Do metapopulations and management matter for relict headwater bull trout populations in a warming climate?

Mountain headwater streams have emerged as important climate refuges for native cold-water species due to their slow climate velocities and extreme physical conditions that inhibit non-native invasions. Species persisting in refuges often do so as fragmented, relict populations from broader historical distributions that are subject to ongoing habitat reductions and increasing isolation as climate change progresses. Key for conservation planning is determining where remaining populations will persist and how habitat restoration strategies can improve biological resilience to enhance the long-term prospects for species of concern. Studying bull trout, a headwater species in the northwestern USA, we developed habitat occupancy models using a data set of population occurrence in 991 natal habitat patches with a suite of novel geospatial covariates derived from high-resolution hydroclimatic scenarios and other sources representing watershed and instream habitat conditions, patch geometry, disturbance, and biological interactions. The best model correctly predicted bull trout occupancy status in 82.6% of the patches and included effects for: patch size estimated as habitat volume, extent of within-patch reaches <9°C mean August temperature, distance to nearest occupied patch, road density, invasive brook trout prevalence, patch slope, and frequency of high winter flows. The model was used to assess 16 scenarios of bull trout occurrence within the study streams that represented a range of restoration strategies under three climatic conditions (baseline, moderate change, and extreme change). Results suggested that regional improvements in bull trout status were difficult to achieve in realistic restoration strategies due to the pervasive nature of climate change and the limited extent of restoration actions given their high costs. However, occurrence probabilities in a subset of patches were highly responsive to restoration actions, suggesting that targeted investments to improve the resilience of some populations may be contextually beneficial. A possible strategy, therefore, is focusing effort on responsive populations near more robust population strongholds, thereby contributing to local enclaves where dispersal among populations further enhances resilience. Equally important, strongholds constituted a small numerical percentage of patches (5%-21%), yet encompassed the large majority of occupied habitat by volume (72%-89%) and their protection could have significant conservation benefits for bull trout.

Broad-scale eDNA sampling for describing aquatic species distributions in running waters: Pacific lamprey Entosphenus tridentatus in the upper Snake River, USA.

One of the most fundamental yet challenging tasks for aquatic ecologists is to precisely delineate the range of species, particularly those that are broadly distributed, require specialized sampling methods, and may be simultaneously declining and increasing in different portions of their range. An exemplar is the Pacific lamprey Entosphenus tridentatus, a jawless anadromous fish of conservation concern that is actively managed in many coastal basins in western North America. To efficiently determine its distribution across the accessible 56,168 km of the upper Snake River basin in the north-western United States, we first delimited potential habitat by using predictions from a species distribution model based on conventionally collected historical data and from the distribution of a potential surrogate, Chinook salmon Oncorhynchus tshawytscha, which yielded a potential habitat network of 10,615 km. Within this area, we conducted a two-stage environmental DNA survey involving 394 new samples and 187 archived samples collected by professional biologists and citizen scientists using a single, standardized method from 2015 to 2021. We estimated that Pacific lamprey occupied 1875 km of lotic habitat in this basin, of which 1444 km may have been influenced by recent translocation efforts. Pacific lamprey DNA was consistently present throughout most river main stems, although detections became weaker or less frequent in the largest and warmest downstream channels and near their headwater extent. Pacific lamprey were detected in nearly all stocked tributaries, but there was no evidence of indigenous populations in such habitats. There was evidence of post-stocking movement because detections were 1.8-36.0 km upstream from release sites. By crafting a model-driven spatial sampling template and executing an eDNA-based sampling campaign led by professionals and volunteers, supplemented by previously collected samples, we established a benchmark for understanding the current range of Pacific lamprey across a large portion of its range in the interior Columbia River basin. This approach could be tailored to refine range estimates for other wide-ranging aquatic species of conservation concern.

Pliocene-Early Pleistocene Geological Events Structure Pacific Martens (Martes caurina).

