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RNA sequencing (RNA-Seq) is a popular method for measuring gene expression in non-model organisms, including wild populations. While RNA-Seq can measure gene expression variation among wild-caught individuals and can yield important biological insights into organism function, sampling methods may also influence gene expression estimates. We examined the influence of multiple technical variables on estimated gene expression in a non-model fish, the westslope cutthroat trout (Oncorhynchus clarkii lewisi), using two RNA-Seq library types: 3’ RNA-Seq and whole mRNA-Seq. We evaluated effects of dip netting versus electrofishing, and of harvesting tissue immediately versus 5 minutes after euthanasia on estimated gene expression in blood, gill, and muscle. We detected 30% more genes with whole mRNA-Seq than with 3’ RNA-Seq and found that 58% of genes were significantly differently expressed between 3’ RNA-Seq and whole mRNA-Seq. Our findings indicate that 3’ RNA-Seq and whole mRNA-Seq are robust to the technical variables related to the field sampling approaches tested here with a lack of differential gene expression among sampling methods and tissue collection time after euthanasia. However, we found that gene expression varied based on which RNA-Seq library type was used on the same set of samples. Our study suggests researchers could safely rely on different fish sampling strategies in the field and save money and analyze more individuals using 3’ RNA-Seq, but should use whole mRNA-Seq when working with a species without good genomic resources, and when maximizing the number of genes identified and detecting alternative splicing are important.

Continuous height-resolved observations of aerosol profiles over the central Arctic throughout a full year were performed for the first time. Such measurements covering aerosol layering features are required for an adequate modeling of Arctic climate conditions, especially with respect to a realistic consideration of cloud formation and here, in particular, of ice nucleation processes. MOSAiC (Multidisciplinary drifting Observatory for the Study of Arctic Climate) offered this favorable opportunity to monitor aerosol and clouds over the central Arctic over all four seasons, from October 2019 to September 2020. In this article, a summary of MOSAiC lidar observations aboard the icebreaker Polarstern of tropospheric aerosol products is presented. Particle optical properties, i.e., light-extinction profiles and aerosol optical thickness (AOT), and estimates of cloud-relevant aerosol properties (cloud condensation nucleus, CCN, and ice-nucleating particle concentrations, INPs) are discussed, separately for the lowest part of the troposphere (near the surface at 250 m height), within the lower free troposphere (2000 m height), and regarding INPs also near the tropopause (cirrus level, 8–10 km height). In situ observations of the particle number concentration and INPs aboard Polarstern are included in the study. Strong differences between summer and winter aerosol conditions were found. During the winter months (Arctic haze period) a strong decrease of the aerosol light extinction coefficient (532 nm) with height up to about 4–5 km height was found with values of 20–100 Mm-1 close to the surface and an order of magnitude less at 1500–2000 m height. Lofted aged wildfire smoke layers caused a re-increase of the aerosol concentration from the middle troposphere up to stratospheric heights and were continuously observable from October 2019 to May 2020. In summer (June to August 2020), much lower particle extinction coefficients, frequently as low as 1–5 Mm-1, were observed. Aerosol removal, controlled by cloud scavenging processes (widely suppressed in winter, very efficient in summer) in the lowermost 1–2 km of the atmosphere, seem to be the main reason for the strong differences between winter and summer aerosol conditions. In line with this pronounced annual cycle in the aerosol optical properties, CCN concentrations (0.2 % supersaturation level) ranged from 50–500 cm-3 in the atmospheric boundary layer (ABL) in winter and 1–40 cm-3 in summer. In the lower free troposphere, however, the CCN level was roughly constant throughout the year with values mostly from 30–100 cm-3. A strong contrast between winter to summer was also given in terms of ABL INPs which control ice production in low-level clouds. INP concentration of 0.01–0.2 L-1 prevailed in the ABL in winter at typical ice-nucleating cloud temperatures of -25 °C and assuming soil dust as the main INP type, and were roughly 2 orders of magnitude lower in the ABL in summer at typical cloud top temperatures of -10 °C. In the summer ABL, marine aerosol (biogenic components) is most probably the main INP type, continental INP contributions (e.g., soil dust INPs) are suppressed by efficient wet removal during long-range transport. A strong reduction in the INP population was also found in the lower free troposphere at 2000 m height from winter to summer (2 orders of magnitude), mostly due to the change in the prevailing ice-nucleation temperatures. Estimated INP concentration accumulated from 0.004–0.02 L-1 during the winter months. The highlight of the MOSAiC lidar studies was the detection of a persistent wildfire smoke layer in the upper troposphere and lower stratosphere from October 2019 to May 2020. The smoke particles (organic aerosol) triggered continuously cirrus formation at INP concentrations mostly from 1–20 L-1 close to the tropopause during the entire winter period.

