Southeastern Naturalist G. A. Marvin and P.V. Cupp Jr. 2018 Vol. 17, No. 1 166 2018 SOUTHEASTERN NATURALIST 17(1):166–175 Chemical Detection of Intraguild Predators (Gyrinophilus, Pseudotriton) by Streamside Plethodontid Salamanders (Eurycea) Glenn A. Marvin1,* and Paul V. Cupp Jr.2 Abstract - To examine whether chemical cues from intraguild predators may affect microhabitat selection by plethodontid salamanders of the genus Eurycea, we tested metamorphosed individuals for the ability to discriminate among odors from 3 larger salamander species. Metamorphosed individuals of Eurycea and 2 of the larger species (Gyrinophilus porphyriticus [Spring Salamander] and Pseudotriton ruber [Red Salamander]) are semiaquatic and often inhabit streamside environments, whereas individuals of the third of the larger species, Plethodon glutinosus (Northern Slimy Salamander), are strictly terrestrial and primarily inhabit woodlands. In the lab, we placed each Eurycea individual in a petri dish with 2 substrate choices. One substrate had chemical cues (i.e., skin secretions and wastes deposited for 6 d) from an adult individual of 1 of the 3 large salamander species, whereas the other substrate had chemical cues from an adult individual of a small Plethodon species (P. dorsalis [Northern Zigzag Salamander] or P. ventralis [Southern Zigzag Salamander]). We recorded the location of each individual on either substrate at 3-min intervals for 2 h. For individuals of both Eurycea cirrigera (Southern Two-lined Salamander) and E. longicauda (Long-tailed Salamander), we tested different experimental groups with the odor of 1 large salamander species. Our results indicate that Southern Two-lined Salamander individuals in Kentucky avoid chemical cues from Spring Salamander and Red Salamander, but not Northern Slimy Salamander. Individuals of both Southern Two-lined Salamander and Long-tailed Salamander in Alabama avoid chemical cues from Red Salamander, but not Northern Slimy Salamander. Spring Salamander and Red Salamander often prey on salamanders, whereas Plethodon rarely do; thus, we conclude that individuals of different Eurycea species and populations distinguish the odors of salamander species that are potential predators. Introduction Animals use a variety of strategies to avoid predation. Prey may use predatoravoidance mechanisms to avoid the foraging microhabitats of predators and/or antipredator mechanisms that reduce the probability of predation when they are within the perceptual field of predators (Brodie et al. 1991). For many prey, the recognition of chemical cues such as predator odors (i.e., kairomones) and alarm chemicals released from injured prey may allow them to avoid predation (Ferrari et al. 2010). Detection of such chemicals may be especially important for prey species that live in habitats with low visibility (e.g., Hickman et al. 2004). For instance, many amphibian 1Department of Biology, University of North Alabama, Box 5048, 1 Harrison Plaza, Florence, AL 35632. 2Department of Biology, Eastern Kentucky University, Richmond, KY 40475. *Corresponding author - gamarvin@una.edu. Manuscript Editor: John Placyk Southeastern Naturalist 167 G. A. Marvin and P.V. Cupp Jr. 2018 Vol. 17, No. 1 species avoid chemicals that indicate the presence of a predator. In aquatic habitats, larval and adult salamanders of several species detect and avoid chemical cues from predatory fish (e.g., Epp and Gabor 2008, Petranka et al. 1987). In terrestrial habitats, some salamander species avoid areas with chemical cues from predatory snakes (e.g., Cupp 1994) and places where they detect alarm chemicals (e.g., Marvin and Hutchison 1995). However, avoidance responses to chemicals are not ubiquitous and may differ among prey populations and species (Marvin et al. 2004), perhaps due to variation in environmental factors such as the severity of predation pressure or the types of predators that are encountered. The detection and avoidance of chemicals from intraguild predators can affect the selection of microhabitats by both invertebrate and vertebrate animal species (e.g., Choh et al. 2010, Huang and Pike 2012). However, few studies have examined the effect of odors from intraguild predators on microhabitat preference in amphibians (e.g., Roudebush and Taylor 1987). Intraguild predation is common among animals and may often play an important role in population dynamics and community structure (e.g., Hairston 1987, Polis et al. 1989). For plethodontid salamanders, the avoidance of odors from intraguild predators may help to explain the structure of some streamside communities (Hairston 1986, Roudebush and Taylor 1987). Although some effects of intraguild predation on larvae of the streamside plethodontid salamander genera Eurycea, Gyrinophilus, and Pseudotriton have been examined (Beachy 1994, Gustafson 1993), the importance of intraguild predation among metamorphosed individuals is unexplored. In this study, we examined avoidance behavior of metamorphosed individuals of Eurycea to