2007 NORTHEASTERN NATURALIST 14(2):293–299 Dietary Selection Among Different Size Classes of Larval Ambystoma jeffersonianum (Jefferson Salamanders) Jeff H. Bardwell*,1,2, Christopher M. Ritzi2, and James A. Parkhurst1 Abstract - This study examines changes in the frequency/abundance of prey selection among five size classes of 183 Ambystoma jeffersonianum (Jefferson Salamanders) within a natural, unmanipulated environment. Significant differences were found in prey selection among size classes in vertebrate and macroinvertebrate (specifically coleopteran and dipteran) prey groups, but not microinvertebrates. Predator-size thresholds were noted as diet shifted from predominantly microinvertebrates to increasingly larger macroinvertebrates to the final dietary selection of other vertebrates. This study augments the catalogue of ingested Ambystoma prey and re-examines the nature of ontogenous dietary selection. Introduction Among North American salamanders, aquatic Ambystoma larvae possess several characteristics that make them ideal for studying trophic interactions: first, Ambystoma grow to relatively large sizes and are common throughout the continental United States; second, they have a high reproductive fecundity; and third, larvae are isolated from adults for months within their natal pools (Martof et al. 1980, Petranka 1998). Larval Ambystoma diet selection has previously been studied within natural and, with various degrees of manipulation, artificial environments. Experiments using natural pools emulate realistic conditions of predator populations, prey availability, and prey selection, but are limited in availability, time, and replication (Benoy et al. 2002, Brophy 1980, Cortwright 1988, Smith and Petranka 1987). Artificial pools containing imported water and detritus from natural areas allow increased replication and manipulation of predator/prey ratios while maintaining a facsimile of natural conditions (Morin et al. 1983, Walls and Williams 2001). Other studies have created controlled lab environments where predators are presented with predetermined quantities and types of prey (Leff and Bachmann 1988, Sih and Petranka 1988). The two latter methods artificially manipulate predator/prey population numbers, but allow for the examination of more precise questions and increased statistical replications. Ontogenous diet trends among salamanders have been well studied as they relate to prey size: foraging larval salamanders, including Ambystoma jeffersonianum (Green) (Jefferson Salamander), are limited by their mouth 1Department of Fisheries and Wildlife Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24060. 2Current address - Department of Biology, Sul Ross State University, Alpine, TX 79830. *Corresponding author - urodela1@yahoo.com. 294 Northeastern Naturalist Vol. 14, No. 2 gape (Smith and Petranka 1987, Zaret 1980). Most ontogenous larval Ambystoma studies have compared predator size and diet selection based on prey size and species (Brophy 1980, Holomuzki and Collins 1987, Johnson et al. 2003, Leff and Bachmann 1988, Smith and Petranka 1987). Studies have also included other prey variables such as capture time, seasonal variations, and electivity indices (Dodson 1970, Holomuzki and Collins 1987, Leff and Bachmann 1988). The objectives of this project were to (1) examine the relative difference of frequency and abundance of prey selected by larval Jefferson Salamanders sampled within an unmanipulated, natural population via stomach content analysis, and (2) quantify the trends and significant differences in diet selection among size classes within the sample to observe the nature of ontogenous selectivity. Field Site Description The study site chosen was an old, diked farm pond (approximately 0.2– 0.4 ha) within a small tongue of fragmented woodlands protruding from the main tract of mesic hardwood forest located on Virginia Tech’s Kentland Farm Research Facility in Montgomery County, VA (37º13'45"N, -80º24'50"E). Jefferson Salamanders were the dominant predators, as the pond did not contain bullfrogs, newts, turtles, or fish. Methods Salamander larvae were sampled during two collection trips within a span of two weeks time in June/July 2004. Because prey resources within the pond were observed in similar abundances for both sampling trials, the sum total of samples per site within a single season were combined for analysis, a method used in several prior studies involving larval Ambystoma diet (Benoy et al. 2002, Brophy 1980, Dodson and Dodson 1971, McWilliams and Bachmann 1989, Smith and Petranka 1987, Walls and Williams 2001). The assumption that ambient prey abundances do not significantly change between sampling periods has yet to be tested for Jefferson Salamanders. Salamanders