INVESTIGATIONS of competitive interactions

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1 Copeia, 2004(1), pp Effects of Conspecifics on the Burrow Occupancy Behavior of Spotted Salamanders (Ambystoma maculatum) JONATHAN V. REGOSIN, BRYAN S. WINDMILLER, AND J. MICHAEL REED The spotted salamander (Ambystoma maculatum) is a pool-breeding species thought to depend on small mammal burrows for survival in terrestrial habitats. We investigated burrow occupancy patterns using laboratory and field experiments where salamanders were housed alone or in pairs, in arenas with either one or two burrows. In the single-burrow field experiment, intruders were significantly less likely than residents to occupy burrows, and the probability of burrow occupancy declined following nighttime rain. However, spotted salamanders frequently co-occupied burrows (mean burrow co-occupancy rate, 59%). In the single-burrow laboratory experiment, mean burrow occupancy rate was 98%, both when salamanders were housed alone and in pairs. However, salamanders housed in pairs with two burrows co-occupied burrows less frequently than expected by chance, and greater size disparity was associated with lower burrow co-occupancy rates. Our results suggest that spotted salamanders may often fail to effectively defend burrows and exclude conspecifics, although avoidance of occupied burrows could, in some contexts, affect spacing in terrestrial habitats. INVESTIGATIONS of competitive interactions among adult salamanders have largely focused on terrestrial plethodontid species (e.g., Thurow, 1975; Jaeger et al., 1982; Hairston, 1987). Plethodontid salamanders can occur at high densities (Burton and Likens, 1975; Petranka and Murray, 2001) and often engage in territorial defense of refugia (e.g., Jaeger et al., 1981; Keen and Reed, 1985; Marvin, 1998). In contrast, factors influencing the terrestrial spatial distribution of pool-breeding amphibians, including mole salamanders (Ambystoma), are poorly understood. Pool-breeding mole salamanders usually migrate to and from breeding ponds in early spring and spend the remainder of the year in underground refugia (Petranka, 1998). Some Ambystoma species, including the spotted salamander (A. maculatum), appear to be incapable of excavating their own burrows (although they may enlarge existing cavities) (Semlitsch, 1983), and they frequently occupy small mammal burrows in terrestrial habitats (Windmiller, 1996; Madison, 1997). Ambystoma populations are thought to be regulated by density-dependent growth and survival during the larval stage (Wilbur and Collins, 1973; Petranka, 1989; but see Windmiller, 1996), suggesting that, unlike the terrestrial plethodontids, competition for resources in the terrestrial environment may be limited (Martin et al. 1986; Petranka 1989). As a result, resource defense and territorial aggression may be weak or absent because they provide little or no adaptive benefit. If so, why do many pool-breeding salamander species migrate over long distances rather than settle at higher densities in suitable habitat closer to the breeding pond (Semlitsch, 1998)? One hypothesis is that supply of suitable burrows is limited, and avoidance of occupied burrows influences dispersion (Ducey and Ritsema, 1988; Walls, 1990). Intraspecific aggression observed among adult and juvenile Ambystoma is consistent with this hypothesis (Ducey, 1989; Walls, 1990; Smyers et al., 2001). Further support comes from Ducey and Ritsema (1988), who found male spotted salamanders, housed in pairs in laboratory test chambers equipped with two artificial burrows, together in burrows less frequently than expected by chance. Ducey and Heuer (1991) showed that the intensity of intraspecific aggression within four Ambystoma species was context-dependent, varying with food availability. Although prior studies suggest that conspecific aggression and avoidance in the Ambystoma may be relatively weak, more research is needed to understand whether, and under what conditions, conspecifics might influence the spatial distribution of Ambystoma species within terrestrial habitats. Our goal was to use experiments in field enclosures and laboratory arenas to test the hypotheses that spotted salamanders influence each other s burrow occupancy rates and that body size and residency status affect burrow cooccupancy behavior. We predicted that, if competition for burrows were intense and the cost of being displaced from or sharing a burrow were high, then spotted salamanders would exclude competitors from burrows at high rates, regardless of whether one or two burrows were 2004 by the American Society of Ichthyologists and Herpetologists

