2008 NORTHEASTERN NATURALIST 15(1):131–140 Nest Ecology of the Southern Two-lined Salamander (Eurycea cirrigera) in Eastern Illinois Jessica Jakubanis1,2, Michael J. Dreslik1, and Christopher A. Phillips1,* Abstract - Peripherally distributed populations of animals often exhibit different life-history parameters compared to those from more central locations. We report on the ecology of Eurycea cirrigera (Southern Two-lined Salamander) from its western range limit in eastern Illinois. We searched two streams and an intervening ridge from April 2002 until December 2003 to monitor clutch size and seasonal abundance of life stages. Oviposition began in April, and larvae were detected by late May. Mean clutch size was smaller than estimates from other parts of the species’ range and did not covary with snout–vent length of the attending female. Nest-attendance rates we detected tended to be lower than previous estimates. As in other parts of their range, clutches were often found under rocks with at least one other clutch. Longer rocks were 1.30 times more likely to have multiple nests deposited under them. Introduction Populations of animals at the periphery of their ranges are often subjected to extreme environmental conditions that may present unique selection regimes leading to changes in life history and accumulation of genetic differences. Spatial isolation of peripheral populations can increase the rate at which these changes develop. Peripherally isolated populations may therefore harbor variation not present in the main portion of the range and be important for the evolutionary potential of the species. Ecological data from such populations is important to conservation efforts and can serve as a model for conservation of similar species. Eurycea cirrigera Green (Southern Two-lined Salamander) is a forestdependent species with a wide distribution throughout the eastern United States extending west to the Mississippi River in Louisiana, Mississippi, and Tennessee, and the eastern edge of Illinois (Petranka 1998). From April 2002 until December 2003, we investigated nest ecology and distribution of this species in two adjacent streams in Vermilion County, IL, the western extent of the species’ range at this latitude (Mauger et al. 2000, Mierzwa 1989, Phillips et al. 1999). We examined clutch size, factors contributing to clutch size, and variations in adult and larval abundance based on temporal and spatial factors. We compared these parameters to published data from populations closer to the center of the species’ range to test for differences related to existence at the range periphery. 1Division of Biodiversity and Ecological Entomology, Illinois Natural History Survey, 1816 South Oak Street, Champaign, IL 61820. 2Current address - 13904 Encantado Road NE, Albuquerque, NM 87123. *Corresponding author - chrisp@inhs.uiuc.edu. 132 Northeastern Naturalist Vol. 15, No. 1 Materials and Methods Study site We conducted our study at the University of Illinois’ Vermilion River Observatory Research Area (VRO), located at approximately 40°03'5"N and 87°33'21"W. This site is an eastern deciduous mixed forest dominated by Quercus spp. (oak) and Carya spp. (hickory) (Phillippe et al. 2003). Intermittent streams dissect the uplands creating deep, closely spaced ravines separated by high ridges. Ravine systems average 700 to 1500 m in length and are forested in contrast to the level farmland surrounding them. We surveyed two adjacent ravines, Kendeigh and Swenson, each with an approximately 45-m change in elevation. The downstream terminus of each ravine was its confluence with the other. No other salamander species utilized the ravine streams at this site. Methods Starting at the confluence of these two streams (designated “0”) and moving upstream, we placed stakes at 30-m intervals along each stream to the point where water was no longer present in March 2002. The stream segments were survey stations, each identified by the stream name and the number of meters it was from the confluence (e.g., the first station in Kendeigh from the confluence was Kendeigh 30). We marked 23 stations in Swenson (720 m) and 38 (1140 m) in Kendeigh (Fig. 1). We conducted visual-encounter surveys in the upstream direction, turning over rocks and debris. Stations were surveyed sporadically from April 2002 through December 2003. Each station was visited at Figure 1. Distribution of Eurycera cirrigera (Southern Two-lined Salamander) nests, larvae, metamorphs, and adults among stations at Swenson Ravine, 2002 and 2003 combined. 