Etheostoma Spectabile Research Articles

Riverscape genetics of the orangethroat darter complex.

Freshwater darters belonging to the orangethroat darter species complex, or Ceasia, are widely distributed in the Central and Southern United States, with ranges that span both glaciated and unglaciated regions. Up to 15 species have been recognized in the complex, with one, Etheostoma spectabile, having a widespread northern distribution and another, Etheostoma pulchellum, having a sizeable southern distribution. The other species in the complex have much more restricted distributions in unglaciated regions of the Central Highlands. We sampled 384 darters from 52 sites covering much of the range of Ceasia and evaluated patterns of genetic diversity, genetic structure, and pre- and post-glacial patterns of range contraction and expansion. We anticipated finding much stronger signals of genetic differentiation and diversification in unglaciated regions, given the higher species diversity and levels of endemism reported there. Surprisingly, microsatellite genotyping revealed two well-differentiated genetic clusters of E. spectabile in samples from glaciated regions, one confined to the Illinois River basin and another found in the Wabash drainage and Great Lakes tributaries. This suggests that there was expansion from two isolated glacial refugia, with little subsequent post-glacial gene flow. Fish collected from throughout the unglaciated region were less genetically differentiated. Fish assigned to Etheostoma burri and Etheostoma uniporum based on collection sites and morphological characters were not genetically differentiated from E. spectabile samples from the region. Hybridization and introgression occurring in the Central Highlands may confound genetic delineation of species in this region of high endemism and diversity.

Male recognition of conspecific female chemical cues in a diverse clade of freshwater fishes.

Where orangethroat darters (Etheostoma: Ceasia) and rainbow darters (Etheostoma caeruleum) co-occur, males prefer conspecific over heterospecific females. The cues males use to identify conspecific females remain unclear. We conducted behavioral trials to ask whether chemical cues function in conspecific recognition. We found that males from three orangethroat darter species preferentially associate with female scent over a control. Our results support the use of olfaction in conspecific identification in the orangethroat clade and contribute to our understanding of signals that may facilitate species recognition and underlie the evolution of behavioral isolation.

Indirect functional effects of neighbors on food web compartments could not overcome density-dependent limited growth of a grazing minnow

Ecosystems include diverse taxonomic and functional groups of animals with variable levels of niche overlap that can lead to direct and indirect interactions with positive or negative outcomes. Species feeding at low trophic positions might be sensitive to interactions with co-occurring functional groups (neighbors) that influence their food resources through various processes, such as indirect nutrient subsidies and trophic cascades. Employing a target-neighbor experimental design using large, outdoor mesocosms, we tested whether adding neighbors with different functional traits affected food web compartments relative to a control group that had grazing minnows, Campostoma anomalum (Central Stoneroller). Additionally, we tested whether growth and body condition of Central Stoneroller was positively influenced through interactions with each neighbor treatment: additional stonerollers, benthic-insectivorous fish Etheostoma spectabile (Orangethroat Darter), terrestrial insectivorous fish Lepomis megalotis (Longear Sunfish), or filter-feeding mussels (two-species assemblage) that are predicted to increase primary production through nutrient enrichment, a trophic cascade or both. Over the 29-day experiment, the presence of neighbors had generally negative or highly variable effects on food web compartments. Stoneroller growth was not evident during the experiment and body condition generally declined but was not influenced by neighbor treatments. Our results show that adding neighboring functional group did not reduce density-dependent growth of grazing fishes via indirect food web interactions over short temporal scales. This study suggested that indirect interactions among species and their combined influence on ecosystem properties were weak under experimental conditions representing typical prairie stream communities.

A perched culvert and natural obstructions limit fish dispersal in an intermittent prairie stream