2 REGOSIN ET AL. SPOTTED SALAMANDER BURROW OCCUPANCY 153 available. In contrast, given their strong tendency to occupy burrows, if spotted salamanders exhibit only a weak tendency to exclude conspecifics, or if they avoid conspecifics, then we predicted that spotted salamanders would co-occupy burrows when only a single burrow was available but be overdispersed when multiple burrows were available. MATERIALS AND METHODS Capture and housing of salamanders. During May 2001, 40 postbreeding adult male spotted salamanders were captured in pitfall traps in eastern Massachusetts. Salamanders were individually toe-clipped and housed individually in small plastic containers lined with damp paper towels in a refrigerator for an average of 14 days (SD 9, n 40). One experiment was conducted in field enclosures, and two experiments were conducted in laboratory test chambers. Because of a limited supply of study animals, each spotted salamander was a subject only once in each experiment but could have been used in up to three experiments. Prior to entering the experiments, salamanders were removed from the refrigerator, housed individually in small plastic containers lined with damp paper towels for h, and fed three to five 5-mm long segments of fresh earthworms. Salamanders were held in the laboratory for up to 24 h between the termination of one experiment and initiation of the next experiment and were again housed and fed as described. Each laboratory experiment lasted 10 days and was divided into two consecutive five-day trials. During the first laboratory experiment, salamanders were fed 10 small crickets upon release (day 0) into the test chambers and again on day 5, with feedings of five 5-mm long earthworm segments on days 3 and 8. During the second laboratory experiment, salamanders were fed 10 crickets upon release and on day 5, with additional feedings of five crickets on days 2 and 7. In the field experiment, invertebrates could move in and out of enclosures, and no supplemental food was provided. All indoor experiments were conducted under a 13:11 h light:dark cycle, at approximately 21 C. Of our 40 experimental animals, only 20 could be accommodated in the field enclosures at once. Consequently, 20 animals were subjects first in the laboratory single-burrow experiment, second in the laboratory two-burrow experiment, and third in the field experiment. The remaining 20 salamanders were used first in the field experiment, and subsequently in the laboratory two-burrow experiment. Two animals were depredated during field trials (see below) and were replaced by fresh animals to complete the laboratory two-burrow experiment. Thus, with the exception of these two animals, all subjects in the laboratory two-burrow experiment had experience with either field enclosures or laboratory test chambers. None of the subjects for the laboratory single burrow experiment had prior experience in our experiments. Field single-burrow experiment. Ten 1.1-m diameter, circular enclosures were constructed of aluminum flashing buried at least 15 cm deep in white pine (Pinus strobus) dominated forest inhabited by spotted salamanders and located approximately 400 m from a spotted salamander breeding pool. A small artificial burrow was constructed near the center of each enclosure by excavating a 10-cm deep hole (diameter, 3.5 cm), at an approximate 30 angle using a wooden stake. Burrows were large enough to accommodate at least two salamanders, and two salamanders were observed to co-occupy these burrows during preliminary trials conducted the previous summer (BSW, unpubl. data). Enclosures were free of alternative refugia. Using these enclosures, we tested the hypotheses that daily burrow occupancy rates would be lower for salamanders when housed together than when housed alone and that intruders and smaller animals would experience the largest decreases in burrow occupancy rates, compared to the housed-alone control. To assign salamanders to experimental treatments, the 40 subjects were divided into two groups based on size, measured by snout vent length. Small and large salamanders were paired randomly, with the constraint that large individuals were at least 10% larger than their counterpart. If this criterion was not met, the large salamander was returned to the pool of unpaired salamanders and selection continued until all salamanders were paired. Resulting pairs had a mean difference in snout vent length of 15% (SD 5.3; n 20). Because there were a limited number of enclosures, it was necessary to run the experiment twice to accommodate all animals. Each round of the experiment consisted of three trials, with each trial lasting eight days. During the first trial, a single individual was released into each enclosure immediately adjacent to the artificial burrow and was classified as a resident. Ten of the 20 pairs of animals were chosen at random for the first run, and one member of each pair was chosen at random to be the resident, with the constraint that there were five large and five small residents. Each enclosure was checked daily for three days (the habituation period),

