An unusually large genome from an unusually large stonefly: a chromosome-length genome assembly for the giant salmonfly, Pteronarcys californica (Plecoptera: Pteronarcyidae).

Pteronarcys californica (Newport 1848) is commonly referred to as the giant salmonfly and is the largest species of stonefly (Insecta: Plecoptera) in the western United States. Historically, it was widespread and abundant in western rivers, but populations have experienced a substantial decline in the past few decades, becoming locally extirpated in numerous rivers in Utah, Colorado, and Montana. Although previous research has explored the ecological variables conducive to the survivability of populations of the giant salmonfly, a lack of genomic resources hampers exploration of how genetic variation is spread across extant populations. To accelerate research on this imperiled species, we present a de novo chromosomal-length genome assembly of P. californica generated from PacBio HiFi sequencing and Hi-C chromosome conformation capture. Our assembly includes 14 predicted pseudo chromosomes and 98.8% of Insecta universal core orthologs. At 2.40 gigabases, the P. californica assembly is the largest of available stonefly assemblies, highlighting at least 9.5-fold variation in assembly size across the order. Repetitive elements (REs) account for much of the genome size increase in P. californica relative to other stonefly species, with the content of Class I retroelements alone exceeding the entire assembly size of all but two other species studied. We also observed preliminary suborder-specific trends in genome size that merit testing with more robust taxon sampling.

Phylogenomics recovers multiple origins of portable case making in caddisflies (Insecta: Trichoptera), nature's underwater architects.

Caddisflies (Trichoptera) are among the most diverse groups of freshwater animals with more than 16 000 described species. They play a fundamental role in freshwater ecology and environmental engineering in streams, rivers and lakes. Because of this, they are frequently used as indicator organisms in biomonitoring programmes. Despite their importance, key questions concerning the evolutionary history of caddisflies, such as the timing and origin of larval case making, remain unanswered owing to the lack of a well-resolved phylogeny. Here, we estimated a phylogenetic tree using a combination of transcriptomes and targeted enrichment data for 207 species, representing 48 of 52 extant families and 174 genera. We calibrated and dated the tree with 33 carefully selected fossils. The first caddisflies originated approximately 295 million years ago in the Permian, and major suborders began to diversify in the Triassic. Furthermore, we show that portable case making evolved in three separate lineages, and shifts in diversification occurred in concert with key evolutionary innovations beyond case making.

Analyses of 600+ insect genomes reveal repetitive element dynamics and highlight biodiversity-scale repeat annotation challenges.

Repetitive elements (REs) are integral to the composition, structure, and function of eukaryotic genomes, yet remain understudied in most taxonomic groups. We investigated REs across 601 insect species and report wide variation in RE dynamics across groups. Analysis of associations between REs and protein-coding genes revealed dynamic evolution at the interface between REs and coding regions across insects, including notably elevated RE-gene associations in lineages with abundant long interspersed nuclear elements (LINEs). We leveraged this large, empirical data set to quantify impacts of long-read technology on RE detection and investigate fundamental challenges to RE annotation in diverse groups. In long-read assemblies, we detected ∼36% more REs than short-read assemblies, with long terminal repeats (LTRs) showing 162% increased detection, whereas DNA transposons and LINEs showed less respective technology-related bias. In most insect lineages, 25%-85% of repetitive sequences were "unclassified" following automated annotation, compared with only ∼13% in Drosophila species. Although the diversity of available insect genomes has rapidly expanded, we show the rate of community contributions to RE databases has not kept pace, preventing efficient annotation and high-resolution study of REs in most groups. We highlight the tremendous opportunity and need for the biodiversity genomics field to embrace REs and suggest collective steps for making progress toward this goal.

Allelic resolution of insect and spider silk genes reveals hidden genetic diversity.

Arthropod silk is vital to the evolutionary success of hundreds of thousands of species. The primary proteins in silks are often encoded by long, repetitive gene sequences. Until recently, sequencing and assembling these complex gene sequences has proven intractable given their repetitive structure. Here, using high-quality long-read sequencing, we show that there is extensive variation-both in terms of length and repeat motif order-between alleles of silk genes within individual arthropods. Further, this variation exists across two deep, independent origins of silk which diverged more than 500 Mya: the insect clade containing caddisflies and butterflies and spiders. This remarkable convergence in previously overlooked patterns of allelic variation across multiple origins of silk suggests common mechanisms for the generation and maintenance of structural protein-coding genes. Future genomic efforts to connect genotypes to phenotypes should account for such allelic variation.

