A new benthic freshwater diatom, Cymbella tridentina sp. nov., was identified with the aid of light and scanning electron micrographs. The most characteristic morphological features are the valve outline (in particular, the shape of the ventral margin), the number of stigmata, and the length/breadth ratio. The new species was epilithic in carbonate, mountain, flowing springs in 3 Natural Parks of the Alps. The environmental preferences of the new species, assessed by the comparative analysis of the sites where it was found and by seasonal and longitudinal-distribution data available for the type locality, are quite distinctive. The new species appears to be rheophilic and oligotraphentic (not present in N-enriched sites), characteristic of dolomite mountain ranges (even if dolomite is not the dominating carbonate rock in the basin), and to develop year-round with low relative abundances (peaks associated with high discharge in autumn and spring). The combination of these ecological requirements might determine the preferential occurrence of C. tridentina sp. nov. in close-to-pristine, carbonate, mountain, flowing springs. The preference of the new species for the spring zone of running waters, and its possible use as an integrity indicator, is discussed in the wider context of crenobiosis. The importance of oligotrophic headwaters for the integrity of running-water systems and for the conservation of freshwater biodiversity, is highlighted.

Bioirrigation by tube-dwelling macrozoobenthos species causes an advective flow of overlying water through the burrow lumen. Particle image velocimetry (PIV) and color tracer measurements were used to measure flow velocities. The volumetric flow rate was calculated based on measured velocities and visual observation of larval body movements. Investigations were done with Chironomus plumosus (Diptera:Chironomidae) larvae and pupae. During pumping periods, flow velocity was 15.0 ± 10.7 (with color tracers: 13.6 ± 1.7) mm/s and volumetric flow rate was 40 ± 24 (with color tracers: 30.9 ± 3.9) µL/s for larvae. For pupae, these values were only 5.1 ± 0.7 mm/s and 11 ± 1 µL/s, respectively. Pupae conduct no filter feeding, so our results indicate that high volumetric flow rates occur because of filter-feeding. Furthermore, PIV measurements revealed the filtering effect of C. plumosus bioirrigation activity. A projection of the measured volumetric flow rate to Lake Müggelsee in Berlin, Germany (745 4^th-instar larvae/m²), resulted in a flow of 1.3 m³ m⁻² d⁻¹. A volume equivalent to the water body of the lake is being pumped through the sediment every 4.8 d. This emphasizes the importance of burrow ventilation and filter-feeding for ecosystems.

Polyunsaturated fatty acids (PUFAs) are abundant in stream-dwelling aquatic insects and might be essential for their growth and survival. We conducted a controlled study designed to clarify fatty acid (FA) assimilation and metabolism by net-spinning caddisfly larvae (Hydropsyche sp.). Specifically, our goal was to determine if these insects could synthesize essential FAs (18:2ω6, 18:3ω3, 20:4ω6, and 20:5ω3) de novo or elongate them from precursors, or if they must acquire them from their diet. In laboratory experiments, we supplied controlled diets (commercial fish food, oat particles, Cladophora glomerata, and conditioned leaves) to larvae in individual microcosms that simulated stream conditions. Caddisfly FA profiles consistently matched those of the diets provided, and in general, these insects had very limited abilities to synthesize and elongate essential FAs. Concentrations of linoleic (18:2ω6) and linolenic (18:3ω3) acid declined significantly in caddisflies fed diets depleted in these FAs. To test the ability to elongate FAs, caddisflies were fed a diet depleted in arachidonic acid (20:4ω6), but rich in its precursor 18:2ω6. Tissue concentrations of 20:4ω6 declined, suggesting that the ability of caddis larvae to elongate 18:2ω6 to 20:4ω6 is limited or absent. Caddisflies accumulated 20:5ω3 at greater assimilation efficiencies than for any other FA, suggesting a key importance of this FA. Across all experiments, caddisflies gained mass, total FA content, or both when supplied with all food sources except leaf litter. We suggest that caddisflies in streams must obtain essential FAs either from algal material or from predation.

The insect orders Ephemeroptera, Plecoptera, and Trichoptera (EPT) are particularly important for freshwater ecological and biomonitoring studies, but difficulties in their identification to species level impede research. DNA barcoding provides a solution to this problem by linking newly collected specimens to a reference library of authoritatively identified specimens. Here, we consider the ways in which patterns of intraspecific and interspecific genetic divergences in the barcode region can provide rapid insights into the taxonomic identity, morphological features, and geographical distributions of species. Our study led to a >5× increase in the EPT fauna, including 68 caddisfly, 37 mayfly, and 7 stonefly species, recorded from Churchill. DNA barcoding also aided detection of rare taxa, allowed identification of otherwise unidentifiable life stages, revealed several potentially new species of caddisflies and mayflies, and suggested the presence of cryptic species. The new insights into this fauna and the strong congruence between morphological and molecular characters affirm the utility of DNA barcoding for rapid characterization of the diversity of EPT faunas. We also explore the phenology and habitat preferences of Churchill's trichopterans and demonstrate that comprehensive sampling is important for documenting biodiversity through DNA barcoding.

