Variation in specific conductivity and major ions in streams must be understood to assess the effects of changes in ionic strength and salinity on stream biota. I compiled data for randomly selected sites from surveys conducted from 1985 to 2009 by the US Environmental Protection Agency (EPA). I followed EPA methods to estimate reference values for specific conductivity (60 ecoregions) and each major ion (34 ecoregions) as the 25^th percentile of values in 1^st- to 4^th-order streams in Level III ecoregions with data from ≥25 sites (85 ecoregions). The 25^th percentiles of specific conductivity were <200 µS/cm for most eastern and western montane ecoregions, except those dominated by limestone or calcareous till. Arid western ecoregions had higher specific conductivities. Ca² was generally the most abundant cation followed by Mg² , Na , and K . HCO₃^- was generally the most abundant anion followed by SO₄^2- and Cl^-. Ecoregions where SO₄^2- or Cl^- concentrations were greater than HCO₃^- concentration have been affected by acidic precipitation or are influenced by marine air masses, respectively, and have very low specific conductivities. Patterns of variation appear to be associated with 3 processes controlling total and relative concentrations of major ions in freshwaters. In many ecoregions, relative ionic concentrations reflect underlying geology, but in arid ecoregions, relative ionic concentrations show concentration by evaporation. Relative ionic concentrations in coastal ecoregions and those affected by acidic precipitation reflect the ionic content of precipitation. Verification of these factors awaits better quantification of the geological and climatic characteristics of each ecoregion.

Hydrologic indices can be used in macroecological models of fish species richness. I used fish distributions and river flow records from 89 river basins in the Pacific Northwest (USA) to identify robust associations between aspects of flow and fish species richness. I calculated 148 hydrologic indices for each basin, created single-predictor regression models for each hydrologic index, and tested for significant relationships between native fish species richness and specific components of flow. I used multiple regression to test the significance and fit of models that combined hydrologic indices that were not highly collinear indices. Significant linear relationships (p ≤ 0.05) were detected for 85 indices. Coefficients of determination (r²) ranged from 0.04 to 0.33 (median = 0.19, coefficient of variation [CV] = 0.55). I identified 2 multiple-regression models, each including 3 hydrologic indices, as best based on information-theoretic model comparisons. One included median large flood rise rate, CV of 1-d maximum flow, and CV of annual flow. The other included mean annual flow, median high flow timing, and CV of small flood timing. Four major themes emerged from the analyses: 1) fish species richness and a variety of indices of flow magnitude were positively related; 2) the strength of the positive association between fish species richness and flow magnitude was inversely related to flow magnitude and the strongest associations were observed during low-flow winter months; 3) flow variability, indicated by the CVs of hydrologic indices, was negatively associated with fish species richness; 4) indicators of episodic high-flow events (e.g., floods and annual maximum flows) were particularly well represented in the best multiple-regression models of fish species richness. These results can provide a baseline for comparison with other rivers and can be used to help formulate a general, flow-mediated theory of fish species richness in lotic ecosystems.

High flows are major disturbances in streams and cause benthic communities to vary temporally. Meltwater runoff in glacier-fed streams at temperate—arctic latitudes primarily follows a strong seasonal pattern. In contrast, such streams at the equator show less seasonal, but more-pronounced diel variability in discharge that tracks a year-round diurnal melting—nocturnal freezing cycle of glaciers. Consequently, qualitative and quantitative differences in temporal variability of macrobenthos communities should be expected between highlatitude and tropical glacier-fed streams. We explored temporal variability in density, taxon richness, and community composition of benthic macroinvertebrates and analyzed community responses to flow events at 3 sites along a glacier-fed stream in equatorial Ecuador (0.05, 1.6, and 4.3 km from the glacier front). We obtained continuous flow recordings and sampled fauna at approximately quarterly intervals over 30 mo. Temporal variability in the fauna was aseasonal. However, the overall magnitude of the coefficient of variability (CV) at the 3 sites was not lower than the CV at temperate latitudes. The explanatory power of flow did not differ among discharge parameters 3, 6, 9, 21, and 45 d before sampling. The effect of flow (slopes of regressions of faunal metrics vs flow) did not differ among sites, but the amount of variation explained by flow was significant only at the 2 downstream sites. Little synchrony was found in variability among sites, possibly because of differences among sites in physical characteristics (e.g., refugia space), which moderated the effect of disturbances, and taxonomic composition of communities. Our study is the first to show a close link between hydrological and biological fluctuations in an equatorial glacier-fed stream, a prerequisite for subsequent predictions of consequences of tropical glacier melting on diversity, composition, and stability of stream communities.

The factors influencing the habitat requirements and dispersal of juvenile Unionidae (unionids) between post-larval detachment from a fish host and burrowing into the substratum are largely unknown, although bed shear stress (τ_w) has been proposed as a critical factor. We used a laboratory wall jet apparatus to manipulate τ_w experimentally to examine the suspension and burrowing of juvenile Epioblasma triquetra, Villosa iris, Lampsilis fasciola, and Ligumia nasuta. τ_w and unionid suspension were significantly related when τ_w > 0.26 Pa, but this relationship also was influenced by mussel behavior. Greater τ_w was required for suspension when a mussel's foot adhered to a surface. Laboratory experiments confirmed predictions that juvenile unionids cannot become established when τ_w exceeds a critical value. Hydrodynamics and behavior strongly affect dispersal by juvenile mussels and may influence juvenile mussel habitat requirements, and thereby have implications for conservation efforts.