Highly accurate long reads are crucial for realizing the potential of biodiversity genomics.

BACKGROUND: Generating the most contiguous, accurate genome assemblies given available sequencing technologies is a long-standing challenge in genome science. With the rise of long-read sequencing, assembly challenges have shifted from merely increasing contiguity to correctly assembling complex, repetitive regions of interest, ideally in a phased manner. At present, researchers largely choose between two types of long read data: longer, but less accurate sequences, or highly accurate, but shorter reads (i.e., >Q20 or 99% accurate). To better understand how these types of long-read data as well as scale of data (i.e., mean length and sequencing depth) influence genome assembly outcomes, we compared genome assemblies for a caddisfly, Hesperophylax magnus, generated with longer, but less accurate, Oxford Nanopore (ONT) R9.4.1 and highly accurate PacBio HiFi (HiFi) data. Next, we expanded this comparison to consider the influence of highly accurate long-read sequence data on genome assemblies across 6750 plant and animal genomes. For this broader comparison, we used HiFi data as a surrogate for highly accurate long-reads broadly as we could identify when they were used from GenBank metadata. RESULTS: HiFi reads outperformed ONT reads in all assembly metrics tested for the caddisfly data set and allowed for accurate assembly of the repetitive ~ 20 Kb H-fibroin gene. Across plants and animals, genome assemblies that incorporated HiFi reads were also more contiguous. For plants, the average HiFi assembly was 501% more contiguous (mean contig N50 = 20.5 Mb) than those generated with any other long-read data (mean contig N50 = 4.1 Mb). For animals, HiFi assemblies were 226% more contiguous (mean contig N50 = 20.9 Mb) versus other long-read assemblies (mean contig N50 = 9.3 Mb). In plants, we also found limited evidence that HiFi may offer a unique solution for overcoming genomic complexity that scales with assembly size. CONCLUSIONS: Highly accurate long-reads generated with HiFi or analogous technologies represent a key tool for maximizing genome assembly quality for a wide swath of plants and animals. This finding is particularly important when resources only allow for one type of sequencing data to be generated. Ultimately, to realize the promise of biodiversity genomics, we call for greater uptake of highly accurate long-reads in future studies.

Pathways to polar adaptation in fishes revealed by long-read sequencing.

Long-read sequencing is driving a new reality for genome science in which highly contiguous assemblies can be produced efficiently with modest resources. Genome assemblies from long-read sequences are particularly exciting for understanding the evolution of complex genomic regions that are often difficult to assemble. In this study, we utilized long-read sequencing data to generate a high-quality genome assembly for an Antarctic eelpout, Ophthalmolycus amberensis, the first for the globally distributed family Zoarcidae. We used this assembly to understand how O. amberensis has adapted to the harsh Southern Ocean and compared it to another group of Antarctic fishes: the notothenioids. We showed that selection has largely acted on different targets in eelpouts relative to notothenioids. However, we did find some overlap; in both groups, genes involved in membrane structure, thermal tolerance and vision have evidence of positive selection. We found evidence for historical shifts of transposable element activity in O. amberensis and other polar fishes, perhaps reflecting a response to environmental change. We were specifically interested in the evolution of two complex genomic loci known to underlie key adaptations to polar seas: haemoglobin and antifreeze proteins (AFPs). We observed unique evolution of the haemoglobin MN cluster in eelpouts and related fishes in the suborder Zoarcoidei relative to other Perciformes. For AFPs, we identified the first species in the suborder with no evidence of afpIII sequences (Cebidichthys violaceus) in the genomic region where they are found in all other Zoarcoidei, potentially reflecting a lineage-specific loss of this cluster. Beyond polar fishes, our results highlight the power of long-read sequencing to understand genome evolution.

Summer Dynamics of Microbial Diversity on a Mountain Glacier.

