Heat tolerance and thermal scope are evolutionarily constrained in Greenlandic terrestrial arthropods

Heat tolerance and thermal scope are evolutionarily constrained in Greenlandic terrestrial arthropods | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Heat tolerance and thermal scope are evolutionarily constrained in Greenlandic terrestrial arthropods Jonas Wesseltoft, Michael Ørsted, Nadieh Jonge, Michael Hansen, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5640068/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Temperatures in the Arctic currently rise at four times the global average, making it of utmost importance to understand the thermal biology of species in these sensitive environments. For arctic ectotherms in particular, thermal tolerance limits and adaptive potential are mostly unknown. Such knowledge is urgently needed to predict climate change impacts on future distributions of biodiversity in these rapidly changing environments. Here, we provide new data on upper and lower thermal limits of 93 Greenlandic species of insects, arachnids, and collembolans identified using barcode sequencing representing ~8% of described terrestrial Greenlandic arthropod species. We found pronounced differences in heat and cold tolerance among species and a strong phylogenetic signal for both heat tolerance and thermal scope (difference between upper and lower thermal limits), suggesting that terrestrial Greenlandic arthropods are evolutionarily constrained in their capacity to cope with increasing and more variable future temperatures. Further, with projected future increases in microclimatic temperatures induced by climate change, we reveal a marked increase in the number of species that will experience potentially stressful temperatures for prolonged periods of time. Together, our results suggest that climate change will likely result in substantial changes in distributions and abundances of Greenlandic terrestrial arthropods. Biological sciences/Ecology/Ecophysiology Biological sciences/Biological techniques/High-throughput screening Biological sciences/Ecology/Evolutionary ecology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction As the earth continues to warm, knowing the thermal tolerance limits of organisms is critical for predicting how ecosystems will respond and be affected 1–4 . With increasing mean temperatures and more frequent extreme weather events, many species will experience temperatures exceeding their optimal or even permissible temperature range 1,5,6 . This can result in heat (or cold) stress, which has marked negative impacts on multiple life-history traits and can ultimately be lethal 7–9 . When populations and species experience climatic stress, they can either migrate, adapt through evolution 5,10 , or via plasticity in physiological, behavioural and/or morphological traits 5,11,12 . However, recent findings have suggested that while animals, and ectotherms in particular, can adapt to colder environments through evolution and plasticity, their capacity to increase heat tolerance through evolutionary adaptation or plasticity is limited 10,13–15 . The current warming rate in arctic regions is almost four times higher than the global mean 16 making it of outmost importance to investigate the effects of climate change on thermal biology of arthropods in this region 17 . Further a low species diversity, in arctic and sub-artic regions makes it a relatively simple system to investigate 18 . Globally, knowledge of thermal tolerances across species and genera of arthropods, is far from complete 19,20 , and there has recently been a call for more focus on thermal limits and adaptations to climate stress of especially polar arthropods 17,21,22 . Historically, work on thermal biology of arthropods from high latitudes and altitudes has focused on their ability to cope with low temperatures 17,22–24 and only recently have studies on responses to high temperature stress been performed 21,25,26 . Efforts to reconcile the fragmented knowledge of thermal tolerances across species and latitudes into centralized databases clearly illustrate the lack of data, as less than 0.0003% of described terrestrial arthropod species have estimates of thermal stress tolerance levels in the database GLOBTHERM 20 . Thus, more data on more species is urgently needed to expand our understanding of consequences of climate changes globally. In this study performed in Southern Greenland, we adopt a novel catch-test-and-sequence methodology, enabling simultaneous determination of thermal limits using ramping assays and species identification utilizing