The complex topography, climate, and geological history of Western North America have shaped contemporary patterns of biodiversity and species distributions in the region. Pacific martens (Martes caurina) are distributed along the northern Pacific Coast of North America with disjunct populations found throughout the Northwestern Forested Mountains and Marine West Coast Forest ecoregions of the West Coast. Martes in this region have been classified into subspecies; however, the subspecific designation has been extensively debated. In this study, we use genomic data to delineate conservation units of Pacific marten in the Sierra-Cascade-Coastal montane belt in the western United States. We analyzed the mitochondrial genome for 94 individuals to evaluate the spatial distribution and divergence times of major lineages. We further genotyped 401 individuals at 13 microsatellite loci to investigate major patterns of population structure. Both nuclear and mitochondrial DNA suggest substantial genetic substructure concordant with historical subspecies designations. Our results revealed that the region contains 2 distinct mitochondrial lineages: a Cascades/Sierra lineage that diverged from the Cascades/coastal lineage 2.23 (1.48-3.14 mya), consistent with orogeny of the Cascade Mountain chain. Interestingly, Pacific Martes share phylogeographic patterns similar with other sympatric taxa, suggesting that the complex geological history has shaped the biota of this region. The information is critical for conservation and management efforts, and further investigation of adaptive diversity is warranted following appropriate revision of conservation management designations.

Slow climate velocities of mountain streams portend their role as refugia for cold-water biodiversity.

The imminent demise of montane species is a recurrent theme in the climate change literature, particularly for aquatic species that are constrained to networks and elevational rather than latitudinal retreat as temperatures increase. Predictions of widespread species losses, however, have yet to be fulfilled despite decades of climate change, suggesting that trends are much weaker than anticipated and may be too subtle for detection given the widespread use of sparse water temperature datasets or imprecise surrogates like elevation and air temperature. Through application of large water-temperature databases evaluated for sensitivity to historical air-temperature variability and computationally interpolated to provide high-resolution thermal habitat information for a 222,000-km network, we estimate a less dire thermal plight for cold-water species within mountains of the northwestern United States. Stream warming rates and climate velocities were both relatively low for 1968-2011 (average warming rate = 0.101 °C/decade; median velocity = 1.07 km/decade) when air temperatures warmed at 0.21 °C/decade. Many cold-water vertebrate species occurred in a subset of the network characterized by low climate velocities, and three native species of conservation concern occurred in extremely cold, slow velocity environments (0.33-0.48 km/decade). Examination of aggressive warming scenarios indicated that although network climate velocities could increase, they remain low in headwaters because of strong local temperature gradients associated with topographic controls. Better information about changing hydrology and disturbance regimes is needed to complement these results, but rather than being climatic cul-de-sacs, many mountain streams appear poised to be redoubts for cold-water biodiversity this century.

Ecological segregation moderates a climactic conclusion to trout hybridization.

For decades, it has been assumed that introgressive hybridization between introduced rainbow trout and native cutthroat trout in western North America will lead to genomic extinction of the latter. A broad-scale re-examination of their interaction indicates that ecological differences between these species and demographic processes are dictating the location and extent of their hybrid zones, and that runaway introgression between these taxa is unlikely.

Big biology meets microclimatology: defining thermal niches of ectotherms at landscape scales for conservation planning.

Temperature profoundly affects ecology, a fact ever more evident as the ability to measure thermal environments increases and global changes alter these environments. The spatial structure of thermalscapes is especially relevant to the distribution and abundance of ectothermic organisms, but the ability to describe biothermal relationships at extents and grains relevant to conservation planning has been limited by small or sparse data sets. Here, we combine a large occurrence database of >23 000 aquatic species surveys with stream microclimate scenarios supported by an equally large temperature database for a 149 000-km mountain stream network to describe thermal relationships for 14 fish and amphibian species. Species occurrence probabilities peaked across a wide range of temperatures (7.0-18.8°C) but distinct warm- or cold-edge distribution boundaries were apparent for all species and represented environments where populations may be most sensitive to thermal changes. Warm-edge boundary temperatures for a native species of conservation concern were used with geospatial data sets and a habitat occupancy model to highlight subsets of the network where conservation measures could benefit local populations by maintaining cool temperatures. Linking that strategic approach to local estimates of habitat impairment remains a key challenge but is also an opportunity to build relationships and develop synergies between the research, management, and regulatory communities. As with any data mining or species distribution modeling exercise, care is required in analysis and interpretation of results, but the use of large biological data sets with accurate microclimate scenarios can provide valuable information about the thermal ecology of many ectotherms and a spatially explicit way of guiding conservation investments.

