine condition. Crooked spine abnormalities did not greatly affect body size at any of the temperature regimes (20 C: H =l.2, P >0.05: 25 C: H=0.01, P>0.05; 30 c: H=5.74, p<0.025). 97 Fig. 19. Mean body lengths ( :2 SE) of laboratory reared R. pipiens at three different thermal regimes. Larval lengths are compared at specific stages of development (Gosner, 1960). 98 m_ weaned EmEQo_m>mD *0 002m nv VV ”V 1 mm Va on mm on an Vm (ww) qlfiue'l Apog x 99 NNN FeN NON om.o m.m. .MII -- o.ON awn. -- -- .m11 we mm.o N.NN _N NN.o N.NN 6N oo.N o.mN N we ON.o N.NN NNF Np.o N.mN mNF NN.o N.NN mo, 33 «N.o N.NN mN _¢.o N.oN 6N N¢.o N.NN mN Ne mm.o ..¢N mm co.o N.¢N o, Nm.o N.NN Pm N3 oo.N o.NN N om.o m.NN N om.o o._m NN _N -- c.6N _ -- -- o NF.N o.mm m oe oo.¢ o.oN N -- -- o NN.N N.NN 4 mm mm .x. 2 mm .x 2 mm M 2 26.5265; mo mmmum u on u mN 0 ON Aoomp .gmcmoov Newsaopo>mu mo mommpm xnlumLNQEcu «so mm>gm4 omega pm xgopmconmp mga cw vogue; mcmwnwa mcmm mcmsgoemcmgu mo AEEV msumcwp xuom .mmgapmgmasma pcmememwu .mm mpnmh 100 Fig. 20. Larvae (reared at 30 C) demonstrating abnormally developed "crooked spines." Some specimens are upside-down due to helical swimming patterns. I., 2...». .ha . a 1 Io D , to w . I ‘D .n7. 0;. 101 Figure 20 102 Fig. 21. Larval B, pipiens demonstrating edemaceous and emaciated body condition. Specimens demonstrating these anomalous body conditions were most common in the cohort reared at 20 C. 103 Figure 21 104 Table 34. Occurrence of developmental abnormalities in larval Rana Ejpjgn§_laboratory reared at three different thermal regimes Crooked Spine 20 C 25 C 30 C n 203 250 230 % affected 21% 10% 74% obs. f 43 34 170 exp. f 73.4 90.4 83.2 X2=l38.3** (P «0.005) Paralyzed Hindlimb 20 C 25 C 30 C n 203 250 230 % affected 1% 2% 4% obs. f 2 5 10 exp. f 5.0 6.2 - 5.7 X’=7.5* (p<0.05) *Statistically significant. 105 Fig. 22. Mean body lengths ( :2 SE) of laboratory reared larval .3. pipiens having normal or crooked spines at three different thermal regimes. 106 NN mesmwa uomN P Uom beam 05% too—00.5 O _OELOC 4 :1. N aw I V N I O N £3 £2. (tum) LHBUG'I Apog up I N m 1: Table laboratory at three temperatures. 107 35. Body lengths of 683 transforming Rana pipiens reared in the spines are compared Larvae with normal spines and crooked Group N 7' SE Range CV 20 C Normal Spines 160 30.2 0.28 22-40 11.78 20 C Crooked spines 43 29.5 0.49 24-38 10.97 25 C Normal spines 216 26.4 0.16 19-34 9.05 25 C Crooked spines 34 26.4 0.37 23-32 8.19 30 C Normal spines 60 25.1 0.29 21-30 9.05 30 C Crooked spines 11!; 23.9 0.21 15-33 11.42 683 108 Table 36. Laboratory reared Rana pipiens with normal and crooked spines. Body lengths (mm) of transforming larvae reared at three thermal regimes are compared by stages of development L Normal Spine Crooked Spine Thermal Stage of Regime Development N 'X SE N 'X SE 41 22 31.9 1.03 5 29.8 1.77 42 24 29.2 0.58 7 29.4 1.31 43 18 29.2 0.63 11 29.4 0.79 2° C 44 83 29.9 0.30 20 29.4 0.78 45 2 29.0 2.00 -- -- -- 46 -- -- -- -- -- 41 2 23.5 0.50 0 -- -- 42 9 24 6 1.09 25 1 0 51 43 21 26 0 0.47 27 0 0 77 25 C 44 156 26 8 0.18 19 26 8 0 56 45 26 26 0 0.36 25 3 0 65 46 1 20.0 -- -- -- 41 0 -- -— 2 28.0 2 00 42 2 24 0 1.00 33 24 1 0 58 43 23 5 0.34 23 23 4 0 43 3° C 44 42 25 2 0.34 95 24 3 0 24 45 9 25 2 0.86 12 22 9 0 60 DISCUSSION Field Study: Pitfall Trapping Survey Pitfall traps provide an effective technique for sampling adult amphibian populations (Gibbons and Bennett, 1974). The two anuran species most commonly encountered in the area: Rana pipiens (the leopard frog) and Bufo terrestris (the Southern toad) account for a majority (61%) of all anurans trapped during the 13 month study period (Table 2). Adding the data for Gastrophryne carolinensis (the narrow- mouthed toad) increases the catch of these three dominant species to 86% of all anurans. The remaining 14% of the catch is represented by 10 less prominent species. These data generally conform to the concept that communities are composed of populations of few "common" and numer- ous "rare“ species (Odum, 1971). Although repeated sampling data for terrestrial anurans probably approximate p0pulation values that might be obtained in a census, fig- ures for arboreal species are not as accurate. Adapted to arboreal habits, some representatives of the anuran family Hylidae (especially Acris gryllus and Hyla sp.) possess toe discs that allow them to cling to vertical surfaces. Pitfall trapping data, therefore, underestimate their numbers because they may spend little time on the surface of the ground, and because they can climb out of cans. The numbers of Acris gryllus trapped are especially low. Every month of the year the more 109 110 aquatic cricket frogs can be found around the periphery of Pond C Reservoir. They are residents of the grassy shoreline, and their actual numbers should probably be an order of magnitude or more larger. Even if actual densities were larger by several orders of magnitude, however, they would not approach those of the three dominant species. Pitfall traps effectively measure both localized activity and extended movements. Whereas the frequencies of recaptured specimens are low (usually 3% to 17%), numbers trapped are attributed primarily to movements. Localized activity of few specimens would be character- ized by high recapture frequencies. As specimens were not individually marked, some were, doubtlessly, recaptured more than one time. The numbers of recaptures, therefore, are over-estimated for the three most common species. .Excluding members of the family Hylidae, low recapture rates among anurans are interpreted as a manifestation of reduced popu- lation size in the habitats studied (3, clamitans, B. guercicus) or extreme seasonality (migratory, breeding movements instead of activity: 6, carolinensis, R, areolata, Table 2). The only urodele (salamander) trapped in appreciable quantities (50% of the total 291 specimens) was Plethodon glutinosus (the slimy salamander). The totally terrestrial breeding habits of the species excludes it from interest in the present study. Rana_pipiens--Seasonality Monthly trapping of R. pipiens demonstrates that the greatest numbers occurred in the spring and early summer (Table 3). The body length at transformation is 20 to 33 mm (Wright and Wright, 1949). 111 Although frogs the size of mature adults were caught, the mean body lengths for R, pipiens trapped from March to June closely approximated the size of emergent transformees. A listing of the ranges in body lengths show that recently metamorphosed young ( <31 mm) were present every month of the year. In February 1972, however, all 11 specimens trapped were recaptures (Table 3). When total captures are separated into initial captures and recaptures, the proportion of specimens trapped for the first time (initial captures) corresponded to the total numbers trapped, and the mean body lengths of initial captures was consistently smaller than values of total captures. The high proportion of R. pipiens trapped initially each month (low numbers of recaptures) indicates that pitfall data represent directional movement instead of local activity. An aver- age of 17% of trapped specimens were recaptures. Since data do not account for multiple recaptures, recapture estimates are probably some- what high. A very strong negative correlation (-O.68) between mean body length and numbers of R, pipiens trapped biweekly is evidenced by the short body lengths of leopard frogs sampled during the spring period of peak activity. After July, when numbers of trapped specimens deClined, the data demonstrate a higher proportion of adult 3, pipiens in pitfalls. Emergent young were trapped after September (as evidenced by the minimum lengths of specimens captured), even though mean body lengths are in the size ranges of adult specimens. The strong negative correlation between numbers trapped biweekly and body length reiterate that transformees dominate populations in the 112 spring and adults dominate populations in the late summer and winter. This relationship poses interesting speculation concerning population size structure and the carrying capacity of the environment. Appro- priate analysis might reveal a homeostatic level of frog biomass per unit area. There may be a calculable carrying capacity associated with a habitat which can function under large populations of small frogs or small populations of large frogs. The mechanisms and feed back systems regulating such a hypothetical, homeostatic level would be an appr0priate t0pic for further study. A maximum overall recapture of 301 (17%) of the B, pipiens trapped suggests that most specimens were not encountered repeatedly, and that data represent accurate population trends rather than much activity of a few specimens. Monthly occurrence of immature R. pipiens (Table 4) portrays the pattern of emerging young in the vicinity of the reservoir, and reiterates that continual breeding and metamorphosis occur there. Since the breeding migration of R, pipiens occurs over a much longer period of time than that of B, terrestris, one cannot accurately determine exactly when eggs were deposited. For this reason it is not possible to test for size differences of emergent transformees among thermally distinct areas. Leopard frogs are known to breed throughout the year in the Southern parts of the country (although primarily in the spring). 8, terrestris, however, demonstrates a sudden (explosive) breeding migration that usually occurs within a few nights of heavy rainfall (during March on the S. R. P.). This contention is further 113 substantiated below (cf., "Reservoir Sampling of Larval Bufo terrestris“). Bufo terrestris--Seasonality In contrast to the activity and emergence of R, pipiens through- out most of the year, B, terrestris were encountered nine months of the year, and emergent transformees trapped only May to July. Monthly trapping data (Table 5) demonstrate a bimodal activity pattern of migrating adults (March and April) and emergent young (May, June and July). The mean body lengths of initial captures from May to July, considerably lower than other months, give evidence of metamorphosis at that time. The previously marked individuals captured in May to July did not include emergent young. B, terrestris is not highly aquatic. It occurs in large numbers on breeding migrations of adults to and from water and on emergent migrations of transformees. The low number of specimens recaptured (272) represents the same proportion (17%) found for R, pipiens. Although low, this pro- portion also is slightly over-estimated due to successive recaptures of some specimens. Since recaptures were few, trapping data are inter- preted as a representation of large numbers of B, terrestris (movement) rather than multiple recaptures (activity) of a few specimens. Gastrophryne carolinensis--Seasonality Uncommon except during the breeding season from May to October (Wright and Wright, 1949). E, carolinensis were trapped in nine of the 13 months of study. Although third in abundance of 13 anuran Species, 114 B, carolinensis (more terrestrial and more restricted in monthly occurrence than B, terrestris) demonstrated only 3% recaptures. The bimodial activity pattern represents overlapping breeding migrations of adults (June to August) and emergence migrations of transformed young (August to October; Table 6). Since emerging transformees (8.5-12.0 mm; Wright and Wright, 1949) were trapped during four months, B, carolinensis were shown to breed and develop in the vicinity of the reservoir. On several occasions eggs were observed in the reservoir proper and in shallow, temporary rain pools. Although the minimum body length of specimens trapped was 10 mm, sufficient transformees were not caught to allow for a comparison of body lengths