Median lethal temperature (LT50) data from water-only exposures with the early life stages of freshwater mussels suggest that some species may be living near their upper thermal tolerances. However, evaluation of thermal sensitivity has never been conducted in sediment. Mussels live most of their lives burrowed in sediment, so understanding the effect of sediment on thermal sensitivity is a necessary step in evaluating the effectiveness of the water-only standard method, on which the regulatory framework for potential thermal criteria currently is based, as a test of thermal sensitivity. We developed a method for testing thermal sensitivity of juvenile mussels in sediment and used the method to assess thermal tolerance of 4 species across a range of temperatures common during summer. Stream beds may provide a thermal refuge in the wild, but we hypothesized that the presence of sediment alone does not alter thermal sensitivity. We also evaluated the effects of 2 temperature acclimation levels (22 and 27°C) and 2 water levels (watered and dewatered treatments). We then compared results from the sediment tests to those conducted using the water-only standard methods. We also conducted water-only LT tests with mussel larvae (glochidia) for comparison with the juvenile life stage. We found few consistent differences in thermal tolerance between sediment and water-only treatments, between acclimation temperatures, between waterlevel treatments, among species, or between juvenile and glochidial life stages (LT50 range = 33.3–37.2°C; mean = 35.6°C), supporting our hypothesis that the presence of sediment alone does not alter thermal sensitivity. The method we developed has potential for evaluating the role of other stressors (e.g., contaminants) in a more natural and complex environment.

Identifying mechanisms of ultraviolet radiation (UVR) tolerance is an important step in understanding the direct and indirect effects of UVR exposure on aquatic organisms. We used laboratory exposure experiments and a solar exposure experiment to evaluate mechanisms of UVR tolerance in the freshwater snail, Physa acuta. Survival following short-wavelength UVB exposure was higher in the presence of simultaneous exposure to longer-wavelength UVA and visible light than with UVB alone, indicating that P. acuta possessed photoenzymatic repair. Survival also increased as a function of body size and shell thickness, suggesting that the shell provides photoprotection. Last, snails actively selected locations further below the water surface in the presence than in the absence of solar UVR, suggesting behavioral avoidance as another mechanism of UVR tolerance. These defense mechanisms can be influenced by environmental conditions, so effects of UVR exposure may vary widely among populations and will be strongest in systems with a combination of low temperature, high transparency, and low Ca concentrations.

Restoration of riparian vegetation is widely recognized as a tool in stream rehabilitation, but information on whether local riparian characteristics can mitigate the effects of catchment-level stressors on in-stream processes is limited. We measured community metabolism in 21 streams in the Canterbury region of New Zealand along 2 independent gradients of agricultural intensity and riparian cover (from closed canopied to open canopied) to assess relative effects of landscape and local factors on stream trophic state. We measured stream metabolism with the single-station open-channel diel O₂ method. We found a correlation between gross primary production (GPP) and ecosystem respiration (ER), indicating a gradient of trophic states across sites. Streams were strongly heterotrophic with P:R values varying from 0.01 to 0.25. GPP and ER increased with % agriculture and % macrophyte cover, but decreased with % shade from riparian vegetation. Hierarchical partitioning analysis indicated that % agriculture was the only landuse variable to have a significant independent effect on GPP and ER. Among local variables, % shade and % macrophyte cover had significant independent effects on GPP. Percent shade was the only local variable to have a significant independent effect on ER. Percent shade had a stronger effect on both GPP and ER than did % agriculture, and a trade-off exists between the importance of agricultural and forest cover on stream metabolism at different spatial scales. Our results highlight the role of local riparian conditions in controlling trophic state and the importance of riparian buffers as a tool to mitigate eutrophication in streams and rivers.

Assessments of biotic nutrient limitation in aquatic ecosystems typically rely on concentrations and ratios of potentially limiting nutrients. While successful in lakes, this approach has been less effective in streams, which often are dominated by benthic autotrophs (e.g., algae and vascular plants). We compared water-column nutrient concentration (C) to 3 alternative metrics and assessed their ability to predict autotrophic nutrient limitation in streams. One metric, nutrient flux (F) is the product of nutrient concentration and stream discharge. The other 2 metrics, autotrophic uptake length (S_w,a) and autotrophic uptake velocity (V_f,a), are derived from nutrient-spiraling theory and incorporate nutrient uptake. To evaluate the ability of each metric to predict nutrient limitation, we analyzed nutrient diffusing substrata (NDS) data from the Lotic Intersite Nitrogen eXperiment (LINX) (phases I and II; n = 80 streams across North America). We calculated NDS response ratios (RRs) to quantify the strength of autotrophic nutrient limitation in each stream, and we regressed RRs against each of the 4 metrics for N and P. For the LINX I analysis, S_w,a was the best predictor of autotrophic N limitation. F and V_f,a also performed well, and C was a poor predictor. In contrast, all 4 metrics were poor predictors of N limitation for the LINX II analysis when evaluated for individual streams, by land use, and within regions. None of the metrics were able to predict P limitation for either LINX study. Accuracy could be enhanced by developing new methods to quantify autotrophic nutrient uptake and limitation in stream reaches, but consistency with nutrient-spiraling theory and improved predictability of autotrophic N limitation make S_w,a a potentially useful metric for evaluating N limitation in streams.