Poorly constructed road crossings block upstream movement of fish into stream reaches that provide critical habitat or connect isolated populations. Although removing these barriers is often a conservation priority, quantifying fish passage following removal has not been well studied, particularly in intermittent streams. In this study, we sought to understand how barriers influence the dispersal of fishes in intermittent prairie streams. We used passive integrated transponder tags and antenna stations to quantify fish movement of 3 prairie-stream fishes (Central Stoneroller Campostoma anomalum [Rafinesque, 1820], Southern Redbelly Dace Chrosomus erythrogaster [Rafinesque, 1820], and Creek Chub Semotilis atromaculatus [Mitchill, 1818]) through a road crossing in an intermittent prairie stream for 3 y before and 3 y after removing a perched culvert. We verified that no upstream fish movement occurred through the culvert, despite large aggregations of tagged fish in the intermittent reach below the culvert. In contrast, tagged individuals of all 3 species were detected above the road crossing in each of the 3 y following removal. We also tracked the recovery of these species, plus the Orangethroat Darter Etheostoma spectabile (Agassiz, 1854), following a severe drought in 2018 in 2 spring-fed tributary reaches, 1 without a downstream barrier and 1 with the removed downstream culvert. Surveys of the tributary reaches showed rapid recovery of fish densities following drought in the tributary without a downstream barrier. However, recovery at the site above the removed culvert appeared to be limited by a natural waterfall. Our observations suggest barrier removal allowed access to spawning habitat within the intermittent reach immediately upstream of the site, but other obstacles or shear distance to perennial spring-fed tributary reaches also limited recolonization following severe drought. Increased connectivity between perennial and intermittent reaches in prairie streams likely benefits fishes by increasing their resilience following disturbance and providing habitat during critical life stages.

Effects of Stormwater Management and an Extended Culvert on Stream Health in Dug Run, Allen County, Ohio, USA

Changes in stream hydrology and habitat—associated with urbanization—have impacted diversity, abundance, and movement of both macroinvertebrates and fish. In 2008 the University of Northwestern Ohio began developing the western half of the campus, incorporating stormwater management practices. This provided an opportunity to examine 3 sections of the Dug Run stream that flows through campus: 1 section on the western half of campus that filters stormwater through the soil, and 2 sections on the eastern half of campus which are affected by both urbanization and a culvert that extends under a building and a road. Significant differences in macroinvertebrate Stream Quality Monitoring (SQM) index scores ( p < 0.001), fish diversity ( p < 0.010), and abundance of Orangethroat Darters (Etheostoma spectabile) ( p < 0.001) were observed between the western and the 2 eastern sections of Dug Run. Lower SQM index scores and lower Orangethroat Darter abundances were found in the urbanized sections of the stream, while lower fish diversity numbers were found upstream of the culvert. The western portion of campus, designed to filter stormwater runoff through the soil, was the only section studied with sensitive macroinvertebrates, a higher SQM index score, and a greater abundance of Orangethroat Darters.

Influence of fishes on macroinvertebrate communities and insect emergence production in intermittent stream refuges

Abstract Drying intermittent stream networks often have permanent water refuges that are important for recolonisation. These habitats may be hotspots for interactions between fishes and invertebrates as they become isolated, but densities and diversity of fishes in these refuges can be highly variable across time and space. Insect emergence from streams provides energy and nutrient subsidies to riparian habitats. The magnitude of such subsidies may be influenced by in‐stream predators such as fishes. We examined whether benthic macroinvertebrate communities, emerging adult insects, and algal biomass in permanent grassland stream pools differed among sites with naturally varying densities of fishes. We also manipulated fish densities in a mesocosm experiment to address how fishes might affect colonisation during recovery from hydrologic disturbance. Fish biomass had a negative impact on invertebrate abundance, but not biomass or taxa richness, in natural pools. Total fish biomass was not correlated with total insect emergence in natural pools, but orangethroat darter (Etheostoma spectabile) biomass was inversely correlated with emerging Chironomidae biomass and individual midge body size. The interaction in our models between predatory fish biomass and date suggested that fishes may also delay insect emergence from natural pools, altering the timing of aquatic–terrestrial subsidies. There was an increase over time in algal biomass (chlorophyll‐a) in mesocosms, but this did not differ among fish density treatments. Regardless, fish presence in mesocosms reduced the abundance of colonising insects and total invertebrate biomass. Mesocosm invertebrate communities in treatments without fishes were characterised by more Chironomidae, Culicidae, and Corduliidae. Results suggest that fishes influence invertebrates in habitats that represent important refuges during hydrologic disturbance, hot spots for subsidy exports to riparian food webs, and source areas for colonists during recovery from hydrologic disturbance. Fish effects in these systems include decreasing invertebrate abundance, shifting community structure, and altering patterns of invertebrate emergence and colonisation.

Genomic Resources for Darters (Percidae: Etheostominae) Provide Insight into Postzygotic Barriers Implicated in Speciation.