2008 J. Jakubanis, M.J. Dreslik, and C.A. Phillips 133 least twice in each year. Site visits varied because of weather constraints. We spent more time surveying stations in Swenson because of the greater number of rocks to be checked under for the presence of salamanders. We conducted two types of land-based surveys once per month, using visual-encounter survey techniques (i.e., turning bark, logs, leaf litter, and rocks) along 1-m wide transects. Streamside surveys started at a randomly chosen station marker, were parallel to the stream, 10 m from the stream edge, and proceeded for two station lengths (60 m). Inter-ravine surveys started at a randomly chosen station marker in Swenson Creek and proceeded over the ridge to Kendeigh Creek, following a transect that was the shortest distance between the two streams. Reproduction At each rock with one or more egg clutches, we recorded: station number; nest-rock length, width (length x width = surface area), and thickness (to the nearest mm using a plastic ruler); number of adults, metamorphs, and larvae within a 40-cm radius; number of separate clutches; and number of eggs per clutch. Occasionally, the number of eggs could not be determined because eggs were stacked on top of each other or hidden below a layer of algal growth; in these areas we recorded only the presence of eggs. Population structure We determined age cohorts as follows: adults were yellow with no gills or tail fin; metamorphs were gray or developing yellow pigment, and occasionally had gill and tail fin remnants; and larvae were gray, with gills and a tail fin. For all adults and metamorphs captured, we measured snout–vent length (SVL, tip of the nose to the posterior end of the cloaca) to the nearest mm using a plastic ruler. Adults were given a ravine-specific toe-clip, which allowed us to monitor movements between ravines as well as abundance. Dip nets were used to capture larvae in water with low visibility, and a subsample of larvae was collected, euthanized in MS-222, and preserved in 10% formalin. Preserved larvae were measured later in the laboratory with digital calipers to the nearest 0.01 mm. Statistical analysis We used ANCOVA to determine the effects of year, ravine, and rock surface area (independent variables) on clutch size (dependent variable), with the SVL of the attending female as the covariate. Rock surface area was categorized into five 100-cm2 size classes. When the covariate SVL was not significant or no female was attending, we reduced the ANCOVA to an ANOVA and retained year and rock size as categories. We used multivariate logistic regression to determine if the rock parameters of thickness, greatest axis (length or width), and surface area (length x width) were related to the presence of more than one nest per rock. We used a Komolgorov-Smirnov (K-S) cumulative probability test to determine if nests and salamanders were uniformly distributed among stations and years for Swenson Ravine. Finally, 134 Northeastern Naturalist Vol. 15, No. 1 we used χ2 tests to determine if the number of nests, larvae, metamorphs, and adults were equal across seasons. All tests were performed in SPSS® ver 12.0 or 14.0, and nominal alpha values were set at 0.05. Results We spent a total of 313 person-hours during in-stream searches: 38 and 66 person-hours at Kendeigh in 2002 and 2003, respectively; 54 and 155 personhours at Swenson in 2002 and 2003, respectively. A total of 5.18 person-hours were spent performing streamside searches: 0 and 0.33 person-hours at Kendeigh in 2002 and 2003, respectively; 1.5 and 3.35 person-hours at Swenson in 2002 and 2003, respectively. A total of 24.47 person-hours were spent conducting inter-ravine surveys: 13.57 person-hours in 2002 and 10.9 personhours in 2003. Reproduction From April 2002 through December 2003, we detected 441 clutches; 210 and 220 from Swenson in 2002 and 2003, respectively; one and ten from Kendeigh in 2002 and 2003, respectively. Thirty-six clutches contained eggs that were stacked on top of each other or hidden beneath a layer of algal growth, preventing accurate determination of clutch size. When those nests are removed from the analyses, mean clutch size was 29.8 eggs (± 18.8 [SD]; range = 1–117; n = 405). Clutch size did not co-vary with attending adult SVL (F1,101 = 0.65, p = 0.427), nor did SVL interact with rock size (F4,101 = 1.27, p = 0.287), year (F1,101 = 0.80, p = 0.374), or the combination of rock size and year (F4,101 = 1.28, p = 0.287); thus the model was collapsed to an ANOVA. Larger nest rocks did not have larger clutches associated with them (F4,101 = 0.830, p = 0.509), and this pattern held true between years (F4,101 = 2.20, p = 0.075). Clutch size was larger in 2003 than in 2002 (F1,101 = 10.50, p = 0.002). The smaller number of clutches detected at Kendeigh (n = 11) precluded any analysis of spatial distribution. At Swenson, nests