Uptake and storage by biofilms facilitate the ability of streams to act as sinks for inorganic nutrients. Quantifying P fluxes over short time periods is important for understanding physiological responses to nutrient inputs, but few researchers have examined spatial and temporal variability in patterns of short-term biofilm nutrient uptake. We sampled intact stream biofilm assemblages seasonally (autumn—summer) across a spatial productivity gradient (8 streams in 2 provinces [Plateau and Piedmont] in Pennsylvania, USA) and tested responses to a P source over the first few minutes of exposure to resolve changes in initial assimilatory kinetics in short-term radiotracer experiments. We estimated P uptake and efflux rates with local regression (LOESS) and found distinct breakpoints in P fluxes. Mean maximum uptake occurred at 1.65 and 2.60 min, whereas maximum efflux occurred at 4.51 and 4.62 min in Piedmont and Plateau provinces, respectively. Biofilm nutrient content and accumulation rates were strong predictors of P uptake and efflux. P uptake and efflux differed between provinces (p = 0.005) and among seasons (p = 0.001). Our results show that considerable and rapid exchange processes occur at early time periods (<5 min). The magnitude of these exchange processes seems to diminish over longer periods (15–30 min), indicating that nutrient processing is a nearly instantaneous physiological, dynamic process. Thus, experimental time periods that are hours long or longer may obscure essential short-term responses. Spatio-temporal effects (productivity and seasonality) are strong determinants of P fluxes.

Local aggregations of unionoids (mussel beds) represent subpopulations constituting a riverine metapopulation and are presumed to be linked by dispersal of parasitic mussel larvae (glochidia) on host fishes. We examined movement patterns of Masu Salmon (Oncorhynchus masou masou), the obligate host fish for the freshwater pearl mussel Margaritifera laevis, during the mussel's parasitic period to determine the dispersal potential of glochidia via host fish in the Shubuto River system, Hokkaido, Japan. We conducted a mark—recapture study in a 650-m river section to describe the distance and directionality of host fish movement, and we used fyke net sampling to quantify the number of host fish moving from mainstem rivers to tributaries. Most individuals of Masu Salmon captured in the study were infected with glochidia of M. laevis. The displacement distance of infected fishes ranged from 37.5 m downstream to 512.5 m upstream (mean = 36.5 m upstream from marking location). The dispersal kernel based on a diffusion—advection model represented a leptokurtic distribution with a fat-tailed upstream bias, but most recaptured fish remained near the initial capture location. Movement of fish from mainstem rivers to tributaries was strongly affected by differences in water temperature among tributaries. Most individuals moved to cooler tributaries, but tributaries did not support populations of M. laevis. These results suggest that glochidial dispersal via host fish is important for local recruitment and upstream dispersal, but dispersal to tributaries may be a source of mortality for M. laevis.

Organisms often experience trade-offs between behaviors necessary for food acquisition and predator avoidance. In mesocosms and microcosms, we manipulated the risk and resource environments of grazing mayflies (Baetis bicaudatus) common in streams near the Rocky Mountain Biological Laboratory (RMBL) in western Colorado, USA. We exposed mayflies to treatments with or without chemical cues from predatory brook trout (Salvelinus fontinalis) and high or low levels of benthic algal resources to test the nonconsumptive effects of predators and food availability on grazer foraging behavior, predator-avoidance behavior, growth rates, and the effect of grazers on accrual of algal food resources. Total drift (daytime nighttime) of Baetis was significantly reduced under conditions of fish cues and high food levels. However, daytime drift, a risky food-search behavior, was negligible in all cases except the no-fish-cue/low-food treatment. Therefore, predator-induced nocturnal periodicity of drift behavior was influenced by variation in food levels. At low-food levels, Baetis growth rates and grazer effect on algal accrual were reduced, but neither measure of grazer performance (growth rates nor grazer impacts) was affected by fish cues or their interaction with food levels. We conclude that mayflies faced with potential trade-offs between food acquisition and predator-avoidance behaviors exhibit risky behavior (high daytime drift) only when predator-risk cues are absent and resources are low. Moreover, we observed no short-term costs of predatorinduced suppression of foraging movements under low-resource conditions. These results imply that previously observed negative effects of trout on size and fecundity of mature Baetis are not explained by slower growth rates.

Availability and quality of food resources limit consumer performance and modulate food webs, which in turn, can affect ecosystem functioning. Availability and quality of food resources can be especially important in streams, where consumers depend on allochthonous organic matter whose availability and quality can differ markedly both spatially and throughout the year. Most studies of the relationships between food quality and detritivore performance have been based on standardized food types and have not been designed to allow consideration of interactions between resource quantity and quality. Our goal was to evaluate the effects of quality and quantity of resource on shredder performance. We raised larvae of the caddisfly Sericostoma vitattum in the laboratory with food taken from a stream reach. We controlled food type (conditioned Alnus glutinosa leaves, natural leaf packs from riffles, and deposits within thick debris jams) and quantity (low: 2 mg individual^-1 d^-1; high: 10 mg individual^-1 d^-1). Consumption rate tended to be higher for lower- than for higher-quality materials, and the differences were more marked at high resource quantity. However, this compensatory mechanism was not enough to compensate growth in the case of lowest-quality food. In addition, food quality and quantity interacted to determine the energy allocation to body condition or growth. Our results show that the interaction between resource quality and quantity can influence invertebrate condition and life cycles. Stored organic matter in large jams, despite their poor quality, could enable consumers to survive until the next high-input season.