Because unionid mussels have a parasitic larval stage, they are able to disperse upstream and downstream as larvae while attached to their host fish and with flow as juveniles after excystment from the host. Understanding unionid population ecology requires knowledge of the processes that affect juvenile dispersal prior to establishment. We examined presettlement (transport and dispersion with flow) and early postsettlement (bed shear stress) hydraulic processes as negative censoring mechanisms. Our approach was to model dispersal using particle tracking through a 3-dimensional flow field output from hydrodynamic models of a reach of the Upper Mississippi River. We tested the potential effects of bed shear stress (τ_b) at 5 flow rates on juvenile mussel dispersal and quantified the magnitude of these effects as a function of flow rate. We explored the reach-scale relationships of Froude number (Fr), water depth (H), local bed slope (S), and unit stream power (QS) with the likelihood of juvenile settling (λ). We ran multiple dispersal simulations at each flow rate to estimate λ, the parameter of a Poisson distribution, from the number of juveniles settling in each grid cell, and calculated dispersal distances. Virtual juveniles that settled in areas of the river where τ_b > critical shear stress (τ_c) were resuspended in the flow and transported further downstream, so we ran simulations at 3 different conditions for τ_c (τ_c  =  ∞ [no resuspension], 0.1, and 0.05 N/m²). Differences in virtual juvenile dispersal distance were significantly dependent upon τ_c and flow rate, and effects of τ_b on settling distribution were dependent upon τ_c. Most simulations resulted in positive correlations between λ and τ_b, results suggesting that during early postsettlement, τ_b might be the primary determinant of juvenile settling distribution. Negative correlations between λ and τ_b occurred in some simulations, a result suggesting that physical or biological presettlement processes might determine juvenile settling distributions. Field data are needed to test these hypotheses. Results support the idea that flow patterns and τ_b can act as negative censoring mechanisms controlling settling distributions. Furthermore, a river reach probably has a quantifiable threshold range of flow rates. Above the upper threshold, τ_b probably is the primary determinant of juvenile settling distribution. Relationships of λ with H, Fr, S, and QS were relatively weak. Important physical processes that affect dispersal probably are not captured by approximations based on large-scale hydraulic parameters, such as Fr and H.

Macroinvertebrate taxon tolerances to human disturbance have been key components of bioassessment since the early 20^th century. Initially, tolerances were used to evaluate organic pollution, but more recently, their use has been generalized to evaluate overall disturbance. Numeric tolerance values have been assigned to individual taxa in many previous studies, but their origins can be unknown, and the values can lack empirical basis. We used macroinvertebrate data collected at 1106 stream and river sites in 12 western states sampled during the Environmental Monitoring and Assessment Program (EMAP) in 2000 to 2004 to develop macroinvertebrate tolerance values for western US lotic ecosystems. We used principal components analysis to create a 9-variable synthetic disturbance variable (SDV) for overall human disturbance at those sites. The SDV included measures of nutrients, site-scale physical habitat, and catchment-scale land use. For taxa found at ≥20 sites, we calculated tolerance values at multiple levels of taxonomic resolution as the weighted (mean standard deviation) of the SDV at the sites with relative abundances as the weights. We then used the taxon tolerance values and relative abundances at each site to calculate an assemblage tolerance index (ATI) score at several levels of taxonomic resolution. These results should help inform discussions of the level of taxonomic resolution needed for bioassessments. We also discuss how the tolerance values could be used to improve the sensitivity of bioassessment metrics.

We present a visually based method for the taxonomic identification of benthic invertebrates that automates image capture, image processing, and specimen classification. The BugID system automatically positions and images specimens with minimal user input. Images are then processed with interest operators (machine-learning algorithms for locating informative visual regions) to identify informative pattern features, and this information is used to train a classifier algorithm. Naïve Bayes modeling of stacked decision trees is used to determine whether a specimen is an unknown distractor (taxon not in the training data set) or one of the species in the training set. When tested on images from 9 larval stonefly taxa, BugID correctly identified 94.5% of images, even though small or damaged specimens were included in testing. When distractor taxa (10 common invertebrates not present in the training set) were included to make classification more challenging, overall accuracy decreased but generally was close to 90%. At the equal error rate (EER), 89.5% of stonefly images were correctly classified and the accuracy of nonrejected stoneflies increased to 96.4%, a result suggesting that many difficult-to-identify or poorly imaged stonefly specimens had been rejected prior to classification. BugID is the first system of its kind that allows users to select thresholds for rejection depending on the required use. Rejected images of distractor taxa or difficult specimens can be identified later by a taxonomic expert, and new taxa ultimately can be incorporated into the training set of known taxa. BugID has several advantages over other automated insect classification systems, including automated handling of specimens, the ability to isolate nontarget and novel species, and the ability to identify specimens across different stages of larval development.