Comparative genomic approaches are increasingly being used to study the evolution of reproductive barriers in nonmodel species. Although numerous studies have examined prezygotic isolation in darters (Percidae), investigations into postzygotic barriers have remained rare due to long generation times and a lack of genomic resources. Orangethroat and rainbow darters naturally hybridize and provide a remarkable example of male-driven speciation via character displacement. Backcross hybrids suffer from high mortality, which appears to promote behavioral isolation in sympatry. To investigate the genomic architecture of postzygotic isolation, we used Illumina and PacBio sequencing to generate a chromosome-level, annotated assembly of the orangethroat darter genome and high-density linkage maps for orangethroat and rainbow darters. We also analyzed genome-wide RADseq data from wild-caught adults of both species and laboratory-generated backcrosses to identify genomic regions associated with hybrid incompatibles. Several putative chromosomal translocations and inversions were observed between orangethroat and rainbow darters, suggesting structural rearrangements may underlie postzygotic isolation. We also found evidence of selection against recombinant haplotypes and transmission ratio distortion in backcross hybrid genomes, providing further insight into the genomic architecture of genetic incompatibilities. Notably, regions with high levels of genetic divergence between species were enriched for genes associated with developmental and meiotic processes, providing strong candidates for postzygotic isolating barriers. These findings mark significant contributions to our understanding of the genetic basis of reproductive isolation between species undergoing character displacement. Furthermore, the genomic resources presented here will be instrumental for studying speciation in darters, the most diverse vertebrate group in North America.

Male-driven reproductive and agonistic character displacement in darters and its implications for speciation in allopatry.

Selection against hybridization can cause mating traits to diverge between species in sympatry via reproductive character displacement (RCD). Additionally, selection against interspecific fighting can cause aggressive traits to diverge between sympatric species via agonistic character displacement (ACD). By directly affecting conspecific recognition traits, RCD and ACD between species can also incidentally cause divergence in mating and fighting traits among populations within a species [termed cascade RCD (CRCD) and cascade ACD]. Here, we demonstrate patterns consistent with male-driven RCD and ACD in 2 groups of darters (orangethroat darter clade Ceasia and rainbow darter Etheostoma caeruleum). In both groups, males that occur in sympatry (between Ceasia and E. caeruleum) have higher levels of preference for mating and fighting with conspecifics over heterospecifics than do males from allopatry. This is consistent with RCD and ACD. We also found patterns consistent with CRCD and cascade ACD among species of Ceasia. Ceasia males that are sympatric to E. caeruleum (but allopatric to one another) also have heightened preferences for mating and fighting with conspecific versus heterospecific Ceasia. In contrast, Ceasia males that are allopatric to E. caeruleum readily mate and fight with heterospecific Ceasia. We suggest that RCD and ACD between Ceasia and E. caeruleum has incidentally led to divergence in mating and fighting traits among Ceasia species. This study is unique in that male preferences evolve via both RCD (male preference for conspecific females) and ACD (male preference to fight conspecific males) which leads to subsequent divergence among allopatric lineages.

Male and female contributions to behavioral isolation in darters as a function of genetic distance and color distance.

Determining which reproductive isolating barriers arise first between geographically isolated lineages is critical to understanding allopatric speciation. We examined behavioral isolation among four recently diverged allopatric species in the orangethroat darter clade (Etheostoma: Ceasia). We also examined behavioral isolation between each Ceasia species and the sympatric rainbow darter Etheostoma caeruleum. We asked (1) is behavioral isolation present between allopatric Ceasia species, and how does this compare to behavioral isolation with E. caeruleum, (2) does male color distance and/or genetic distance predict behavioral isolation between species, and (3) what are the relative contributions of female choice, male choice, and male competition to behavioral isolation? We found that behavioral isolation, genetic differentiation, and male color pattern differentiation were present between allopatric Ceasia species. Males, but not females, discerned between conspecific and heterospecific mates. Males also directed more aggression toward conspecific rival males. The high levels of behavioral isolation among Ceasia species showed no obvious pattern with genetic distance or male color distance. However, when the E. caeruleum was included in the analysis, an association between male aggression and male color distance was apparent. We discuss the possibility that reinforcement between Ceasia and E. caeruleum is driving behavioral isolation among allopatric Ceasia species.

