Summary 1. The aquatic macrophyte Podostemum ceratophyllum has been shown to increase stream productivity, abundance and biomass of benthic invertebrates, and local occurrences of some stream fishes. However, experimental evidence that fishes preferentially associate with Podostemum is lacking, and the value of Podostemum as a predictor of stream fish assemblage composition has not been studied. 2. We conducted two short‐term (2 week), small‐scale (36 m2) experimental manipulations of Podostemum cover in the Conasauga River (Georgia and Tennessee, U.S.), and found higher abundances of benthic insectivorous fishes in patches with augmented (>80%) compared to reduced (7%) Podostemum cover. In an observational study, we quantified associations among percent cover of Podostemum, fish species richness, land cover, shoal length and base‐flow turbidity at 20 randomly selected shoals from a 39‐km reach that spanned a gradient of decreasing forest land cover. 3. Richness of all fish species and of lotic fishes peaked in the centre of the study reach, and richness was weakly correlated with predictor variables. Occupancy models for individual species also indicated that longitudinal position was a strong covariate for 13 of 19 species examined, with little support that Podostemum cover influenced occupancy. 4. Local associations may reflect choices by benthic fishes to utilise Podostemum, whereas downstream decline in fish species richness and Podostemum cover may reflect altered capacity of the system to support native species.

Connectivity among stream fish populations allows for exchange of genetic material and helps maintain genetic diversity, adaptive potential and population stability over time. Changes in species demographics and population connectivity have the potential to permanently alter the genetic patterns of stream fish, although these changes through space and time are variable and understudied in small‐bodied freshwater fish. As a spatially widespread, common species of benthic freshwater fish, the variegate darter (Etheostoma variatum) is a model species for documenting how patterns of genetic structure and diversity respond to increasing isolation due to large dams and how scale of study may shape our understanding of these patterns. We sampled variegate darters from 34 sites across their range in the North American Ohio River basin and examined how patterns of genetic structure and diversity within and between populations responded to historical population changes and dams within and between populations. Spatial scale and configuration of genetic structure varied across the eight identified populations, from tributaries within a watershed, to a single watershed, to multiple watersheds that encompass Ohio River mainstem habitats. This multiwatershed pattern of population structuring suggests genetic dispersal across large distances was and may continue to be common, although some populations remain isolated despite no apparent structural dispersal barriers. Populations with low effective population sizes and evidence of past population bottlenecks showed low allelic richness, but diversity patterns were not related to watershed size, a surrogate for habitat availability. Pairwise genetic differentiation (FST) increased with fluvial distance and was related to both historic and contemporary processes. Genetic diversity changes were influenced by underlying population size and stability, and while instream barriers were not strong determinants of genetic structuring or loss of genetic diversity, they reduce population connectivity and may impact long‐term population persistence. The broad spatial scale of this study demonstrated the large spatial extent of some variegate darter populations and indicated that dispersal is more extensive than expected given the movement patterns typically observed for small‐bodied, benthic fish. Dam impacts depended on underlying population size and stability, with larger populations more resilient to genetic drift and allelic richness loss than smaller populations. Other darters that inhabit large river habitats may show similar patterns in landscape‐scale studies, and large river barriers may impact populations of small‐bodied fish more than previously expected. Estimation of dispersal rates and behaviours is critical to conservation of imperilled riverine species such as darters.

Larval fish ecology is poorly characterized because sampling is difficult and tools for phenotypically identifying larvae are poorly developed. While DNA barcoding can help address the latter problem, ‘universal’ primers do not work for all fish species. The Roanoke River in the southeastern United States includes seven darters (Family Percide: Tribe Etheostomatini). We made 393 collections of larval fishes in 2015 and 2018, examined darter larvae for morphometric and pigmentation traits, developed PCR primers amplifying darter DNA, and evaluated three gear types for collecting larval darters. Amplified DNA sequences for 1351 larvae matched archived mitochondrial cytochrome oxidase I sequences for darters occurring in the ecosystem. Larval darters were classified to genus with 100% accuracy using the ratio of pectoral fin length to body length; however, identification to species using morphometrics alone was subject to a misclassification rate of 11.8%, which can be resolved by considering pigmentation patterns. Gear-types varied considerably in their capture efficacy for larval darters; most Percina larvae were collected in drift nets. Larval Percina species appeared in the drift before Etheostoma species in both study years. Application of molecular genetic and phenotypic tools to larval fish identification can advance understanding of larval darter ecology.

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