The cold-water climate shield: delineating refugia for preserving salmonid fishes through the 21st century.

The distribution and future fate of ectothermic organisms in a warming world will be dictated by thermalscapes across landscapes. That is particularly true for stream fishes and cold-water species like trout, salmon, and char that are already constrained to high elevations and latitudes. The extreme climates in those environments also preclude invasions by most non-native species, so identifying especially cold habitats capable of absorbing future climate change while still supporting native populations would highlight important refugia. By coupling crowd-sourced biological datasets with high-resolution stream temperature scenarios, we delineate network refugia across >250 000 stream km in the Northern Rocky Mountains for two native salmonids-bull trout (BT) and cutthroat trout (CT). Under both moderate and extreme climate change scenarios, refugia with high probabilities of trout population occupancy (>0.9) were predicted to exist (33-68 BT refugia; 917-1425 CT refugia). Most refugia are on public lands (>90%) where few currently have protected status in National Parks or Wilderness Areas (<15%). Forecasts of refuge locations could enable protection of key watersheds and provide a foundation for climate smart planning of conservation networks. Using cold water as a 'climate shield' is generalizable to other species and geographic areas because it has a strong physiological basis, relies on nationally available geospatial data, and mines existing biological datasets. Importantly, the approach creates a framework to integrate data contributed by many individuals and resource agencies, and a process that strengthens the collaborative and social networks needed to preserve many cold-water fish populations through the 21st century.

Flow regime, temperature, and biotic interactions drive differential declines of trout species under climate change.

Broad-scale studies of climate change effects on freshwater species have focused mainly on temperature, ignoring critical drivers such as flow regime and biotic interactions. We use downscaled outputs from general circulation models coupled with a hydrologic model to forecast the effects of altered flows and increased temperatures on four interacting species of trout across the interior western United States (1.01 million km(2)), based on empirical statistical models built from fish surveys at 9,890 sites. Projections under the 2080s A1B emissions scenario forecast a mean 47% decline in total suitable habitat for all trout, a group of fishes of major socioeconomic and ecological significance. We project that native cutthroat trout Oncorhynchus clarkii, already excluded from much of its potential range by nonnative species, will lose a further 58% of habitat due to an increase in temperatures beyond the species' physiological optima and continued negative biotic interactions. Habitat for nonnative brook trout Salvelinus fontinalis and brown trout Salmo trutta is predicted to decline by 77% and 48%, respectively, driven by increases in temperature and winter flood frequency caused by warmer, rainier winters. Habitat for rainbow trout, Oncorhynchus mykiss, is projected to decline the least (35%) because negative temperature effects are partly offset by flow regime shifts that benefit the species. These results illustrate how drivers other than temperature influence species response to climate change. Despite some uncertainty, large declines in trout habitat are likely, but our findings point to opportunities for strategic targeting of mitigation efforts to appropriate stressors and locations.

Cottus schitsuumsh, a new species of sculpin (Scorpaeniformes: Cottidae) in the Columbia River basin, Idaho-Montana, USA.

Fishes of the genus Cottus have long been taxonomically challenging because of morphological similarities among species and their tendency to hybridize, and a number of undescribed species may remain in this genus. We used a combination of genetic and morphological methods to delineate and describe Cottus schitsuumsh, Cedar Sculpin, a new species, from the upper Columbia River basin, Idaho-Montana, USA. Although historically confused with the Shorthead Sculpin (C. confusus), the genetic distance between C. schitsuumsh and C. confusus (4.84-6.29%) suggests these species are distant relatives. Moreover, the two species can be differentiated on the basis of lateral-line pores on the caudal peduncle, head width, and interpelvic width. Cottus schitsuumsh is also distinct from all other Cottus in this region in having a single small, skin-covered, preopercular spine. Haplotypes of mtDNA cytochrome oxidase c subunit 1 of C. schitsuumsh differed from all other members of the genus at three positions, had interspecific genetic distances typical for congeneric fishes (1.61-2.74% to nearest neighbors), and were monophyletic in maximum-likelihood trees. Microsatellite analyses confirmed these taxonomic groupings for species potentially sympatric with C. schitsuumsh and that fish used in morphological comparisons were unlikely to be introgressed. Its irregular distribution, in the Spokane River basin in Idaho and portions of the Clark Fork River basin in Montana, may have resulted from human-assisted translocation.