Passive Integrated Transponder Tags: Review of Studies on Warmwater Fishes With Notes on Additional Species

Abstract Although numerous studies have assessed retention and survival of passive integrated transponder (PIT) tags, data are scattered and information gaps remain for many diminutive fishes. Our study objectives were to 1) systematically review PIT tag studies and summarize retention, growth, and survival data for warmwater fishes; and 2) conduct a laboratory study to evaluate the retention, survival, and growth effects of intracoelomic-placed, half duplex PIT tags on six small-bodied species common to warmwater streams. Our systematic review suggested small sample sizes were common within PIT tag retention and survival studies (39% with n ≤ 20) and that many experiments (15%, 14 of 97) failed to use control fish as part of their evaluations. Studies focused primarily on short-term changes (15 d to 2 y) in tag retention and survival. Tag retention was equal to or greater than 90% in 85% of the experiments reviewed and median survival was 92%. Growth was reported by fishes in the majority of reviewed studies. We found similar results after PIT tagging (peritoneum tagging using 12- or 23-mm half duplex tags) adult Cardinal Shiner Luxilus cardinalis, Central Stoneroller Campostoma annomalum, Greenside Darter Etheostoma blennioides, Orangethroat Darter Etheostoma spectabile, Slender Madtom Noturus exilis, and juvenile Smallmouth Bass Micropterus dolomieu. Tag retention for all species was high, with only one tag loss recorded after 60 d. Survival was also high (≥88%) for all of our species with the exception of Orangethroat Darter (56% survival). No significant difference in mean growth between treatment and control groups was found. Both our results and the findings of the literature review suggested generally high tag retention and low mortality in tagged fishes (across 31 species reviewed). However, within our study (e.g., Orangethroat Darter) and from the literature, examples of negative effects of PIT tagging on fishes were apparent, suggesting methodological testing is prudent before using PIT tags in field studies. We suggest future studies would benefit from addressing the behavioral implications that may be associated with tagging and examination of longer-term tag retention. Furthermore, standard reporting (i.e., sample sizes) in PIT tag studies would be beneficial, and use of control subjects or groups for statistical comparisons is needed.

Breakdown of a Hybrid Swarm between Two Darters (Percidae),Etheostoma radiosumandEtheostoma spectabile,with Loss of One Parental Species

A local hybrid swarm between Etheostoma radiosum and Etheostoma spectabile, found in June 1985 in a short reach of Little Glasses Creek, southern Oklahoma, consisted by eye of 35% phenotypic E. radiosum, 18% phenotypic E. spectabile, and 47% hybrids. In July 1985, investigation of an additional longer reach downstream of the initial discovery site showed that hybrids also were present there, although fewer. Thus, in 1985 both parental species, and substantial numbers of hybrids, were present in approximately a 300 m reach of the stream. No further collections were made until 2003, when sampling in the former hybrid reach and substantially further downstream showed E. radiosum to be the only species of Etheostoma present, and neither E. spectabile nor hybrids (by eye) were detected. From 2003 to 2013 the reach was surveyed 11 times, with a total of 363 darters identified phenotypically as E. radiosum, and none as E. spectabile or hybrids. Of 20 individuals sampled in December 2011 for molecular analyses, 1...

Intrasexual competition underlies sexual selection on male breeding coloration in the orangethroat darter, Etheostoma spectabile.

Elaborate, sexually dimorphic traits are widely thought to evolve under sexual selection through female preference, male–male competition, or both. The orangethroat darter (Etheostoma spectabile) is a sexually dichromatic fish in which females exhibit no preferences for male size or coloration. We tested whether these traits affect individual reproductive success in E. spectabile when multiple males are allowed to freely compete for a female. The quality and quantity of male coloration were associated with greater success in maintaining access to the female and in spawning as the primary male (first male to participate). On the other hand, sneaking behavior showed little correlation with coloration. Male breeding coloration in E. spectabile may therefore demonstrate how intrasexual competition can be a predominant factor underlying the evolution of male ornaments.

Fish Communities in Pools and Riffles of Fox Creek, Tallgrass Prairie National Preserve, Chase County, Kansas

We sampled fishes in Fox Creek at Tallgrass Prairie National Preserve, Chase Co., Kansas in June 2012 to record fish species present and to compare richness, abundance, and evenness between pools and riffles. We collected 31 fish species from 94 pools and 14 riffles. The most common species in riffles were Orangethroat Darter (Etheostoma spectabile), Redfin Shiner (Lythrurus umbratilis), and Central Stoneroller (Compostoma anomalum), and the most common species in pools were Redfin Shiner, Central Stoneroller, and Orangespotted Sunfish (Lepomis humilis). Species richness and abundance were higher in pools than riffles, however evenness was not statistically significantly different. Since this study was conducted in the middle of a severe drought, these results could provide important data on how fish communities are assembled during periods of low-flow in tallgrass prairie streams.