odors from larger salamander species that are either potential predators or unlikely predators. We tested the responses of individuals from 2 species and different populations to substrates with chemical cues from adults of the plethodontid species Gyrinophilus porphyriticus Green (Spring Salamander) and Pseudotriton ruber (Latrelle) (Red Salamander), which often eat smaller salamanders, and Plethodon glutinosus (Green) (Northern Slimy Salamander), which primarily eat invertebrate prey. Both larval and metamorphosed individuals of Spring Salamander and Red Salamander are known predators of smaller salamanders including Eurycea species; salamanders can be a major component of their diets (Beachy 1994, Bishop 1941, Bruce 1972, Dunn 1926, Gustafson 1993). Although adults of large Plethodon species may occasionally eat very small salamanders (Oliver 1967, Powders 1973, Powders and Tietjen 1974), their diets primarily consist of invertebrate animals (Davidson 1956, Hamilton 1932, Oliver 1967, Pope 1950). Whereas metamorphosed individuals of the Eurycea species, as well as Spring Salamanders and Red Salamanders, are semiaquatic and often occupy streamside habitats, Northern Slimy Salamanders are strictly terrestrial and primarily occur in woodlands (Petranka 1998). However, individuals of each streamside species may also be found in wooded areas away from streams on moist or rainy evenings, thus providing opportunities for predation by terrestrial salamanders. We tested the hypothesis that metamorphosed individuals of Eurycea cirrigera (Green) (Southern Two-lined Salamander) and E. longicauda (Green) (Long-tailed Salamander) avoid substrates Southeastern Naturalist G. A. Marvin and P.V. Cupp Jr. 2018 Vol. 17, No. 1 168 with chemical cues from probable intraguild predators (i.e., adults of Spring Salamander or Red Salamander), but do not avoid substrates with chemical cues from likely non-predatory salamanders of the genus Plethodon. Methods Animal collection and care We collected salamanders in Kentucky during March and April, 1988 and 1989. We collected adult individuals of Southern Two-lined Salamander and Spring Salamander in Rockcastle and Adair counties, Red Salamander in Rockcastle and McCreary counties, Northern Slimy Salamander in Rockcastle and Jackson counties, and P. dorsalis Cope (Northern Zigzag Salamander) in Madison County. Due to resource constraints, we did not collect and test individuals of other Eurycea species in Kentucky. In Alabama during 2009 and 2010, we collected individuals of Southern Two-lined Salamander, Long-tailed Salamander, Red Salamander, Northern Slimy Salamander, and Southern Zigzag Salamander in Lauderdale County. Based on the body sizes of Long-tailed Salamander individuals (30–48 mm standard body length), most individuals (n = 28) were likely juveniles, but some (n = 8) were probably small adults (Anderson and Martino 1966). We used the same ratio of juveniles to small adults (i.e., 14 to 4) in different experimental groups. We did not include larger individuals of Long-tailed Salamanders because we presumed that smaller individuals might be eaten more readily by, and thus, might experience greater predation risk from, predatory salamanders. Individuals of other species from Alabama were adults. We did not collect individuals of Spring Salamander in Alabama because they were rare at the collection locality. Richard Highton (University of Maryland, College Park, MD, pers. comm.) verified the presence in Lauderdale County of Northern Slimy Salamander and Southern Zigzag Salamander (i.e., confirmed that they were not P. mississippi (Highton) [Mississippi Slimy Salamander] or Northern Zigzag Salamander, species which are very difficult to distinguish visually from Northern Slimy and Southern Zigzag, respectively). We did not determine the sex of salamanders. We kept individual salamanders in separate housing containers (~14 cm × 14 cm × 3.5 cm) within environmental chambers at 10–15 °C with a 12-h light:12-h dark photoperiod. We provided all salamanders with clean paper towel substrates once a week. For at least 3 weeks prior to experiments, we fed individuals of Northern Zigzag Salamander and Southern Zigzag Salamander vestigial-wing Drosophila to satiation once each week, and we fed individuals of large salamander species small Eisenia fetida Savigny (English Redworms) to satiation once each week. We minimized the potential effect of variation in predator diet (i.e., with salamanders as prey) on kairomone avoidance by providing a predator diet of invertebrate animals (e.g., Madison et al. 1999). For 6 d before experiments, we kept all salamanders individually in plastic petri dishes (14.5 cm diameter × 2.5 cm) with moist filter paper lining the bottoms. We added ~2.5 ml of distilled water to each dish to moisten the filter paper. We subsequently used halves of these filter papers soiled with skin secretions and wastes from an Southeastern Naturalist 169 G. A. Marvin and P.V. Cupp Jr. 2018 Vol. 17, No. 1 individual of