among areas or with the literature. AcrisBgryllus--Seasonality Acris gryllus (the cricket frog) is commonly found along the shoreline of Pond C Reservoir. As these small, active frogs easily held onto the sides of pitfalls and escaped the traps, pitfall data do not accurately characterize their abundance (Table 7). They were encountered throughout the 13 month period of study, but remained localized within the emergent vegetation along shore. Specimens were not caught in traps located in wooded areas. Data for specimens that escaped pitfalls are not presented. Emergent transformees were trapped from July to December; A, gryllus emerge at 9 to 15 m body length from April to October (Wright and Wright, 1949). Trapping of immatures in Pond C Reservoir did not 115 represent modifications in breeding habits (from July to December). The low occurrence of recaptures (2.9%) reiterates the inefficiency of pitfalls for sampling this species, as well as the absence of pro- nounced breeding migrations. Most adults probably remain closer to the water than where pitfalls were placed. The seasonality shown coincides with the normal pattern for the species, not greatly affected by the thermally polluted reservoir. Pseudacris triseriata--Seasonality Pseudacris triseriata feriarum (the upland chorus frog) commonly breeds from February until May and emerges until mid-June at 8 to 12 mm (Wright and Wright, 1949). It was not caught in great numbers. Unlike Acris gryllus and Hyla sp., B, triseriata could not easily escape pit- falls. Trapping data are therefore considered to be representative of actual population density (Table 8). A unimodal seasonal activity peak of adults (April to May) precedes emergence of young (June). The seasonal occurrence of B, terrestris, B, carolinensis, B, gryllus and B, triseriata are comparable with patterns demonstrated by populations elsewhere on the Savannah River Plant where the water is not thermally affected (Gibbons, in preparation). Data presented for B, pipiens, however, demonstrate activity every month, and emergence of transformees for 11 months of the year. Breeding of adults and emergence of transformees therefore occur with greater frequency at Pond C Reservoir than at Karen's Pond or Risher Pond on the Savannah River Plant. Further comparisons are presented below. 116 Rana,pipiens--Distribution Pitfall catch per trap is presented for B, pipiens (Table 9). Traps are listed in order of position along the thermal gradient (cool to hot), and are grouped according to thermal zones (Table 1, Fig. 2). It is clearly evident that more specimens were trapped at the cool end as opposed to the hot end of the thermal gradient. An overall low frequency of recaptured specimens (17%) substantiates that the high numbers trapped represent many individuals rather than successive recaptures of a few. Variability in catch per trap, however, is very high and numbers do not closely conform to the thermal gradient. High variability among traps is due mainly to positioning effects. As trap sites were systematically spaced at regular intervals along the thermal gradient, they represent an array of habitat types: woods, grassland, marshy seepage areas and several ecotones. Catch per trap area was highest in pitfalls associated with nearby cool seepage ponds (areas A, B, I, J, and Z) or reservoir shallows receiving cool seepage where larval deve10pment was known to occur (areas V and K) (Fig. 2). When reactor activity was peri- odically discontinued, reservoir water levels declined 0.3 to 1.0 m. At various times, developing larval B, pipiens were secured from the exposed shorelines of the reservoir near trap sites V, W, E, and K: doubtless they occurred elsewhere as well, although undetected. Trapping area 2, although associated with a seepage pond, did not demonstrate a particularly high occurrence of B, pipiens. This site was considerably removed from other ponds; terrain there was xeric, without apparent seepage, and at the hot extreme of the thermal gradient. 117 Pitfall catch, therefore, represents a combination of variable activity at the different sampling areas, emergence of young from cool peripheral seepage ponds, and emergence of young from reservoir shallows. Total catch of B, pipiens was lowest at trapping sites F, G, H, X and M. Trapping areas F, G, H and X were all surrounded by woods; area M was partly wooded and very xeric. Unfavorable habitats (elevated and wooded) at trapping areas F and G are indicated by the absence of adults caught there. Minimum values of mean body lengths for specimens trapped indicate the presence of emergent transformees at all trap areas. Rather than newly emergent young, however, specimens in some areas must represent the migration of young frogs from other reservoir areas. No- where within 50 m of trap area M (for instance) is there an aquatic habitat in which larval amphibians might develop. Data for initial captures exclude those specimens recaptured. Trap sites associated with seepage ponds or reservoir shallows (A, B, I, J, V, and W) catch the greatest numbers of specimens. Recapture rates for specific traps ranged from 6% (U) to 31% (W). Mean body lengths for recaptures were generally larger than for initial captures. These data suggest that more adults were recaptured than immatures. Two-way breeding migrations would expose adults to a greater likelihood of capture. Bufo terrestris--Distribution Bufo terrestris (the Southern toad) was caught most frequently (52%) in pitfall trap areas A, B and C (all in thermal zone I, Table 10). Emergent transformees (5 to 11 mm) were removed only from trap areas A 118 and B. Although the numbers trapped are much higher in thermal zone I, they do not decline steadily in traps progressing toward the hot extreme of the thermal gradient (Table 17). B, terrestris and B, carolinensis are less aquatic than other anurans for which data are presented. They are normally located around water in great numbers only during breeding migrations (B, terrestris in the spring, B, carolinensis in the summer). During most of the summer and autumn they remain active and may be encountered even in dry, ele- vated habitats. Greater numbers trapped at the "cooler" extreme of the thermal gradient (zone I) probably results from the natural drainage there. Elevation there is less than anywhere else along the cool arm of the reservoir. In March 1972, B, terrestris larvae were found and widely collected along the reservoir shore (cf. "Reservoir Sampling of Larval Bufo terrestris"). Specimens are believed to have occurred in many areas where they were not trapped in pitfalls. Initial captures of specimens were also most numerous in zone 1. Very few emergent transformees were encountered; their influence was insufficient to affect the mean body size of specimens trapped. Emer- gent young were trapped only as initial captures; none were recaptured. The infrequency of captures and absence of recaptures show that newly transformed B, terrestris migrate away from the water and into surround- ing terrestrial habitats. Pitfall captures of emergent toads must, therefore, greatly underestimate their numbers. Since they are small and move only short distances at a time, they may well have avoided pitfalls or have been eaten by adults therein. 119 Recaptures (as total captures) were least in trap sites U, V, X and 2. There is no apparent reason for lowest trapping success to occur among traps on the same side of the reservoir. Habitats at these trap sites are not greatly similar. Trapping area V (that caught the highest proportion of B. pipiens) was a site of considerable larval amphibian development (cf. "Reservoir Sampling with Dipnets and Minnow Traps"). Cooled by shoreline seepage there, the reservoir shallows should have provided an especially favorable habitat for larval toads. Trapping area Z was closely related to a nearby seepage pond, but this site also yielded low levels of B, terrestris. If the emergent young emigrated along depressed terrain, however, they would have missed the pitfalls at area 2 (which were elevated on a sandy mound). A high water table and considerable seepage at site V neces- sitated placement of pitfalls at greater distances from the reservoir than elsewhere. As the development of B, terrestris there is documented below, the low occurrence of specimens in trapping area V is attributed to positioning of pitfalls outside natural emigration routes. Dense brush was located directly in front of the pitfall-drift fence section at area V. Emigrating young probably follow the shoreline until a suitable pathway is found. Although the body lengths of newly transfbrmed B, pipiens did not differ from the values reported by Wright and Wright (1949), B, terrestris did not conform to their data. The authors list the body lengths of recently metamorphosed B, terrestris as 6.5 to 11 mm. Many specimens trapped in pitfalls were as small as 5 mm (Table 10). Data 120 are not sufficient to test the statistical significance of this apparent difference. The minimum body lengths of B, terrestris removed from Karen's Pond (Table 21) was 7 mm, which is closer to the expected size range. Although the smaller size of B, terrestris from Pond C Reservoir might be explained by random variation, data suggest that the body size of newly transformed toads in the vicinity of the heated reservoir is less than might be expected. Gastrophryne carolinensis--Distribution Gastrophryne carolinensis (the Eastern narrow-mouthed toad) was also trapped in all pitfalls. A majority (52%) of the specimens occurred in pitfall areas A, B, C and Z. They were not equally dense in all traps near seepage ponds, but 25% occurred in trap area Z (near a cool seepage pond there, Table 11). Emergent young were trapped in pitfall areas A, U, C, V, H, J and 2. Habitat types at these sites are highly variant. The high proportion of transformees caught at area 2 is expressed in the very low mean length there. The low overall recapture rate (2%), as well as that for trapping area 2 (3%), demonstrates the explosive nature of breeding migrations in B, carolinensis. Of the anuran species discussed, B, carolinensis manifests the shortest seasonal activity (nine months), remaining around the water for the shortest period of time (Table 2). Because of the high occurrence of specimens at trap area Z, any direct conformity between the thermal gradient and numbers trapped is obscured. 