Effects of pulse and press drying disturbance on benthic stream communities

Natural disturbance is an integral component of most ecosystems and occurs in 3 different forms: pulse, press, and ramp. In lotic ecosystems, seasonal drought is a major form of disturbance, particularly in intermittent headwater streams, which often are reduced to pools that serve as refuges for biota. We used simulated intermittent stream pools to compare the effects of control, pulse, and press drying on growth and survival in 3 fish species (Lepomis megalotis, Campostoma anomalum, and Etheostoma spectabile) commonly found together in drought-prone streams in the Ozark Highlands, USA. We also compared effects on benthic community structure, including periphyton and chironomid density and sediment in deep (permanently watered) and shallow (intermittently dewatered) habitat. Only one species, L. megalotis, showed a significant reduction in length and mass growth in press drying compared with control treatments. Drying and type of drying had no effect on survival of any fish species. Drying and type of drying had strong overall effects on periphyton growth in shallow habitats, where ash-free dry mass decreased and the autotrophic index (the ratio of chlorophyll a to total biomass) increased significantly in drying relative to control and in press relative to pulse treatments. Drying negatively affected sediment accumulation in shallow habitat and chironomid density in deep habitat. Drying in intermittent streams has species-dependent effects on fish growth and benthic structure, and pulse and press drying differ in their effects on periphyton in these systems. These effects may have important consequences in seasonally drying streams as anthropogenic influence on stream drying increases.

Body shape variation in two species of darters (Etheostoma, Percidae) and its relation to the environment

AbstractEnvironmental variation can shape phenotypic variation in organisms, but most evidence for trait differentiation comes from analyses of dichotomous habitat types that differ in only one or few key environmental factors. In reality, environmental variation is often more subtle, gradual and multifarious. Here, we investigated geographic variation in body shape of two darter species (Etheostoma spectabileandEtheostoma flabellare; Percidae) that occur along river gradients. This study addressed three specific questions: Is there intraspecific geographic variation in the two species across different sites in the Ozark Highlands of Oklahoma (USA)? Is phenotypic variation across sites correlated with abiotic environmental conditions? Do the two species share site‐specific (i.e. convergent) phenotypic variation in areas where they occur together? Our results indicated significant body shape variation in both species. Population differences in body shape were particularly correlated with variation in substrate composition. The combined analysis of both species indicated a small but significant effect of convergence on body shape wherever they are sympatric; shared variation, however, was not related to any environmental variables included in the analysis. While it remains unclear whether phenotypic variation in these species is due to heritable differentiation or environmentally induced plasticity, our results indicate that even subtle and gradual environmental variation can induce substantial variation in phenotypes on a relatively small spatial scale.

Sexually asymmetric colour-based species discrimination in orangethroat darters

Female mate preferences, male mate preferences and male–male competition can all potentially play roles in promoting speciation by sexual selection. The orangethroat darter, Etheostoma spectabile , is a sexually dichromatic fish in which males compete for access to females. We quantified male and female responses to conspecific versus heterospecific fish under normal light, dim light and blue light, the last of which impaired colour perception. We found that behavioural isolation was likely mediated primarily by male behavioural discrimination against heterospecific fish, with females showing no evident mate preferences. Furthermore, male aggression towards conspecific rivals was reduced in blue light, suggesting that the evolution of male coloration may contribute to speciation through male–male interactions. • We examined behavioural isolation in two Etheostoma darters with dichromatic colour vision. • Male, but not female, E. spectabile discriminated between conspecific and heterospecific males. • Male E. caeruleum did not respond to heterospecific fish of either sex. • Perception of reddish coloration was necessary for conspecific male–male aggression.

Patterns of Male Breeding Color Variation Differ across Species, Populations, and Body Size in Rainbow and Orangethroat Darters

Sexually dimorphic coloration has been widely suggested to play a role in sexual selection and speciation. Animal colors can originate from several different biochemical pathways, which may underlie different patterns of selection and diversification. Darters of the speciose genus Etheostoma exhibit substantial diversity in male breeding coloration. We used digital photography and image software to comprehensively quantify male coloration in the Rainbow Darter (Etheostoma caeruleum) and the Orangethroat Darter (E. spectabile). Color traits differed across species, populations, and body sizes, with size differences contributing the most to individual color variation. The bluish colors were overall more strongly correlated with size than the reddish colors. Conversely, the reddish colors tended to be less correlated with size and better indicators of species and population identity. Finally, we determined that the bluish colored tissue contained a chromoprotein pigment, and that the reddish colored tissue c...