Spring Salamander, Red Salamander, Northern Slimy Salamander, Northern Zigzag Salamander, or Southern Zigzag Salamander as substrate choices in experiments. Substrate choice experiments The methods we employed for our substrate-choice experiments were very similar to those of Cupp (1994). We conducted our experiments from April to June at room temperature (~19–21 °C) under overhead red light (40 W) between the 2nd and 5th h of the scotophase. For an experimental trial, we transferred each Eurycea individual to a clean glass petri dish (14.5 cm diameter × 2.5 cm) with a bottom that was lined with 2 soiled filter paper halves. One substrate had the odor of an individual of a large salamander species (Spring Salamander, Red Salamander, or Northern Slimy Salamander), whereas the other substrate had the odor of an individual of a small Plethodon species (Northern Zigzag Salamander or Southern Zigzag Salamander). We provided substrate permeated with the odor of a small Plethodon as the alternative choice in each trial to ensure that individuals of Eurycea would not simply select a clean, “odorless” substrate over a soiled substrate infused with salamander odor. We tested each individual of Eurycea with only 1 of the 3 large species’ substrate odor (as summarized below). We tested individuals only with odors of salamanders from the same state. Opaque partitions separated neighboring test chambers. We released each individual at the center of a petri dish, placed lids on dishes, and then waited 10–15 min before recording data. From behind a blind, we observed and recorded each individual’s position on a substrate (hereafter, referred to as the individual’s substrate choice or response) at 3-min intervals during a 2-h trial. Thus, we recorded 40 responses for each individual in an experimental trial. If a salamander’s body straddled the 2 substrates, we recorded the response for the substrate on which the animal's snout rested. We tested individuals of Southern Two-lined Salamander in Kentucky in different experimental groups (A, B, and C) with substrate odor of Northern Zigzag Salamander versus substrate odor of Spring Salamander (group A, n = 19), Red Salamander (group B, n = 20), or Northern Slimy Salamander (group C, n = 20). We tested individuals of Southern Two-lined Salamander in Alabama with substrate odor of Southern Zigzag Salamander versus substrate odor of Red Salamander (group D, n = 20) or Northern Slimy Salamander (group E, n = 20). We tested individuals of Long-tailed Salamander in Alabama with substrate odor of Southern Zigzag Salamander versus substrate odor of Red Salamander (group F, n = 18) or Northern Slimy Salamander (group G, n = 18). To compare a pair of substrate-odor choices (a small Plethodon species versus a large salamander species), we conducted 2 trials for each individual of Eurycea in the experimental group with 1 week between trials. We hereafter refer to the 2 trials for an individual as the 1st and 2nd trials. In the 1st trial, we placed the substrate with the odor of the large salamander species on the right side of the petri dish and the small species’ odor on left. In the 2nd trial, we placed the substrate with odor of the large salamander species on the left side and with the small species’ odor on right. For each individual of Eurycea in Southeastern Naturalist G. A. Marvin and P.V. Cupp Jr. 2018 Vol. 17, No. 1 170 an experimental group, we calculated an avoidance index as the difference between the number of responses to the substrate with odor of the small species in the 1st trial and the number of responses to the substrate with odor of the large species in the 2nd trial. For the avoidance index (which could range from -40 to 40), a value close to zero indicated no odor avoidance while a high positive value indicated avoidance of the large salamander species’ odor. Statistical analyses We square-root transformed data when necessary to meet assumptions of parametric tests. When assumptions were not met following data transformation, we conducted non-parametric tests. For individuals of Southern Two-lined Salamander in Kentucky, we performed analysis of variance (ANOVA) to compare avoidance indices to the odors of Spring Salamander, Red Salamander, and Northern Slimy Salamander. For individuals of either Southern Two-lined Salamander or Longtailed Salamander in Alabama, we used a t-test to compare avoidance indices (square-root–transformed) to the odors of Red Salamander and Northern Slimy Salamander. We employed a Wilcoxon matched-pairs signed-ranks test to assess position bias in each experimental group. For each individual in an experimental group, we compared the number of responses to the substrate with odor from the small salamander species in the 1st trial to the number of responses to the substrate with odor from the small salamander species in the 2nd trial. Results For individuals of Southern Two-lined Salamander in Kentucky, avoidance indices were significantly different in response to the odors of Spring