were captured primarily using a stationary seine aided by herding. This technique was accomplished by stomping and splashing through the water toward the net. Other methods (i.e., random sweeping patterns, hand sampling, and aiming toward ripples) yielded inconsistent harvests. Captured salamanders were euthanized in 70% ethanol and identified using larval taxonomic keys (Altig and Ireland 1984). Prey selection A ventral sagittal incision was made in each specimen, and the stomach extracted. Stomachs were placed into petri dishes, bisected, and flushed using 70% ethanol. Contents were examined using a dissecting microscope and identified to the lowest taxonomic level possible using field guides (Conant and Collins 1998) and taxonomic keys (Thorp and Covich 2001). 2007 J.H. Bardwell, C.M. Ritzi, and J.A. Parkhurst 295 Each taxonomic category of stomach contents for each salamander was quantified by frequency and abundance (Table 1). Prey cover classes, similar to those first used by Nudds and Bowlby (1984) to analyze fish dietary component abundances, were used for volumetric estimation of each stomach’s prey contents. Eleven cover-class values were assigned using a scaled percentage distribution to decrease visual estimation error. An estimated range of zero contents would get a cover class of zero. An estimated range of 0–2.5% proportional abundance of stomach contents received a cover-class value of 0.0125; 2.5–10%, 0.0625; 10–21%, 0.155; 21–35%, 0.28; 35–50%, 0.425; 50–65%, 0.575; 65–79%, 0.72; 79–90%, 0.845; 90– 97.5%, 0.9375; and 97.5–100%, 0.9875. Dietary significance The predator-population sample consisted of a total length (TL) size range of 3.0–7.5 cm and was subdivided into five size classes (SC), each spanning 0.5 cm, except SC 1 and SC 5. The nature of stomach-content data measurements (Magnusson et al. 2003) lends itself to skewed and bimodal distributions. Therefore, cover-class volumetric estimations (each data point was limited to one of eleven possible cover classes) were left untransformed for statistical analysis (Table 2). A Kruskal-Wallis test was performed using SPSS ver. 11.5, using a Nemenyi post hoc test on significant factors (Zar 1999). Results Prey selection Diet selection analysis for Jefferson Salamanders was based on the proportional differences of each prey category within the stomach contents of the entire sample (n = 183), independent of the predator’s size. To achieve Table 1. Ambystoma jeffersonianum (Jefferson Salamander) diet frequency and mean abundance; L = larval prey, A = adult prey, * = new dietary record for A. jeffersonianum. Prey category Frequency Mean abundance Microinvertebrates (Nematoda) 172 0.94 Macroinvertebrates 161 0.16 Coleoptera L/A 11 0.28 Diptera L 106 0.06 Hemiptera/Homoptera L/A 7 0.43 Odonata L 2 0.28 Orthoptera A 1 0.43 Megaloptera L 3 0.01 Lepidoptera* L 1 0.56 Unidentified insects 16 0.06 Vertebrates 2 0.85 Rana sylvatica A 1 0.85 Ambystoma jeffersonianum L 1 0.94 Empty 11 - Total (n) 183 296 Northeastern Naturalist Vol. 14, No. 2 greater resolution, large-prey categories were split into more basal taxonomic groups. Microinvertebrates were not split into subgroups, but identified as Nematoda and/or unidentifiable unicellular green algae in binucleate paired clumps. Other zooplankton beyond nematodes were not observed within the stomach contents. Macroinvertebrates were identified to order: Coleoptera, Diptera, Hemiptera, Lepidoptera, Megaloptera, Odonata, Orthoptera, and unidentified. Vertebrates were identified to species: Jefferson Salamander and Rana sylvatica (LeConte) (Wood Frog). All stomachs that contained food were distended similarly and contained various amounts of microinvertebrates inversely proportional to the amount of macro-invertebrate or vertebrate prey ingested. Dietary significance The five size classes of larvae were divided as follows: SC 1: 3.0–4.5 cm (n = 21); SC 2: 4.5–5.0 cm (n = 25); SC 3: 5.0–5.5 cm (n = 46); SC 4: 5.5–6.0 cm (n = 65); and SC 5: 6.0–7.5 cm (n = 26). Only two main prey groups exhibited significantly different abundances among size classes (􀁟 = 0.05): macroinvertebrates (0.022) (coleopterans [0.047] and dipterans [0.041]) and vertebrates (0.016). Several groups expressed trends toward significant abundance differences among size classes (􀁟 = 0.1), including microinvertebrates (0.084) and unidentified insects (0.058). All other prey groups lacked significant differences among size classes (Table 2). Discussion Prey selection This study lends further support to the finding that microinvertebrates (daphnia, copepods, ostracods, or nematodes) appear consistently in larval Ambystoma diets (Brophy 1980, Freda 1983, Wilson and Meret 2003, Table 2. Results of a Kruskal-Wallis test with Nemenyi