Cultural eutrophication of surface waters has become a major source of water-quality impairment throughout the US. In response, the US Environmental Protection Agency (USEPA) has devised a national strategy for the development of regional nutrient criteria. Our study is part of New York State's effort to revise its narrative nutrient standard for N and P and is based on the USEPA's recommended weight-of-evidence approach. The objective of our investigation was to identify nutrient thresholds based on a final weighted average of results from percentile analysis, nonparametric deviance reduction (changepoint), and cluster analysis. The thresholds were determined from shifts in biological community structure (benthic macroinvertebrate and diatom) related to water-column nutrient data from 40 large river sites throughout New York State. USEPA's percentile analysis yielded possible criteria of 0.023 mg total P (TP)/L, 0.51 mg total N (TN)/L, 0.16 mg NO₃-N /L, and 2.4 mg chlorophyll a (chl a)/m³. Threshold responses in benthic macroinvertebrate metrics at the 50^th percentile occurred at concentrations between 0.009 and 0.07 mg TP/L, 0.41 and 1.2 mg TN/L, 0.18 and 0.55 mg NO₃-N/L, and 2.1 mg chl a/m³. Cluster analysis yielded 3 groups of sites based on macroinvertebrate and diatom taxa. The median nutrient values of the medium-nutrient-condition site clusters were used to set criteria for TP and TN. For site clusters based on macroinvertebrate data these values were 0.037 mg TP/L and 0.68 mg TN/L. For clusters based on diatom data these were 0.037 mg TP/L and 0.78 mg TN/L. Based on the weight-of-evidence approach and results from all 3 methods, the proposed guidance values for nutrients in large rivers are 0.03 mg TP/L, 0.7 mg TN/L, 0.3 mg NO₃-N/L, and 2.2 mg chl a/m³. These values are similar to those derived by others and provide meaningful nutrient endpoints that would be protective of aquatic life in large rivers.

Perhaps no subject is more intriguing and complex than climate change and its effects on ecosystems and their biological communities. Changes in precipitation and snowmelt patterns, sea level rise, increased intensity of storms and wet-weather events, thawing permafrost, changes in vegetation and wildlife composition and distribution, and other effects present significant policy and land management challenges. Scientific modeling and analyses demonstrate that the effects of a changing climate are complex, with high variability over time, space, and species, including effects on benthic organisms. The variable effects of a changing climate complicate decision making, rendering scenario planning, adaptive management, and other management learning tools increasingly important. The very complexity of effects presents particularly difficult challenges for policy makers and resource managers because the available science often is highly uncertain. Thus, other decision support tools are needed to help managers anticipate and respond to local and regional conditions. Effective policy and management require relevant science to inform decision making. Key needs include more assessments of ecosystem and species trends and their possible linkage to climate change and a better understanding of the interplay of multiple variables, how different management regimes might affect ecosystems and species survival, and how to apply risk management tools and adaptive management to resource issues.

We examined whether presence of vegetation and seasonal changes in flow affected N chemistry and denitrification rates within sandbars of a 7^th-order sandy alluvial river (Wisconsin River, USA). We addressed these questions with a broad-scale approach of measuring parafluvial water chemistry and denitrification rates in multiple sandbars distributed along a 15-km river reach during summer 2004 and 2005. After recession of spring flooding, parafluvial chemistry in unvegetated bars was characterized by moderate dissolved O₂ (DO) and elevated NO₃⁻-N concentrations (>3.5 mg N/L), whereas vegetated bars tended to be hypoxic (<2 mg/L DO) and depleted in NO₃⁻-N (0.2 mg N/L) relative to unvegetated bars and surface water (0.47 mg N/L). As flow declined over the summer, NO₃⁻-N also declined in both bar types, whereas SO₄²⁻ was relatively constant in unvegetated bars but decreased in vegetated bars. Amendment experiments demonstrated that denitrification was limited primarily by NO₃⁻-N and secondarily by organic C in both bar types, but the strength of this limitation varied over time and was greater in vegetated bars, a result suggesting loss of denitrification capacity. Thus, spatial and temporal patterns of water chemistry and denitrification activity among multiple sandbars indicated that unvegetated bars shifted from N transformers/NO₃⁻-N sources early in the summer to N retainers/NO₃⁻-N sinks as discharge declined, whereas vegetated bars always supported anaerobic processes and probably shifted from NO₃⁻-N to SO₄²⁻ sinks. We hypothesize that the contribution of vegetated islands to overall riverine N retention is small because establishment of vegetation reduces hydrologic linkages between bars and surface water. Modern changes in the flow regime of the Wisconsin River have increased establishment of riparian vegetation on exposed bars, a pattern suggesting that parafluvial N retention is being reduced while riverine N loading is increasing.

