*2.1. Study Area*

The southern Appalachian region of the southeastern United States is globally significant for the exceptionally high biodiversity it supports, especially for amphibians, freshwater species, and temperate broadleaf and mixed forest species [32]. The diverse topography, warm, rainy climate, and lack of past glaciation contributes to the high biodiversity found in the southern Appalachian region [33]. The topography within the southern Appalachian region spans from steep mountainous terrain in the Blue Ridge ecoregion to rolling foothills in the Piedmont ecoregion [34]. Temperate broadleaf and mixed forests typify the landscape, yet the area is undergoing rapid urbanization and land use change [34]. Surface waters within the region include lotic systems (e.g., headwater streams, tributaries, rivers), impoundments, and wetlands inclusive of small, ephemeral wetlands that are essential for supporting biodiversity within the forested landscape [35]. Land use and land cover change (e.g., deforestation, urbanization), climate change, and other anthropogenic stressors threaten these terrestrial and aquatic ecosystems and biodiversity within this region, much as they do elsewhere [33,36–38].

As part of a larger study to improve knowledge about wetlands in an area undergoing rapid land use change, Pitt et al. [35] used remote sensing and local ecological knowledge to map 10506 small, ephemeral, and/or isolated wetlands in forests within the Piedmont and Blue Ridge ecoregions of South Carolina. Of these wetlands, 4611 were not mapped by the NWI (National Wetlands Inventory), likely due to the small size of the wetlands and coarse resolution of the available remote sensing data, and thus would likely be excluded from regulatory protections and land management and conservation planning [35]. We selected 41 of the newly mapped (i.e., non-NWI) small and ephemeral wetlands for intensive field-based study over a two-year period (Figure 1). We collected abiotic and biotic data from each of the 41 target wetlands over a minimum of 3 site visits per year (i.e., ≥6 site visits total) between January and June and once in November. Site visits were timed to maximize detectability based

on amphibian breeding phenology. Similarly, the two-year time period was intended to maximize detectability of species that may exhibit inter-annual variability in their activity or abundance.

**Figure 1.** Map of the study sites.