3 154 COPEIA, 2004, NO. 1 and the position of each resident salamander (in or out of burrow) was recorded. On the third day of the trial, to ensure that salamanders located the burrow, any salamander not observed occupying the burrow was relocated immediately adjacent to the burrow, and occupancy rates were recorded for five more days. On day 8, five large individuals were released into enclosures containing small animals, and five small individuals were released into enclosures containing large animals, one into each enclosure; these animals were classified as intruders. To maintain consistency across trials, intruders not occupying the burrow on day 3 were relocated adjacent to the burrow. Burrows were checked daily for eight days, with the individual or individuals occupying each burrow recorded. If one or more of the animals was not observed in the burrow, leaf litter was carefully moved and then replaced within the enclosure to determine the identity and location of the salamander(s) not occupying the burrow. The third trial, initiated on day 16, involved removing the resident individual from the enclosure to obtain data on the burrow occupancy rates of intruders when housed alone. To maintain consistency with the first trial, and to eliminate potential effects of prior burrow use or burrow familiarity, existing burrows were sealed and new burrows excavated in each enclosure. Each intruder was then assigned randomly to a new enclosure and released alone. The trial then proceeded for eight days as described above for trial one. This design controlled for possible effects of treatment order on burrow occupancy rates because half of the individuals received the alone treatment before the co-occupancy treatment, and half received treatments in the reverse order. The entire threetrial procedure was repeated with nine pairs of salamanders. Each day, the weather during the previous night was recorded as rained or not, because spotted salamanders are more active when it rains (Petranka, 1998). Enclosures were checked for burrow occupancy during midday ( h), when spotted salamanders are not active above ground. Our data consisted of daily observations of each individual s burrow occupancy (in or out of burrow) under varied experimental treatments and weather conditions. In addition to using a paired t-test to compare each individual s burrow occupancy rate when housed with another salamander and when housed alone, we used the Generalized Estimating Equations (GEE) method (Liang and Zeger, 1986) to analyze these discrete (in our case, binary), correlated responses in a repeated measures logistic model (PROC GENMOD; SAS OnlineDoc, vers. 8, unpubl.). Specifically, we modeled the effects of treatment (alone, resident, or intruder), body size, and prior night s weather (rain or clear) on daily probability of burrow occupancy. Within-subject correlations among daily burrow occupancy decisions were modeled as exchangeable, meaning that the strength of the correlation was assumed not to vary across days (SAS OnlineDoc, vers. 8, unpubl.). Empirical standard error estimates were used to determine P-values for the parameters. In determining P-values for treatment effects, withinsubject correlations among observations were statistically controlled for by this procedure. A number of recent ecological studies have employed GEEs to analyze correlated response data (Brand and George, 2001; Johnson and Igl, 2001; McLaughlin, 2001). Laboratory single-burrow experiment. We compared burrow occupancy rates of individuals housed together and housed alone, without variation in weather and food availability experienced in the field. The experiment was conducted on 20 animals that had not yet been placed into field enclosures. Twenty plastic test chambers measuring cm were filled with 10 cm of moist dirt. Half-cylinder shaped burrows 3.5 cm diameter and 12 cm long were constructed in the dirt in one-half of the rectangular test chamber. Hardware cloth, fashioned into half-cylinders, was used to support burrow roofs. Damp leaf litter was placed on top of the dirt floor of each chamber. This experiment proceeded as described above for the first two trials of the field enclosures; the third trial was not needed because all animals received the housed-alone treatment first, and there was no three-day habituation period. On day five