In this paper, we review the history of Hydropsychinae genus-level classification and nomenclature and present new molecular evidence from mitochondrial cytochrome c oxidase subunit I (COI) and nuclear large subunit ribosomal ribonucleic acid (28S) markers supporting the monophyly of the genus Hydropsyche. Both molecular and morphological characters support a broad conservative definition of Hydropsyche. Caledopsyche, Hydatomanicus, and Occutanspsyche are synonymized with Hydropsyche. The following species groups are established: Hydropsyche bronta Group (generally corresponding with Ceratopsyche and Hydropsyche morosa and newae Groups), Hydropsyche colonica Group (generally corresponding with Orthopsyche), Hydropsyche instabilis Group (generally corresponding with Hydropsyche s.s.), and Hydropsyche naumanni Group (generally corresponding with Occutanspsyche). Molecular data recovered Hydromanicus as paraphyletic, and Cheumatopsyche and Potamyia as sister taxa. The genus names Plectropsyche and Streptopsyche are reinstated.

Knowledge of functional aspects of communities in intermittent streams currently is lacking, and the role of terrestrial detritus as a resource in open-canopy streams is uncertain. Our main objective was to characterize and quantify the macroinvertebrate shredder assemblages in 3 intermittent open-canopy streams on the island of Öland in the Baltic Sea, Sweden, by estimating secondary production. Estimates of annual shredder secondary production ranged from 0.005 to 13.6 g ash-free dry mass (AFDM) m⁻² y⁻¹ among sites. Shredder production and amount of organic matter were positively related, but shredder production was more strongly influenced by the duration of the summer dry period. Production decreased with increase in the length of the summer dry period, but shredder production was still high at sites with long dry phases and was comparable to estimates of shredder production in permanent forested streams, results suggesting that terrestrial organic matter might be an important energy resource in open-canopy streams. Shredder species richness decreased with increase in length of the summer dry phase, and shredder species composition at sites with a long dry period was dominated by drought-tolerant taxa. Our results suggest that ecosystem functions might shift from being sustained by many taxa to being sustained by fewer taxa as stream ecosystems experience moderate droughts of similar length (2–4 mo).

Downstream drift of benthic invertebrates is one of the defining characteristics of lotic environments. We collected 90 drift samples from adjacent sand-bed and riffle sections in a Mediterranean river during low flow conditions. Our goal was to compare drift fluxes (densities and instantaneous drift) and model the drift response across the velocity gradient in each habitat. Velocity distributions did not differ significantly between the habitats, but bedload transport rates and suspended sediment concentrations were significantly higher in the sand-bed habitat, where substrate was finer and less stable than in the riffle habitat. Benthic invertebrate densities differed by an order of magnitude between the habitats, with low and relatively homogeneous densities in the sand-bed. However, mean drift density did not differ significantly between the habitats. Quantile regression indicated that the upper limits of the drift response across the velocity gradient differed between habitats. Sample values below these upper limits suggest that substrate stability and benthic density act to constrain drift in some locations. Instantaneous drift was much higher in the sand-bed than in the riffle habitat, with up to 42% of the benthos present in the drift at any given moment. The taxonomic composition of benthic and drift samples suggested that animals from the riffle contributed to drift in the sand-bed habitat. The high loss rates in the sand-bed habitat suggest a rapid turnover of animals, supported by invertebrates arriving from the upstream riffle.

Freshwater mussels (order Unioniformes) fulfill an essential role in benthic aquatic communities, but also are among the most sensitive and rapidly declining faunal groups in North America. Rising water temperatures, caused by global climate change, industrial discharges, drought, or land development, could further challenge imperiled unionid communities. The aim of our study was to determine the upper thermal tolerances of the larval (glochidia) and juvenile life stages of freshwater mussels. Glochidia of 8 species of mussels were tested: Lampsilis siliquoidea, Potamilus alatus, Ligumia recta, Ellipsaria lineolata, Lasmigona complanata, Megalonaias nervosa, Alasmidonta varicosa, and Villosa delumbis. Seven of these species also were tested as juveniles. Survival trends were monitored while mussels held at 3 acclimation temperatures (17, 22, and 27°C) were exposed to a range of common and extreme water temperatures (20–42°C) in standard acute laboratory tests. The average median lethal temperature (LT50) among species in 24-h tests with glochidia was 31.6°C and ranged from 21.4 to 42.7°C. The mean LT50 in 96-h juvenile tests was 34.7°C and ranged from 32.5 to 38.8°C. Based on comparisons of LT50s, thermal tolerances differed among species for glochidia, but not for juveniles. Acclimation temperature did not affect thermal tolerance for either life stage. Our results indicate that freshwater mussels already might be living close to their upper thermal tolerances in some systems and, thus, might be at risk from rising environmental temperatures.