We analyzed basal sources, trophic levels, and connectance in dry-season food webs on 4 rivers in the upper Burdekin catchment in the dry tropics of northeastern Australia. The region is characterized by episodic summer rainfall, and most of the annual river flow occurs in a short period. In the dry season, rivers typically contract into a series of water holes of varying permanence and hydrologic connectivity. We used stable-isotope and stomach-content analyses to identify trophic levels of macroinvertebrates and fish, and we used a mixing model (SIAR) to identify foodweb basal sources at each site. We found substantial variability among sites in basal-source contributions, trophic position of individual taxa, and foodweb structure, and sites from the same river often were as different as sites from different rivers. Important basal sources at different sites included allochthonous tree litter, autochthonous algae and macrophytes, and Fe-fixing bacteria. Many relationships between consumers and basal sources were not resolved in the mixing model, mainly because of extensive omnivory or isotopic overlap among sources. Nevertheless, our results show high variability of dry-tropics river communities that extends beyond previously described macroinvertebrate assemblages to the broader food web. However, the main components of the upper trophic levels were similar across sites, such that different lower trophic levels supported similar assemblages of top consumers. These tropical rivers were defined by omnivory and ecological opportunism, which may be adaptations to seasonal hydrological variability.

The ecological effects of water hyacinth (Eichhornia crassipes), an introduced macrophyte, in freshwater systems depend on the growth and extent of floating mats. We studied macroinvertebrates associated with roots of water hyacinth in the Waccamaw River, a blackwater, tidal river in northeastern South Carolina, USA. In this system, water hyacinth is limited to a few protected bays and backwaters where the ecological effect is unknown. Our goal was to assess whether water hyacinth roots provided unique habitat. Plants representing ambient conditions, plants with defaunated roots, and a root analog (cotton mop strands = mop) were secured to floating frames in open water adjacent to water hyacinth mats. Samples were collected every 2 wk for 2 mo, and invertebrates were identified and quantified. Colonization of defaunated roots began within 2 wk, and invertebrate assemblages differed between roots and mops. The most common taxa on water hyacinth roots were Branchiopoda, Oligochaeta, Talitridae, and Chironomidae (Diptera), whereas Oligochaeta and Chironomidae were predominant on mops. Berosus sp.(Hydrophilidae) was the top-ranked taxon by proportional biomass on roots and mops. Total abundance and taxon richness of macroinvertebrates were greater on roots than on mops. Collector-gatherers were the most abundant functional feeding group (FFG) on mops, whereas distributions of abundance were relatively even among FFGs on ambient and defaunated roots. Predators dominated invertebrate biomass of all treatments, and shredder biomass was higher on roots than on mops. These data suggest that water hyacinth roots provide habitat for a diverse assemblage of macroinvertebrates, a function that should be weighed and assessed with other impacts before management actions are initiated.

Most population genetic studies in streams infer long-term patterns of gene flow by calculating fixation indices (e.g., F_ST) among sampled populations. In more-recent analytical methods, the need to assign individuals to populations a priori (clustering algorithms) is relaxed, and spatial autocorrelation analysis of allele frequencies (SA) is used to infer finer-scale and potentially short-term dispersal distances. We applied multiple methods to study the population genetic structure of the riverine caddisfly Stenopsyche marmorata (Trichoptera:Stenopsychidae) from 4 adjacent catchments in northeastern Japan. We genotyped larval individuals (N = 532) from 30 sites at 8 polymorphic microsatellite loci. Fixation indices suggested low levels of genetic differentiation among populations (global F_ST = 0.062, p < 0.01), and significant isolation-by-distance (IBD) indicated populations were in drift-migration equilibrium. Bayesian clustering separated S. marmorata into distinct upland (>250 m asl) and lowland populations, with different F_ST values (upland F_ST = 0.048, p < 0.01; lowland F_ST = 0.029, p < 0.01) and significant IBD only among upland populations. Allele frequencies were significantly positively autocorrelated (Moran's I > 0, p < 0.05) at distances up to 18 km along streams and up to 12 km across terrestrial habitat. These values were similar to directly observed flight distance in a single generation for this species in the field. We conclude that the multiple-method approach revealed: 1) unexpected population subdivision between upland and lowland areas that may result from local adaptation, differences in phenology, and historical colonization by multiple lineages; and 2) fine-scale estimates of dispersal that match direct observations of flight and suggest gene flow is more pronounced along water courses in this species.

Aquatic insects are widely used as indicator taxa to assess the ecological state of streams and to evaluate the success of stream restoration projects. Information on intraspecific genetic diversity and population connectivity is often lacking for such indicator taxa. However, these parameters are of critical importance for restoration plans and conservation management because: 1) species sometimes consist of several cryptic species and 2) species can recolonize only those restored habitats within a reachable distance from their source populations. Gene flow generally cannot be observed directly, and molecular markers provide a reasonable alternative to assess the dispersal potential and evaluate species' genetic diversity. We investigated the genetic diversity and dispersal potential of the predatory stonefly Dinocras cephalotes using 323 specimens from 29 populations in the Sauerland, a low mountain range in Germany. We used a 658 base pair (bp) fragment of the mitochondrial cytochrome c oxidase subunit I gene (COI) and found 2 distinct and diverse haplotype groups, which were shared across most populations. The groups were separated by a minimum intraspecific P-distance of 4.3%, suggesting historic isolation and possible presence of cryptic species. However, complementing analyses of the nuclear Wingless gene and 3 newly developed microsatellite markers clearly showed that individuals from both COI haplotype groups are interbreeding, and therefore, D. cephalotes is considered a single valid species. Population comparisons indicated high connectivity among all populations, with only a few individual populations showing signatures of isolation. Based on the molecular data, we conclude that dispersal is primarily achieved by the adult females of D. cephalotes.