post hoc analysis (􀁟 = 0.05) to determine the degree of selection between size classes per prey category: results denoted by insignificant groups (i.e., 1–3), insignificant groups with zero abundance values (underlined: i.e., 1–3) and significant differences between groups (i.e., 1 < 2–3–4–5) Prey category 􀁲2 df P-value Size class selection Microinvertebrates (Nematoda) 8.204 4 0.084 Macroinvertebrates 11.476 4 0.022 1 < 2–3–4–5 Coleoptera 9.637 4 0.047 1–3 < 2 < 4–5 Diptera 9.996 4 0.041 1–2–4–5 < 3 Hemiptera/Homoptera 4.706 4 0.319 Odonata 5.989 4 0.200 Orthoptera 1.815 4 0.770 Megaloptera 3.229 4 0.520 Lepidoptera 6.320 4 0.176 Unidentified insects 9.144 4 0.058 Vertebrates 12.143 4 0.016 1–2–3–4 < 5 Ambystoma jeffersonianum 6.038 4 0.196 Rana sylvatica 6.038 4 0.196 2007 J.H. Bardwell, C.M. Ritzi, and J.A. Parkhurst 297 Wurst and Mull 1999), as reflected by the high frequency and mean abundance data we observed (Table 1). Excluding the dipterans (specifically chironomids and chaoborids), most aquatic insects usually are recorded at low frequencies and abundances or not at all (Brophy 1980, Dodson and Dodson 1971, Smith and Petranka 1987). This is consistent with the low frequencies and mean abundances historically recorded for most macroinvertebrates and, by contrast, the high frequency and low mean abundance of dipterans. This indicates that, although Jefferson Salamanders may eat a large number of macroinvertebrates, the small size of most prevents all but the largest orders from comprising a substantial mean volumetric abundance within the salamanders’ diet (Table 1). Aquatic amphibians also occurred in low frequency, as previously indicated in the literature (Dodson and Dodson 1971, Freda 1983). However, when consumed, a substantial proportion of the diet was comprised of a single vertebrate prey item. Although larval Jefferson Salamanders did not eat vertebrates often, when they did so, the dietary reward to that individual was significant. Dietary significance Although microinvertebrates did not vary statistically among size classes, a trend toward reduced consumption with increasing predator size was suggested. This trend is supported by documented decreases in Daphnia (Leff and Bachmann 1988), ostracod, and copepod (Brophy 1980) consumption as larval Ambystoma size increased. SC 1 (3.0–4.5 cm) predominantly ate microinvertebrates. Macroinvertebrate consumption rose significantly from SC 1 (3.0–4.5 cm) to SC 2 (4.5–5.0 cm), suggesting that 4.5 cm may be a threshold for general macroinvertebrate consumption. Utilization of two macroinvertebrate orders (coleopterans and dipterans) increased significantly from SC 2 (4.5–5.0 cm) to SC 4 (5.5–6.0 cm) and SC 3 (5.0–5.5 cm), respectively, whereas the remaining macroinvertebrate orders did not differ among size classes. Holomuzki and Collins (1987) saw consumption of dipterans in “small” Ambystoma expand to include five orders (Odonata, Ephemeroptera, Trichoptera, Hemiptera, and Coleoptera) as they grew larger, three of which were present in SC 2–5 (4.5–7.5 cm). Finally, vertebrate, including conspecifics, consumption did not occur until SC 5 (6.0–7.5 cm). Vertebrate consumption is rare in larval Ambystoma diet studies and, if it occurs at all, is limited to one or two occurrences per hundreds of specimens (Holomuzki and Collins 1987, Smith and Petranka 1987). Vertebrate consumption appears limited by the size, morphology, and, possibly, the seasonal collection times of the Ambystoma samples. Exceptions to these trends involve cannabalistic A. tigrinum (Green) (Tiger Salamander) and A. mavortium Baird (Western Tiger Salamander) morphs (Smith and Petranka1987, Wurst and Mull 1999). The ecological significance of dietary ontogenous shifts extends beyond a catalogue of Jefferson Salamander diet. These salamanders, as top 298 Northeastern Naturalist Vol. 14, No. 2 predators within their aquatic lotic habitats, mold the entire trophic dynamic of these isolated pool communities as they grow. To varying extents, the survival success of phytoplankton and planktonic diptera may be manipulated by the “smallest” Jefferson Salamander, the synchronized seasonal breeding of zooplankton and lotic invertebrates by the “medium” sized Jefferson Salamander, and vertebrate larvae mortality by the “largest” Jefferson Salamander, whose mouth gapes have grown wide enough for such prey. Acknowledgments First and foremost, we thank our volunteer field crew—Kelly Berger, Ransom Hughes, and Joshua Evans—for slogging through knee-deep mud and submerged snags. Thanks to Chris d’Orgeix, Don Mackler, and Mike Pinder for their valuable assistance in site location, sample identification, and permit acquisition, respectively. 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