Ecological thresholds that lead to alternative community states can be exceeded through gradual perturbation or as a result of sudden disturbance. Many Great Plains streams have experienced dramatic changes in their hydrologic regime resulting from water and landuse changes that began as early as 1880. These changes, combined with the presence of many invasive species, have substantially altered the fish communities in this area. We quantified temporal changes in fish communities in 3 large river basins in relation to putative anthropogenic stressors, including increased sediment supply derived from row-crop agriculture (beginning in 1880), habitat fragmentation caused by reservoir construction (beginning in the 1950s), and reduced discharge caused by groundwater withdrawal (beginning in the 1960s). We hypothesized that these abiotic regime shifts, coupled with species invasions, would shift the system from a fish community dominated by lotic (flowing water) species to one dominated by lentic (still water) species. Further, we predicted that the timing and intensity of community change would vary across basins that experienced different types and levels of stressors. Restructuring of fish communities across the 3 river basins was driven primarily by similar increases in lentic species, with only a few declines in several large-river species. Current fish communities in these basins share <50% of the species recorded in historic collections, and these differences were driven by species extirpations and invasions. The greatest levels of community divergence over time occurred in western Kansas basins that experienced the most intense groundwater withdrawals and fragmentation by reservoirs. An alarming result from this analysis was the recent (after 1991) expansion of several invasive species in the Arkansas and lower Kansas River basins and the decline or extirpation of several native species where flow regimes are less heavily altered. Accelerating changes in the biota and habitat identified by our retrospective analysis highlight potential complications for restoring the habitat and native fish communities to a previous state.

Nonlinear ecological responses to anthropogenic forcing are common, and in some cases, the ecosystem responds by assuming a new stable state. This article is an overview and serves as the introduction to several articles in this BRIDGES cluster that are directed toward managers interested in dealing with nonlinear responses in freshwaters, particularly streams. A threshold or breakpoint occurs where the system responds rapidly to a relatively small change in a driver. The existence of a threshold can signal a change in system configuration to an alternative stable state, although such a change does not occur with all thresholds. In general, a mechanistic understanding of ecological dynamics is required to predict thresholds, where they will occur, and if they are associated with the occurrence of alternative stable states. Thresholds are difficult to predict, although a variety of univariate methods has been used to indicate thresholds in ecological data. When we applied several methods to one type of response variable, the resulting threshold values varied 3-fold, indicating that more research on detection methods is necessary. Numerous case studies suggest that the threshold concept is important in all ecosystems. Managers should be aware that human actions might result in undesirable rapid changes and potentially an unwanted alternative stable state, and that recovery from that state might require far more resources and time than avoiding entering the state in the first place would have required. Given the difficulties in predicting thresholds and alternative states, the precautionary approach to ecosystem management is probably the most prudent.

The goal of this paper is to help managers better understand implications of using aggregate community metrics, such as taxon richness or Indices of Biotic Integrity (IBI), for detecting threshold responses to anthropogenic environmental gradients. To illustrate, we offer an alternative analytical approach, Threshold Indicator Taxa ANalysis (TITAN), geared toward identifying synchronous changes in the distribution of multiple taxa as evidence of an ecological community threshold. Our approach underscores the fundamental reality that which taxa are affected by stressors is important, both from a conservation standpoint and because taxon-specific life-history traits help us understand relevant mechanisms. First, we examine macroinvertebrate community response to an impervious cover gradient using a well-studied biomonitoring data set to show that representative community metrics are relatively insensitive to synchronous threshold declines of numerous individual taxa. We then reproduce these response relationships using a simulated community data set with similar properties to demonstrate that linear or wedge-shaped responses of community metrics to anthropogenic gradients can occur as an artifact of aggregating multiple taxa into a single value per sampling unit, despite strong nonlinearity in community response. Our findings do not repudiate the use of community metrics or multimetric indices, but they challenge assumptions that such metrics are capable of accurately reflecting community thresholds across a broad range of anthropogenic gradients. We recommend an alternative analysis framework that begins with characterization of the responses of individual taxa and uses aggregation only after distinguishing the magnitude, direction, and uncertainty in the responses of individual members of the community.

Ecological thresholds have much potential as a tool to use for watershed management. Not all systems exhibit abrupt nonlinear responses, but the threshold concept is still useful for describing stressor responses or changes in state variables. For example, the minimum detectable negative response is an initiation-of-impact threshold that might allow for planning and management before population-scale change occurs in taxa. An extirpation threshold, which is the point where a system loses a vital component, such as a species or function, also exists. A number of taxon-specific thresholds to landuse change in watersheds have been identified in previous research. We apply these values to make watershed-level and spatially explicit forecasts regarding imperilment and loss of biodiversity in the face of watershed alterations. We show that unchecked development in the Potapsco River watershed of Maryland could result in the loss of nearly 60% of the benthic macroinvertebrate taxa by the time impervious surface cover reaches 15% of the watershed. Application of analytical thresholds to projected increases in residential development in the Middle Patuxent River watershed by 2030 indicates substantial future changes in aquatic biodiversity, with up to 50% of benthic macroinvertebrate taxa disappearing from some stream reaches and few reaches immune from projected biodiversity loss or impairment.