Reproductive isolation between two darter species is enhanced and asymmetric in sympatry.

Robust reproductive isolation was found between the rainbow darter Etheostoma caeruleum and the orangethroat darter Etheostoma spectabile, as more offspring were produced when conspecific males and females were crossed as compared with heterospecific crosses. Furthermore, fewer eggs resulted from heterospecific crosses involving sympatric E. spectabile females than those using allopatric E. spectabile females, while a similar pattern was not observed in heterospecific crosses using E. caeruleum females. These results suggest that reinforcement, i.e. selection for pre-zygotic reproductive barriers driven by reduced hybrid fitness, may have contributed to the evolution and maintenance of reproductive barriers between these potentially hybridizing species in sympatry.

Nonlinear response of stream ecosystem structure to low‐level phosphorus enrichment

Summary Anthropogenic inputs of nitrogen (N) and phosphorus (P) create environmental conditions that alter biological organisation and ecosystem functioning in fresh waters. We studied 38 wadeable streams spanning a N and P gradient to contrast responses of algal and fish assemblages to nutrient enrichment. Surface water total P (TP) and total N (TN) concentrations represented a wide range (TP: 7–2380 μg L−1; TN: 127–15 860 μg L−1) and were correlated across our study sites. Total P explained significantly more variance in periphyton carbon (C)‐to‐nutrient (C : P, C : N) and N : P ratios than TN. Abrupt, nonlinear declines in these ratios were observed between 20 and 50 μg L−1 TP and 500–1000 μg L−1 TN; beyond these values, ratios exhibited minimal additional decline. Algae assemblage structure was strongly linked to surface water TP, TN and catchment‐scale nutrient sources (wastewater treatment plant (WWTP) discharges and % pasture cover). In particular, there were synchronous declines in frequency and cell densities of many alga species associated with TP concentrations >21 μg L−1 (90% CI of 18–48 μg L−1) as well as simultaneous increases in tolerant species associated with increasing enrichment. Fish assemblage structure was most strongly associated with % pasture, WWTP discharges and fine sediment cover, yet also showed significant but weaker correlations with surface water and periphyton nutrient variables. However, two benthic fish species, Etheostoma spectabile and Campostoma anomalum, significantly declined with TP >28 μg L−1 (90% CI, 24–56 μg L−1) and 34 μg L−1 (90% CI, 21–56 μg L−1), respectively. Conversely, the tolerant minnow Cyprinella lutrensis and invasive carp Cyprinus carpio increased nonlinearly with increasing surface water TP. Our results provide new insights into interpretation and analysis of assemblage‐level responses to nutrient enrichment. Our findings indicate that a numerical criterion for surface water TP of c. 20 μg L−1 would be needed to maintain natural algae assemblages and at least two specialist fishes within our study region. Proliferation of weedy alga species and increased abundance of invasive fishes are also likely when surface water concentrations exceed these thresholds. While many streams likely exceed these thresholds, managers should consider potential low‐level enrichment effects when developing criteria for ecosystems to protect existing nutrient‐limited streams.

Habitat Use and Separation among Congeneric Darter Species

AbstractFive darter species (Greenside Darter Etheostoma blennioides, Rainbow Darter E. caeruleum, Fantail Darter E. flabellare, Johnny Darter E. nigrum, and Orangethroat Darter E. spectabile) were collected from seven streams and rivers in two Indiana watersheds to determine patterns of microhabitat selection with respect to depth, water velocity (flow), and substrate size. Heterogeneous habitats create a variety of niche dimensions, promoting the coexistence of congeneric species. Greenside and Rainbow darters were most commonly found among intermediate substrate sizes (cobble + boulder) and in locations with higher velocities and deeper water. Fantail and Orangethroat darters associated with intermediate to large substrate sizes (cobble + bedrock) were found in shallower water with lower velocities. Fantail Darters were only observed in the Whitewater River watershed. In contrast, Johnny Darters were observed only within the White River watershed among small substrate sizes (silt + sand), moderate velocities, and deeper water. Based on nonmetric multidimensional scaling and multinomial logistic regression, substrate size was the most important variable in characterizing microhabitat selection. Ultimately, habitats produced patterns of segregation and coexistence among these five darters in Indiana streams. Although ecological patterns of microhabitat use are additionally influenced by competition, morphology, and life history traits, heterogeneous habitat features within ecosystems appear to be structuring fish assemblages.