121 Dominant Anurans--Distribution The numbers of specimens caught per trap area among the three dominant species of anurans exhibit a general trend of more specimens at the "cool" end and fewer specimens at the "hot" end of the thermal gradient (Table 12). The highly aquatic B, pipiens shows this trend best, followed by the more terrestrial B, terrestris. Total catch is greatest in zone I for all three species. Data for B, carolinensis. however, disrupt the gradient response shown by the other two anurans. The combined numbers of the three dominant species do not show a clear gradient response, although the greatest numbers (in overall ranking) were caught in the trap areas at zone I (A, B, C) and the fewest in a trap area in zone VI (L). Localized effects of terrain and habitat greatly influence anuran responses to the thermal gradient. Overall values for zone III are especially low because of wooded trap areas F and G. Ranking of traps by capture success for the three anuran species demonstrates these differences, and result in a disordered array of overall ranks. These differences are due to location effects: variable affinities for shoreline areas V and W, semi-wooded area U, and cool seepage pond Z particularly. Analysis of the thermal gradient, therefore, is shown to be difficult even though the temperature differential is very real. Localized effects of specific trap habitats and chance occurrence of a species in any given seepage pond considerably influence overall numbers per trap. 122 Acrisgryllus--Distribution As already mentioned, pitfalls are less effective for sampling most tree frogs and their relatives (family Hylidae) than other anurans, because they possess expanded toe diScs that are adapted for clinging to : vertical surfaces. The Southern cricket frog (Acris gryllus) was the only major anuran subject to this trapping bias. Although B, triseriata is also a member of the same family, it possesses reduced toe discs that do not allow specimens to escape by scaling the vertical surfaces of pitfalls. B, gryllus would often leave the pitfalls before they could be measured and recorded. Although the actual numbers trapped are known to be greatly underestimated, the relative abundance among traps should represent valid comparisons. Specimens should have escaped from all pitfall traps with equal ease. Traps catching the highest numbers of specimens (I, J, W, E) are similar in placement near littoral, emergent vegetation (Table 13). Although traps I and J are both associated with cool seepage ponds, trap Z (also near a seepage pond) was approximately 2 m removed from aquatic vegetation, and caught few numbers. B, gryllus remains localized in vegetation along the reservoir or seepage ponds throughout the year. Traps away from vegetation (as at wooded sites F, G, H, X, Y) caught few specimens. Positioning of the pitfall at site V from the shallow shoreline resulted in few specimens being trapped there also. These data, along with a low recapture rate (3%), reiterate the reduced mobility exhibited by this species. 123 Emergent young (9-15 mm) were encountered throughout the gradient. Because of the extensive shoreline seepage along the reservoir and the limited mobility demonstrated by B, gryllus within these areas, the species is not considered to be as severely affected by the thermal loading as wider distributed anurans having larger, more mobile larvae. Adults continuously litter the shoreline along the periphery of the cool arm of Pond C Reservoir. The monthly sampling program revealed that larvae of B, gryllus did occur in parts of the reservoir proper (cf. "Reservoir Sampling with Dipnets and Minnow Traps"). Pseudacris triseriata-~Distribution Pitfall catch for B, triseriata, the upland chorus frog, was least among the five dominant anuran species trapped (Table 2). The low numbers of specimens trapped (172) and the low recapture frequency (4%) suggest that the distribution of this species is less extensive than other anurans. Larger numbers of specimens trapped at area 2 may well indicate a preference for cool ponds over the heated reservoir. Lower numbers of chorus frogs at other pitfall trapping areas (A, B, I and J), where cool seepage ponds also occur, however, indicate that reproduction is extremely localized (Table 14). Since emergent transformees were trapped at area 2, they are known to have completed development there. Emergent young were also caught at pitfall trapping area A (where few specimens were encountered). Larger numbers of specimens caught at pitfall site V and the low overall 124 mean body length of specimens trapped there suggest that B, triseriata also may well breed and deve10p in Pond C Reservoir. Immature Rana_pipiens To analyze the occurrence of immature B, pipiens along the thermal gradient by season, the numbers of immatures ( <47 mm body length) initially caught per trap are listed by month (Table 15). The symbol (°) indicates the presence of at least one recently transformed specimen ( <30 mm). The high negative correlation already shown between the numbers of specimens trapped biweekly and their body lengths is sufficient evidence to assume that the presence of many immature larvae represents large numbers of emergent transformees. This is shown to be true. Only four monthly records of more than 10 captures per trap area fail to include transformees: trap A for July, trap C for February and June, and trap E for April. The July catch at trap site A contains no specimens 3<30 mm, although they were encountered there during the two months before and month following. This does not mean, however, that the specimens trapped did not include transformees. As already stated, Wright and Wright (1949) report the body lengths of newly metamorphosed B, pipiens as 30-33 mm. Many of the 57 immature specimens trapped were twithin this range. To assure that specimens were indeed transformees, a conservative criterion (‘<30 mm) was used to identify transformees. The monthly totals of immature B, pipiens caught per trap certainly included larger transformees. None of the figures indicated by the symbol (') (Table 15), however, can be doubted to represent recently metamorphosed frogs. 125 The catch for February and June at site C and for April at site E do not include specimens «<30 mm, although transformees were trapped in adjoining months. Either the metamorphosed larvae were actually not encountered or they were larger than those elsewhere. Larvae laboratory-reared at cooler temperatures have been shown to attain larger body size than those at warmer ones (Etkin, 1968: cf. "Rearing of Larval Rana pipiens"). Although overall peak metamorphic activity was demonstrated from May to July, larvae have been shown to occur year around at certain trap sites. Emergence of young at trap area Y continued for seven successive months. The pitfalls at site Y are very close to the reservoir pr0per. Considerable seepage into the reservoir allows devel- 0pment of young there. The absence of other breeding ponds at trap area Y indicates that frogs did emerge from the reservoir proper. Although trap area Y is located near the hot extreme of the thermal gradient (Fig. 2, Table l), the shoreline there is cooled by a localized pothole of underwater seepage. When the reservoir water level drops :>6 cm, it exposes a protective shelf of sand that gives the appearance of an atoll (Fig. 12). The success of larval development there is extremely probabilistic, depending upon a combination of stresses: fluctuating water level and critical thermal tolerance (reactor activity). Because of increased developmental rates at elevated temperatures, emergence at Y early in the year may represent more rapid development there, or earlier breeding there. When compared 126 to the heated reservoir, cool seepage ponds might well retard developmental rates but cause larvae to grow and metamorphose at a larger size. The absence of emergent transformees at trap area A in July might well represent the increased size of larvae reared at cool temperatures. Body lengths of newly transformed B, pipiens trapped in pitfalls were apparently not different from the size range reported for trans- formees by Wright and Wright, 1949 (22-33 mm). Although the body size of recently metamorphosed frogs may be influenced by the increased tem- peratures of the heated reservoir, data from the pitfall study do not demonstrate such an effect. Minimum body lengths recorded for B, pipiens are not smaller than the values reported by Wright and Wright (1949). Cool seepage areas (A, B, I, J, Z) demonstrate the occurrence of numerous transformed larvae. Traps surrounded by dry woody habitats (D, F, G, H, M) caught fewer young overall and much fewer emergent transformees. The Gradient Effect Since the temperatures in the cool arm of Pond C Reservoir may vary from lethal (near 50 C) at the mouth to ambient at the opposite extremity, one might expect the distribution of specimens in the designated thermal zones to reflect the degree of stress sustained along the shoreline. It was of interest to determine whether or not adult or immature anurans occur uniformly along the heated reservoir. 127 To summarize population responses to the overall thermal gradient, the mean numbers of the five dominant species trapped in pitfalls were compared among thermal zones (Tables 16-19). Data represent the mean numbers of anurans trapped among pitfalls at each zone. Probabilistic breeding success among species at certain periph- eral seepage ponds and variant habitat requirements have been shown to be sufficient to distort generalized clinal phenomena. Local condi- tions at specific trap sites along the thermal gradient appear to elicit responses from the anurans studied that override to some degree, the manifestations of the thermal gradient. Although numerous recently transformed B, pipiens were caught at protected area Y in February and-March, 1971 (Table 15), none were trapped during the same months of 1972. The effect of conditions there (degree of seepage and protection conferred by a surrounding sandy shelf) are doubtless influenced by the reservoir water level (Fig. 12). Successful development of larvae there occurs only because of conditions that protect the microenvironment from otherwise lethal temperatures. Doubtless the possibility of increased reactor activity (resulting in increased temperatures) makes the probability of successful deve10pment there low. The occurrence of favorable conditions such as these (though tenuous) disrupt the continuity of an otherwise well delineated thermal gradient. Unpredictable occurrences of this sort are wherein lie the hope of organisms to otherwise uninhabitable ecosystems. Adults of all five dominant anuran species were trapped through- out the periphery of Pond C Reservoir. The occurrence of B, pipiens, 128 B, terrestris and B, gryllus was lowest at the hot extreme of the thermal gradient (zone VI). In the same zone, however, especially high numbers of adult B, carolinensis and B, triseriata were trapped (as were most of the few immatures of these species). These data probably represent random, successful breeding there. Receiving little visible shoreline seepage, cool pond Z became virtually dry when the water level of the reservoir dropped appreciably. Neither was the pond ever contiguous with the reservoir. Probably for these reasons, cool pond 2 was unique in having no fish. Larval anurans therefore might be free of predatory pressures that occur in all other aquatic habitats in the vicinity. Due to supposed bad taste, larval anurans are believed not to be subject to predation. Palpitated specimens of the snake genera BBBBiB_sp. and Thamnophis sp., however, reveal that larval anurans naturally comprise part of their diets. Bluegill and immature largemouth bass might well feed upon larval anurans. Because of unexpected successful development of B, pipiens at protected area Y (zone V, Fig. 2), 1e0pard frogs were most numerous in pitfalls at zones I, II and V. Initial captures of immature B, pipiens and B, terrestris demonstrate an occurrence frequency virtually identi- cal to that of the adults. As might be expected, the young emerge in greatest numbers at zones where most of the corresponding adults were found. If many adults had been trapped in migration at the reservoir, yet no young were encountered, then data might circumstantially suggest embryonic/larval mortality. Data do not support such a contention; perhaps they are insufficient. Severe embryonic mortality was 129 documented at reservoir area K: B, pipiens and B, terrestris (cf. "Reservoir Sampling with Dipnets and Minnow Traps" and "Reservoir Sampling of Larval Bufo terrestris"). As already indicated, the greatest numbers of B, carolinensis were caught at trap area 2 (zone VI) which was adjacent to prairie-like terrain. Numerous young emerged from nearby cool pond Z. The prepon- derance of specimens around the hottest thermal zone, therefore, is interpreted as an isolated Opportunistic occurrence rather than a response to the reservoir's thermal conditions there (which were usually