Migratory life cycles and population structure of 2 putatively diadromous Australian fishes were examined using otolith chemistry (⁸⁷Sr/⁸⁶Sr) and genetics (microsatellites and mitochondrial deoxyribonucleic acid [mtDNA]). Australian whitebait (Lovettia sealii) is widely considered to be one of only a few anadromous fish species in the southern hemisphere. The congolli or tupong (Pseudaphritis urvillii) is reported to undertake an unusual form of sexually segregated catadromous migration, where females switch habitats between marine and freshwater, while males remain in marine or estuarine environments. Sr-isotope profiles of L. sealii showed this species does not move into fully freshwater habitats during its life cycle, suggesting it should be considered semianadromous or estuarine-dependent, rather than truly anadromous. This life-history strategy is unique among the Galaxiidae. Lovettia sealii is regionally divided into at least 3 well differentiated genetic stocks: northern and southern Tasmanian coasts and mainland Australia. Sr-isotope profiles of P. urvillii showed that females are catadromous, with the early life history spent in the marine environment and a single migratory transition from marine to freshwater occurring at an early point in the life history. Lack of bidirectional adult migration between freshwater and the sea suggests that female P. urvillii are semelparous, returning to the marine habitat to mate with resident males after an extended period of freshwater residence. Pseudaphritis exhibit weak genetic structure across their mainland range. An isolation-by-distance relationship describes the genetic structure of this species, a pattern it shares with several other nearshore-restricted catadromous fishes.

Climate change will cause relict alpine glaciers to disappear within decades. Associated high-altitude streams will face significant hydrological changes that might affect population genetic diversity of lotic species. In a recent study of glacier-fed streams in the Pyrenees, Finn et al. (2013) predicted that a large proportion of regional genetic diversity of the mayfly Baetis alpinus would be lost as glacial meltwater sources disappear. We expanded the analysis of Finn et al. to include genetic data (mitochondrial barcoding region) collected from B. alpinus occupying recently deglaciated locations including multiple basins in 2 mountain ranges (Sierra Nevada and Picos de Europa) on the Iberian Peninsula and 1 additional, deglaciated Pyrenean basin. We hypothesized that regional genetic diversity at scales of entire mountain ranges (γ diversity) and population structure within ranges (β diversity) would be lower in recently deglaciated mountain ranges than in the still-glaciated Pyrenees. For 4 cryptic lineages of the B. alpinus species complex in 3 mountain ranges, we found significantly lower genetic diversity in recently deglaciated than glaciated regions. β and γ genetic diversity were correlated, suggesting that population structure (β) strongly influences total regional diversity. Results support the hypothesis of Finn et al. (2013) that disappearance of alpine glaciers will result in substantial loss of genetic diversity. The distinctive hydrological environment created by glacial meltwater might drive an interaction between reproductive barriers (between highly and minimally glacier-influenced reaches within basins) and physical barriers (isolating highly glacier-influenced reaches among basins) that amplifies regional genetic diversity in mountain ranges still containing alpine glaciers.

Natural distributions of most freshwater taxa are restricted geographically, a pattern that reflects dispersal limitation. Macrobrachium rosenbergii is unusual because it occurs naturally in rivers from near Pakistan in the west, across India and Bangladesh to the Malay Peninsula, and across the Sunda Shelf and Indonesian archipelago to western Java. Individuals cannot tolerate full marine conditions, so dispersal between river drainage basins must occur at limited geographical scales when ecological or climatic factors are favorable. We examined molecular diversity in wild populations of M. rosenbergii across its complete natural range to document patterns of diversity and to relate them to factors that have driven evolution of diversity in this species. We found 3 clades in the mitochondrial deoxyribonucleic acid (mtDNA) data set that corresponded geographically with eastern, central, and western sets of haplotypes that last shared a common ancestor ∼1 × 10⁶ y ago. The eastern clade was closest to the common ancestor of all 3 clades and to the common ancestor with its congener, Macrobrachium spinipes, distributed east of Huxley's Line. Macrobrachium rosenbergii could have evolved in the western Indonesian archipelago and spread westward during the early to mid-Pleistocene to India and Sri Lanka. Additional groups identified in the nuclear DNA data set in the central and western clades probably indicate secondary contact via dispersal between regions and modern introductions that have mixed nuclear and mtDNA genes. Pleistocene sea-level fluctuations can explain dispersal across the Indonesian archipelago and parts of mainland southeastern Asia via changing river drainage connections in shallow seas on wide continental shelves. At the western end of the modern distribution where continental shelves are smaller, intermittent freshwater plumes from large rivers probably permitted larval dispersal across inshore areas of lowered salinity.

We used mitochondrial cytochrome c oxidase subunit I to infer the demographic history of Acroneuria frisoni Stark & Brown, a widespread eastern North American stonefly. We sequenced 1510 base pairs from 348 individuals from 36 populations, obtained 160 haplotypes, and tested 3 major hypotheses: 1) Wisconsinan and Illinoian glaciations were vicariant events that separated western and eastern populations; 2) the Mississippi River floodplain was a postglacial barrier to dispersal of eastern and western populations; and 3) at least 2 genetically distinct glacial refugia existed, east and west of the Mississippi River. We also investigated other barriers, pathways to dispersal, and the relative importance of refugia to repopulation of northern areas. Species-distribution modeling of its Wisconsinan-glacial-age distribution placed A. frisoni at the far southern end of the current distribution and in other areas further south and west than it is currently known, and separated western and eastern populations. Eastern and western populations accounted for 70% of the variation in the data set (analysis of molecular variance), and the Mississippi River was a major barrier to gene flow (Bayesian phylogenetic analysis). We identified a western, Ozark Mountains clade (OZK), a Midwest clade (MDW) in central and western Tennessee, and an Appalachian Mountains clade (APP) in Tennessee and Pennsylvania. Members of the MDW clade recolonized once-glaciated and glacially influenced areas north of the Ohio River. Members of the APP clade appear to be confined to the Tennessee River valley and the Ridge and Valley province of the Appalachian Mountains. The Mississippi River floodplain prevented members of the OZK clade from recolonizing northern areas, except in southern Illinois. Comparison to other species with similar distributions points to refuge use and recolonization similar to that of a megalopteran. Our data will guide reintroduction of A. frisoni into areas of the Midwest from which it has been extirpated.