Glaciers are rapidly receding under climate change. A melting cryosphere will dramatically alter global sea levels, carbon cycling, and water resource availability. Glaciers host rich biotic communities that are dominated by microbial diversity, and this biodiversity can impact surface albedo, thereby driving a feedback loop between biodiversity and cryosphere melt. However, the microbial diversity of glacier ecosystems remains largely unknown outside of major ice sheets, particularly from a temporal perspective. Here, we characterized temporal dynamics of bacteria, eukaryotes, and algae on the Paradise Glacier, Mount Rainier, USA, over nine time points spanning the summer melt season. During our study, the glacier surface steadily darkened as seasonal snow melted and darkening agents accumulated until new snow fell in late September. From a community-wide perspective, the bacterial community remained generally constant while eukaryotes and algae exhibited temporal progression and community turnover. Patterns of individual taxonomic groups, however, were highly stochastic. We found little support for our a priori prediction that autotroph abundance would peak before heterotrophs. Notably, two different trends in snow algae emerged-an abundant early- and late-season operational taxonomic unit (OTU) with a different midsummer OTU that peaked in August. Overall, our results highlight the need for temporal sampling to clarify microbial diversity on glaciers and that caution should be exercised when interpreting results from single or few time points. IMPORTANCE Microbial diversity on mountain glaciers is an underexplored component of global biodiversity. Microbial presence and activity can also reduce the surface albedo or reflectiveness of glaciers, causing them to absorb more solar radiation and melt faster, which in turn drives more microbial activity. To date, most explorations of microbial diversity in the mountain cryosphere have only included single time points or focused on one microbial community (e.g., bacteria). Here, we performed temporal sampling over a summer melt season for the full microbial community, including bacteria, eukaryotes, and fungi, on the Paradise Glacier, Washington, USA. Over the summer, the bacterial community remained generally constant, whereas eukaryote and algal communities temporally changed through the melt season. Individual taxonomic groups, however, exhibited considerable stochasticity. Overall, our results highlight the need for temporal sampling on glaciers and that caution should be exercised when interpreting results from single or few time points.

Long-read HiFi sequencing correctly assembles repetitive heavy fibroin silk genes in new moth and caddisfly genomes.

Insect silk is a versatile biomaterial. Lepidoptera and Trichoptera display some of the most diverse uses of silk, with varying strength, adhesive qualities, and elastic properties. Silk fibroin genes are long (>20 Kbp), with many repetitive motifs that make them challenging to sequence. Most research thus far has focused on conserved N- and C-terminal regions of fibroin genes because a full comparison of repetitive regions across taxa has not been possible. Using the PacBio Sequel II system and SMRT sequencing, we generated high fidelity (HiFi) long-read genomic and transcriptomic sequences for the Indianmeal moth (Plodia interpunctella) and genomic sequences for the caddisfly Eubasilissa regina. Both genomes were highly contiguous (N50  = 9.7 Mbp/32.4 Mbp, L50  = 13/11) and complete (BUSCO complete  = 99.3%/95.2%), with complete and contiguous recovery of silk heavy fibroin gene sequences. We show that HiFi long-read sequencing is helpful for understanding genes with long, repetitive regions.

Toward a genome sequence for every animal: Where are we now?

In less than 25 y, the field of animal genome science has transformed from a discipline seeking its first glimpses into genome sequences across the Tree of Life to a global enterprise with ambitions to sequence genomes for all of Earth's eukaryotic diversity [H. A. Lewin et al., Proc. Natl. Acad. Sci. U.S.A. 115, 4325-4333 (2018)]. As the field rapidly moves forward, it is important to take stock of the progress that has been made to best inform the discipline's future. In this Perspective, we provide a contemporary, quantitative overview of animal genome sequencing. We identified the best available genome assemblies in GenBank, the world's most extensive genetic database, for 3,278 unique animal species across 24 phyla. We assessed taxonomic representation, assembly quality, and annotation status for major clades. We show that while tremendous taxonomic progress has occurred, stark disparities in genomic representation exist, highlighted by a systemic overrepresentation of vertebrates and underrepresentation of arthropods. In terms of assembly quality, long-read sequencing has dramatically improved contiguity, whereas gene annotations are available for just 34.3% of taxa. Furthermore, we show that animal genome science has diversified in recent years with an ever-expanding pool of researchers participating. However, the field still appears to be dominated by institutions in the Global North, which have been listed as the submitting institution for 77% of all assemblies. We conclude by offering recommendations for improving genomic resource availability and research value while also broadening global representation.

Representation and participation across 20 years of plant genome sequencing.