supralethal to animals). Pitfall trapping data for B, triseriata and B, gryllus are insufficient to compare trends among adults and ini- tially captured immatures, but as with other anurans, adults were trapped through the study area. Body Lengths of Pitfall-Trgpped Bufo terrestris and'Ranagpjpjens on the Savannah RiverIPTant Body lengths of specimens trapped in pitfalls are compared among three habitats on the Savannah River Plant for the two dominant species (Tables 20 and 21). Although the months of greatest activity were sim- ilar for both species, emergent young were consistently trapped earliest at the heated reservoir. In addition, the body lengths of emergent young were consistently smallest at the heated reservoir for both species. Such a consistent trend between the two anurans alludes to possible growth inhibition in the heated study areas. To be conclusive, data must relate body size to age; there is no technique, however, to age amphibians. The size differences observed might represent random 130 variation. Nonetheless, the minimum lengths recorded are assumed to represent the size of transformees. Data summaries represent striking similarities among the three habitats. Other than the presence of smallest transformees at heated Pond C Reservoir, the mean body lengths and variances are strikingly similar for both species. The absence of recently metamorphosed B, terrestris at Risher Pond is attributed to predation by fish and low densities there. Considerable variation may occur among years, but data do not show whether apparent size differentials are real or artifact. Unfortunately, data are not available from all areas for the same year. Field Stugy: Reservoir Sampling with ijnets andfiMifinow Trapg Larvae of nine amphibian species were identified among the 15 consecutive monthly dipnet samples and in minnow trap samples taken in and around the heated reservoir. Cumulative samples included at least one specimen of four anuran larvae (B, pipiens, B, terrestris, B, gryllus and B, cinerea) in all areas. Species diversity was greatest in pond 2, which was never contiguous with the reservoir proper, and least in heated reservoir area K. Very few specimens of the two salamander species were ever encountered. Because of certain differences among sampling areas, they are not amenable to rigorous quantitative comparisons. Most data presented, therefore, are qualitative. The peripheral cool ponds (Z, A, I and B) were well circumscribed bodies of water that differed in size less than 131 an order of magnitude. The shoreline at reservoir area V, however, was considerably larger. Although maximum water depths at the ponds were generally not greater than 0.5 m, they were by no means uniform. Different water volumes, amounts of seepage, and vegetation types also distinguish the areas from one another and precluded a strictly quanti- tative comparison. To prevent the removal of entire p0pulations where species diversity was low, only a few representative specimens were removed. Generally, specimens could be identified in the field, so that not all larvae needed to be preserved. An expression of quantitative comparisons is evident in the mean numbers of species removed per monthly sample. Cool pond Z revealed an average of 3.1 species per month, whereas faunally depaup- erate, hot reservoir area K revealed 0.6 species per month: less than one species per monthly sample. Other areas sampled revealed densities intermediate to these two extremes. Another quantitative expression is found in the number of samples in which no larvae occurred. Data show that amphibian larvae of some species (usually B, pipiens) were always present in cool ponds Z, A and I. Over half (57%) of the samples taken at heated area K, however, revealed no specimens. These data attest to the thermally stressed conditions there. Although eggs of B. carolinensis were sampled at area K, no larvae were ever observed. Presumably all specimens in area K died. Although species diversity over the 15 month study period was identical for sampling areas B and V, much greater numbers were encountered at cool pond B than at reser- voir shore V. These differences are not apparent from the summarized monthly data (Table 22). 132 Comparing the monthly samples of larval amphibians among the six areas, species diversity at non-contiguous cool pond Z is 100% greater than corresponding figures from heated area K and 25% greater than those from heated area V. Data for the other cool ponds (occa- sionally contiguous with the reservoir) fall somewhere between these two extremes. Over the 15 month study, the average number of species trapped per month at pond 2 was 500% greater than heated area K and almost 300% greater than heated area V, with intermediate values for the other cool areas. In addition, more than half of the monthly samples taken at heated area K revealed no larvae. Fourteen percent of the samples from heated area V and depauperate cool pond B revealed no specimens. All other areas yielded at least one species for each monthly sample. The disparity of data between areas Z and K are outstanding. Greatest overall results (diversity and commonness) at area Z may well be partly due to the absence of fish there. The absence of elevated temperatures from reservoir overflow at pond Z doubtless contributes to stability and resulting greater diversity. Sparse samples at area K are interpreted as a result of thermally stressed conditions there. The minnow traps continuously maintained in all areas during the same 15 months, provide comparable data. All anuran species caught in minnow traps were also removed in monthly dipnet samples. Lgpomis macrochirus (bluegill) were minnow trapped in all areas except pond Z. Specimens captured at heated area K usually died in the traps. Fish and anuran larvae demonstrate an inability to withstand 133 sustained high temperature levels. Massive fish kills as noted at area K did not occur elsewhere. Embryonic and larval mortality for B, terrestris and B, pipiens were observed several times. Except in shallow cool seepage areas, temperatures at area K were apparently lethal except during periods of heavy rainfall. Sampling data reveal greatest species diversity, population sizes, and frequency of occurrence (commonness) of larval amphibians at cool pond Z. Other cool ponds were less favorable, and heated reservoir areas considerably less favorable to larval amphibians. The vast differences observed among areas are partly indicative of thermally stressed conditions in the heated reservoir. Total Lengths of Larval Rana pipiens Many specimens removed from minnow traps and taken in the 15 successive monthly samples were measured (total length, mm) and staged according to the level of deve10pment (Gosner, 1960). To determine whether size differences were related to the thermal regime from which larvae were removed, larval lengths are compared (by specific develop- mental stage) for each of three sampling areas (Fig. 13). Pond 2 was cool, unaffected by thermal effluent. Shore areas V were characterized by higher temperatures (cf. Table 1, Fig. 2). The fewest numbers of specimens were removed from the hottest area (K), where larvae were not commonly encountered. Most larvae trapped there were removed, yet data for only 27 larvae were available. Since specimens were always avail- able at cool pond Z, few were removed. At intermediately heated area V. 134 however, most specimens were removed. Low survivorship of larvae at heated area K reduced the probability of capturing specimens a second time. Therefore, they were not released. At all stages of development, larvae removed from the hottest area K were smallest. Larvae removed from cool pond 2 were usually the largest. Small sample sizes may account for inconsistencies at advanced stages 35 to 38. Overall larval lengths are reduced in these stages as the larvae near transformation (stage 46). Differences, therefore, may be less distinct. Sample sizes are small at some stages for sampling area K, and inconsistencies should be expected. The overall trend, however, shows largest larvae in the coolest area (B) and smallest larvae in the warm- est area (K). These data support the contention that growth in amphib- ian larvae is inversely related to temperature (within the range of temperatures studied). Field Study: Reservoir Sampligg of Larval Bufé terrestris Larvae sampled in this study are believed to have originated primarily from the massive spring breeding migration prior to 22 March 1972. Concomitant with the greatest monthly rainfall (2.3 cm), a record number of adult B, terrestris (562) were trapped in pitfall traps around the reservoir on this day. Data from another pitfall study of amphibian activity conducted the same year at Karen's Pond (a 0.1 ha vernal pond on the Savannah River Plant, 13.0 km from the reservoir), revealed the 135 greatest yearly catch of B, terrestris on the same day. Pitfall captures (Table 23) indicated that little movement occurred on other days. Successive samples, therefore, were from discrete populations at each location except K (Table 24), and comparisons could be made of stages and lengths as functions of time and temperature. As already mentioned, high egg and embryonic mortality is the overriding effect of thermal loading in hotter sections of the reservoir. Area K was the most severely stressed location from which larval amphib- ians were removed in any of the sampling programs. Initial (=>95%) heat death of B, terrestri§_eggs among three clutches there on 29 March and 17 clutches of B, pipiens on 22 January document the great extent of embryonic mortality. Since only living embryos were encountered else- where along the reservoir, severe mortality at area K is attributed to heat death. All viable embryos at area K occurred in egg masses situ- ated where cool stream seepage entered the reservoir, cooling temper- atures there. Ruibal (1959) reported similar marginal survival of Rana pipiens eggs in seepage areas of hypersaline desert streams. The presence of dead embryos at heated site K suggests either that adult toads deposited eggs in water temperatures exceeding their maximum thermal tolerance (33 C; Volpe, 1953), or that they deposited eggs in water only temporarily at sublethal temperatures. Hathaway (1928) reported that larvae of Bufo americanus could withstand a maximum temperature of 36.3 C for 24 hours. Heated effluent entered the reser- voir continuously for several days before the appearance of eggs at area K. Although the eggs were deposited within a temperature range 136 of 29-36 C, the temperatures probably remained closer to 36 C, falling to 29 C only during heavy rainfall. 