The karst areas in the Dinaric region of the Western Balkan Peninsula are a hotspot of freshwater biodiversity. Many investigators have examined diversification of the subterranean freshwater fauna in these karst systems. However, diversification of surface-water fauna remains largely unexplored. We assessed local and regional diversification of surface-water species in karst systems and asked whether patterns of population differentiation could be explained by dispersal—diversification processes or allopatric diversification following karstrelated microscale vicariance. We analyzed mitochondrial cytochrome c oxidase subunit I (mtCOI) sequence data of 4 caddisfly species (genus Drusus) in a phylogeographic framework to assess local and regional population genetic structure and Pliocene/Pleistocene history. We used BEAST software to assess the timing of intraspecific diversification of the target species. We compared climate envelopes of the study species and projected climatically suitable areas during the last glacial maximum (LGM) to assess differences in the species climatic niches and infer potential LGM refugia. The haplotype distribution of the 4 species (324 individuals from 32 populations) was characterized by strong genetic differentiation with few haplotypes shared among populations (16%) and deep divergence among populations of the 3 endemic species, even at local scales. Divergence among local populations of endemics often exceeded divergence among regional and continental clades of the widespread D. discolor. Major divergences among regional populations dated to 2.0 to 0.5 Mya. Species distribution model projections and genetic structure suggest that the endemic species persisted in situ and diversified locally throughout multiple Pleistocene climate cycles. The pattern for D. discolor was different and consistent with multiple invasions into the region. Patterns of population genetic structure and diversification were similar for the 3 regional endemic Drusus species and consistent with microscale vicariance after the onset of intensified karstification in the Dinaric region. Karstification may induce microscale vicariance of running surface-water habitats and probably promotes allopatric fragmentation of stream insects at small spatialscales.

Several freshwater fishes have disjunct (geographically discrete and widely spaced) distributions across northern Australia. We used mitochondrial deoxyribonucleic acid (mtDNA) data and phylogeographic analyses to examine the origin of these common disjunct distributions and to test the hypothesis that they were a result of a single biogeographic event. These disjunct distributions are not perfectly shared among species, but we selected 3 species that have wide ranges with the most similar disjunct geographic overlap: Spotted Blue Eye (Pseudomugil gertrudae), Pennyfish (Denariusa australis), and MacCulloch's Rainbow Fish (Melanotaenia maccullochi). Despite similarity in their present-day disjunct distributions, spatial genetic patterns varied considerably among the 3 species in terms of measures of molecular diversity, number of mtDNA lineages within each species, inter- and intra-regional spatial distribution of individual lineages within species, and degree of partitioning of genetic variation among regions. Pseudomugil gertrudae and D. australis each contained 1 to 2 divergent lineages at particular sites in 1 of the regions (Top End), but both species also contained lineages in this region that were more closely related to conspecific populations in other regions. Two regional populations (Top End and Northern Cape York Peninsula) of M. maccullochi consisted exclusively of highly divergent lineages that probably reflect cryptic species. When the divergent lineages within each species were excluded from temporal analyses, a single vicariant event among regions could not be rejected. Our results indicate that several regional populations are long-term relicts for M. maccullochi and that several sites within Top End are associated with localized long-term refugia for P. gertrudae and D. australis. A single biogeographic event in the mid- to late-Pleistocene may have created broadscale separation of most populations of these species.

The Australian Monsoonal Tropics (AMT) is a unique location for the study of phylogeography and intraspecific genetic variation in freshwater fish. We assessed the phylogeographic structure of 5 species from 2 genera across the region. The species included 3 neosilurids (Plotosidae, Neosilurus hyrtlii, Neosilurus ater, and Neosilurus pseudospinosus) and 2 members of the genus Oxyeleotris (Eleotridae, O. selheimi and O. lineolata). We used mitochondrial deoxyribonucleic acid (mtDNA) and phylogenetic analyses to explore the phylogeographic histories of these species. Overall, phylogeographic patterns were inconsistent. Some species were highly structured, and phylogeographic breaks were detected (e.g., N. hyrtlii, N. pseudospinosus, and O. selheimi), but other species showed no obvious divergences across the AMT (N. ater and O. lineolata). All species sampled in the Gulf of Carpentaria had shallow phylogenies, consistent with the expectation that historically, Lake Carpentaria would have provided connectivity through this region. All species also showed evidence of recent connectivity across drainage divides on the eastern and western coasts of the Cape York Peninsula. Some species in the Kimberley region were highly structured, consistent with expectation that these ancient and geologically stable catchments would promote divergence in allopatry. Conservation efforts should now be directed toward ensuring that the intraspecific biodiversity identified in our study and others are protected in the future.