The field of plant genome sequencing has grown rapidly in the past 20 years, leading to increases in the quantity and quality of publicly available genomic resources. The growing wealth of genomic data from an increasingly diverse set of taxa provides unprecedented potential to better understand the genome biology and evolution of land plants. Here we provide a contemporary view of land plant genomics, including analyses on assembly quality, taxonomic distribution of sequenced species and national participation. We show that assembly quality has increased dramatically in recent years, that substantial taxonomic gaps exist and that the field has been dominated by affluent nations in the Global North and China, despite a wide geographic distribution of study species. We identify numerous disconnects between the native range of focal species and the national affiliation of the researchers studying them, which we argue are rooted in colonialism-both past and present. Luckily, falling sequencing costs, widening availability of analytical tools and an increasingly connected scientific community provide key opportunities to improve existing assemblies, fill sampling gaps and empower a more global plant genomics community.

Long Reads Are Revolutionizing 20 Years of Insect Genome Sequencing.

The first insect genome assembly (Drosophila melanogaster) was published two decades ago. Today, nuclear genome assemblies are available for a staggering 601 insect species representing 20 orders. In this study, we analyzed the most-contiguous assembly for each species and provide a "state-of-the-field" perspective, emphasizing taxonomic representation, assembly quality, gene completeness, and sequencing technologies. Relative to species richness, genomic efforts have been biased toward four orders (Diptera, Hymenoptera, Collembola, and Phasmatodea), Coleoptera are underrepresented, and 11 orders still lack a publicly available genome assembly. The average insect genome assembly is 439.2 Mb in length with 87.5% of single-copy benchmarking genes intact. Most notable has been the impact of long-read sequencing; assemblies that incorporate long reads are ∼48× more contiguous than those that do not. We offer four recommendations as we collectively continue building insect genome resources: 1) seek better integration between independent research groups and consortia, 2) balance future sampling between filling taxonomic gaps and generating data for targeted questions, 3) take advantage of long-read sequencing technologies, and 4) expand and improve gene annotations.

The biogeographic history of eelpouts and related fishes: Linking phylogeny, environmental change, and patterns of dispersal in a globally distributed fish group.

Modern genetic data sets present unprecedented opportunities to understand the evolutionary origins of diverse taxonomic groups. When the timing of key events is known, it is possible to investigate biogeographic history in the context of major phenomena (e.g., cooling of a major ocean). In this study, we investigated the biogeographic history of the suborder Zoarcoidei, a globally distributed fish group that includes species inhabiting both poles that produce antifreeze proteins to survive chronic subfreezing temperatures. We first generated a multi-locus, time-calibrated phylogeny for the group. We then used biogeographic modeling to reconstruct ancestral ranges across the tree and to quantify the type and frequency of biogeographic events (e.g., founder, dispersal). With these results, we considered how the cooling of the Southern and Arctic Oceans, which reached their present-day subfreezing temperatures 10-15 million years ago (Mya) and 2-3 Mya, respectively, may have shaped the group's evolutionary history, with an emphasis on the most speciose and widely distributed family, eelpouts (family Zoarcidae). Our phylogenetic results clarified the Zoarcoidei taxonomy and showed that the group began to diversify in the Oligocene ~31-32 Mya, with the center of origin for all families in north temperate waters. Within-area speciation was the most common biogeographic event in the group's history (80% of all events) followed by dispersal (20%). Finally, we only found evidence, albeit limited, for ocean cooling underpinning diversification of eelpouts living in the high Antarctic over the last 10 million years.

Engaging the next generation of editorial talent through a hands-on fellowship model.

Peer-review and subject-matter editing is the backbone of scientific publishing. However, early-career researchers (ECRs) are given few opportunities to participate in the editorial process beyond reviewing articles. Thus, a disconnect exists: science needs high-quality editorial talent to conduct, oversee and improve the publishing process, yet we dedicate few resources to building editorial talent nor giving ECRs formal opportunities to influence publishing from within. ECRs can contribute to the publishing landscape in unique ways given their insight into new and rapidly developing publishing trends (e.g. open science). Here, we describe a two-way fellowship model that gives ECRs a "seat" at the editorial table of a field-leading journal. We describe both the necessary framework and benefits that can stem from editorial fellowships for ECRs, editors, journals, societies, and the ​broader scientific community.

Geography is more important than life history in the recent diversification of the tiger salamander complex.