0n the night of the massive breeding migration, the cooling influence of rainfall did permit initial survival of larvae. Many, however, died when lethal thermal conditions were restored. Mean March water temperature fluctuations (maximum-minimum) recorded at area K during rains were 14 C, as opposed to 8 C when no rainfall was recorded. Samples from 29 March and 5 April attest to the survival of a small number of embryos from clutches laid subsequent to 22 March (Table 24). The reduction from 29 March to 5 April in both stages and lengths of the two samples from site K indicates that they represent two distinct cohorts each week. The absence of successive samples from a single population at area K is interpreted as a manifestation of thermal stress and attests to larval mortality, in addition to that observed in embryos. Since only living larvae were encountered elsewhere in the reservoir, extreme mortality at area K is attributed to heat death. Within the range of thermal tolerance at the other areas, both lengths and stages of larvae sampled reflect the level of thermal load- ing sustained. In the first sample (29 March), both lengths (Table 25) and stages of development (Fig. 14) differed significantly among the three areas. Stages from area B were appreciably ahead of the others in development as eggs were deposited there earliest of all. Toads migrating to the reservoir would be expected to breed in peripheral ponds before reaching the reservoir proper. More rapid development in warmer sites A and V allowed individuals in successive samples from , 137 these areas to surpass larvae at site B in size and stage. Mean stages and lengths did not differ significantly among the three sampling areas in the second weekly sample (5 April) due to this reordering. Larvae from the three reservoir stations on 5 April clearly demonstrate in- creased development and growth among larvae in heated parts of the thermal gradient. Within two weeks of development at area D, larvae were nearing transformation (stage 46). Specimens from area B were consistently larger than those from areas A or V at comparable stages of development. The test data strongly suggest that metamorphic lengths attained by larvae reared at cool temperatures are significantly larger than those attained in the areas receiving the influence of heated effluent (Table 26). Fish and larval amphibians maintained at lower temperatures in the laboratory have been shown to develop more slowly and to attain a larger size than those at warmer temperatures (Etkin, 1955; Kokurewicz, 1969; Muto, 1972; Wilbur and Collins, 1973). Variation due to measuring is greater for toad larvae than for larger anuran species (g_g, Rana pipiens). If the lengths of metamorphic toads were larger, the size differences among sampling areas would likely be more clearly demonstrated. The adaptation of anurans to nocturnal breeding migrations during rainfall fortuitously confers a double advantage to survival in a thermally polluted reservoir. Not only are nocturnal temperatures lower than those during the day, but substantial amounts of rainfall that commonly accompany anuran breeding migrations further reduce water temperatures in the reservoir. Toads lay eggs at night or early morning 138 when water temperatures are low. Embryos thereby pass through the most thermally sensitive stages (uncleaved egg and gastrulation; Atlas, 1935; Grainger, 1959; Hoadley, 1938; King, 1903; Moore, 1942b; Volpe, 1953) before water temperatures increase and approach the maximum temperature tolerance of early embryos (Brown, 1967; Zweifel, 1968). Anurans depositing eggs in a heated reservoir, however, are denied one of the inherent advantages of breeding in reduced nocturnal temperatures. High egg and embryonic mortality, therefore, is the obvious overriding effect of thermal loading in hotter sections of such a reservoir. Data from these natural field populations corroborate trends demonstrated among larval amphibians in the laboratory. Since condi- tions prevailing in field situations are much more variable than those maintained in laboratory experiments, thermal responses of organisms and p0pulations in nature are not as easily documented. Since field conditions are not nearly as well controlled as those in the laboratory, statistical significance is not as easily documented. Variability in field conditions at the study areas probably accounts for the fact that only two of three analyses were statistically significant. Although water temperatures were monitored at specific locations, toad larvae are known to thermoregulate behaviorally. They have been shown to selectively aggregate in warmer areas of a pond. Perhaps they also select cooler, optimum temperatures. Brattstrom (1962, 1963) documented several cases of behavioral thermoregulation in larval anurans and dis- cussed its adaptive significance. For this reason, field studies are needed to determine the extent to which thermal parameters derived from 139 the laboratory can be extrapolated to actual conditions in nature. Field data should be instructive in determining thermal limits that can be imposed on biological systems without destroying stability. In addition to the overriding effect of outright heat death, precocious development and retarded growth in thermally loaded areas, receding water levels, predation and shortages of food and cover could impose other threats of mortality. During the period of time that the toads were developing in the reservoir, cessation of flow from the thermal canal probably would have resulted in the death of all larvae except those in cool pond B or the inland pool at site V. Reactor shutdown results in receding water levels that leave larvae exposed on dry ground. The biological effects of fluctuating water levels are discussed by Kroger, 1973. Since temperatures in the reservoir exceed the thermal tolerance of most organisms, blue-green algae comprise most of the flora, with the exception of scattered patches of green algae (Spirogyra sp.). The sterile vegetative conditions in the reservoir proper provide little food and cover for anuran larvae. Starvation and predation, therefore, are likely secondary sources of mortality. Predaceous birds (kingfish- ers, herons, egrets) are residents of the reservoir and have been observed feeding along the shore where larval B, terrestris occurred. Banded water snakes (Natrix sipedon) and eastern garter snakes (Thamn0phis sirtalis) were also trapped along the reservoir. Both are known to prey upon larval amphibians. 140 Laboratory Stugy: Rearigg of Larval Rana pipiens A full report of specimens (Table 27) shows the fate of the 1,600 original embryos. Data for each replicate tank were categorized into four classes: accidental deaths, "natural" mortality, transforma- tion through late metamorphic stages, and specimens unaccounted for. Each group is discussed separately below. Accidental Deaths The major cause of accidental, experimental mortality was inadvertent mashing of larvae with either the stainless steel siphon covers or weights that stabilized undergravel filters. Increased pre- cautions reduced subsequent accidental deaths from initially higher levels. Large rocks used to hold down undergravel filters (Fig. 11) were replaced with culture dishes (Fig. 10). Since the larvae could be viewed through the glass culture dishes, specimens weren't crushed beneath these weights as they were with the rocks. For unexplained reasons, certain aquaria were slightly more hazardous to larval survival than others (§_g, 20 C--Tank C, 25 C--tank B; Table 27). Accidental deaths were lowest (0.5%) at 35 C where the short-lived embryos were subjected to very little laboratory manipulation in the nine days that some survived. In the three other thermal regimes, accidental deaths were similarly low (ranging from 4.0% to 5.2%). Variability among the three warmer temperatures is attributed to randomness; the total loss to accidental deaths (N==58, 3.6%) is considered negligible. 141 Mortality Observed mortality was greatest (313, 78.2%) in the group of embryos reared at 35 C. Since no specimens survived for more than nine days, mortality is known to be complete, although not fully accounted for. Only two specimens were accidentally killed within this short period of time: others were lost to causes discussed below. Volpe (1953) reports the maximum thermal tolerance of Rana pipiens as 34 C. Although some embryos can sustain limited exposure to 37 C (Briggs, 1947), high mortality at 35 C for an extended period is not surprising. Temperatures approaching the maximum thermal tolerance of a species may be lethal if prolonged through time. Embryonic amphibians are known to be thermally sensitive in certain embryonic stages (early cleavage and gastrulation), but more thermally tolerant in advanced stages. Neither of the "egg" clutches in replicate tanks A and B (with larvae introduced at stage 11: "mid-gastrula") or tanks B and C (with larvae introduced at stage 18: "muscular response") proved to be appreciably more thermally tolerant. Mortality was second greatest (134) at 30 C. Somewhat reduced mortalities at 20 C and 25 C (112 and 111, respectively) might suggest that thermal stress was greater at 30 C than either of the two other temperatures. Although this might be true, the differences among the 12 replicate aquaria are not statistically significant (X2==l7.96, P>0.05). 142 Transformees A majority (57%) of the 1,200 specimens introduced into aquaria at 20 C, 25 C and 30 C did develop into late metamorphic stages. The numbers of larvae transforming in the 12 replicate aquaria at these three thermal regimes were not significantly different from one another (X2==l8.63, P:>0.05). The differences in numbers observed, therefore, can be attributed to random variation. Early in the study, specimens were removed, measured and preserved upon reaching developmental stage 45 or 46. Mortality at these stages was high initially, and many transformees at stage 45 or 46 were found dead in the tanks. Since they were believed to have drowned, subsequent transformees were removed at an earlier stage of development (mostly 44). Fifty-two specimens were removed at stage 45 and only three specimens were removed at stage 46. Few specimens, therefore, remained in the tanks through final metamorphosis at stage 46, and the occurrence of dead transformees was virtually eliminated. Larvae of stages 43 to 44 were frequently found to have emerged from the water, holding onto the screen siphon covers. Occasionally, Rana pipiens in the field were caught in pitfalls at stages prior to stage 46 (having naturally emerged from the water). Larval mortality among anurans has been reported to be greatest at uncleaved egg and gastrulation (Atlas, 1935; Grainger, 1959; King, 1903; and Volpe, 1953) and metamorphosis (Dent, 1968). The results of these experiments corroborate high mortality at transformation. This occurrence, however, may well represent a response to laboratory rearing 143 and may not be observed in nature. Assuming that mortality at late metamorphic stages is an experimental aberration (not representing natural mortality), specimens dead at stage 45 or 46 were considered as transformees and not attributed to mortality. Had the larvae devel- oped in the field, presumably they would have already left the water by the time they reached stage 45. Growth in B. pipiens ceases at stage 42; final body size is attained by this time (Etkin, 1964), although the tail is continuously resorbed until metamorphosis is complete at stage 46. Larval body lengths, therefore, are considered to be comparable at all stages thereafter. Missing_0ata Data for the four replicate aquaria (A, B, C, D) were similar among temperatures in all classes (accidental deaths, mortality and transformations) except that for which no account of specimens could be given (Table 27). Numbers of larvae in this category were greatest at 35 C and 20 C, and considerable variability was evident at these temperatures. The numbers of larvae were significantly different among the 12 aquaria (X2==72.93, p <0.005) in which larvae survived more than nine days. Of the 400 embryos originally placed in aquaria at 35 C, 21% were not accounted for. Early embryonic stages are small and specimens nay have been immobilized in the screen wire of siphon or heater covers. Decomposition occurred much more rapidly at 35 C than at other thermal regimes, and checking specimens twice a day may not have been sufficient to provide an accurate record. Dead or dying larvae were at times found entangled in the spinach, and could have been removed accidentally. 