cytochrome c oxidase I (COI) barcoding. We present heat and / or cold tolerance data on 93 species of terrestrial Greenlandic insects, spiders and collembolans covering ~8 % of the described terrestrial Greenlandic arthropod species. Our results reveal marked inter-specific differences in thermal tolerances and a clear phylogenetic signal for both upper thermal limits and thermal scope suggesting evolutionary constraints. These results provide evidence that rapid climate changes in Greenland will likely have strong impact on the future distribution and abundance of arthropods in this region. Results Catch-test-and-sequence method describes thermal limits for ~8% of Greenlandic terrestrial arthropods COI barcode sequencing of the tested arthropods allowed us to obtain high resolution taxonomic information on 552 of the 701 collected animals (~79%) across 11 taxonomic orders (Table 1 & Figure 1). A total of 99 species were described in this study comprising 8.25 % of the described terrestrial arthropod fauna in Greenland 18 . CT max and / or CT min estimates were obtained for 93 of the recorded species. For six species we could not obtain measures of thermal tolerance either because they were physically too big to test in the vials ( Apis mellifera and Vespula rufa ) or water entered the vials drowning the individuals during testing. The highest degree of species coverage was observed in the least diverse orders where between 50-100% of the described Greenlandic terrestrial arthropods were included in our study while the most diverse orders had a coverage between 8% and 15% (Table 1). Table 1: Coverage of thermal tolerances and species across taxonomic orders. All reference to Greenlandic (GL) arthropods is according to Böcher et. al. 18 . % indicates percentage of described GL species found in the current study. Orders above 100% of described GL species are on account of lower taxonomic resolution (e.g. Lamyctes sp.). Order # of described GL species # and % of GL species recorded in current study # of species with CT max measures obtained in current study # species with CT min measures obtained in current study # of species where thermal scope could be calculated Araneae 75 11 (15%) 9 7 5 Chilopoda 1 2 (200%) 2 0 0 Coleoptera 36 (native) 36 (introduced) 9 (25%) (native) 0 (introduced) 9 2 2 Diptera ~370 36 (10%) 28 17 10 Entomobryomorpha 2 1 (50%) 0 1 0 Hemiptera 41 11 (27%) 7 10 6 Hymenoptera ~ 200 16 (8%) 6 11 5 Lepidoptera 58 9 (16%) 8 4 4 Neuroptera 2 2 (100%) 1 1 0 Trichoptera 8 2 (25%) 0 2 0 Trombidiformes 2 2 (100%) 1 1 1 TOTAL 830 101 (12%) 71 56 33 Heat and cold tolerance vary drastically within and among orders We then tested the inter-order differences of critical thermal limits and thermal scope and found that the median values of CT max for each species differed significantly across taxonomic orders (Kruskal-Wallis, Χ 2 = 35.558, df = 8, p < 0.001) (Figure 2A). The same was seen for thermal scope, which was also significantly different across orders (Χ 2 = 15.354, df = 6, p = 0.0177) (Figure 2C). However, differences across orders were not observed for CT min (Χ 2 = 12.969, df = 9, p = 0.164) (Figure 2B). For CT max , Araneae ranked as having the highest median values across orders, while the lowest was observed for Diptera. A post-hoc pairwise Dunns tests revealed that Araneae and Lepidoptera had significantly higher median CT max compared to species of Diptera (p = <0.001 and 0.0028). Other orders did not differ in CT max (Figure 2A). For thermal scope, Araneae and Diptera had the highest and lowest median values, respectively and only these two were found to be significantly different (p = 0.0021) (Figure 2C). Phylogenetic signals show evolutionary constraint of upper thermal limits in arctic arthropods We found a disparity between phylogenetic groups in CT max and thermal scope when these are presented with phylogenetic relations (Figure 3A). This corresponds with the results reported above showing that Araneae had the highest CT max with some species having individuals still being active at temperatures above 50˚C. In contrast Dipterans had the lowest CT max typically below 35˚C. The comparatively higher CT max for Lepidopterans seen in Figure 2, was also observed here across species (and life stages) (Figure 3A). For CT min , no apparent trend is observed (Figure 3B) which aligns with results showing no significant effect of order for CT min (Figure 2B). Finally, for thermal scope (Figure 3C) Araneae species have the highest values, while Lepidoptera have a lower measure of thermal scope, and the Dipterans the lowest values. To test for phylogenetic