Elevation gradients allow scientists to observe changes in fauna over a range of abiotic conditions. A variety of trends in aquatic insect diversity patterns across elevation have been reported. However, many of these studies are confounded because they include streams at lower elevations, which are often larger in size and more polluted than their higher-elevation counterparts. Moreover, such studies always relied solely on morphological delineation of taxa, thereby potentially overlooking cryptic diversity. We reduced these limitations by sampling only minimally impacted wadeable streams across an elevation gradient and by combining morphological taxonomy with deoxyribonucleic acid (DNA) barcoding to identify taxa. We collected numerically abundant Ephemeroptera, Plecoptera, and Trichoptera (EPT) from single streams at ∼200-m elevation intervals across >1000-m transects in 3 watersheds draining the eastern slope of the Colorado Rocky Mountains. Based on morphology alone, we identified 49 numerically abundant EPT morphospecies across 26 sites. Using DNA barcoding, we found 69 distinct lineages that probably represent distinct species. EPT species richness was highest at midelevations, and rates of turnover along elevation transects showed no consistent elevation trend or trend among ecological zones defined by vegetation. β-diversity across sites at comparable elevations in different watersheds showed a negative trend with increasing elevation that was marginally significant for DNA barcode taxa (p = 0.051) but not for morphospecies. Furthermore, significant (p < 0.05) differences in taxon richness, turnover, and lateral β-diversity values generated by DNA barcoding underscore the ability of molecular tools to quantify patterns in aquatic insect diversity across elevations.

Benthic macroinvertebrate community composition is used to assess wetland and stream condition and to help differentiate the effects of stressors among sites. Deoxyribonucleic acid (DNA) barcoding has been promoted as a way to increase taxonomic resolution and, thereby, to increase the sensitivity of bioassessment metrics. We compared the ability of several commonly used bioassessment metrics calculated with data derived from morphology and from DNA barcoding to detect differences in stream condition of 6 paired sites in southern California with relatively subtle impacts to habitat. At each site, we sampled an upstream (reference) reach and a downstream (impact) reach with armored stream banks. We counted and identified ∼600 organisms/ sample based on morphology (generally to species, but to genus for midges). We then extracted mitochondrial (mt)DNA from each individual and sequenced the ∼658-base pair (bp) barcoding region of the cytochrome c oxidase subunit I (COI) gene. Most (91%) organisms yielded sequences >350 bp in length, but high failure rates for all taxa collected from 1 stream required that we exclude it from analysis. Sixteen metrics calculated with morphological data showed subtle but not significant differences in community composition between armored and unarmored reaches. The statistical power of 10 of the 16 metrics was substantially higher when calculated with DNA than with morphological data, and we were able to discern differences between armored and unarmored reaches with the DNA data. These differences were associated with increased taxonomic richness detected for midges, mayflies, noninsects, caddisflies, and black flies when DNA data were used. Our results suggest that identifications based on DNA barcoding have the potential to improve power to detect small changes in stream condition.

Species-level identifications are difficult or impossible for many larval aquatic macroinvertebrates. We described the taxonomic composition of macroinvertebrate communities from 5 coastal streams in 3 neighboring catchments in southern California. We compared taxonomic identifications based on deoxyribonucleic acid (DNA) barcoding (cytochrome c oxidase subunit I [COI]) with morphological identifications of the same specimens. We examined 5870 individuals, and barcodes with sequence lengths >350 base pairs (bp) for 91% of those specimens. We used the naturally occurring gaps in divergence frequencies for each order (usually 2% level of genetic divergence) to delimit putative species for all taxonomic groups except Simulium (3%) and Baetis (1%). We identified 200 species across these 5 streams. We identified 104 more species via barcodes than via morphology (200 vs 96, a 108% increase). Richness increases were greatest for Chironomidae (60 more species), Ephemeroptera (10 species), Acari (10 species), and Trichoptera (6 species). Forty-five percent of the genera/species identified morphologically represented >2 species. Many (86) species identified with barcodes were represented by only 1 or 2 specimens and were found at only 1 stream. Thus, species rarity (either spatially or numerically) appears to be a common characteristic of these streams. Barcoding increased total richness at each site by 12 to 40 taxa over morphology alone, and increased the difference between reference and impact sites in terms of lost taxa. These results suggest that macroinvertebrate biodiversity in streams has been underestimated substantially in the past, as has the biodiversity lost in response to environmental stress. The potential of DNA barcoding will not be fully realized until we can assign traits, such as habitat preference, ecological function, and pollution tolerance, at the species level.

Deoxyribonucleic acid (DNA) barcode (cytochrome c oxidase subunit I [COI]) sequences have been used to associate adult and immature stages of stoneflies as a rapid and effective alternative to rearing immatures. However, no molecular associations have been made for Neotropical species, which have very few described immature stages. Kempnyia Klapálek (Perlidae) is an endemic South American genus. Currently, 20 females have been associated with males of the 40 valid species, but only 5 nymphs have been described. We collected stonefly nymphs at Macaé River Basin, Rio de Janeiro State, Brazil, and Kempnyia nymphs were separated those in 6 morphotypes. These morphotypes were associated to nominal Kempnyia species using 38 COI sequences (471 base pairs [bp]) of immatures and identified adults of 10 species occurring in southern and southeastern Brazil. Based on a neighbor-joining analysis of Kimura-2-parameter (K2P) pairwise divergences, we found 6 genetic Kempnyia lineages at Macaé River Basin represented by all morphotypes of nymphs included in our study. These nymphs were associated with males of Kempnyia alterosarum, K. colossica, K. gracilenta, K. petersorum, K. obtusa, and K. varipes. Intraspecific divergences in Kempnyia varied from 0 to 15%, but ∼½ of comparisons had values >3%. These data are in agreement with previous studies with stonefly populations, which tend to show high COI divergences. Based on our results, we described previously unknown immatures of K. alterosarum, K. petersorum, and K. obtusa, and the male genitalia of K. varipes for the first time. We also described a new adult color morph of K. petersorum. Our study increased the number of Kempnyia species recorded at Macaé River Basin from 5 to 11. We recorded for the first time K. alterosarum and K. reticulata from Rio de Janeiro State and K. jatim from Espírito Santo State.