Simultaneous species detection and discovery with environmental DNA metabarcoding: A freshwater mollusk case study.

Environmental DNA (eDNA) sampling is a powerful tool for rapidly characterizing biodiversity patterns for specious, cryptic taxa with incomplete taxonomies. One such group that are also of high conservation concern are North American freshwater gastropods. In particular, springsnails of the genus Pyrgulopsis (Family: Hydrobiidae) are prevalent throughout the western United States where >140 species have been described. Many of the described species are narrow endemics known from a single spring or locality, and it is believed that there are likely many additional species which have yet to be described. The distribution of these species across the landscape is of interest because habitat loss and degradation, climate change, groundwater mining, and pollution have resulted in springsnail imperilment rates as high as 92%. Determining distributions with conventional sampling methods is limited by the fact that these snails are often <5 mm in length with few distinguishing morphological characters, making them both difficult to detect and to identify. We developed an eDNA metabarcoding protocol that is both inexpensive and capable of rapid, accurate detection of all known Pyrgulopsis species. When compared with conventional collection techniques, our pipeline consistently resulted in detection at sites previously known to contain Pyrgulopsis springsnails and at a cost per site that is likely to be substantially less than the conventional sampling and individual barcoding that has been done historically. Additionally, because our method uses eDNA extracted from filtered water, it is non-destructive and suitable for the detection of endangered species where "no take" restrictions may be in effect. This effort represents both a tool which is immediately applicable to taxa of high conservation concern across western North America and a case study in the broader application of eDNA sampling for landscape assessments of cryptic taxa of conservation concern.

The unknown unknown: A framework for assessing environmental DNA assay specificity against unsampled taxa.

Taxon-specific quantitative PCR (qPCR) assays are commonly used for environmental DNA sampling-based inference of animal presence. These assays require thorough validation to ensure that amplification truly indicates detection of the target taxon, but a thorough validation is difficult when there are potentially many non-target taxa, some of which may have incomplete taxonomies. Here, we use a previously published, quantitative model of cross-amplification risk to describe a framework for assessing qPCR assay specificity when there is missing information and it is not possible to assess assay specificity for each individual non-target confamilial. In this framework, we predict assay specificity against unsampled taxa (non-target taxa without sequence data available) using the sequence information that is available for other confamilials. We demonstrate this framework using four case study assays for: (1) An endemic, freshwater arthropod (meltwater stonefly; Lednia tumana), (2) a globally distributed, marine ascidian (Didemnum perlucidum), (3) a continentally distributed freshwater crustacean (virile crayfish; Faxonius virilis, deanae and nais species complex) and (4) a globally distributed freshwater teleost (common carp; Cyprinus carpio and its close relative C. rubrofuscus). We tested the robustness of our approach to missing information by simulating application of our framework for all possible subsamples of 20-all non-target taxa. Our results suggest that the modelling framework results in estimates which are largely concordant with observed levels of cross-amplification risk using all available sequence data, even when there are high levels of data missingness. We explore potential limitations and extensions of this approach for assessing assay specificity and provide users with an R Markdown template for generating reproducible reports to support their own assay validation efforts.

Probabilistic accounting of uncertainty in forecasts of species distributions under climate change.

