A Test of Ecological Character Displacement between the Larvae of Two Cryptic, Sympatric Species of Two-lined Salamander, Eurycea wilderae and Eurycea cirrigera Results Mean temperature for E. lucifuga = 11.81 C Stephen Owensby Kristina Coggins Carlos Camp Introduction We took head measurements associated with feeding (Adams, 2000) from specimens from all sampled populations: Snout-vent length (SVL), head length, head width, eye-to-nostril distance, head depth, gape width, and jaw length. Comparisons were made between species in allopatry and between species at each sympatric site (NC & PC) using MANCOVA, with SVL set as covariate. Discriminant function analysis (DFA) was used to classify species. Character displacement occurs when differences between similar species are amplified in areas of sympatry due to competition (ecological character displacement) or hybridization (reproductive character displacement). Ecological character displacement has been demonstrated in terrestrial salamanders of the genus Plethodon (Adams & Rohlf 2000). Two species of the Two-lined Salamander (Eurycea bislineata) species complex, the Southern Two-lined Salamander (E. cirrigera) and the Blue Ridge Two-lined Salamander (E. wilderae), are parapatrically distributed. The former species occurs in the Piedmont of the southeastern US, and the latter is located in the southern Appalachian Mountains (Lannoo 2005). Abstract Competition has been hypothesized to represent a powerful force in determining the interactions between similar species. One possible result of interspecific competition is resource partitioning, which in turn may result in ecological character displacement. Character displacement is a pattern in which two species with overlapping ecological requirements differ more when they co-occur than when they do not. This pattern has been demonstrated in terrestrial, lungless salamander species that compete for food resources. Because the aquatic larval stages of biphasic species often occur in high densities, competition between sympatric species may be more likely between larvae than between adults. However, the ecology of larval lungless salamanders has not received the research attention as post-metamorphic salamanders. We studied two cryptic species of salamander, Eurycea wilderae (Blue Ridge Two-lined Salamander) and E. cirrigera (Southern Two-lined Salamander), which occur in two different types of environment – the Blue Ridge versus the Piedmont physiographic provinces – in the southeastern US. These two species occur in a narrow zone of sympatry in the foothills of the Blue Ridge Mountains of northeastern Georgia. We tested for food partitioning in the larvae by determining prey items (to order) from specimens collected from allopatric and sympatric localities and comparing them with the available invertebrate fauna within each stream. We similarly tested for character displacement in head measurements known to be associated with feeding dynamics. Larvae at the sympatric sites were identified genetically. Chi-square analyses indicated no significant difference between species in prey items taken in sympatry. Moreover, MANCOVA indicated no consistent interspecific differences in head measurements in sympatric populations. Therefore, we concluded that there is evidence for neither food partitioning nor ecological character displacement between the larvae of these two species. Results MtDNA IDs and nDNA IDs matched for both PC & NC, indicating no hybridization. In each allopatric population, there was a highly significant difference between prey eaten and prey available (FC, Χ2 = 129.4; CC, Χ2 = 992.0). For NC, there was a significant difference between stream fauna and prey of E. wilderae (Χ2 = 157.6) but not of E. cirrigera (Χ2 = 12.0). There was no significance between the prey of E. wilderae and E. cirrigera (Χ2 = 7.8). E. wilderae E. cirrigera These two species occur sympatrically in northeastern Georgia (Camp et al. 2000). The larvae of both species have similar ecologies and may occur in high densities (up to 27 m-2; Milanovich et al., 2015).. Competition between larvae may be a contributing factor maintaining the parapatric distribution of the two species (Cunningham et al., 2009).. Purpose We tested for competition and ecological character displacement between the larvae of E. cirrigera and E. wilderae in an area of sympatry in northeastern Georgia. MANCOVA showed a significant difference between the species in allopatry (F = 3.09, P = 0.008). DFA classified 86.8% of E. wilderae and 78.3% of E. cirrigera correctly for allopatric populations. There was no significant difference between the species at either PC (F = 1.12, P = 0.325) or NC (F = 0.74, P = 0.618). At PC DFA correctly classified 80.6% of E. wilderae but only 55.2% of E. cirrigera correctly. At NC DFA correctly classified 56.7% of E. wilderae and 64.1% of E. cirrigera. Materials & Methods Literature Cited Salamanders were collected from four allopatric sites (two for each species) and from two sympatric sites. MC = Madison Co (E. cirrigera) FC = Fulton Co (E. cirrigera) SC = Union Co, Sosebee Cove (E. wilderae) CC = Union Co, Cooper Creek (E. wilderae) NC = Habersham Co, Nancytown Creek (both) PC = Habersham Co, Piedmont College (both) Three specimens from each allopatric site and all specimens from sympatric sites were genotyped based on a mtDNA gene (cyt-b) and a nDNA gene (POMC).   SC & CC MC FC PC & NC Adams. 2000. Pp 383—394, In Bruce, Jaeger & Houck. The Biology of Plethodontid Salamanders. Plenum Press. Adams & Rohlf. 2000. PNAS 97:4106—4111. Camp, Marshall, Landau, Austin & Tilley..Copeia 2000:572—578. Cunningham, Rissler & Apodaca. 2009. Journal of Animal Ecology 78:52—-62. Keller & Seehausen. 2012. Molecular Ecology 21:782—799. Lannoo. 2005. Amphibian Declines: The Conservation Status of United States Species. Univ. Calif. Press. Milanovich, Maerz & Rosemond. 2015. Freshwater Biology 16:1340—1353.   Discussion While there was a significant difference between E. wilderae prey and the stream fauna at NC, this difference was driven by only a single taxon (Megaloptera). In addition, there was no difference in foods between the species in sympatry. We concluded that there is no food partitioning between in larvae. Moreover, we found no evidence of ecological character displacement in larvae. Therefore, we concluded that competition does not occur between the larvae of the two species. Alternative explanations for the maintetance of parapatry include adaptation to different thermal environments (Keller and Seehausen, 2012) or competition between adults. We collected samples of invertebrates from CC, FC, and NC by sweeping 5 arbitrarily chosen sites, each measuring 10 x 12.5 cm. We dissected salamander stomachs to retrieve prey items. We identified all prey items and stream invertebrates to taxon (usually order) using Pennak (1978). We compared prey eaten to available stream fauna using chi square. For the sympatric site (NC), we also compared the prey items of the two species, adjusting the α level to 0.025.