Nonnative fish eradication via the piscicide rotenone is an effective tool for fisheries management and conservation of native species. However, the long-term effects on nontarget organisms, including benthic invertebrates and zooplankton in alpine lakes, are understudied and are poorly understood. As part of a landscape-scale native fish conservation project, we assessed the effects of 50 ppb of rotenone on the aquatic invertebrate community by comparing pre- and postrotenone treatment density and diversity metrics of benthic invertebrates and zooplankton in 13 alpine lakes and their outlets in Montana, USA. Across study sites, decreases in density and diversity of some invertebrate taxa, including Ephemeroptera, Plecoptera, and Trichoptera, were observed the year following rotenone treatment, and within 3 y, densities and diversities were similar to and sometimes higher than pretreatment values. These results demonstrate resilience of aquatic invertebrate communities following rotenone exposure in alpine lakes and streams and informs fisheries managers for planning rotenone projects and monitoring recovery of nontarget organisms. Additional studies will be useful to evaluate the mechanisms driving invertebrate recovery rates, including downstream drift from nontreated areas and terrestrial adult dispersal.

The impact of aerosols on clouds is a well-studied, although still poorly constrained, part of the atmospheric system. New particle formation (NPF) is thought to contribute 40 %–80 % of the global cloud droplet number concentration, although it is extremely difficult to observe an air mass from NPF to cloud formation. NPF and growth occurs frequently in the Canadian Arctic summer atmosphere, although only a few studies have characterized the source and properties of these aerosols. This study presents cloud condensation nuclei (CCN) concentrations measured on board the CCGS Amundsen in the eastern Canadian Arctic Archipelago from 23 July to 23 August 2016 as part of the Network on Climate and Aerosols: Addressing Uncertainties in Remote Canadian Environments (NETCARE). The study was dominated by frequent ultrafine particle and/or growth events, and particles smaller than 100 nm dominated the size distribution for 92 % of the study period. Using κ-Köhler theory and aerosol size distributions, the mean hygroscopicity parameter (κ) calculated for the entire study was 0.12 (0.06–0.12, 25th–75th percentile), suggesting that the condensable vapours that led to particle growth were primarily slightly hygroscopic, which we infer to be organic. Based on past measurement and modelling studies from NETCARE and the Canadian Arctic, it seems likely that the source of these slightly hygroscopic, organic, vapours is the ocean. Examining specific growth events suggests that the mode diameter (Dmax) had to exceed 40 nm before CCN concentrations at 0.99 % supersaturation (SS) started to increase, although a statistical analysis shows that CCN concentrations increased 13–274 cm−3 during all ultrafine particle and/or growth times (total particle concentrations >500 cm−3, Dmax nm) compared with background times (total concentrations cm−3) at SS of 0.26 %–0.99 %. This value increased to 25–425 cm−3 if the growth times were limited to times when Dmax was also larger than 40 nm. These results support past results from NETCARE by showing that the frequently observed ultrafine particle and growth events are dominated by a slightly hygroscopic fraction, which we interpret to be organic vapours originating from the ocean, and that these growing particles can increase the background CCN concentrations at SS as low as 0.26 %, thus pointing to their potential contribution to cloud properties and thus climate through the radiation balance.

RNA sequencing (RNA-Seq) is becoming a popular method for measuring gene expression in non-model organisms, including wild populations sampled in the field. While RNA-Seq can be used to measure gene expression variation among wild-caught individuals and can yield important biological insights into organismal function, technical variables may also influence gene expression estimates. We examined the influence of multiple technical variables on estimated gene expression in a non-model fish species, the westslope cutthroat trout (Oncorhynchus clarkii lewisi), using two RNA-Seq methods: 3’ RNA-Seq and whole mRNA-Seq. We evaluated the effects of dip netting versus electrofishing, and of harvesting tissue immediately versus 5 minutes after euthanasia on estimated gene expression in blood, gill, muscle, and liver. We found higher RNA degradation in the liver compared to the other tissues. There were fewer expressed genes in blood compared to gill and muscle. We found no difference in gene expression among sampling methods or due to a delay in tissue collection. However, we detected fewer genes with 3’ RNA-Seq than with whole mRNA-Seq and found statistically significant differences in gene expression between 3’ RNA-Seq and whole mRNA-Seq. The magnitude and direction of these differences does not appear to be dependent on gene type or length. Our findings indicate that RNA-Seq is robust to the technical variables related to the field sampling techniques tested here but varies based on the tissue sampled and the RNA-Seq library used. This study advances understanding of usefulness of RNA-Seq to study gene expression variation in evolution, ecology, and conservation.