144 Excluding the group maintained at 35 C, the number of unaccountable specimens appears to be negatively related to temperature (or more appropriately, negatively related to development time, which is shown to be a function of temperature; Fig. 15). Larvae maintained in the laboratory for longer periods of time would have been exposed to a greater chance of loss than those transforming more rapidly. Specimens were lost due to several factors. A few active specimens were able to get inside the stainless steel, siphon screen covers. Not protected there from the current pull of interconnecting siphons among replicate tanks (Fig. 10) or incurrent siphons for the external filters, some larvae were doubtless drawn in, immobilized, and died in the siphons, tubes or external filters. Extended development time of larvae at 20 C probably increased losses there by exposing larvae to repeated, increased amounts of physical manipulation. Possible debilitating effects of inhibited development at 20 C will be discussed below. They probably represent manifestations of stress that result from decreased temperatures. Larvae demonstrate a preference for close quarters (in corners, below spinach, behind siphon screens, 232:) and occasionally managed to maneuver themselves below the undergravel filters. Specimens recovered dead were not numerous, and were considered "accidental deaths"; these data were deleted from analyses. Rarely larvae were accidentally removed from tanks with old spinach. Usually they were returned to aquaria unharmed: at least one active larva, however, went down the drainpipe of the sink. 145 Doubtless some specimens for which data were not available were lost to these and other hazards. Two additional transformed specimens were found on the floor of the laboratory on 23 November 1971. Appar- ently they had escaped the unidentified tank(s) through the corner openings in the plexiglas aquarium cover (Fig. 10, Table 27). They probably escaped from the series maintained at 30 C or 25 C, since these two groups were transforming at the time. Other larvae may have escaped unnoticed as well. Once escaped specimens were discovered, the cover openings were occluded with paper toweling throughout the remainder of the experiment. The total number of escaped transformees is believed, therefore, to have been small. Unaccountable specimens were consider- ably reduced (104, 9% of 1,200) in the three thermal regimes in which larvae did transform. The majority (66) of these, however, were missing from the group reared at 20 C. The prolonged, l7-month deve10pment time for some larvae at 20 C is believed to be responsible. Larvae in this series were maintained for more than twice the development time at either of the two warmer temperature regimes. The time required for B, pipiens to complete normal development reportedly requires 90-120 days (Etkin, 1968), 75-90 days (Rugh, 1951), and 67-86 days (Wright and Wright, 1949). Repeated activity of daily laboratory maintenance (feed- ing larvae, removing spinach, filtering water) subjected the larvae at 20 C to the greatest probability of loss or accident. Doubtless some larvae were inadvertently removed or injured in the mechanical process of moving weights, siphon screens, 232: For unknown reasons, some replicate tanks in a series (20 C--tank D, 25 C--tank A) had very low incidences of unaccounted-for specimens (Table 27). 146 Accidental mortality and unaccountability of data were considered to be unbiased effects, essentially random in nature. Excluding these figures in a comparison of transformation and mortality, therefore, 30-37% of the larvae were lost to mortality and 63-70% trans- formed (Table 28). .Specimens unaccounted for and accidental mortalities are deleted from further comparisons among groups or temperatures. Growth and Transformation Mean body lengths and numbers of larval B, pipiens transforming monthly (Fig. 15) are shown to change through time. Although all spec- imens were placed in aquaria on 2 September 1971, this month is not represented on the figure. *Emergent ydung (transformed larvae) were not Eecured until October. As indicated on the figure, the final sam- ples in series 20 C and 30 C were both terminated before all larvae transformed. A malfunctioning thermoregulator allowed water tempera- tures in the 30 C group to exceed 40 C on 11 February 1972; the eight larvae remaining were thus killed. Presumably the larvae would have completed development through final metamorphosis within the following month (March). The series maintained at 20 C was deliberately termi- nated after being maintained for 18 continuous months without trans- forming. The fact that 21 specimens still survived at the 20.0 series after 18 months suggests that larvae there might have persisted for several weeks or months more. Although these terminated larvae were not completely transformed, most were developed sufficiently to indicate their body lengths at metamorphosis. Larvae reared at 25 C transformed or died without interruption. Lengths of transforming larvae reared at the three temperatures are compared below. 147 Two maj0r points are immediately evident. Pronounced size disparities in body length were exhibited among larvae reared at the three different thermal regimes. Secondly, outstanding differences lie in the extended amount of time required to complete development at the coolest temperature regime (20 C). Although the larvae maintained at 25 C required less than two additional months to develop than those at 30 C, 21 of those larvae reared at 20 C were arrested after :>lO addi- tional months and may have required considerably more time. Development at 20 C therefore was quite depressed in comparison. (Survivorship is estimated above.) Conditions responsible for this protracted develop- ment may be in part due to two factors: first the unusually low tem- perature and secondly in the constancy of the temperature cycle. Larval t development at constant temperatures has been shown to be slower than at fluctuating regimes. Rana pipiens may develop more rapidly when exposed to temperature fluctuations than at constant temperatures, even when the temperature of the constant thermal regime equals the maximum tempera- ture achieved by the fluctuating cycle (Ryan, 1941). Similar to other biological and chemical processes, deve10pment is known to vary with temperature. In addition to other workers, Adolph (1931) and Garside (1966) have shown that amphibians and fish develop more rapidly, and to a smaller size when reared at elevated temperatures. In this study, the aberrant response of growth to temperature is pro- nounced at both the cooler (20 C) and warmer (30 C) regimes. Not only were smallest specimens recovered from the warmest temperature, and the largest specimens recovered from the coolest temperature, but the body 148 size of transformees at all three thermal regimes generally increased in time (Fig. 15). Increased body size among later transforming larvae probably reflects the increased length of the feeding period which is under endocrine control. In crowded conditions, growth among larval anurans has been shown to be reduced by the presence of a variety of intraspecific and interspecific inhibitors (Akin, 1966; Licht, 1967; Richards, 1958, 1962: and Rose, 1960). Although the constant filtering of water with both outside and undergravel filters, and the daily removal of old spinach and feces were designed to have obviated this hazard, it is possible that the larvae were subject to the influence of an intraspecific regulator of density and development. None of the algae cells described by Richards (1958) were observed in the fecal matter of ten larvae that demonstrated inhibited development (:>l5 months) at 20 C. The density of larvae maintained (originally 100 specimens per tank) was probably greater than the optimum number for the volume of water. Amstislavskaya (1971) discussed retarded growth and accelerated differentiation rates among Rana terrestris maintained in crowded condi- tions. The inhibition of some larvae by larger, healthier ones until the large ones transformed, would partially explain why the development time was so greatly lengthened, and why relatively few numbers of larvae transformed each month at 20 C. After the more rapidly deve10ping larvae transformed, the larvae heretofore suppressed (affected by the metabolite) would have opportunity to develop and yet suppress develop- ment in other, smaller larvae present. Should inhibitory substances 149 have been involved, they might have reacted synergistically with endocrine factors that regulate the onset of metamorphosis. Continuous filtration of water by both undergravel and outside filters in the study, however, probably precluded inhibition by anti-metabolites. Conditions of low temperature and abundant food may confer ecological advantages that are detected by biological feed-back mechanisms and result in prolongation of the larval stage (Wilbur and Collins, 1973). Larvae of the genus ngg_are extremely variable in the extent of larval development. Rana clamitans may require from 70 to 360 days for trans- formation (Martof, 1956): B, catesbeiana may require less than one or three years (Dent, 1968; Willis e3_gl,, 1956). The increased body sizes among larvae in the final samples may represent growing specimens whose development had been inhibited for some time (perhaps weeks), but finally were allowed to complete devel- opment when the suppressive, antibiotic producing larvae were lost from the population. In amphibians, the larval stage is most characterized by growth and feeding activity. Whatever the causes of extended devel- opment at 20 C, the reason for increasing body sizes in time is likely the result of continued feeding, thus growth, during this time. The numbers of transformees from each of the 12 replicate tanks did not differ significantly from one another. The frequency observed (Table 29) was not greater than might be expected if the actual fre- quencies were identical (1_g, 57 larvae from each tank; X2==l8.63, P:>0.05). Minor differences of sample sizes among replicate tanks, therefore, are attributable to randomness. 150 As already noted, maximum body growth is attained by developmental stage 42. Thereafter, the tail is continually resorbed but the body length does not change (Etkin, 1964). Since the trans- forming larvae removed were past the growth stages, body lengths among stages 42 to 46 represent comparably accurate measures of total body growth at metamorphosis. At these stages the body and degenerating tail can be readily distinguished by a difference in pigmentation. Considerable variation occurred among mean body lengths of larvae from replicate aquaria at each thermal regime. When tested with the Kruskal-Wallis nonparametric analysis of variance (Sokal and Rohlf, 1969), only the mean lengths of larvae reared in replicate tanks at 30 C were not significantly different among themselves. Mean body lengths among replicate tanks at 20 C and 25 C differed significantly among both groups (Table 29). Differences in body lengths, therefore, did not follow‘a trend consistent with the positioning of the two separate egg clutches (tanks A and B, C and D). By comparison, how- ever, the differences among mean body length at three temperatures was ngB 1 significant (Table 30). Although the "within group" variances are different, "between group" variances are greater by more than 20 orders of magnitude. The mean body lengths of larvae reared at 25 C and 30 C were more similar to each other than either were to those reared at 20 C. This similarity is evident both in the body lengths and the transfor- mation rates. Rather than demonstrating thermal stress, these data probably represent the influence of sustained, unusually cool temper- ature in the group reared at 20 C. Both of the warmer temperatures 151 more closely resemble the thermal ranges commonly encountered by larval Rana pipiens in South