1 | INTRODUCTION
Freshwater aquatic organisms in fluvial systems have gene flow constraints that are associated with the directional flow of water, habitat heterogeneity and the dendritic nature of the stream network. Understanding the influences on the evolution of spatial genetic structure of populations in riverine environments is a unique problem that involves a specific set of questions, which are described as riverscape genetics (Davis et al., 2018). Based on the degree of connectedness of the stream network, the distribution of habitat and life history of the organism, a directional hierarchy in genetic diversity of local populations is expected. This is referred to as the stream hierarchy model (Meffe & Vrijenhoek, 1988). Any modification to the riverscape structure such as dams, loss of habitat, flooding or alteration of the drainage system can disrupt the patterns of gene flow and lead to changes in the expectations of the population genetic structure (Davis et al., 2018; Meffe & Vrijenhoek, 1988).
The connectivity of stream networks and the species assemblage present within the upper Mississippi River basin of central North America are the result of a complex geologic and climatic history. Recurrent, alternating cycles of glacial advance and retreat by the Laurentide Ice Sheet during the Quaternary period, 2.6 million to 10,500 years ago, reshaped and fragmented the regional landscape and altered drainage patterns and flow regimes (Cupples & Van Arsdale, 2014; Lowe & Walker, 2014; Galloway et al., 2011; Knox, 2007; Figure 1). To the west of the Mississippi River, the southernmost extent of these glacial advances occurred during the Pre-Illinoian Glacial Stage (2.6 - 0.3 million years ago), reaching the present-day Missouri River valley in central Missouri (Burr & Page, 1986; Hobbs, 1999; Thornbury, 1965; Figure 1). The Ozark Plateau and associated stream networks to the south of the Missouri River remained relatively unaffected by the glacial disturbances (Pflieger, 1975).
The distribution of aquatic species in central North America was notably affected by the habitat changes in the north, resulting in displacement or extirpation of local populations (Berendzen et al., 2010; Burr & Page, 1986). As glacial fronts expanded and retreated the riverscape was dramatically altered causing changes to the drainage pattern, flow regime and habitat (Thornbury, 1965). West of the Mississippi River, the Ozark Plateau provided a refugium for many fish species during the periodic glacial advances of the Quaternary period (Berendzen et al., 2003; Berendzen et al., 2010; Burr & Page, 1986; Echelle et al., 2014; Near et al., 2001). Following the retreat of the last glacial maximum (19,000 to 10,500 years ago), new dispersal opportunities were created among the newly formed stream connections, which contributed to the expansion of populations northward and the establishment of the contemporary fish assemblage (Berendzen et al., 2010; Burr & Page, 1986; Ray et al., 2006).
Etheostoma caeruleum , the rainbow darter, is a member of this assemblage with a broad distribution in small rivers, creeks, and streams in the Mississippi River drainage across eastern North America (Ray et al., 2006; Strange & Burr, 1997; Figure 2). The species is a small, sexually dimorphic fish that displays a preference for shallow, fast-flowing water within gravel or rubble riffles (Harding et al., 1998; Mueller et al., 2020). Mature  individuals exhibit high site-fidelity to their natal habitats throughout their life cycle, with breeding males migrating up to 1 km during spring spawning periods (Hicks & Servos, 2017). The rainbow darter is a benthic invertivore that actively forages for a variety of macroinvertebrates that inhabit riffles. Their preferred food sources are much less abundant in the deeper pools separating riffle structures (Mueller et al., 2020). In addition, E. caeruleum  is considered a sentinel species and is relatively sensitive to habitat and water quality changes due to channel siltation, chemical runoff, and related effects of anthropogenic activity (Haponski et al., 2009; Tonnis, 2006).
West of the Mississippi River, E. caeruleum has a disjunct distribution inhabiting streams in the upper Mississippi River basin and the Ozark Plateau, but is absent in the intervening region (Figure 2). In tributaries of the upper Mississippi River, local populations of the rainbow darter have a patchy, disjunct distribution across the riverscape (Davis et al., 2015). Areas of local concentration are often separated by large river distances. This pattern results from the direct impact of glacial processes on the landscape causing the spatial distribution of suitable habitat to be highly fragmented and heterogeneous. Modern river drainages in this region, flow through varying depths of glacial drift overlaying the Tertiary bedrock topography (Burr & Page, 1986; Thornbury, 1965). In contrast, the drainage patterns of the Ozark Plateau have maintained their basic configuration since the late Paleozoic era (Pflieger, 1975). Modern river drainages in this region are characterized by clear, cool high gradient streams with course substrate in deeply dissected valleys (Pflieger, 1975; Thornbury, 1965). Local populations of the rainbow darter in this region are widespread and nearly continuous, resulting from the abundant distribution of suitable habitat. The rainbow darter is one of the most abundant fish species in the Ozarks (Pflieger, 1975).
In this study we compare the fine-scale population genetic structure of rainbow darter populations in two similar river systems, one located in the glaciated environment of the upper Mississippi River basin and one in the unglaciated environment of the Ozark Plateau. We test the hypothesis that climatic and geologic processes during the Quaternary period influenced the observed genetic signature and disrupted expectations under riverscape genetic models. Under the stream hierarchy model, the population genetic structure is consistent with the structure of the stream network, distance between local populations, connectedness of the habitat and life history of the organism (Brauer et al., 2018; Meffe & Vrijenhoek, 1988). Since the rainbow darter exhibits high site fidelity and short dispersal distances, the expectation is that local populations will be partially isolated with limited geneflow and evidence of isolation by distance in an upstream to downstream pattern. We predict that the population genetic structure of E. caeruleumin the glaciated environment in the North will not fit expectations under the stream hierarchy model and will exhibit low levels of genetic diversity. Conversely, we predict that the population genetic structure of E. caeruleum in the unglaciated environment in the South will fit expectations under the stream hierarchy model and exhibit higher levels of genetic diversity. In this study, we utilize genome-wide SNPs generated using RADseq to infer fine-scale population genetic structure in each river system.