Estimating the effective population size and effective number of breeders per year (Nb ) can facilitate early detection of population declines. We used computer simulations to quantify bias and precision of the one-sample LDNe estimator of Nb in age-structured populations using a range of published species life history types, sample sizes, and DNA markers. Nb estimates were biased by ~5%-10% when using SNPs or microsatellites in species ranging from fishes to mosquitoes, frogs, and seaweed. The bias (high or low) was similar for different life history types within a species suggesting that life history variation in populations will not influence Nb estimation. Precision was higher for 100 SNPs (H ≈ 0.30) than for 15 microsatellites (H ≈ 0.70). Confidence intervals (CIs) were occasionally too narrow, and biased high when Nb was small (Nb

Hybridization between introduced and native fauna is a risk to native species and may threaten the long-term persistence of numerous taxa. Rainbow Trout Oncorhynchus mykiss has been one of the most widely introduced species around the globe and often hybridizes with native Cutthroat Trout O. clarkii in the Rocky Mountains. Previous work has shown that hybridization negatively affects reproductive success, but identification of the traits contributing to that reduction has been elusive. In this study, we used a combination of field and laboratory techniques to assess how hybridization with Rainbow Trout affects seven traits during several stages of Westslope Cutthroat Trout development: embryonic survival, ova size, ova energy concentration, sperm motility, juvenile weight, juvenile survival, and burst swimming endurance. Rainbow Trout admixture was correlated with an increase in embryonic survival and ova energy concentration but with a decrease in juvenile weight and burst swimming endurance. The...

Invasive hybridization and introgression pose a serious threat to the persistence of many native species. Understanding the effects of hybridization on native populations (e.g., fitness consequences) requires numerous species-diagnostic loci distributed genome-wide. Here we used RAD sequencing to discover thousands of single-nucleotide polymorphisms (SNPs) that are diagnostic between rainbow trout (RBT, Oncorhynchus mykiss), the world’s most widely introduced fish, and native westslope cutthroat trout (WCT, O. clarkii lewisi) in the northern Rocky Mountains, USA. We advanced previous work that identified 4,914 species-diagnostic loci by using longer sequence reads (100 bp vs. 60 bp) and a larger set of individuals (n = 84). We sequenced RAD libraries for individuals from diverse sampling sources, including native populations of WCT and hatchery broodstocks of WCT and RBT. We also took advantage of a newly released reference genome assembly for RBT to align our RAD loci. In total, we discovered 16,788 putatively diagnostic SNPs, 10,267 of which we mapped to anchored chromosome locations on the RBT genome. A small portion of previously discovered putative diagnostic loci (325 of 4,914) were no longer diagnostic (i.e., fixed between species) based on our wider survey of non-hybridized RBT and WCT individuals. Our study suggests that RAD loci mapped to a draft genome assembly could provide the marker density required to identify genes and chromosomal regions influencing selection in admixed populations of conservation concern and evolutionary interest.

Cross-breeding between invasive and native species (hybridization) is one of the potential ways that climate change can impact biodiversity; unfortunately there is little data on this phenomenon. Now, research shows that rapid climate-warming has exacerbated interactions between native trout (Oncorhynchus clarkii lewisi) and the non-native species (Oncorhynchus mykiss) through invasive hybridization in western North America.

Quantifying the effectiveness of management actions to mitigate the effects of changing climatic conditions (i.e., climate adaptation) can be difficult, yet critical for conservation. We used population genetic data from 1984 to 2011 to assess the degree to which ambient climatic conditions and targeted suppression of sources of nonnative Rainbow Trout Oncorhynchus mykiss have influenced the spread of introgressive hybridization in native populations of Westslope Cutthroat Trout O. clarkii lewisi. We found rapid expansion in the spatial distribution and proportion of nonnative genetic admixture in hybridized populations from 1984 to 2004, but minimal change since 2004. The spread of hybridization was negatively correlated with the number of streamflow events in May that exceeded the 75th percentile of historic flows (r = −0.98) and positively correlated with August stream temperatures (r = 0.89). Concomitantly, suppression data showed a 60% decline in catch per unit effort for fish with a high pro...