Ecological thresholds have been applied conventionally to characterize relationships between stressors and biological responses. We demonstrated how ecological thresholds can be used to assess recovery in a system where stressors have been removed. We applied a relatively new statistical approach, the significant zero crossings (SiZer) model, which approximates a response function and its derivatives and then shows how these functions vary across the range of an explanatory variable. We applied the model to a long-term macroinvertebrate data set collected from the Arkansas River, a Colorado stream undergoing restoration from historical mining pollution. SiZer analysis identified distinct threshold responses of benthic communities through time and related these shifts to improvements in water quality. With respect to restoration success, a recovery threshold was observed ∼10 y after clean up was initiated. Recovery trajectories in the Arkansas River were relatively complex, with multiple thresholds, and these responses were strongly influenced by temporal variation in heavy metal concentrations and stream discharge. A unique aspect of SiZer is that it easily handles multiple thresholds and displays significant change points along a predictor variable graphically. In addition, SiZer allows investigators to examine potential thresholds at different levels of resolution and provides a transparent representation of the threshold and how it responds to variability of the data.

Research on aquatic hyphomycetes has been dominated by process-oriented approaches. The main objectives have been accurate estimates of fungal biomass and production and measuring fungal impact on plant litter decomposition. In some cases, these estimates have been complemented by community assessments based on spore counts. Many other ecological and evolutionary topics, commonly studied in macroorganisms, were largely inaccessible, in part because of the low morphological complexity of fungal structures and the near impossibility of identifying them in situ unless attached to propagules. Molecular methods rely on extraction, amplification (polymerase chain reaction) and characterization (denaturing gradient gel electrophoresis, digitized fluorescent restriction-fragment length polymorphism, sequencing) of deoxyribonucleic acid (DNA), which occurs in all cells regardless of their reproductive status. Molecular methods allow more comprehensive characterizations of fungal diversity and evolution. Enzymatic activities can be explored at the level of gene presence (DNA amplification and sequencing), gene transcription (reverse transcription of messenger ribonucleic acid [mRNA]), by quantifying the total amount of specified enzymes in a sample via global antibodies, or by estimating their effect on model compounds. Selected actual and potential applications of these techniques are reviewed.

The mayfly fauna of Madagascar is highly diverse and largely endemic. Many species remain undescribed, and many species are known from only the larval or adult life stage. The high biodiversity in Madagascar and in other areas has led to an increasing reliance on DNA-based approaches to taxonomy, i.e., to define species boundaries and to associate different developmental stages. We used the general mixed Yule-coalescent (GMYC) model to combine population- and species-level sequence variation of mitochondrial deoxyribonucleic acid (mtDNA) to detect species boundaries in Baetidae mayflies (Ephemeroptera). Starting with a database of 240 sequences (57 species), significant clustering of newly sequenced larvae allowed us to establish 1 new species and 1 new combination and to associate adult and larval stages for both. A molecular phylogeny using additional nuclear (18S) and mtDNA (rrnS, rrnL) gene regions recovered the new species and new combination as a monophyletic group, distinct from other Afrotropical lineages. Therefore, we established a new genus, Adnoptilum, endemic to Madagascar. Adnoptilum gen. n. can be distinguished from other species in both the imaginal and adult stages and appears to belong to the Bugilliesia complex. We conclude that routine sampling of population- and species-level genetic diversity, combined with coalescent-based methods of species delineation, has great potential to become a standard procedure for the study of poorly known taxonomic groups.

Mitochondrial deoxyribonucleic acid (mtDNA) sequence data have been both heralded and scrutinized for their ability or lack thereof to discriminate among species for identification (DNA barcoding) or description (DNA taxonomy). Few studies have systematically examined the ability of mtDNA from the DNA barcode region (658 base pair fragment of the 5′ terminus of the mitochondrial cytochrome c oxidase I gene) to distinguish species based on range-wide sampling of specimens from closely related species. Here we examined the utility of DNA barcode data for delimiting species, associating life stages, and as a potential genetic marker for phylogeographic studies by analyzing a range-wide sample of closely related Chilean representatives of the caddisfly genus Smicridea subgenus Smicridea. Our data revealed the existence of 7 deeply diverged, previously unrecognized lineages and confirmed the existence of 2 new species: Smicridea (S.) patinae, new species and Smicridea (S.) lourditae, new species. Based on our current taxonomic evaluation, we considered the other 5 lineages to be cryptic species. The DNA barcode data proved useful in delimiting species within Chilean Smicridea (Smicridea) and were suitable for life-stage associations. The data also contained sufficient intraspecific variation to make the DNA barcode a candidate locus for widespread application in phylogeographic studies.

Morphological and genetic data are increasingly used in combination to test taxonomic hypotheses of species status. We compared the morphological and genetic diversity of the spring-dwelling crane fly, Pedicia occulta (Diptera), a widely distributed western Palearctic species considered to be monotypic throughout its range. We studied 11 morphological characters from 360 male individuals from the entire recorded range of P. occulta, and analyzed a subset of 77 individuals from the southeastern Carpathians, Rila, Pirin, and Rhodope for genetic variation at the mitochondrial cytochrome c oxidase I subunit (COI) gene. In contrast to expectations, our data on morphology and genetic structure of populations showed a structured pattern in the Carpathian region. Two well-separated groups were identified with nonmetric multidimensional scaling based on morphology. COI data revealed 2 divergent lineages (13.4% sequence distance) that corresponded to the 2 morphological groups and to differences in phenology and behavior. Sequence data showed additional genetic subdivision within one of these morphological groups, perhaps indicating the existence of additional cryptic entities. Our results also point out the importance of the Carpathian spring and mountain aquatic ecosystems as Pleistocene refugia. Our study emphasizes the power of combining morphological, genetic, and behavioral approaches in taxonomic hypothesis testing.