of the experiment, large and small salamanders were paired at random, subject to the constraint that they differ by at least 10% in snout vent length. One member of each pair was selected randomly to be an intruder, subject to the constraint that half of the intruders were large and half were small. Intruders were released into the middle of the test chamber occupied by the resident with which they were paired, and occupancy rates were recorded for the five-day trial. Laboratory two-burrow experiment. We tested whether pairs of salamanders introduced simultaneously, or sequentially as intruders and residents, into test chambers containing two burrows would occupy separate burrows more often than expected by chance. This experiment was

4 REGOSIN ET AL. SPOTTED SALAMANDER BURROW OCCUPANCY 155 conducted in the test chambers described in the first laboratory experiment, but this time each chamber contained two burrows located in opposite corners of the rectangular chamber. Two salamanders were chosen at random from the available pool of animals (with no restriction on size), and released into the middle of each test chamber. Locations of each salamander were recorded daily for five days. Salamanders were recorded as co-occupying a burrow, occupying a burrow alone, or not occupying a burrow. On day 5, one animal in each test chamber was chosen at random and removed. These animals were then randomly assigned singly to the test chambers still occupied by a single resident individual and released into the center as intruders. The location of each animal was recorded as above for the next five days. This experiment was run on 20 pairs of salamanders. Test chambers were lightly misted nightly to maintain moisture. For both laboratory experiments, the binomial probability test (Sokal and Rohlf, 1981) was used to determine whether burrow co-occupancy rates were less than expected by chance given the observed burrow occupancy rates of each individual. In addition, logistic regression was used to test the hypothesis that pairs of individuals differing more in size were less likely to co-occupy burrows. We predicted lower co-occupancy rates when animals differed more in size because, under these conditions, smaller animals might have an increased tendency toward avoidance, and aggression or threat displays by larger individuals might be more effective. RESULTS Field single-burrow experiment. Of the 19 trials, five were disrupted by a predator and aborted (one of these trials was aborted during the alone treatment only). Daily burrow occupancy rates were high for individuals when alone (mean 0.90; SD 0.25) and when paired (mean 0.79; SD 0.30) and did not differ significantly by treatment (paired t-test, t , P 0.12). In paired trials, salamanders cooccupied burrows 59.2% of the time (SD 28.9; n 15). The repeated measures logistic model indicated that residents were significantly more likely than intruders to occupy a burrow (P 0.002), whereas residents did not differ from salamanders housed alone in their probability of occupying a burrow (Table 1). The mean change in burrow occupancy rate (for each eight-day trial) relative to the alone treatment was 17.1% for intruders (SD 32.2; n TABLE 1. PARAMETER ESTIMATES FROM REPEATEDMEA- SURES LOGLINEAR MODEL OF THE DAILY PROBABILITY OF BURROW OCCUPANCY DURING 16-DAY EXPERIMENTAL TRIALS. Positive parameter estimates for significant predictors indicate an increase in the probability of burrow occupancy, and negative estimates indicate a decrease. For example, spotted salamanders were more likely to occupy a burrow if it did not rain the previous night. Parameter Estimate SE P-value Intercept Housed alone Intruder No rain previous night Large salamander a 1.72 a 1.03 b 0.16 c a Relative to resident. b Relative to rained previous night. c Relative to small (see Materials and Methods) ) but only 5.3% for residents (SD 41.7; n 15). Spotted salamanders were significantly more likely to occupy a burrow on days following a clear night than on days following a rainy night (P 0.001, Table 1). Laboratory single-burrow experiment. Mean burrow occupancy rates for spotted salamanders occupying a chamber alone and together with a conspecific were identical (mean 0.98 for both, SD 0.09 and 0.06, respectively, n 20 for both). Only three of 20 salamanders were observed outside of a burrow during daylight hours over the entire series of two five-day trials. Eight of 10 pairs co-occupied the burrow on all five days of the two-salamander trial, with the remaining two pairs having co-occupancy rates of Laboratory two-burrow experiment. When two spotted salamanders were housed together in test chambers with two burrows, both individuals had daytime burrow occupancy rates of 100%, whether introduced together (n 38) or as intruder and resident (n 36). Using this occupancy rate, and assuming that both burrows in each test chamber were of equal quality, spotted salamanders would be expected to cooccupy burrows 50% of the time if burrow occupancy decisions were independent. Because an individual s burrow occupancy decision on a given day of the study is not likely to be independent of prior decisions made by that individual, we analyzed co-occupancy rates on the final day of each trial. During this experiment, two salamanders developed skin lesions and were removed from the experiment, resulting in