signal in each of these thermal limit measures, Pagel’s λ is calculated which reveals a strong and highly significant phylogenetic signal for thermal scope (λ = 0.99, p < 0.001), and CT max (λ = 0.49, p < 0.001) suggesting that closely related species have a more similar thermal scope and CT max than more distantly related species. In contrast, no phylogenetic signal was observed for CT min (λ < 0.001, p = 1). Independent evolution of upper and lower thermal limits A Phylogenetic Generalized Least Squares model (PGLS) revealed that there was a clear positive association between CT max and thermal scope across all samples with a slope of 1.06 (SE = 0.09, t = 12.03, p < 0.001) (Figure 4A). CT min was also found to be strongly associated with thermal scope, however negatively given the nature of CT min as low values are evident of high cold tolerance, resulting in a slope of -0.89 (SE = 0.15, t = -5.77, p < 0.001) (Figure 4B). As opposed to CT max , this PGLS model for CT min and thermal scope differed from a linear model with a higher intercept for thermal scope (46.10 and 43.12), but otherwise similar coefficients with the linear model having a slope of -1.11 (SE = 0.26, t = -4.24, p = 0.001). However, no correlation was observed between CT min and CT max with a slope of -0.08 (SE = 0.01, t = -0.77, p = 0.44) (Figure 4C), indicating independent evolution of the two traits. Additionally, no significant relationships were observed between the medians of length of specimens and both CT max and CT min at species level (R 2 < 0.001, p = 0.89 and (R 2 = 0.010, p = 0.47) respectively. Projected future microclimatic temperatures may exceed upper thermal tolerance limits Comparing hourly microclimatic measurements from the summer of 2023 in Narsarsuaq with air temperatures in Narsarsuaq in the same period shows microclimatic temperatures far exceeding those measured in the air (Figure 5 and Figure S2). Using the CT max estimates reported here, we show that during the entire summer period of 2023 only two species ( Delia platura and Calliphora uralensis ) are likely to have experienced temperatures above their CT max (Table S1). However, during this period, these stressful temperatures occurred regularly for the two species at 89% and 77% of days, respectively, in the investigated period. Interestingly, when associating the CT max measures to the projected microclimatic temperatures (end-of-century SSP370 scenario), the number of species that will potentially experience temperatures above their upper thermal limit will increase dramatically to 17 (24% of species with CT max estimates in our study) and on average they will be exposed to stressful temperatures (defined as time spend exposed to temperatures above their estimated CT max ) 35.77% of days across the investigated period (Table S1). Discussion In this study, we investigated thermal tolerances of a wide range of terrestrial arthropods from Narsarsuaq in Southern Greenland. Our results 1) contribute to reducing the knowledge gap on thermal tolerance limits in the Greenlandic terrestrial arthropods, 2) highlight the power of incorporating molecular species identification in arthropod field surveys, 3) reveal evidence of evolutionary constraints in thermal tolerance traits in arctic arthropod species as seen for heat but not cold tolerance in species from other latitudes 10,14 and 4) reveal that periodically a quarter of the investigated species will be exposed to temperatures above their CT max under expected future microclimatic conditions. With the present work, we have established an inventory of thermal tolerance measures of south Greenlandic arthropods and a resource with photos and size measurements of investigated species for use in future studies on Greenlandic arthropods. By combining rapid thermal tolerance measurements with COI barcoding, we enabled a high-throughput identification of 101 species and simultaneous measures of upper and lower thermal limits. Thus, this study provides a novel framework for rapid biodiversity and eco-physiological field assessments and offers new insights into the capacity of 93 Greenlandic arthropod species to withstand low and high temperatures. This constitutes a significant portion of the described Greenlandic terrestrial arthropod species diversity (~8%) and globally represents a large increase in the number of arthropod species where information on thermal tolerances is available 20 . Although molecular methods for species identification are routinely used 27–29 , there is a large unutilized potential in incorporating barcoding approaches with phenotypic and