Species delimitation (grouping individuals into distinct taxonomic groups) is an essential part of evolutionary, conservation, and molecular ecology. Deoxyribonucleic acid (DNA) barcodes, short fragments of the cytochrome c oxidase subunit I (COI) gene, are being used in environmental bioassessments to assign specimens to putative species, but no method for delimiting DNA barcodes into species-level entities is universally accepted. We investigated the effect of delimitation methods on outcomes of bioassessments based on DNA barcodes. We used 2 tree-construction methods (neighbor joining [NJ], maximum likelihood [ML]) and 4 classes of species-delimitation criteria (distance-based, bootstrap support, reciprocal monophyly, and coalescentbased) with a DNA barcode data set consisting of 3 genera and 2202 COI sequences. We compared species delimitations for Baetis (Ephemeroptera:Baetidae), Eukiefferiella (Diptera:Chironomidae), and Simulium (Diptera: Simuliidae) from different streams. We assessed congruence among trees and compared species abundances and estimated species richness among methods. NJ followed by use of a standard barcoding distance cutoff (2%) yielded the greatest number of putative species. All other delimitation methods yielded similar, but lower, richness. Differences in species delimitations produced by various methods might have been caused by confounding factors, such as possible parthenogenesis in Baetis and rare haplotypes in abundant species of Baetis and Simulium. Eukiefferiella presented the fewest discrepancies among delimitations. Each method can be regarded as producing a separate line of evidence contributing to the delimitation of separately evolving lineages. The increased resolution offered by DNA barcoding can yield important insights into the natural history of organisms, but the power of these observations is limited without the use of multigene and multilocus data sets.

Diatoms are main bioindicators used to assess the ecological quality of rivers, but their identification is difficult and time-consuming. Next Generation Sequencing (NGS) can be used to study communities of microorganisms, so we carried out a test of the reliability of 454 pyrosequencing for estimating diatom inventories in environmental samples. We used small subunit ribosomal deoxyribonucleic acid (SSU rDNA), ribulose-1, 5-bisphosphate carboxylase (rbcL), and cytochrome oxidase I (COI) markers and examined reference libraries to define thresholds between the intra- and interspecific and intra- and intergeneric genetic distances. Based on tests of 1 mock community, we used a threshold of 99% identity for SSU rDNA and rbcL sequences to study freshwater diatoms at the species level. We applied 454 pyrosequencing to 4 contrasting environmental samples (with one in duplicate), assigned taxon names to environmental sequences, and compared the qualitative and quantitative molecular inventories to those obtained by microscopy. Species richness detected by microscopy was always higher than that detected by pyrosequencing. Some morphologically detected taxa may have been persistent frustules from dead cells. Some taxa detected by molecular analysis were not detected by morphology and vice versa. The main source of divergence appears to be inadequate taxonomic coverage in DNA reference libraries. Only a small percentage of species (but almost all genera) in morphological inventories were included in DNA reference libraries. DNA reference libraries contained a smaller percentage of species from tropical (27.1–38.1%) than from temperate samples (53.7–77.8%). Agreement between morphological and molecular inventories was better for species with relative abundance >1% than for rare species. The rbcL marker appeared to provide more reproducible results (94.9% species similarity between the 2 duplicates) and was very useful for molecular identification, but procedural standardization is needed. The water-quality ranking assigned to a site via the Pollution Sensitivity diatom index was the same whether calculated with molecular or morphological data. Pyrosequencing is a promising approach for detecting all species, even rare ones, once reference libraries have been developed.

Lack of nonmicrosatellite nuclear markers that are informative at the intraspecific level has limited inferences about the phylogeography and phylogenetics of most aquatic insect taxa. Genomic resources are becoming available at an unprecedented rate and scale. Sequencing a single transcriptome (complementary deoxyribonucleic acid [cDNA] library corresponding to all messenger ribonucleic acid [mRNA] expressed at a single point in time) can generate tens of thousands of sets of overlapping DNA segments that together represent a consensus region of DNA (contigs) averaging >500 base pairs [bp] in length. A challenge posed by these large data sets is extraction of markers with population-level utility. We demonstrate proof-of-concept for using comparative transcriptomics to develop primers for multiple nuclear loci with population-level utility in Hesperoperla pacifica efficiently and cost effectively. We created a cDNA library from total RNA and assembled the resulting transcriptomes de novo with the computer program Trinity. We used a Basic Local Alignment Search Tool (BLAST)-based algorithm to identify and filter putative orthologs (homologous gene sequences in different species). Filtering with a minimum sequence length of 600 bp and nucleotide identity of ≥85% yielded >1800 ortholog alignments. We examined some of these alignments for suitability for polymerase chain reaction (PCR) primer design. We screened 40 primers for their ability to amplify the correct-size PCR product and to detect genetic variation in 4 populations across the Great Basin (USA). The large number of loci that can be identified with this technique will lead to more-robust estimates of population parameters and more-rigorous tests of phylogeographic hypotheses than can be done with fewer loci. The advantages of nuclear-sequence-based studies include better comparisons with mitochondrial DNA phylogenies because of shared statistical techniques and models of mutation and the potential for direct comparisons of genetic structure across taxa.