Forecasts of species distributions under future climates are inherently uncertain, but there have been few attempts to describe this uncertainty comprehensively in a probabilistic manner. We developed a Monte Carlo approach that accounts for uncertainty within generalized linear regression models (parameter uncertainty and residual error), uncertainty among competing models (model uncertainty), and uncertainty in future climate conditions (climate uncertainty) to produce site-specific frequency distributions of occurrence probabilities across a species' range. We illustrated the method by forecasting suitable habitat for bull trout (Salvelinus confluentus) in the Interior Columbia River Basin, USA, under recent and projected 2040s and 2080s climate conditions. The 95% interval of total suitable habitat under recent conditions was estimated at 30.1-42.5 thousand km; this was predicted to decline to 0.5-7.9 thousand km by the 2080s. Projections for the 2080s showed that the great majority of stream segments would be unsuitable with high certainty, regardless of the climate data set or bull trout model employed. The largest contributor to uncertainty in total suitable habitat was climate uncertainty, followed by parameter uncertainty and model uncertainty. Our approach makes it possible to calculate a full distribution of possible outcomes for a species, and permits ready graphical display of uncertainty for individual locations and of total habitat.

eDNAssay: A machine learning tool that accurately predicts qPCR cross-amplification.

Environmental DNA (eDNA) sampling is a highly sensitive and cost-effective technique for wildlife monitoring, notably through the use of qPCR assays. However, it can be difficult to ensure assay specificity when many closely related species co-occur. In theory, specificity may be assessed in silico by determining whether assay oligonucleotides have enough base-pair mismatches with nontarget sequences to preclude amplification. However, the mismatch qualities required are poorly understood, making in silico assessments difficult and often necessitating extensive in vitro testing-typically the greatest bottleneck in assay development. Increasing the accuracy of in silico assessments would therefore streamline the assay development process. In this study, we paired 10 qPCR assays with 82 synthetic gene fragments for 530 specificity tests using SYBR Green intercalating dye (n = 262) and TaqMan hydrolysis probes (n = 268). Test results were used to train random forest classifiers to predict amplification. The primer-only model (SYBR Green results) and full-assay model (TaqMan probe-based results) were 99.6% and 100% accurate, respectively, in cross-validation. We further assessed model performance using six independent assays not used in model training. In these tests the primer-only model was 92.4% accurate (n = 119) and the full-assay model was 96.5% accurate (n = 144). The high performance achieved by these models makes it possible for eDNA practitioners to more quickly and confidently develop assays specific to the intended target. Practitioners can access the full-assay model online via eDNAssay (https://NationalGenomicsCenter.shinyapps.io/eDNAssay), a user-friendly tool for predicting qPCR cross-amplification.

Molecular species delimitation refines the taxonomy of native and nonnative physinine snails in North America.

Being able to associate an organism with a scientific name is fundamental to our understanding of its conservation status, ecology, and evolutionary history. Gastropods in the subfamily Physinae have been especially troublesome to identify because morphological variation can be unrelated to interspecific differences and there have been widespread introductions of an unknown number of species, which has led to a speculative taxonomy. To resolve uncertainty about species diversity in North America, we targeted an array of single-locus species delimitation methods at publically available specimens and new specimens collected from the Snake River basin, USA to generate species hypotheses, corroborated using nuclear analyses of the newly collected specimens. A total-evidence approach delineated 18 candidate species, revealing cryptic diversity within recognized taxa and a lack of support for other named taxa. Hypotheses regarding certain local endemics were confirmed, as were widespread introductions, including of an undescribed taxon likely belonging to a separate genus in southeastern Idaho for which the closest relatives are in southeast Asia. Overall, single-locus species delimitation was an effective first step toward understanding the diversity and distribution of species in Physinae and to guiding future investigation sampling and analyses of species hypotheses.

Modeling predicts that redd trampling by cattle may contribute to population declines of native trout.