Both historical and contemporary processes can influence population genetic structure and phylogeographic patterns at different spatial scales. We studied the genetic structure of a common alpine caddisfly, Allogamus uncatus, across the central European Alps using mitochondrial cytochrome oxidase c subunit I (COI) sequence data to evaluate broad- and fine-scale spatial patterns in population genetic structure and examined how these patterns relate to historical and contemporary landscape processes. Both historical and contemporary influences on population genetic structure could be distinguished. At the broad-scale, our results showed the existence of 6 geographically structured haplogroups across the central Alps that were derived from 4 deeply diverged (up to 5.5%) older lineages. This broad-scale pattern in genetic structure of A. uncatus probably was the result of recolonization from multiple glacial refugia peripheral to the Alps. These data support the idea that phylogeographic patterns involving multiple refugia might be more common among alpine insects than previously thought. Within haplogroups, a finer-scale genetic structuring was found at the valley scale. Thirty of the 34 haplotypes found occurred in single valleys, demonstrating a high level of endemism and indicating little contemporary gene flow between valleys. This pattern might be typical for alpine specialists and other montane species with disjunct distributions. We suggest that these different scales of population genetic structure of A. uncatus probably were influenced by the fact that the species is a cold-tolerant alpine specialist. Hence, historical differences in availability of glacial refugia should be considered in conservation programs directed to preserve genetic variation in montane aquatic species.

We used ecological information and current and historic species distribution models for the last glacial maximum (LGM) to develop hypotheses regarding the Pleistocene history of the montane, autumn-emerging caddisfly Chaetopterygopsis maclachlani. We used mitochondrial sequence data from the cytochrome oxidase I (mtCOI) gene of 282 specimens from populations across Europe to analyze the population structure and phylogeography of C. maclachlani and to test our hypotheses. We examined the population genetic structure with median-joining networks, analysis of molecular variance (AMOVA), exact tests of population differentiation, and Mantel tests of isolation by distance. Furthermore, we used tests for selective neutrality (Tajima's D, Fu's F_S) to infer potential population growth and expansion and Bayesian Skyline Plots to analyze past population dynamics. We found strong population structure with 47 different haplotypes that could be separated into a southeastern and a western clade. The western clade seems to have survived at least the LGM in an extra-Mediterranean refugium, independent of the southeastern clade. Within the 2 main clades, we found haplotype overlap between mountain ranges and a high proportion of private haplotypes (89%). As predicted for a montane species with very limited adult dispersal capabilities, many regions and populations are currently isolated and appear to be diversifying into independent lineages. We discuss speciation within the Chaetopterygini and the diversification of C. maclachlani in particular.

Pennyfish, Denariusa bandata, are small freshwater fish widely distributed in northern Australia in 4 highly disjunct regions and in the Fly River and Bensbach River drainages in southern Papua New Guinea (PNG). Alternating phases of exposure of the Australia–New Guinea Continental Shelf (ANGCS) during stands of lowered sea levels during Pleistocene glacial phases created a land bridge and fresh/brackish water habitats that intermittently connected Australia and PNG. Some biogeographic theories and empirical evidence for several freshwater crustaceans suggest that wetlands on the exposed ANGCS during the last glacial maximum were an important conduit for dispersal and gene flow in freshwater species between Australia and PNG and across northern Australia and that connectivity was severed by the most recent sea level rise in the region (∼6–8 thousand years before present). We used mitochondrial deoxyribonucleic acid (mtDNA) sequence data to test phylogeographic hypotheses concerning the origin of the disjunct distribution in D. bandata in relation to dispersal–vicariant processes and exposure of the ANGCS during Pleistocene sea level changes. Rather than a late Pleistocene origin associated with the last glacial maximum, the coalescence of the regional populations was in the early- to mid-Pleistocene, and the molecular data indicated that the eastern regional populations split before the northern populations split (i.e., sequential vicariance), whereas the northern populations split contemporaneously (i.e., simultaneous vicariance). The complex mtDNA genealogy for D. bandata also indicated a phylogeographic history in which ancestral lineages were retained in the northwestern part of its distribution, and ancestral haplotype diversity was retained in the Fly River (PNG) population because several divergent clusters of PNG haplotypes were more closely related to Australian haplotypes than to each other.