Salamander, Red Salamander, and Northern Slimy Salamander (F2,56 = 6.38, P < 0.01; Fig. 1). Avoidance indices were significantly greater to the odor of either Spring Salamander or Red Salamander than to the odor of Northern Slimy Salamander (t = 3.45 and 2.52, P < 0.01 and P = 0.029, respectively), but there was no significant difference between avoidance indices to the odors of Spring Salamander and Red Salamander (t = 0.95, P = 0.34; Fig. 1). For individuals of Southern Two-lined Salamander in Alabama, avoidance indices to the odor of Red Salamander were significantly greater than to the odor of Northern Slimy Salamander (t38 = 3.18, P < 0.01; Fig. 1). For individuals of Long-tailed Salamander in Alabama, avoidance indices to the odor of Red Salamander were significantly greater than to the odor of Northern Slimy Salamander (t34 = 5.10, P < 0.001; Fig. 1). Individuals of Eurycea showed no statistically significant position bias in any of the experiments (Table 1). Discussion Given the great diversity of plethodontid salamander species that inhabit the woodlands and streams of the southeastern US, an increased knowledge of the variety of interactions among these species can improve our understanding of these ecosystems. For example, intraguild predation has been implicated as an important factor determining microhabitat use and population size in some Southeastern Naturalist 171 G. A. Marvin and P.V. Cupp Jr. 2018 Vol. 17, No. 1 Figure 1. Avoidance indices for individuals of Eurycea cirrigera (Southern Two-lined Salamander) and E. longicauda (Long-tailed Salamander) during 2-choice lab experiments when presented with substrates with the odor of a small salamander species (Plethodon dorsalis Cope [Northern Zigzag Salamander] in Kentucky or P. ventralis [Southern Zigzag Salamander] in Alabama) or a large, potentially predatory salamander species (Gyrinophilus porphyriticus [Spring Salamander], Pseudotriton ruber [Red Salamander], or P. glutinosus [Northern Slimy Salamander]). Box plots show min, max, median, mean (dotted line), and percentiles (10th, 25th, 75th, and 90th). Spring = G. porphyriticus, Red = P. ruber, Slimy = P. glutinosus. An asterisk (*) = P < 0.03 (t-test for each Eurycea species in Alabama, or ANOVA with Holm-Sidak multiple comparison method for Southern Two-lined Salamander in Kentucky). Table 1. Results of statistical tests for position bias by individuals of Eurycea cirrigera (Southern Two-lined Salamander) and E. longicauda (Long-tailed Salamander) during 2-choice lab experiments when presented with substrates with the odor of a small salamander species (Plethodon dorsalis Cope [Northern Zigzag Salamander] in Kentucky or P. ventralis [Southern Zigzag Salamander] in Alabama) or a large, potentially predatory salamander species (Gyrinophilus porphyriticus [Spring Salamander], Pseudotriton ruber [Red Salamander], or P. glutinosus [Northern Slimy Salamander]). To test for position bias in each experimental group, we compared left-side versus right-side choices for substrate with odor of the small species between 2 experimental trials. Statistical values are from Wilcoxon matched-pairs signed-ranks tests. Position bias test Eurycea species (state) Potential predator n W T+ Z P Southern Two-lined Salamander (KY) Spring Salamander 19 12.0 91.5 0.3 0.799 Red Salamander 20 64.0 117.5 1.4 0.167 Northern Slimy Salamander 20 -6.0 102.0 -0.1 0.927 Southern Two-lined Salamander (AL) Red Salamander 20 -60.0 38.0 -1.6 0.130 Northern Slimy Salamander 20 -21.0 66.0 -0.5 0.644 Long-tailed Salamander (AL) Red Salamander 18 37.0 78.5 1.1 0.303 Northern Slimy Salamander 18 -2.0 51.5 -0.1 0.952 Southeastern Naturalist G. A. Marvin and P.V. Cupp Jr. 2018 Vol. 17, No. 1 172 streamside salamander communities (Hairston 1987). Although the impacts of intraguild predation by larval individuals of Gyrinophilus and Pseudotriton on growth and survivorship of larval salamanders of the genus Eurycea have been examined (Beachy 1994, Gustafson 1993), the potential ef fect of intraguild predation on microhabitat selection by metamorphosed individuals is unknown for these plethodontid salamanders. Our results indicate that metamorphosed individuals of Southern Two-lined Salamander in Kentucky avoid substrates with chemical cues from the predatory salamander species Spring Salamander and Red Salamander. Likewise, metamorphosed individuals of Southern Two-lined Salamander and Long-tailed Salamander in Alabama apparently avoid substrates with chemical cues from Red Salamander. These results indicate that metamorphosed individuals of different Eurycea species and populations detect and avoid odors from predatory salamanders. We infer that this ability has selective value by reducing predation risk via enhanced predator avoidance in the field. However, because species interactions in a guild involve both competition and predation, additional research is needed to determine to what degree the behavioral response to the odor of another species is a reflection of predation and/or