Unrestricted livestock grazing can degrade aquatic ecosystems, and its effects on native vertebrate species are generally mediated by changes to physical habitat. Recently, high estimated rates of cattle trampling on artificial redds within federal grazing allotments in southwestern Montana, USA, has raised concern that direct mortality from trampling may contribute to imperilment of native westslope cutthroat trout (Oncorhynchus clarkii lewisi). To explore the implications of cattle trampling, we built two mathematical models. First we used a temperature-driven model of egg-to-fry mortality representative of the developmental stages during which embryos would be vulnerable to trampling. Cattle trampling was an additional source of mortality (beyond natural mortality), and we modeled egg-to-fry mortality across a range of trampling rates (25-125% per month) for scenarios assuming low (0.60), moderate (0.81), and high (0.95) natural mortality. We then used a matrix model to determine how trampling affected population growth (lambda), assuming initially stable (lambda = 1.008) or slow-growing populations (lambda = 1.025 and 1.05). Cattle trampling concentrated over a few days when the embryos were most sensitive caused greater egg-to-fry mortality than when the same amount of trampling occurred over one month. Trampling caused a large increase in egg-to-fry mortality when that natural mortality was low, but the overall population-level effect was far less than might have been anticipated from the rate of trampling itself. Nonetheless, small reductions in population growth rate could be biologically significant for populations with little or no demographic resilience, and trampling rates as low as 25% could lead to negative population growth. The rapid reduction in resilience with increased trampling rates (>50%) means that even growing populations are less likely to recover from periodic fluctuations. The overall risk posed by trampling will depend on whether cutthroat trout populations face concurrent threats that have already reduced their abundance and resilience. Biologists can potentially use the egg-to-fry model and thermograph data to identify dates when limiting cattle presence in or near stream habitats would likely reduce mortality from trampling. Evaluation of grazing policies on federal lands may be needed to ensure that species conservation and land use concerns are equitably balanced.

Fuel reduction management practices in riparian areas of the Western USA.

Two decades of uncharacteristically severe wildfires have caused government and private land managers to actively reduce hazardous fuels to lessen wildfire severity in western forests, including riparian areas. Because riparian fuel treatments are a fairly new management strategy, we set out to document their frequency and extent on federal lands in the western U.S. Seventy-four USDA Forest Service Fire Management Officers (FMOs) in 11 states were interviewed to collect information on the number and characteristics of riparian fuel reduction treatments in their management district. Just under half of the FMOs surveyed (43%) indicated that they were conducting fuel reduction treatments in riparian areas. The primary management objective listed for these projects was either fuel reduction (81%) or ecological restoration and habitat improvement (41%), though multiple management goals were common (56%). Most projects were of small extent (93% < 300 acres), occurred in the wildland-urban interface (75%), and were conducted in ways to minimize negative impacts on species and habitats. The results of this survey suggest that managers are proceeding cautiously with treatments. To facilitate project planning and implementation, managers recommended early coordination with resource specialists, such as hydrologists and fish and wildlife biologists. Well-designed monitoring of the consequences of riparian fuel treatments on fuel loads, fire risk, and ecological effects is needed to provide a scientifically-defensible basis for the continued and growing implementation of these treatments.

Invasion versus isolation: trade-offs in managing native salmonids with barriers to upstream movement.

Conservation biologists often face the trade-off that increasing connectivity in fragmented landscapes to reduce extinction risk of native species can foster invasion by non-native species that enter via the corridors created, which can then increase extinction risk. This dilemma is acute for stream fishes, especially native salmonids, because their populations are frequently relegated to fragments of headwater habitat threatened by invasion from downstream by 3 cosmopolitan non-native salmonids. Managers often block these upstream invasions with movement barriers, but isolation of native salmonids in small headwater streams can increase the threat of local extinction. We propose a conceptual framework to address this worldwide problem that focuses on 4 main questions. First, are populations of conservation value present (considering evolutionary legacies, ecological functions, and socioeconomic benefits as distinct values)? Second, are populations vulnerable to invasion and displacement by non-native salmonids? Third, would these populations be threatened with local extinction if isolated with barriers? And, fourth, how should management be prioritized among multiple populations? We also developed a conceptual model of the joint trade-off of invasion and isolation threats that considers the opportunities for managers to make strategic decisions. We illustrated use of this framework in an analysis of the invasion-isolation trade-off for native cutthroat trout (Oncorhynchus clarkii) in 2 contrasting basins in western North America where invasion and isolation are either present and strong or farther away and apparently weak. These cases demonstrate that decisions to install or remove barriers to conserve native salmonids are often complex and depend on conservation values, environmental context (which influences the threat of invasion and isolation), and additional socioeconomic factors. Explicit analysis with tools such as those we propose can help managers make sound decisions in such complex circumstances.

Integrative taxonomy refutes a species hypothesis: The asymmetric hybrid origin of Arsapnia arapahoe (Plecoptera, Capniidae).