Carolina. Differences in mean total lengths and variances at each tank are affected by the confounding effect of combining data from larvae of different stages. Unlike body lengths, tail lengths (thus total lengths) of larvae are highly correlated to the stage of development ’ (Table 36). Differences among stages caused by variable tail lengths confound those attributable to temperature. The coefficients of variation might be interpreted as indices of stress. The intermediate thermal regime (25 C) experienced the lowest variance and probably represented the most nearly "optimum" of the three temperatures. Greater coefficients of variations at 20 C and 30 C also reflect the stresses incurred there: temperatures approaching the maximum thermal tolerance at 30 C and Severely inhibited development at 20 C (Table 31). The range of body lengths is greater for larvae reared at 20 C and 30 C (18 mm; Table 31), than those at 25 C (15 mm). In addition, the variances at 20 C and 30 C are considerably . greater than that at 25 C. These differences probably represent the depressed development of larvae at a constantly reduced temperature (20 C). The low variances of lengths recorded at 25 C may well rep- resent near-optimum development of larvae there. The maximum disparity of values recorded at 20 C and 30 C indicate the aberrant patterns of development at those temperatures. “Mean body lengths of larvae transforming monthly show that the ranges of minimum and maximum values at each thermal regime were from 152 6 to 8 mm (Table 31). Unlike the other two "populations," larvae transforming at 20 C increased steadily in time both in mean body lengths monthly and maximum length attained (40 mm). Although the mean body lengths of larvae reared at 25 C increased m0nthly, the maximum body size (34 mm) was attained earlier, in the fifth of approximately eight months. Larvae at 30 C also inCreased in mean body length monthly, attaining the maximum value (33 mm) in the third of six months. Both variances and coefficients of variations for mean lengths are least at 25 C, further substantiating the favorability of this thermal regime in comparison to the others. Transformation and Survivorship Transformation (survivorship excluding mortality) at 25 C and 30 C differs markedly from that at 20 C in rate and form (Fig. 16). All specimens at 25 C had metamorphosed by April; those at 30 C probably would have transformed in March or April. Specimens at 20 C, however, would have taken considerably longer (more than 10 additional months). When data (n==302) for "natural" mortality (not accidental, experimental) are aaded to the numbers of transformations, all curves are smoothed-out (Fig. 17, N==985). The resulting survivorship curves at 25 C and 30 C are more similar than the transformation rates indicate. After the initial few months, those two groups represent virtually identical trends displaced by a time peridd of approximately one month. Data for 20 C follows a slower, more consistent rate of decrease. Survivorship at 30 C demonstrated the most rapid decline but closely 153 resembled that at 25 C. The transformation curves (survivorship excluding mortality) for the 25 C and 30 C series resemble a geometric progression, and the curve for larvae maintained at 20 C more closely approximates an arithmetic (additive) progression, being more linear in appearance. Although the groups reared at 20 C and 30 C were both termi- nated prematurely, an approximation of the probable overall survivorship can be estimated by extrapolating the curves to zero. When this is done, the termination dates are March 1972, for the group at 30 C and June 1973, for the group at 20 C. If these termination dates are used to compare survivorship at the three thermal regimes, all larvae would .have died within 88 weeks at 20 C, 28 weeks at 25 C and 24 weeks at 30 C. Data are plotted with the assumption that all larvae living at the time would eventually complete metamorphosis. Rather than comparing the groups at the estimated times of zero survivorship, actual values are compared at specific intervals (Table 32). When one calculates the time intervals required to reach successive survivorship intervals (90%, 75%, 50%, 25%, 10%), the survivorship of larvae at 25 C and 30 C are again found to be quite similar both in the amount of time between levels and their respective proportions. The amount of time (weeks) required for each group to reach the next arbitrary level of survivorship are comparable. Again the series reared at 20 C is shown to be distinctly different, requiring much more time, and in different proportions than the other two thermal regimes. The vast differences between the rates of survivorship and 154 transformation at 25 C and 30 C as opposed to 20 C are obvious. More detailed quantification and elaborate comparison of these values probably would not be meaningful. J Larvae reared at 20 C differ markedly from the other groups. These data suggest that somewhere between 20 C and 25 C lies a constant temperature below which ”normal“ development is greatly retarded. The response observed could represent a laboratory effect. Constant thermal regimes, although necessary in quantifying and comparing rate functions and standardizing iaboratory conditions, are not commonly encountered in nature. In thermal ranges where temperatures inhibit the rates of biochemical processes, natural responses (growth, transformation) may ‘be severely distorted. Plasticity inherent in amphibian biology is highly variable even outside thermal considerations. Body weight and time of metamorphosis naturally demonstrate much variability (Adolph, 1931: Brockelman, 1969: Martof, 1956; Pollister and Moore, 1937; and Wilbur, 1972). Percent Survivorship‘ A comparison of survivorship among thermal regimes is made in. regards to the percent of accountable surviving specimens as a percent of the time that animals were alive in each group (Fig. 18). Such a comparison demonstrates the overall survivorship patterns held to a constant time reference. Actual time periods at different thermal regimes vary from nine days at 35 C to :>17 months at 20 C. If the patterns of survivorship were consistent without regards to temperature, or if survivorship varied greatly with temperature, these relationships 155 should be apparent; they are not. Since the larvae reared at 20 C: and 30 C were terminated, the percent of larvae surviving at termination are indicated at 100% total time. In this respect, all curves are not strictly comparable: they might have been slightly different had the larvae at 20 C and 30 C fully terminated. Since so few specimens were left at termination (10% at 20 C and 4% at 30 C), differences are shown to be small in comparison to the total number; corresponding changes in curves are therefore considered negligible. ' Whereas no larvae transformed at 35 C, the curve for this group represents mortality only. This fact may account for the disparity exhibited between specimens at this temperature and the other three. At temperatures where transformation occurred (20 C, 25 C, 30 C), the survivorship curves closely approximate each other. The slope of the survivorship curve f0r larvae at 20 C was the most consistent. The slower, regular pattern contrasts with the more sigmoid patterns at 25 C and 30 C. Because of variance heterogeneity among data, the mathematical values of slopes are not compared. The gradually declining survivorship at 20 C reflects the inhibited development rates that are concommitant with a longer period of growth (Wilbur and Collins, 1973). As data from 35 C represent mortality only, the curve might not I be expected to demonstrate a trend similar,to those at the other thermal regimes, where a majority of data represent transformed larvae. It is included in the interest of completion. Fifty percent of the embryos died in 76% of the time (7 days) and 50% in the final two days. Heavy mortality in two days probably represents one of the more thermally 156 sensitive stages that embryos had attained. Larval amphibians are known to be extremely sensitive to temperatures changes in early cleavage and gastrulation (Atlas, 1935: Grainger, 1959; King, 1903; Moore, 1942b; Volpe, 1953). The remaining specimens apparently reached another sensi- tive stage and suffered thermal death in the final two days. Another possibility is that larvae demonstrate only limited tolerance to tem- peratures as high as 35 C. Extended exposure probably results in greater mortality. Briggs (1947) reports 12% survival among larval B, pipiens subjected to 37 C for a period of four minutes. Survivorship at all three temperatures declines gradually and steadily for the first 30% of the time (Fig. 18). Thereafter the rates at all three temperatures decline more rapidly. At this time,survivor- ship is 84% of the larvae at 20 C and 95% of the larvae at 25 C and 30 C. For the first 30% of development time required for premetamorphic devel— 0pment, therefore, specimens die at a fairly constant rate at all tem- peratures. The significance of this phenomenon (if any) is not clear. Larvae begin to transform soon thereafter and survivorship curves reflect metamorphosis in addition to mortality. Division of these data into components (mortality and transformation), verify that the mortality rates were steady and that differences among temperatures represent the greater numbers of larvae transforming. Similarity between populations reared at 25 C and 30 C is reiterated in initial and subsequent sorvivorship patterns. The curve for larvae at 20 C does not represent a massive transformation in short time, but gradual loss of larvae throughout (Fig. 18). 157 Because of the complex life pattern of B, pipiens, it is difficult to present survivorship data for the organism from embryo to adult. Only the larval stage is aquatic and, thus, commonly subjected to thermal stress. For this reason, the larval stage is treated separately here. Metamorphosis results in the loss of speci- mens from larval populations and the aquatic habitat as well. Thus, transformation is equated with mortality. Such an interpretation is appropriate for studies focusing on aquatic larval forms that emigrate from water upon metamorphosis. Growth 111 BodLSize Body lengths have been shown to be inversely related to the temperature at which specimens are reared: 20 C:>25 C:>30 C (Table 30). Since transforming larvae were removed or died at several developméntal stages, a valid comparison of lengths must be made among specific. stages. The trend demonstrated by body lengths of larvae reared at specific stages follows the same pattern as overall data at each tem- perature (Fig. 19, Table 33). As sample variances are heterogeneous, the ranges of the standard errors do not necessarily indicate statis- tically significant differences. Trends, nonetheless are consistent and are shown to be'significantly different when subjected to the Kruskal-Wallis nonparametric analysis of variance. From Table 33 it can be seen that most specimens removed from each temperature belong to the same developmental stage, and are thus comparable. As already mentioned above ("Reservoir sampling of larval B, terrestris"), this conclusion conforms to those of other workers (Atlas, 1935: Etkin, 1955; 158 Herreid and Kinney, 1967; Johnson, 1970; Kokurewicz, 1969: Krough, 1914; Mc Laren, 1965; Muto, 1969a, 1972; Parker, 1965; Purcell, 1968; Rugh, 1951; Ryan, 1941). Growth inhibition is directly related to faster developmental rates. Both responses are characteristic of environments at higher temperatures. Wilbur and Collins (1973) have described the relationship of growth and development parameters to formulate an ecological model of amphibian metamorphosis. Abnormalities Major