5 156 COPEIA, 2004, NO. 1 of the resident and intruder (a measure of their joint size, P 0.84) were significant predictors of co-occupancy rate. Only three of 18 residents (16.7%) switched burrows between the first and last days of the trial. In two of these cases the burrow vacated by the resident was occupied by the intruder, indicating possible displacement. DISCUSSION Fig. 1. Burrow occupancy patterns on the final day of the two-burrow laboratory trials. The percent difference in size (snout vent length) between members of pairs of salamanders housed together is shown on the x-axis. (A) Spotted salamanders co-occupied burrows less frequently than expected by chance (P 0.02). (B) Pairs of spotted salamanders more similar in size were more likely to co-occupy burrows (P 0.036). 19 successful simultaneous release trials and 18 successful intruder versus resident trials. When salamanders were introduced simultaneously into the test chamber, only four of 19 pairs (21.1%) co-occupied a burrow on the final day of the trial, which is significantly ove-dispersed (Binomial test: P 0.02). The observed co-occupancy rate was constant across the fiveday trial. When salamanders were introduced sequentially to compare intruders and residents, daily burrow co-occupancy rate ranged from 36.8% to 44.4% over the course of the fiveday trial, and the co-occupancy rate on the final day was not significantly different from that expected by chance (Binomial test: P 0.8). However, logistic regression indicated that salamanders of similar size were more likely to co-occupy a burrow than were salamanders of disparate sizes (P 0.036; Concordance 82.5; Fig. 1). Neither the resident s snout vent length (P 0.55) nor the combined snout vent lengths Throughout all experimental trials, spotted salamanders were found in burrows at least 79% of the time, results consistent with the idea that underground refugia are important for terrestrial survival of this species (Windmiller, 1996; Madison, 1997). Prior observations of conspecific aggression and biting (Ducey and Ritsema 1988; Walls 1990) led us to predict low levels of burrow co-occupancy regardless of whether one or two burrows were provided. However, in our laboratory test chambers with a single burrow, resident spotted salamanders failed to exclude conspecific intruders from burrows, and burrow co-occupancy rates approached 100%. Results from the field experiment (also with a single burrow) were consistent with this pattern (mean co-occupancy rate 59.2%). In the field, 14 of 15 (93.3%) pairs were found co-occupying the burrow at least once during the eight-day trial, and there was no trend of decreasing probability of co-occupancy over time, as might be expected if one animal excluded the other. These results contrast with those of numerous territoriality studies showing a strong tendency toward aggressive conspecific exclusion (Brown and Orians, 1970). For example, terrestrial red-backed salamander (Plethedon cinereus) residents expelled intruders in 74% of encounters, with only 8% of encounters ending in a draw ( Jaeger et al., 1982). Despite a lack of consistent territorial exclusion, when pairs of salamanders were introduced simultaneously into laboratory test chambers with two artificial burrows, they were found together less frequently than expected by chance. These results are consistent with those of Ducey and Ritsema (1988) that, when given multiple refugia, spotted salamanders tend to avoid conspecifics. Our experiments suggest, however, that the influence of conspecifics on burrow occupancy is context-dependent, perhaps because experimentally altering refuge availability alters the costs and benefits of escalating a contest for a given burrow. Exclusion either provides relatively modest benefits or is difficult, as evidenced by the high co-occupancy rates observed in the one-burrow experiments. Similarly, Ducey and Heuer (1991) found con-