Rapid and inexpensive methods for genomewide single nucleotide polymorphism (SNP) discovery and genotyping are urgently needed for population management and conservation. In hybridized populations, genomic techniques that can identify and genotype thousands of species-diagnostic markers would allow precise estimates of population- and individual-level admixture as well as identification of 'super invasive' alleles, which show elevated rates of introgression above the genomewide background (likely due to natural selection). Techniques like restriction-site-associated DNA (RAD) sequencing can discover and genotype large numbers of SNPs, but they have been limited by the length of continuous sequence data they produce with Illumina short-read sequencing. We present a novel approach, overlapping paired-end RAD sequencing, to generate RAD contigs of300-400 bp. These contigs provide sufficient flanking sequence for design of high-throughput SNP genotyping arrays and strict filtering to identify duplicate paralogous loci. We applied this approach in five populations of native westslope cutthroat trout that previously showed varying (low) levels of admixture from introduced rainbow trout (RBT). We produced 77 141 RAD contigs and used these data to filter and genotype 3180 previously identified species-diagnostic SNP loci. Our population-level and individual-level estimates of admixture were generally consistent with previous microsatellite-based estimates from the same individuals. However, we observed slightly lower admixture estimates from genomewide markers, which might result from natural selection against certain genome regions, different genomic locations for microsatellites vs. RAD-derived SNPs and/or sampling error from the small number of microsatellite loci (n = 7). We also identified candidate adaptive super invasive alleles from RBT that had excessively high admixture proportions in hybridized cutthroat trout populations.

Evolutionary and ecological consequences of hybridization between native and invasive species are notoriously complicated because patterns of selection acting on non-native alleles can vary throughout the genome and across environments. Rapid advances in genomics now make it feasible to assess locus-specific and genome-wide patterns of natural selection acting on invasive introgression within and among natural populations occupying diverse environments. We quantified genome-wide patterns of admixture across multiple independent hybrid zones of native westslope cutthroat trout and invasive rainbow trout, the world's most widely introduced fish, by genotyping 339 individuals from 21 populations using 9380 species-diagnostic loci. A significantly greater proportion of the genome appeared to be under selection favouring native cutthroat trout (rather than rainbow trout), and this pattern was pervasive across the genome (detected on most chromosomes). Furthermore, selection against invasive alleles was consistent across populations and environments, even in those where rainbow trout were predicted to have a selective advantage (warm environments). These data corroborate field studies showing that hybrids between these species have lower fitness than the native taxa, and show that these fitness differences are due to selection favouring many native genes distributed widely throughout the genome.

Invasive hybridization is one of the greatest threats to the persistence of Westslope Cutthroat Trout Oncorhynchus clarkii lewisi. Large protected areas, where nonhybridized populations are interconnected and express historical life history and genetic diversity, provide some of the last ecological and evolutionary strongholds for conserving this species. Here, we describe the genetic status and distribution of Westslope Cutthroat Trout throughout Glacier National Park, Montana. Admixture between Westslope Cutthroat Trout and introduced Rainbow Trout O. mykiss and Yellowstone Cutthroat Trout O. clarkii bouvieri was estimated by genotyping 1,622 fish collected at 115 sites distributed throughout the Columbia, Missouri, and South Saskatchewan River drainages. Currently, Westslope Cutthroat Trout occupy an estimated 1,465 km of stream habitat and 45 lakes (9,218 ha) in Glacier National Park. There was no evidence of introgression in samples from 32 sites along 587 km of stream length (40% of the stream kilometers currently occupied) and 17 lakes (2,555 ha; 46% of the lake area currently occupied). However, nearly all (97%) of the streams and lakes that were occupied by nonhybridized populations occurred in the Columbia River basin. Based on genetic status (nonnative genetic admixture ≤ 10%), 36 Westslope Cutthroat Trout populations occupying 821 km of stream and 5,482 ha of lakes were identified as “conservation populations.” Most of the conservation populations (N = 27; 736 km of stream habitat) occurred in the Columbia River basin, whereas only a few geographically restricted populations were found in the South Saskatchewan River (N = 7; 55 km) and Missouri River (N = 2; 30 km) basins. Westslope Cutthroat Trout appear to be at imminent risk of genomic extinction in the South Saskatchewan and Missouri River basins, whereas populations in the Columbia River basin are widely distributed and conservation efforts are actively addressing threats from hybridization and other stressors. A diverse set of pro-active management approaches will be required to conserve, protect, and restore Westslope Cutthroat Trout populations in Glacier National Park throughout the 21st century. Received September 10, 2015; accepted March 30, 2016 Published online August 12, 2016