physiological data for a wide range of species. This combination offers the possibility of covering much of the knowledge gap with relatively little effort, as in this case, where the field work was performed within a week. This is particularly important when working with limited resources or in areas with adverse working conditions or a short growing season as is the case in polar regions 30 . In this study, individual imaging of the collected specimen was used as a tool for curation of the taxonomic classification provided using the DNA barcoding. In fact, for 49 individuals it was determined based on the images that the taxonomic order provided by the DNA barcoding was erroneous. This illuminates an inherent weakness in blindly relying upon sequence data for species identification even when including a surface bleaching to avoid cross contamination. Interestingly, we observed an abundance (9 cases) of the parasitic wasp Ichneumon sarcitorius among the erroneous identifications. This could explain the wrong identification in some cases, as it is reasonable to suspect the investigated individuals could be carrying wasp DNA, which was not removed by the bleach treatment. This suggest that images can be a crucial part of species identification when performed on a scale as in the present study. We observed a clear and significant trend that members of the order Araneae had both the highest CT max and the lowest CT min . Alongside Araneae, Lepidoptera were also found to have a high CT max , but this was mainly driven by the larval individuals from the Noctuidae family which have previously been shown to have higher CT max in the larval stage 31 . Similarly, Bahrndorff et al. 21 reported a CT max value for adult Eurois occulta which was 3˚C lower than reported here, while presenting comparable values for several other species across different orders also tested here including Nysius groenlandicus, Psammotettix lividellus and Nabis flavimarginatus 21 . For the Pardosa genera, our estimates of CT max were higher and lower for CT min compared to findings in Anthony et al., 2019 32 . Lepidoptera and Araneae typically have high upper thermal limits with estimates in the literature in the range 45 – 50˚C 31,33 and 42 - 49˚C 32,34 respectively. Our results for these taxa were in alignment with these findings and generally upper thermal limits across the other taxonomic orders corresponds to what has previously been reported in the literature. A general finding in our study is that Diptera has low and variable heat tolerance with CT max values in the range 22 – 43˚C. This aligns with literature findings 7,21,35–38 highlighting the large intra-order variability and the importance of high taxonomic resolution in thermal tolerance studies. Likewise, Coleoptera and Hymenoptera have been reported to have CT max between 38 and 45˚C 39–41 , aligning with our findings. In comparing and interpretating CT max and CT min values across studies, it is however important that conditions including ramping rates, start temperature and pretest rearing temperatures can have marked impact on the estimates 42,43 . Estimates of upper thermal limits obtained in this study were shown to be significantly dependent upon the phylogenetic relations across the tested species (Figure 3A), meaning that closely related species were more likely to have similar measures of upper thermal tolerance than distantly related species. This suggests that the upper thermal tolerance of the arctic arthropods investigated here show little capacity to adapt to higher temperatures through evolutionary adaptation. While this have been shown for other species of arthropods 13,14,44 , it has not previously been shown across arctic terrestrial arthropods, further highlighting the knowledge gap in these rapidly changing sensitive environments. Importantly, thermal scope of the tested arthropods (Figure 3C), similarly exhibited a strong phylogenetic signal suggesting that this trait is also phylogenetically constrained. Arctic and subarctic (and polar in general) climates vary dramatically on a diurnal and seasonal basis (Figure S1A and S2) and with climate change, we expect to see increases in the frequency of climatic extremes and more unpredictable thermal fluctuations 45–47 . Our data suggest that the ability to evolutionary adapt to high temperature fluctuations is limited, which could have serious implications for the abundance and distribution of the future Greenlandic terrestrial arthropods. We find that both upper thermal tolerance and thermal scope is conserved across species of the same order despite quite different