were uniformly distributed in 2002 (K-S Z = 1.09, p = 0.157), in contrast to 2003 when nests were more abundant upstream in (K-S Z = 2.02, p > 0.001). We detected more nests in 2003 than 2002 (K-S Z = 1.85, p = 0.001). Out of 441 clutches, we detected 117 (26.5%) under the same rock with at least one other clutch. No clutches were detected under the same rock in Kendeigh Ravine. In Swenson Ravine, 32.7% (n = 72) of clutches were under the same rock in 2002 compared to 21.4% (n = 45) in 2003. The overall model for the logistic regression was significant (-2LL = 479.1) and predicted 73.9% of the cases correctly. Neither the thickness of a rock (B = -0.001 ± 0.031, Wald = 0.002, p = 0.963, df = 1) nor the estimated surface area (B = 0.021 ± 0.011, Wald = 3.52, p = 0.061, df = 1) had an effect on whether or not multiple nests were deposited, after controlling for other predictors. However, the length of the greatest axis of the rock did effect whether multiple nests were oviposited (B = -0.259 ± 0.093, Wald = 7.71, 2008 J. Jakubanis, M.J. Dreslik, and C.A. Phillips 135 p = 0.005, df = 1). Multiple nests were 1.30 ± 0.91 times more likely to be deposited under longer rocks. Nests were not found uniformly throughout the year (χ2 = 807.7, p < 0.001, df = 3). All nests were found in the spring and early summer, with 83% of the nests found in the spring (Fig. 2). Overall nest attendance across years was 35.8%, with attendance always being highest for Swenson Ravine in any year. In 2002, 22.7% and 0% of nests in Swenson and Kendeigh ravines, respectively, were found in association with an adult. In 2003, 50.5% and 20.0% of nests in Swenson and Kendeigh ravines, respectively, were found in association with an adult. Population Structure From April 2002 to December 2003, over all age cohorts, we recorded 2202 salamanders in Kendeigh (379 and 77 in 2002 and 2003, respectively) and Swenson (1674 and 72 in 2002 and 2003, respectively) streams. Larvae dominated most samples, especially in upstream stations (Fig. 1). This upstream distribution of larvae was non-uniform over the length of the stream for 2002 (K-S Z = 3.30, p > 0.001), 2003 (K-S Z = 1.49, p = 0.02), and overall (K-S Z = 1.49, p = 0.02). Metamorphs were the least common stage class encountered (Fig. 1), and their distributional pattern was bimodal. A more upstream mode was present in 2002 (K-S Z = 2.11, p > 0.001), and a more downstream mode was present in 2003 (K-S Z = 2.46, p > 0.001). Adults were the second most abundant stage encountered and were more abundant upstream in 2002 (K-S Z = 1.39, p = 0.033), 2003 (K-S Z = 1.64, p = 0.007), and overall (K-S Z = 1.40, p = 0.031; Fig 1). Figure 2. Abundance of Eurycea cirrigera (Southern Two-lined Salamanders) by season, pooled across ravines and years. 136 Northeastern Naturalist Vol. 15, No. 1 Larvae were not found equally across the seasons (χ2 = 1152.7, p < 0.001, df = 3). Most larvae were encountered in the spring and summer (97.1%), with 88.4% of individuals detected in the summer (Fig. 2). The distribution of metamorphs was unequal across seasons (χ2 = 307.2, p < 0.001, df = 3), with metamorphs being encountered in greatest abundance during the summer (Fig. 2). Finally, adults were not equally distributed in abundance across seasons either (χ2 = 396.3, p < 0.001, df = 3). Their abundance was highest in both the spring and summer. In Swenson, adults were significantly larger in 2003 than 2002 (2-tailed t-test, p < 0.001). Small sample sizes for metamorphs in Swenson and Kendeigh overall precluded statistical analysis of size differences. Fifty-seven and seven larvae were sacrificed and measured from Swenson and Kendeigh, respectively (Table 1). We observed six adult and two metamorph Southern Two-lined Salamanders during inter-ravine surveys conducted from November 10, 2002 through November 19, 2003. Adults were found in fall, whereas the metamorphs were found in July. Five of the adults were found on the Swenson side of the ravine, whereas one was found on the Kendeigh side. The two metamorphs were observed on top of the ridge dividing the ravines. None of the eight salamanders were marked. No salamanders were encountered in the streamside surveys. A total of 187 adult Southern Two-lined Salamanders were toe-clipped. Nineteen adult salamanders were re-captured once during this study. Only one had a toe-clip indicating it had moved from the neighboring ravine. Table 1. Snout–vent length (SVL) of three age cohorts of Eurgea cirrigera (SouthernTwo-lined Salamanders) encountered in two streams at Vermilion River Observatory Research Area, IL. Ravine Year SVL Statistic Larvae Metamorphs Adults Swenson 2002 Mean 17.72 25.81 40.4 SD 8.36 3.33 4.75 Range 6.68–32.11 