abnormalities encountered in larvae included crooked spine, paralyzed hindlimb, and edemaceous or emaciated body condition. All of these anomalies appeared to some degree in all three thermal regimes where metamorphosis occurred. Edemaceous and emaciated body conditions were difficult to determine and accurately quantify, and will not be compared. These two designations were necessarily more subjective than the presence or absence of a paralyzed hindlimb or crooked spine. Although specimens in each group were affected to some extent, edemaceous larvae were more apparent at 20 C and emaciated larvae at 25 C and 30 C. Specimens at 30 C were severely affected by crooked spines and paralyzed hindlimbs. Data are presented for larvae demonstrating a crooked spine or paralyzed hindlimb. Crooked Spine Specimens with crooked spines were affected to various degrees of severity. Some larvae possessed a slight curve in the basal part of 159 the tail; severely affected specimens had tails convoluted more than 360°. Pelvic girdles of these specimens may have been deleterously affected, as distorted backs remained after metamorphosis was complete. The permanent curve in the tail of the larvae impaired swimming motion, to the degree that the spine was bent. Severely afflicted specimens were capable only of circular swimming; slightly affected specimens demonstrated awkward, irregular, helical swimming motion (Fig. 20). The occurrence of high numbers of specimens with crooked spines at 30 C is interpreted as an indication of stress incurred there. Mechanisms involved in the appearance of crooked spine in larvae at the three thermal regimes are not fully understood. Several workers have related its occurrence in some stocks of experimental lab- oratory cultured B. pipiens (Jane Kaltenback, George Nace, personal communication). Others have published studies with illustrations of larvae demonstrating this same phenomenon (Dent, 1968; Etkin, 1964; Gordon gt_gl,, 1945). Very little appears to be known about its occurrence or the influence of external factors on this phenomenon. Moore (1942) observed laboratory reared, larval B. pipiens with "crooked tails." He attributed the occurrence of this abnormality (and others) to elevated temperatures. It seems that the condition does not appear until advanced stages of metamorphosis: affected specimens in this study were not recorded prior to stage 41. The presence of specimens with crooked spines during climax metamorphosis may indicate that this phenomenon is somehow related to tail resorption preceding transforma- tion. 160 Crooked spines occurred in 170 (74%) of the larvae transforming at 30 C, 34 (10%) of the larvae transforming at 25 C and 43 (21%) of the larvae transforming at 20 C (Fig. 20, Table 34). The unlikely occurrence of affected specimens appearing in the pr0portions reported indicate that some mechanism is involved other than random variation (X2==138.4, P3<0.OO5). These data show that the appearance of crooked spines is partially influenced by heat. The highest proportion of ' affected larvae occurred at the highest temperature: 30 C. The lowest proportion, however, occurred at the intermediate temperature: 25 C. The fact that some larvae at all three thermal regimes possessed crooked spines suggests that although its demonstration is thermally influenced, its propensity is doubtless genetic. Low levels (lo-21%) might be expected to occur exclusive of thermal influence, and probably represent inherent variability. Some of the larvae demonstrate this anomaly at any temperature, but elevated temperature increases the likelihood of its occurrence. It is interesting to note that the hundreds of larval Rana pipiens sampled in the field studies never included a single specimen affected with a crooked spine. The absence of such affected larvae probably attests to negative natural selection pressures exerted upon these organisms in nature. A further inquiry into the mechanisms involved await histological analysis of specimens. When the mean body lengths of specimens are compared at each temperature, there is no apparent difference between affected and unaffected larvae (Table 35). Stage by stage analysis of body lengths at the three temperatures also reveals that body lengths differ 161 according to thermal regime only. Values for larvae with normal and crooked spines are clearly comparable at all stages at each temperature except perhaps for stages 44 and 45 (Table 36).. These differences, however, are not considered significant and they follow no distinct trend. Mean tail lengths differ at specific stages and temperatures, but not in a consistent manner. The variance of these comparisons were generally greater in specimens with a crooked spine. This param- eter was measured to detect possible differences in tail resorption rates; no distinct trends were apparent. Gordon e$_gl, (1945) reported the occurrence of "peculiar tail abnormalities characterized by a twisting confined to the basal region" in experimental B, pipiens. The phenomenon was observed in most thiourea treated specimens and rarely in untreated controls. Radio- graphic and hiStological studies demonstrated that these tails reveal no abnormal skeletal growth but rather an overgrowth of connective tissue. Muto and Hasegawa (1969) reported that larvae of the frog 3222. 'japonica cultured at temperatures "higher than normal" demonstrated wavy tails that closely resemble those described herein. They showed that no wavy tails developed in larvae reared at room temperature or at 20 C, although some of the larvae reared at 25 C and all of those at 30 C possessed such abnormalities. Histological studies of Muto and Hasegawa (1969) and those of Gordon e3_gl, (1945) revealed that the wavy tails represented a winding of the notochord as it increased in length more rapidly than the surrounding muscle tissues. Unlike Muto and.Hasegawa's 162 data, abnormally developed tails in laboratory reared B, pipiens occurred at all temperatures to some extent. The absence of affected specimens at lower temperatures in Muto and Hasegawa's study suggests that the occurrence of wavy tails is environmental rather than genetic. Curved tails have also been shown to occur in embryonic herring subjected to high temperature (Kudinskii, 1969). The morphological structure causing the abnormality in this case was not defined. Paralyzed Hindlimbs To a lesser extent, the frequency of paralyzed hindlimbs was related to temperature. Although only 17 (2.5%) of the 683 transformed larvae demonstrated this anomaly, a chi-square analysis revealed that the frequency of occurrence was significantly nonrandom among temper- atures. Since the pr0portion of affected larvae was apparently asso- ciated with the level of thermal influence (Table 34), it is interpreted as a thermal response. The occurrence of paralyzed hindlimbs is appar- ently a more subtle thermal response than crooked spine. Data for paralyzed hindlimbs are considered insufficient to reveal specific conclusions. Muto (1969a, 1969b, 1970a, 1970b) has reported heat induced malformations in the skeleton (hindlimbs and forelimbs) of Bufo vglgaris reared at 30 C. Edema and retarded development were observed at 30 C (Muto, 1969a). Such anomalous development patterns were not observed in B, pipiens in this study. 163 Crooked spines are shown to be strongly related to thermal loading, and paralyzed hindlimbs significantly so. Both of these anomalies inflict obvious disadvantages on affected larvae. One might not expect to encounter such clearly disadvantageous traits in large numbers; selection against such nonadaptive characters would likely keep them in low numbers. Their ecological significances, therefore, might be quite far reaching. Among several hundreds of larval BEES. pipiens removed from heated areas in the heated reservoir, never were any specimens seen or collected that demonstrated either of these maladies. This might suggest that these anomalies are responses to constant (laboratory) temperature, and thus not likely to be observed in nature. The rather common occurrence of this trait among laboratory populations, however, suggests that it should be expected in field situations as well. Another possible interpretation is that affected larvae succumb to selective forces (j_e, eaten by prey) and simply may not survive long enough to be collected. SUMMARY AND CONCLUSIONS Pitfall Trapping_Survey 1. Of 13 anuran species trapped in pitfalls peripheral to Pond C Reservoir, three dominant species (B, pipiens, B, terrestris, and B, carolinensis) exhibited massive yearly migrations of breeding adults and emergent young. 2. Emergent young of B, terrestris and B, carolinensis were trapped along the reservoir during the same months that they were trapped elsewhere on the Savannah River Plant. Emerging B, pipiens, however, were not restricted to Seasonal occurrence characteristic of unheated areas. Recently transformed specimens were encountered when- ever larvae were trapped (during 11 months of the year). Breeding patterns are apparently modified by the increased temperatures at the reservoir. 3. Captures ole, pipiens in pitfalls along the reservoir indicate that adults do breed and larvae develop in certain parts of the heated reservoir. 4. Although considerable development of larval amphibians occurs in cool seepage ponds flanking the reservoir, the numbers of .B. pipiens and B, terrestris encountered reflect the severity of the thermal gradient along the reservoir's cool arm. 164 165 Reservoir Sampling with Dipnets and Minnow Traps 1. Continued sampling of larvae in the vicinity of Pond C Reservoir confirms that larval anurans of five species were found to develop in the heated waters. 2. Abundance, species diversity and density of larval anuran populations generally reflected the level of thermal loading sustained. 3. Egg and embryonic mortality was severe in areas receiving the greatest amounts of thermal loading, and negligible in cool seepage ponds peripheral to the reservoir. 4. Within the ranges of thermal tolerance, the lengths of specimens among heated and cool microhabitats varied inversely with the degree of thermal loading. . Reservoir Sampligg of Larval Bufo terrestris l. The cooling effect of shoreline seepage allows the Southern toad (B, terrestris) to successfully breed and develop in restricted . parts of Pond C Reservoir receiving lethal levels of thermal loading. 2. Heavy mortality of toad eggs and embryos occurs in the heated reservoir. Within the ranges of thermal tolerance, however, rates of larval growth and development differ in proportion to the level of thermal loading. Larval B, terrestris grow to a larger size but develop more slowly at cooler temperatures. 3. Constantly elevated temperatures in the reservoir prevent anurans from benefiting from reduced thermal levels naturally associated with nocturnal breeding. 166 Laboratory Rearing of Larval Rana pipiens 1. Embryonic B, pipiens reared at a constant temperature regime of 35 C suffered total mortality within nine days. 2. Within the limits of thermal tolerance, larvae reared at warmer temperatures (30 C and 25 C) developed more rapidly but grew less than those reared at the coolest temperature (20 C), where larvae demon- strated markedly inhibited rates of development. 3. The occurrence of abnormalities (crooked spine and paralyzed hindlimbs) was greater at 30 C than20 C or 25 C. 4. Even aquatic ecosystems receiving lethal levels of thermal loading may contain habitable microenvironments created by local seepage or other physical conditions. 5. Anurans are shown to deposit eggs in the heated reservoir at lethal or near-lethal temperatures, where mortality may be very high (90%). 6. 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