6 REGOSIN ET AL. SPOTTED SALAMANDER BURROW OCCUPANCY 157 text-dependent intraspecific aggression by four Ambystoma species, increasing as food availability decreased. Burrow co-occupancy rates were lower in the field than in the laboratory single-burrow experiments. This difference may be partially accounted for by a reduced tendency to occupy burrows in the field, apparently associated with rainfall. If aboveground movement is reduced in the laboratory setting, perhaps because of suboptimal conditions, then laboratory studies may underestimate avoidance behavior. Thurow (1975) suggested that some salamander studies in the laboratory that failed to detect territorial behavior might not have allowed sufficient time for residency establishment prior to the introduction of intruders. In our study, the residency period was five days in the laboratory and eight days in the field, durations that are similar to residency periods used by other researchers who successfully detected evidence of territoriality in other species (e.g., Cupp, 1980; Jaeger, 1981; Keen and Reed, 1985). Consequently, it seems unlikely that our results are an artifact of a limited residency period. However, some caution is advisable in extrapolating from experimental results because artificial burrows used in this and previous experiments (Ducey and Ritsema 1988; Ducey 1989) were of short length and may result in more crowding of co-occupants than would be the case in a natural burrow system. In the single-burrow field experiment, intruders were significantly less likely than were residents to occupy a burrow, indicating a residency advantage (Maynard Smith and Parker, 1976; Parker and Rubinstein, 1981). Residency advantage in territorial contests has been reported in a variety of plethodontid species (e.g., Cupp, 1980; Jaeger, 1981; Jaeger et al., 1982), but our results are the first reported evidence of residency advantage for adult Ambystoma. In the intruder versus resident two-burrow laboratory experiment, we found that pairs of salamanders that were more similar in size were more likely to be found co-occupying a burrow, perhaps because aggression or threat displays might be more effective with greater size disparity. These results, along with the tendency toward conspecific avoidance observed in the two-burrow laboratory experiment, and results from previous studies (e.g., Martin et al., 1986; Ducey and Ritsema, 1988; Smyers et al., 2001), suggest that conspecific avoidance may influence the spacing and consequently migration distances, of spotted salamanders in the terrestrial environment. However, the tendency toward conspecific aggression and avoidance among the Ambystoma appears to be weak when compared to terrestrial plethodontids. Therefore, more research is needed to determine the extent to which conspecifics play a role in influencing the spatial distribution of Ambystoma in terrestrial habitats. ACKNOWLEDGMENTS We thank J. Fahey, S. Lewis, D. Marshall, C. Orians, and M. Romero for advice and assistance, and D. Delahanty, M. Romero, and three anonymous reviewers for commenting on the manuscript. Salamanders were collected under MDFW permit SCRA. Research was approved by the IACUC of Tufts University and was partially funded through grants from the Massachusetts Environmental Trust, Tufts Institute for the Environment, Sigma Xi, and the Roosevelt fund of the American Museum of Natural History. We are indebted to the Sudbury Conservation Commission for allowing this research on land that they manage. LITERATURE CITED BRAND, L. A., AND T. L. GEORGE Response of passerine birds to forest edge in coast redwood forest fragments. Auk 118: BROWN, J. L., AND G. H. ORIANS Spacing patterns in mobile animals. Annu. Rev. Ecol. Syst. 1: BURTON, T. M., AND G. E. LIKENS Salamander populations and biomass in the Hubbard Brook Experimental Forest, New Hampshire. Copeia 1975: CUPP JR., P. V Territoriality in the green salamander, Aneides aeneus. Ibid. 1980: DUCEY, P. K Agonistic behavior and biting during intraspecific encounters in Ambystoma salamanders. Herpetologica 45: , AND J. HEUER Effects of food availability on intraspecific aggression in salamanders of the genus Ambystoma. Can. J. Zool. 69: , AND P. RITSEMA Intraspecific aggression and response to marked substrates in Ambystoma maculatum. Copeia 1988: HAIRSTON SR., N. G Community ecology and salamander guilds. Cambridge Univ. Press, Cambridge. JAEGER, R. G Dear enemy recognition and the costs of aggression between salamanders. Am. Nat. 117: , R. G. JOSEPH, AND D. E. BERNARD Foraging tactics of a terrestrial salamander: sustained yield in territories. Anim. Behav. 29: , D. KALVARSKY, AND N. SHIMIZU Territorial behavior of the red-backed salamander: expulsion of intruders. Ibid. 30: JOHNSON, D. H., AND L. D. IGL Area require-

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