The North American tiger salamander species complex, including its best-known species, the Mexican axolotl, has long been a source of biological fascination. The complex exhibits a wide range of variation in developmental life history strategies, including populations and individuals that undergo metamorphosis; those able to forego metamorphosis and retain a larval, aquatic lifestyle (i.e., paedomorphosis); and those that do both. The evolution of a paedomorphic life history state is thought to lead to increased population genetic differentiation and ultimately reproductive isolation and speciation, but the degree to which it has shaped population- and species-level divergence is poorly understood. Using a large multilocus dataset from hundreds of samples across North America, we identified genetic clusters across the geographic range of the tiger salamander complex. These clusters often contain a mixture of paedomorphic and metamorphic taxa, indicating that geographic isolation has played a larger role in lineage divergence than paedomorphosis in this system. This conclusion is bolstered by geography-informed analyses indicating no effect of life history strategy on population genetic differentiation and by model-based population genetic analyses demonstrating gene flow between adjacent metamorphic and paedomorphic populations. This fine-scale genetic perspective on life history variation establishes a framework for understanding how plasticity, local adaptation, and gene flow contribute to lineage divergence. Many members of the tiger salamander complex are endangered, and the Mexican axolotl is an important model system in regenerative and biomedical research. Our results chart a course for more informed use of these taxa in experimental, ecological, and conservation research.

Rock glaciers and related cold rocky landforms: Overlooked climate refugia for mountain biodiversity.

Mountains are global biodiversity hotspots where cold environments and their associated ecological communities are threatened by climate warming. Considerable research attention has been devoted to understanding the ecological effects of alpine glacier and snowfield recession. However, much less attention has been given to identifying climate refugia in mountain ecosystems where present-day environmental conditions will be maintained, at least in the near-term, as other habitats change. Around the world, montane communities of microbes, animals, and plants live on, adjacent to, and downstream of rock glaciers and related cold rocky landforms (CRL). These geomorphological features have been overlooked in the ecological literature despite being extremely common in mountain ranges worldwide with a propensity to support cold and stable habitats for aquatic and terrestrial biodiversity. CRLs are less responsive to atmospheric warming than alpine glaciers and snowfields due to the insulating nature and thermal inertia of their debris cover paired with their internal ventilation patterns. Thus, CRLs are likely to remain on the landscape after adjacent glaciers and snowfields have melted, thereby providing longer-term cold habitat for biodiversity living on and downstream of them. Here, we show that CRLs will likely act as key climate refugia for terrestrial and aquatic biodiversity in mountain ecosystems, offer guidelines for incorporating CRLs into conservation practices, and identify areas for future research.

Insects in high-elevation streams: Life in extreme environments imperiled by climate change.

Climate change is altering conditions in high-elevation streams worldwide, with largely unknown effects on resident communities of aquatic insects. Here, we review the challenges of climate change for high-elevation aquatic insects and how they may respond, focusing on current gaps in knowledge. Understanding current effects and predicting future impacts will depend on progress in three areas. First, we need better descriptions of the multivariate physical challenges and interactions among challenges in high-elevation streams, which include low but rising temperatures, low oxygen supply and increasing oxygen demand, high and rising exposure to ultraviolet radiation, low ionic strength, and variable but shifting flow regimes. These factors are often studied in isolation even though they covary in nature and interact in space and time. Second, we need a better mechanistic understanding of how physical conditions in streams drive the performance of individual insects. Environment-performance links are mediated by physiology and behavior, which are poorly known in high-elevation taxa. Third, we need to define the scope and importance of potential responses across levels of biological organization. Short-term responses are defined by the tolerances of individuals, their capacities to perform adequately across a range of conditions, and behaviors used to exploit local, fine-scale variation in abiotic factors. Longer term responses to climate change, however, may include individual plasticity and evolution of populations. Whether high-elevation aquatic insects can mitigate climatic risks via these pathways is largely unknown.

Key rules of life and the fading cryosphere: Impacts in alpine lakes and streams.

Alpine regions are changing rapidly due to loss of snow and ice in response to ongoing climate change. While studies have documented ecological responses in alpine lakes and streams to these changes, our ability to predict such outcomes is limited. We propose that the application of fundamental rules of life can help develop necessary predictive frameworks. We focus on four key rules of life and their interactions: the temperature dependence of biotic processes from enzymes to evolution; the wavelength dependence of the effects of solar radiation on biological and ecological processes; the ramifications of the non-arbitrary elemental stoichiometry of life; and maximization of limiting resource use efficiency across scales. As the cryosphere melts and thaws, alpine lakes and streams will experience major changes in temperature regimes, absolute and relative inputs of solar radiation in ultraviolet and photosynthetically active radiation, and relative supplies of resources (e.g., carbon, nitrogen, and phosphorus), leading to nonlinear and interactive effects on particular biota, as well as on community and ecosystem properties. We propose that applying these key rules of life to cryosphere-influenced ecosystems will reduce uncertainties about the impacts of global change and help develop an integrated global view of rapidly changing alpine environments. However, doing so will require intensive interdisciplinary collaboration and international cooperation. More broadly, the alpine cryosphere is an example of a system where improving our understanding of mechanistic underpinnings of living systems might transform our ability to predict and mitigate the impacts of ongoing global change across the daunting scope of diversity in Earth's biota and environments.