modes of life as seen in the spiders with the brush and herb-living mesh-weavers ( Dictyna major and Emblyna borealis) and the ground-dwelling wolf spiders ( Pardosa groenlandica and Pardosa furcifera) 18 . Microhabitat temperatures in their habitats are expected to differ between the two groups 48,49 but despite this, they all exhibit exceptionally high thermal tolerances (Figure 4A and 4C). Our results and literature findings thus support that the phylogenetic constraints observed here are likely not caused by shared environmental history and evolutionary pressures as suggested to be a causative agent of phylogenetic signals by Freckleton and Jetz 50 . Finally, by projecting future diurnal microclimatic temperatures according to climate predictions, we highlight the issue of increased potential exposure to stressful warm temperatures. A quarter of the tested species reported here will potentially experience future microclimatic temperatures exceeding their upper critical thermal limit. This finding is contrary to that of Deutsch et. al. 51 , who argued that terrestrial ectotherms at temperate latitudes were far from their critical limits and thus not susceptible to being negatively affected by global warming as opposed to tropical species who had a smaller thermal safety margin and thus would be more negatively affected. However, as also pointed out by others 5,52 , the use of such measures of thermal safety margins can be problematic as they often rely on air temperature, which, as shown here, is not representative of the micro-environment experienced by small arthropods. Although behavioural thermoregulation and plasticity may protect arthropods against future warmer and more variable temperatures, our finding suggests that warming macro- and micro-climates in Arctic and sub-Arctic regions will have profound effects on the ecology, behaviour and distribution of the arthropod community 53 . While individual studies reporting thermal limits for a wide range of arthropod species is essential to expand the limited knowledge base, it is also crucial to coordinate a joint effort to conglomerate all this information into large, united databases, as information is currently scattered and hard to assess for researchers and practitioners. Thus, to enable more powerful analysis that illuminate trends, not just locally, but globally and over a longer time scale, it is essential to make data as comparable and easily accessible as possible. The data and knowledge provided here are highly valuable for future studies aiming at predicting the potential changes in diversity, abundance and distribution of arthropods with a changing climate and how this will impact on complex ecological networks. Conclusion Changes in abundance and distribution of arctic terrestrial arthropods are important to understand as they play crucial roles in ecosystems, including pollination, nutrient cycling (decomposition), seed dispersal 54 , and functioning as disease vectors for both plants and animals, including humans 55,56 . Our study provides important insights into the thermal tolerances of a large proportion of Greenlandic arthropods and reveals stark phylogenetic signals for both CT max and thermal scope. The observed phylogenetic signal suggesting evolutionary constraints for these measures along with our results showing that a quarter of the investigated species will likely be exposed microclimates above their upper thermal limits for extended time periods in the future, suggest that climate changes in arctic regions will have profound impacts on arthropod diversity, abundance and distribution. Materials and Methods Sample collection Terrestrial arthropods (insects, arachnids and collembola) were collected in Narsarsuaq, Southern Greenland (61.160°N, 45.424°W). This region is characterized by cool temperatures, long winters and short summers (average -7 °C in January and +11˚C in July, Figure S1A). The area has recently experienced rapidly increasing temperatures with mean annual temperature anomalies exceeding +1˚C on average (range -1.7 to +4.4˚C) since 2000 compared to the 1979-2023 baseline (Figure S1B). Importantly, summers in the area are very thermally variable with maximum temperatures at ground level exceeding 30˚C 57 . Ambient temperature was measured 7.5 cm above ground using TMS-4 loggers (TOMST) at 5 min resolution in two locations in Narsarsuaq (Figure S2). The study site is a heath-like, grass- and shrub covered area, where arthropods were collected from grasses and bushes using a sweep net or a pooter (a flexible tube used to catch animals directly from leaves or the