The invasion of nonnative fishes in freshwater systems is often facilitated by the interaction of biotic and abiotic factors operating at multiple spatial and temporal scales. We evaluated the association of local habitat features (width, gradient, and elevation), watershed characteristics (mean and maximum summer water temperatures, the number of road crossings, and road density), and biotic factors (the distance to the source of hybridization and trout density) with the spread of hybridization between native westslope cutthroat trout Oncorhynchus clarkii lewisi and introduced rainbow trout O. mykiss in the upper Flathead River system in Montana and British Columbia. The presence of hybridization and the proportion of rainbow trout admixture were estimated using seven diagnostic microsatellite loci. We defined logistic and linear regression models including various combinations of spatial and environmental factors and used an information-theoretic approach to evaluate the relative plausibility o...

Introductions of nonnative northern pike Esox lucius have created recreational fisheries in many waters in the United States and Canada, yet many studies have shown that introduced northern pike may alter the composition and structure of fish communities through predation. We estimated the abundance of nonnative northern pike (2002–2003) and applied food habits data (1999–2003) to estimate their annual consumption of native bull trout Salvelinus confluentus and westslope cutthroat trout Oncorhynchus clarkii lewisi juveniles in the upper Flathead River system, Montana. Population estimates were generally consistent among years and ranged from 1,200 to 1,300 individuals. Westslope cutthroat trout were present in the diet of younger (≤600 mm) and older (>600 mm) northern pike during all seasons and bull trout were found only in larger northern pike during all seasons but summer. Bioenergetics modeling estimated that the northern pike population annually consumed a total of 8.0 metric tons (mt) of fi...

We analyzed 13 microsatellite loci to estimate gene flow among westslope cutthroat trout, Oncorhynchus clarkii lewisi, populations and determine the invasion pattern of hybrids between native O. c. lewisi and introduced rainbow trout, Oncorhynchus mykiss, in streams of the upper Flathead River system, Montana (USA) and British Columbia (Canada). Fourteen of 31 sites lacked evidence of O. mykiss introgression, and gene flow among these nonhybridized O. c. lewisi populations was low, as indicated by significant allele frequency divergence among populations (θST = 0.076, ρST = 0.094, P < 0.001). Among hybridized sites, O. mykiss admixture declined with upstream distance from a site containing a hybrid swarm with a predominant (92%) O. mykiss genetic contribution. The spatial distribution of hybrid genotypes at seven diagnostic microsatellite loci revealed that O. mykiss invasion is facilitated by both long distance dispersal from this hybrid swarm and stepping-stone dispersal between hybridized populations. This study provides an example of how increased straying rates in the invasive taxon can contribute to the spread of extinction by hybridization and suggests that eradicating sources of introgression may be a useful conservation strategy for protecting species threatened with genomic extinction.

Hybridization between native and non-native species has serious biological consequences, but our understanding of how dispersal and selection interact to influence invasive hybridization is limited. Here, we document the spread of genetic introgression between a native ( Oncorhynchus clarkii ) and invasive ( Oncorhynchus mykiss ) trout, and identify the mechanisms influencing genetic admixture. In two populations inhabiting contrasting environments, non-native admixture increased rapidly from 1984 to 2007 and was driven by surprisingly consistent processes. Individual admixture was related to two phenotypic traits associated with fitness: size at spawning and age of juvenile emigration. Fish with higher non-native admixture were larger and tended to emigrate at a younger age―relationships that are expected to confer fitness advantages to hybrid individuals. However, strong selection against non-native admixture was evident across streams and cohorts (mean selection coefficient against genotypes with non-native alleles ( s ) = 0.60; s.e. = 0.10). Nevertheless, hybridization was promoted in both streams by the continuous immigration of individuals with high levels of non-native admixture from other hybrid source populations. Thus, antagonistic relationships between dispersal and selection are mediating invasive hybridization between these fish, emphasizing that data on dispersal and natural selection are needed to fully understand the dynamics of introgression between native and non-native species.