competition. Similarly, the recognition and avoidance of chemical cues from different predators has been implicated as a beneficial behavior that reduces predation risk for individuals of many other salamander species. Individuals of D. monticola Dunn (Seal Salamander) avoid chemical cues from predatory D. quadramaculatus (Black-bellied Salamander) (Roudebush and Taylor 1987). Individuals of Northern Zigzag Salamander, P. richmondi Netting and Mittleman (Southern Ravine Salamander), and D. ochrophaeus Cope (Allegheny Mountain Dusky Salamander) avoid substrates with chemical cues from predatory snakes (Cupp 1994). Individuals of E. nana Bishop (San Marcos Salamander) and E. multiplicata (Many-ribbed Salamander) avoid chemical cues from predatory fish (Epp and Gabor 2008, Hickman et al. 2004). Larval individuals of Ambystoma annulatum Cope (Ringed Salamander) may reduce predation risk by decreasing activity in response to the detection of chemical cues from predatory newts (Mathis et al. 2003). Salamanders may also respond to chemical cues by altering foraging and mating behavior to reduce exposure to predators (Fonner and Woodley 2015, Sullivan et al. 2002). For prey in some environments, it may also be beneficial to distinguish among chemical cues from various species and only avoid odors from probable predators (e.g., Crane et al. 2012, Epp and Gabor 2008). Our results indicate that metamorphosed individuals of Southern Two-lined Salamander and Long-tailed Salamander do not avoid substrates with chemical cues from adult individuals of Northern Slimy Salamander, which are large enough to be a potential predator of small salamanders but do not typically prey on individuals of Eurycea species (e.g., Hamilton 1932). Individuals of Eurycea did not avoid substrate with the odor of large, adult Northern Slimy Salamander; thus, their avoidance of substrate with the odor of either Spring Salamander or Red Salamander was probably not simply due to an avoidance of a larger amount of chemicals produced by a larger salamander. Southeastern Naturalist 173 G. A. Marvin and P.V. Cupp Jr. 2018 Vol. 17, No. 1 Our results indicate that metamorphosed individuals of Eurycea species have the ability to discriminate among chemical cues from larger salamander species that are predatory versus those that are non-predatory. This behavior would likely be advantageous for individuals in the field because they would not expend time and energy avoiding sites with odors from large salamanders that are not a likely predatory threat. Similarly, individuals of the terrestrial salamander P. angusticlavius Grobman (Ozark Zigzag Salamander) distinguish between chemical cues from predatory versus non-predatory mammals (Crane et al. 2012). The avoidance of kairomones from intraguild predators can be an important factor that affects microhabitat preference in some prey species (e.g., Choh et al. 2010, Huang and Pike 2012). Likewise, microhabitat selection by metamorphosed individuals of Eurycea may be influenced by kairomones from Spring Salamander and Red Salamander. In many salamander species, individuals deposit odors or pheromones that aid in the location and identification of potential mates and/or the marking of territories (Jaeger and Forrester 1993). For predator–prey interactions among salamander species, perhaps such chemicals can also be recognized by prey to detect the foraging microhabitats of predators. Although the ability to detect predator odors does not necessarily protect an individual from foraging predators, some reduction in predation risk may be gained by selecting microhabitats not frequented by predators. This behavior could be an important factor that influences the selection of a home-range area, territory, refuge, or oviposition site by an individual. For example, females of A. barbouri Kraus and Petranka (Streamside Salamander) apparently benefit from selective oviposition in pools that lack predatory fish (Kats and Sih 1992). Additional research that examines the responses of Eurycea species to predatory salamander odors is needed to investigate whether (1) responses are innate or learned (e.g., whether responses may vary between juveniles and adults), (2) differences in predator diet (e.g., with salamanders as prey versus invertebrate animals as prey) affect responses, (3) ecological factors (e.g., differences in densities of predator or prey species) affect responses, (4) season and time of day affect responses, and (5) detection of odors affects antipredator behaviors, stress levels, activity levels and patterns, and behaviors such as foraging and mating. Acknowledgments Scientific collecting permits were issued by the Kentucky Department of Fish and Wildlife Resources to P.V. Cupp and G.A. Marvin and the Alabama Department of Conservation and Natural Resources to G.A. Marvin. This study was approved by the Institutional Animal Care and Use Committee at Eastern Kentucky University for P.V. Cupp and G.A. 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