Aquatic Insects Are Dramatically Underrepresented in Genomic Research.

Aquatic insects comprise 10% of all insect diversity, can be found on every continent except Antarctica, and are key components of freshwater ecosystems. However, aquatic insect genome biology lags dramatically behind that of terrestrial insects. If genomic effort was spread evenly, one aquatic insect genome would be sequenced for every ~9 terrestrial insect genomes. Instead, ~24 terrestrial insect genomes have been sequenced for every aquatic insect genome. This discrepancy is even more dramatic if the quality of genomic resources is considered; for instance, while no aquatic insect genome has been assembled to the chromosome level, 29 terrestrial insect genomes spanning four orders have. We argue that a lack of aquatic insect genomes is not due to any underlying difficulty (e.g., small body sizes or unusually large genomes), yet it is severely hampering aquatic insect research at both fundamental and applied scales. By expanding the availability of aquatic insect genomes, we will gain key insight into insect diversification and empower future research for a globally important taxonomic group.

Mountain stoneflies may tolerate warming streams: Evidence from organismal physiology and gene expression.

Rapid glacier recession is altering the physical conditions of headwater streams. Stream temperatures are predicted to rise and become increasingly variable, putting entire meltwater-associated biological communities at risk of extinction. Thus, there is a pressing need to understand how thermal stress affects mountain stream insects, particularly where glaciers are likely to vanish on contemporary timescales. In this study, we measured the critical thermal maximum (CTMAX ) of stonefly nymphs representing multiple species and a range of thermal regimes in the high Rocky Mountains, USA. We then collected RNA-sequencing data to assess how organismal thermal stress translated to the cellular level. Our focal species included the meltwater stonefly, Lednia tumana, which was recently listed under the U.S. Endangered Species Act due to climate-induced habitat loss. For all study species, critical thermal maxima (CTMAX  > 20°C) far exceeded the stream temperatures mountain stoneflies experience (<10°C). Moreover, while evidence for a cellular stress response was present, we also observed constitutive expression of genes encoding proteins known to underlie thermal stress (i.e., heat shock proteins) even at low temperatures that reflected natural conditions. We show that high-elevation aquatic insects may not be physiologically threatened by short-term exposure to warm temperatures and that longer-term physiological responses or biotic factors (e.g., competition) may better explain their extreme distributions.

To composite or replicate: how sampling method and protocol differences alter collected stream invertebrates and associated bioassessment metrics.

Aquatic invertebrates are excellent indicators of ecosystem quality; however, choosing a sampling method can be difficult. Each method and associated protocol has advantages and disadvantages, and finding the approach that minimizes biases yet fulfills management objectives is crucial. To test the effects of both sampling methods and sample handling-i.e., to composite samples or leave them as replicates-we collected aquatic invertebrates from the Niobrara River at Agate Fossil Beds National Monument, Nebraska, using three methods and two sample handling protocols. We compared aquatic invertebrate assemblages collected with a Hester-Dendy multi-plate sampler, Hess sampler, and a D-frame dipnet. We calculated six common bioassessment metrics from composite (combined) and replicate (separate) samples. Hess samples contained the highest taxonomic richness (capturing 77% of all taxa observed) and dipnet samples the least (47%). Hester-Dendy samples had the greatest proportion of Ephemeroptera, and Ephemeroptera, Plecoptera, and Trichoptera (EPT). Dipnet samples had the lowest evenness values. In terms of sample handling, composite samples had inflated richness, diversity, and evenness compared with replicate samples, but bioassessment metrics calculated from proportions or averages (i.e., Hilsenhoff's Biotic Index and the proportion of EPT taxa) did not differ between them. The proportion of invertebrate groups from composite samples were not statistically different among sampling methods, but several groups differed between replicate samples collected by different methods. Ultimately, we recommend collecting replicate samples with a Hess sampler when the goal of the study is to detect ecosystem change, among locations or differences in variables of interest.