Molecular tools are commonly directed at refining taxonomies and the species that constitute their fundamental units. This has been especially insightful for groups for which species hypotheses are ambiguous and have largely been based on morphological differences between certain life stages or sexes, and has added importance when taxa are a focus of conservation efforts. Here, we examine the taxonomic status of Arsapnia arapahoe, a winter stonefly in the family Capniidae that is a species of conservation concern because of its limited abundance and restricted range in northern Colorado, USA. Phylogenetic analyses of sequences of mitochondrial and nuclear genes of this and other capniid stoneflies from this region and elsewhere in western North America indicated extensive haplotype sharing, limited genetic differences, and a lack of reciprocal monophyly between A. arapahoe and the sympatric A. decepta, despite distinctive and consistent morphological differences in the sexual apparatus of males of both species. Analyses of autosomal and sex-linked single nucleotide polymorphisms detected using genotyping by sequencing indicated that all individuals of A. arapahoe consisted of F1 hybrids between female A. decepta and males of another sympatric stonefly, Capnia gracilaria. Rather than constitute a self-sustaining evolutionary lineage, A. arapahoe appears to represent the product of nonintrogressive hybridization in the limited area of syntopy between two widely distributed taxa. This offers a cautionary tale for taxonomists and conservation biologists working on the less-studied components of the global fauna.

Decreased total psoas muscle area after neoadjuvant therapy is a predictor of increased mortality in patients undergoing oesophageal cancer resection.

BACKGROUND: Oesophagectomy for locally advanced cancer carries high rates of morbidity and mortality. Patients require a thorough risk assessment alongside preoperative counselling. Total psoas area (TPA) measurements have been used as a surrogate marker of sarcopenia to predict post-operative complications in oesophageal cancer patients. No studies to date have determined whether there is an association between the proportion of TPA lost during neoadjuvant therapy and post-operative outcomes. METHODS: Clinical data and imaging of patients who underwent neoadjuvant therapy followed by open two-stage oesophagectomy between January 2008 and April 2018 were analysed retrospectively. Patients who did not undergo restaging computed tomography scan prior to surgery were excluded from the study. The TPA was measured on two cross-sectional slices at L4 on computed tomography scans pre- and post-neoadjuvant therapy. RESULTS: A total of 53 patients who met inclusion criteria were identified. The mean loss of TPA was 7.3%. Patients who had a decrease of TPA of more than 4% had significantly increased 30-day mortality compared to those who lost 4% or less (24% versus 0%, P = 0.02). Patients aged over 65 years who also had a loss of TPA >4% had significantly increased 30-day mortality (37% versus 2.9%, odds ratio 19, P = 0.008). CONCLUSION: A decrease in TPA of >4% is associated with a significantly higher risk of post-operative mortality in patients undergoing neoadjuvant therapy followed by oesophagectomy. Measuring the loss of TPA during neoadjuvant treatment could be a novel aid to preoperative risk assessment.

Capture enrichment of aquatic environmental DNA: A first proof of concept.

Environmental DNA (eDNA) sampling-the detection of genetic material in the environment to infer species presence-has rapidly grown as a tool for sampling aquatic animal communities. A potentially powerful feature of environmental sampling is that all taxa within the habitat shed DNA and so may be detectable, creating opportunity for whole-community assessments. However, animal DNA in the environment tends to be comparatively rare, making it necessary to enrich for genetic targets from focal taxa prior to sequencing. Current metabarcoding approaches for enrichment rely on bulk amplification using conserved primer annealing sites, which can result in skewed relative sequence abundance and failure to detect some taxa because of PCR bias. Here, we test capture enrichment via hybridization as an alternative strategy for target enrichment using a series of experiments on environmental samples and laboratory-generated, known-composition DNA mixtures. Capture enrichment resulted in detecting multiple species in both kinds of samples, and postcapture relative sequence abundance accurately reflected initial relative template abundance. However, further optimization is needed to permit reliable species detection at the very low-DNA quantities typical of environmental samples (<0.1 ng DNA). We estimate that our capture protocols are comparable to, but less sensitive than, current PCR-based eDNA analyses.