2.1 | Study Region and Sampling Methods
The Volga (Fayette County, IA) and Meramec (Crawford/Dent Counties, MO) rivers were selected as systems for comparison. Both waterways are historically known to contain abundant resident populations of E. caeruleum and have segments that are relatively undisturbed and unimpeded (Davis et al., 2015; Ray et al., 2006). The Volga River is a tributary of the upper Mississippi River within the previously glaciated region of North Central North America. The Meramec River, in contrast, is a tributary of the Mississippi River traversing land within the historically unglaciated Ozark Plateau. Both systems are located to the west of the Mississippi River (Figure 2).
Local populations in each river system were sampled from riffle sites separated by at least 2 km of river distance (Figure 2). This distance was chosen because of the notable high site-fidelity exhibited byE. caeruleum individuals (Hicks & Servos, 2017). Candidate segments and sampling localities in both rivers were identified using freely available GIS data in combination with satellite imagery from Google Earth. This was done to ensure adequate accessibility to each location and a lack of artificial barriers such as low-head dams within the sampling areas. A total of 10 E. caeruleum individuals were collected from each locality using kick-seining techniques and backpack electrofishing where permitted. Pectoral fin tissue was taken from the right side of positively identified fish and stored in 95% ethanol for DNA work; whole specimens were preserved in formalin as vouchers and deposited at the Bell Museum of Natural History Fish collection, University of Minnesota (Table S1).