21.63–32.11 23–48 n 15 7 54 2003 Mean 18.17 24.83 42.8 SD 6.21 1.87 2.49 Range 6.11–41.64 22.75–26.37 30–49 n 42 3 112 Kendeigh 2002 Mean 14.40 - 40.6 SD 5.26 - 3.07 Range 8.33–17.66 - 31–44 n 3 - 18 2003 Mean 20.36 - 42.3 SD 9.27 - 1.53 Range 6.63–26.23 - 26–49 n 4 - 3 2008 J. Jakubanis, M.J. Dreslik, and C.A. Phillips 137 Discussion Reproduction Mean clutch size for Southern Two-lined Salamanders at our study site in Illinois (29.8 ± 0.9 [SE] eggs per clutch) was lower than those reported from Mississippi (mean = 53, n = 7; Marshall 1996), Virginia (mean = 52, n = 26; Wood and McCutcheon 1954), West Virginia (mean = 42, n = 6 ; Brophy and Pauley 2002), Ohio (mean = 39 ± 2.7 [SE], n = 49; Baumann and Huels 1982), and Georgia (mean = 36, n = 37; Guy et al. 2004). Of these locations, only the Ohio site is clearly not on the periphery of the species’ range (Guttman 1989). Categorizing the remaining sites is hindered by incomplete knowledge of the local distribution of Southern Two-lined Salamanders. Based on the Ohio estimates provided by Baumann and Huels (1982), there are significant differences among Ohio and Illinois sites in clutch size. It is possible that our elimination of 36 clutches that contained eggs that were stacked has biased our average clutch size downward, which may account for our lower mean compared to the other studies. However, in all 36 cases, the egg count was already over 120 when we stopped counting. Because the maximum number of ovarian eggs from a female Southern Two-lined Salamander is 115 (Baumann and Huels 1982), it seems likely that all of these uncountable clutches were the product of more than one female. Indeed, our maximum clutch size included in the calculation of our mean (117 eggs) may actually be from more than one female, suggesting that our estimated clutch size might be inflated. The number of eggs per clutch did not differ significantly between years, between ravines, or between years and ravines. Number of eggs per clutch also did not differ significantly with attending adult SVL. A positive relationship between female body size and clutch size was reported for a population in Georgia (Wood and McCutcheon 1954), but no such relationship was found for an Ohio population (Baumann and Huels 1982). We found that females of varying SVLs exploited rocks of varying surface areas and did not appear to select rocks as deposition sites based on rock size alone. Guy et al. (2004) also failed to show a positive relationship between female SVL and nest-rock surface area. More clutches were found in the upstream portions of Swenson in both years. Overall, 188 more clutches were found upstream of the 300 m station marker (66 more in 2002 and 122 more in 2003) compared to downstream of the marker. This finding suggests that Southern Two-lined Salamanders prefer ovipostion sites in upstream portions of Swenson. Environmental factors such water depth, velocity, turbidity, and predator levels may vary as a function of stream location and could explain this trend. In Swenson ravine, there was a high proportion of multiple clutches deposited under the same rock. Although females used rocks of all sizes, multiple nests were much more likely to be oviposited under longer rocks, which agrees with Baumann and Huels (1982) in Ohio. The dimensions of available rocks may be a primary factor determining whether females will 138 Northeastern Naturalist Vol. 15, No. 1 oviposit beneath the same rock. Interestingly, we observed that Swenson Ravine contained more rocks than Kendeigh; in addition, fewer clutches and adults were discovered at Kendeigh. Our results suggest that the population of salamanders in Swenson may be so large that individuals exploit all suitable rocks, forcing some females to share nesting sites. Rock sharing does not appear to be an issue in Kendeigh, despite fewer suitable nesting rocks, possibly because the population in this creek is much smaller. Although the Southern Two-lined Salamander exhibits parental care of nests (Petranka 1998), only 35.8% of 441 clutches we detected had an adult nearby in attendance. Other studies have shown stronger female-clutch associations, but their results may be due to smaller sample sizes. Guy et al. (2004) discovered females guarding 73.0% of 37 observed nests, and Wood and McCutcheon (1954) discovered females guarding 66.7% of 6 observed nests. Population structure We found no significant difference in adult SVL among ravines, years, or ravines and years. All adult salamanders in this study fell within the 6.5–12 cm total-length range provided by Petranka (1998), and animals of varying lengths were found in each ravine throughout both years. Few studies have investigated the within-stream