Whether active or passive, dispersal accompanied by gene flow shapes the genetic makeup of populations and ultimately the evolutionary divergence of species. Our objective was to determine if 2 very different aquatic invertebrates with overlapping distributions show similar dispersal histories in their phylogeographic patterns and genetic uniqueness. Two spring-dwelling invertebrates, Hyalella azteca and Callibaetis americanus, were collected from 6 adjacent closed basins in the Great Basin of western North America. Cytochrome c oxidase subunit I (COI) and the 28S ribosomal subunit were used as genetic markers in Hyalella, and COI with the 16S ribosomal subunit of the mitochondrial genome were examined in Callibaetis. Maximum parsimony (MP) and likelihood (ML) analyses, F_ST values, analysis of molecular variance (AMOVA), Mantel tests, and nested clade phylogeographical analysis (NCPA) were used to evaluate geographical associations. Hyalella azteca appears to have been in the adjacent basins much longer than has Callibaetis. F_ST values in H. azteca reached near fixation. Callibaetis americanus F_ST values were lower suggesting greater gene flow and, consequently, higher dispersal rates. Mantel tests did not detect significant isolation by distance for either species, but NCPA on smaller networks of closely related haplotypes found the genetic structure in C. americanus dominated by restricted gene flow with isolation by distance. Hyalella azteca was characterized more by gradual range expansion followed by fragmentation. These results suggest that these isolated freshwater communities are amalgams of species that entered at different times, with weak dispersers having greater constraints on movement and, thus, reflecting an older geographical story than do species with stronger dispersal capabilities.

The genetic structure of the freshwater fish Ambassis macleayi Castelnau 1878 was explored across the tropical catchments of the Gulf of Carpentaria Basin, northern Australia. The Gulf of Carpentaria provides a unique opportunity to explore simultaneously contemporary and historical gene flow resulting from unique climatic patterns and historical connectivity among catchments via a freshwater lake that existed during lower sea levels. The control region of the mtDNA genome and 4 microsatellite loci were used to detect significant genetic structure among and within catchments. Within catchments, genetic structure suggested that dispersal of A. macleayi is restricted, despite high levels of connectivity during summer monsoonal events. Among catchments, divergence appeared to be deeper than what would be predicted based on the last opportunity for connectivity via the lake of Carpentaria (∼10,000 years before present [ybp]). Overall, these results have important implications for A. macleayi and other members of this genus. If individuals are not proficient dispersers, recolonization after disturbance will be limited. Because of historical isolation among catchments, each catchment harbors unique genetic diversity that should be conserved independently.

Ecological speciation is the process by which barriers to gene flow arise between populations as a result of ecologically based divergent selection. Environmental salinity has been identified as one of the most important ecological drivers influencing distribution, abundance, and species richness of aquatic organisms. Springs of the northern Chihuahuan Desert vary in salinity and are home to the Gammarus pecos (Crustacea:Amphipoda) species complex. We used field experiments to compare salinity tolerance among 9 amphipod populations from geographically discrete habitats differing in ambient salinities and to calculate salinity response distances among populations. Cluster analysis placed populations into 3 groups corresponding to low, medium, and high ambient salinities. Partial Mantel tests revealed significant positive correlations between salinity tolerance and habitat salinity after controlling for other variables, such as geographic distance and neutral genetic differences. Our results provide evidence that ecological speciation could be occurring among amphipod populations at different springs, as indicated by dissimilar physiological responses that are correlated with differences in ambient spring salinities. Gene flow is restricted among populations, the restriction is reinforced by dispersal barriers between springs, and selection might preserve variants that most effectively tolerate local salinity levels. Gammarus diversification in the northern Chihuahuan Desert is driven by vicariance and isolation, along with local selection and adaptation.

When 2 populations are mixed for whatever reason, the outcome can be difficult to predict. In 1993, 2 populations of an atyid shrimp (Paratya australiensis:Atyidae) from different subcatchments of the Brisbane River, Queensland, Australia, were mixed as a result of a translocation. At the time, they were thought to represent slightly divergent populations of a single species. However, subsequent molecular analysis showed that they were significantly divergent. An analysis of patterns at one of the sites in 2001 suggested that the translocated lineage might be sending the resident population extinct in one of the streams. Analysis of mitochondrial deoxyribonucleic acid (DNA) and 3 allozyme loci indicated that the explanation for the outcome might be related to asymmetrical hybridization and nonviability of hybrids between resident females and translocated males. We present data for 2002 and an analysis of samples that had been collected prior to the 2001 study, but not analyzed. We tested 3 hypotheses: 1) the translocated genotypes would continue to increase in relative frequency at sites above the translocation site because their site of origin was at higher altitudes, to which they were expected to be better adapted; 2) at intermediate sites, the relative frequency of translocated alleles in juveniles would increase relative to in their parents; and 3) resident genotypes would survive better than translocated genotypes within a generation, especially at lower and intermediate elevations. Translocated genotypes increased extremely rapidly at the most upstream site. Consistent evidence was present for higher relative frequency of translocated alleles in juveniles relative to their parents, both in the generation following the 2001 study and in the pre-2001 samples. With the exception of in the upstream site, translocated genotypes had lower fitness within a generation than resident genotypes. Thus, the translocated genotypes had a higher reproductive success but were less well adapted to their local environment than residents, a situation that decreased population fitness overall. Such an outcome demonstrates a rarely reported effect of translocations and mixing of divergent lineages.