distribution of Southern Two-lined Salamanders and E. bislineata Green (Northern Two-lined Salamanders). Bruce (1986) suggested that first-year Northern Twolined Salamander larvae are more common downstream, whereas second-year larvae and metamorphs are more common upstream. He suggested that this phenomenon might occur because first-year larvae cannot compensate for downstream drift compared to older animals. In the two ravines at the VRO, all first-year larvae were concentrated in the upstream portion of the creek. Smith and Grossman (2003) suggest that larvae prefer habitats that provide ample substratum covered with little silt. Their data also suggest that larvae avoid deeper regions of creeks as an avoidance response to fish and other predators that frequent such areas. In both Swenson and Kendeigh, more rocks and cover items occur upstream than downstream. Upstream portions of each creek also had fewer deep regions than downstream portions. These characteristics might explain why larvae were more prevalent in upstream portions of the creeks. The presence of more cover might allow larvae protection from downstream drift as well as from predators. Southern Two-lined Salamanders were much more abundant in Swenson than in Kendeigh. Cover objects seemed to be more abundant in Swenson, so this may be a factor, as suggested by Smith and Grossman (2003). However, Swenson has more silt compared to the sandy substrate of Kendeigh and Swenson is deeper than Kendeigh throughout most of its course. In consideration of both of these habitat features, the higher population of salamanders in Swenson is in contrast to the findings of Smith and Grossman (2003). Yet much of Kendeigh was reduced to widely spaced pools during the summer, so this may explain why, despite being shallower than Swenson, it was 2008 J. Jakubanis, M.J. Dreslik, and C.A. Phillips 139 less suitable for larvae. Predatory Semotilus atromaculatus Mitchill (Creek Chubs) occupied the remaining deep pools at Kendeigh, and thus larvae would not only have to compete for limited resources, but would be subject to increased predator densities. Swenson also supported a wider variety of aquatic organisms, particularly an abundance of caddisfly larvae, which were often found on the undersides of rocks, whereas almost none were found in Kendeigh. This may indicate a better water quality in Swenson’s creek than in Kendeigh’s creek, which also may contribute to the scarcity of Southern Two-lined Salamanders in Kendeigh. Of 187 adult Southern Two-lined Salamanders measured, only 19 individuals were recaptured. Because so few recaptures occurred, an accurate estimate of population size is not possible, but it suggests that the density of salamanders is high. Since Southern Two-lined Salamanders exhibit highly variable surface activity, they might violate the equal catchability assumption of most population estimation models (Hyde and Simmons 2001). Surface activity may be seasonal and weather-dependent, and individuals may choose to remain underground during harsh conditions (Jung et al. 2000). Surface activity might vary over time and between habitats and microhabitats, and censuses might provide uncertain data due to variations in animal behavior or differences in observers’ detection skills (Hyde and Simmons 2001). We were unable to determine whether animals at our study site represented one or two populations. Of the 19 salamanders recaptured in this study, only one exhibited a toe clip indicative of having traveled from the neighboring ravine. The presence of this individual may represent an uncommon occurrence between two populations or a natural injury coincident with our marking scheme. Marking errors were unlikely due to consistent double-checking and back-up verification from assistants. Because these animals exhibited low recapture rates, it is possible that surface activity is so variable that the likelihood of recapturing a migrating individual is low, even if migration is common. However, the presence of two metamorphs on the ridge between the ravines suggests migration between streams occurs. Whether the significantly smaller clutch size for Southern Two-lined Salamanders in VRO is related to its peripheral position within the species’ overall range will have to wait until better local range maps are available and more peripheral populations are investigated. Acknowledgments We would like to thank J. Petzing, J. Mui, and our many field assistants for their support of this project. Much gratitude goes to S. Buck for his communication of knowledge of the Vermilion River Observatory. Thanks also to S. Taylor for providing field equipment, and to M. 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