ground). All individuals used in the experiment were caught in a 200 x 200 m area less than 100 m from the laboratory facilities. Animals were collected in the summer season from the 12 th of August until the 17 th of August 2023. All animals were collected during the day between 09:00 and 19:00, and all days throughout the study period showed similar temperature profiles (Figure S2). A subset of the collected arthropods was chosen based on morphological novelty (aiming at having as many different species as possible tested) and transferred to individual 5 mL glass vials with plastic screw-lids within 30 minutes after being collected. The vials with arthropods were kept outside of the laboratory in the shade for maximum one hour before being tested for either heat or cold tolerance. Animals were collected on six separate days constituting six batches. In each batch, between 80 and 138 individuals were tested. In total, 701 individuals were tested for either heat or cold stress tolerance. Critical Thermal Limits Vials with arthropods were placed randomly in a rack before being submerged in a circulating water bath for Critical Thermal maximum (CT max )or Critical Thermal minimum (CT min ) assessments. Liquid in the water baths was kept at 20˚C until vials with animals had been submerged, whereafter the temperature was increased or lowered by 0.1˚C per minute to assess CT max or CT min , respectively. The temperature of onset of coma, i.e. at which the last movement was observed in response to agitation (facilitated by softly knocking on the vials with a metal rod), was recorded as the thermal tolerance limit to heat or cold stress for that individual 43 . Immediately following the critical thermal limit test, each individual was transferred to a 1.5 mL Eppendorf tube before being stored at -20˚C until imaging and DNA extraction (to be used for species identification based on DNA barcoding). Imaging Imaging was performed using an Olympus SZX10 stereo microscope, and Olympus DP74 colour camera (Olympus Corporation). Animals were thawed in small batches of between 1 and 10, before being placed on a 1% agarose plate facilitating proper positioning of the animals. Each animal was pictured from multiple angles, including ventral, dorsal and profile in addition to identifying markers, i.e. wing webbings or cornicles. Additionally, each animal was measured from the tip of the head to the end of the abdomen and a micro-ruler was imaged at the same magnification for scale. Following imaging, each animal was returned to the Eppendorf tube and kept at 5˚C until DNA extraction was performed on the following day. Representative images of each observed species can be found in Appendix 1 while images of all individuals can be accessed at Dryad depository (https://doi.org/10.5061/dryad.1g1jwsv6w). DNA extraction All specimens were washed twice in 200 uL 2.5% bleach solution (Thermo Fisher Scientific) and twice in 800 uL UV treated ELGA water to minimize cross contamination from sample collection and imaging. Total genomic DNA was extracted from all individuals using the DNeasy Blood and Tissue kit (Qiagen) according to the manufacturer's instructions for extraction of DNA from insects. Agilent Tapestation 2200 was used to run Genomic DNA Screentape (Agilent Technologies) to assess DNA quality. Following DNA extraction and quality control, samples were stored at -80˚C. PCR, cleanup and COI barcode sequencing Samples of extracted DNA from all tested animals were diluted 1:10 in 2x UV treated nuclease free water. All samples were amplified using either the HCO1490/LCO2198 primer pair 58 or the BF2/BR3 primer pair 59 (Appendix 2). For the former primer pair, a single touchdown PCR reaction was performed for each sample in a reaction volume of 25 uL (1X PCRBIO Ultramix (PCR Biosystems), 400 nM of each primer and 2 uL template DNA). The following thermocycler settings were used for the touchdown PCR: Initial denaturation at 95˚C for 2 minutes followed by 16 cycles of: 95˚C for 30 sec, 62˚C (-1 °C each cycle) for 1 minute, 72˚C for 1 minute, followed by 25 cycles of: 95˚C for 30 sec, 46˚C for 1 minute, 72˚C for 1 minute followed by a final extension at 72˚C for 5 minutes. For the latter primer pair, a single PCR was performed for each sample in a reaction volume of 25 uL (1X PCRBIO Ultramix (PCR Biosystems), 400 nM of each primer and 2 uL template DNA). The following thermocycler settings were used for the PCR: Initial denaturation at 95˚C for 2 minutes followed by 25 cycles of: 95˚C for 30 sec, 44˚C for 1 minute, 72˚C for 45 seconds, followed by a final extension at 72˚C for 5 minutes. All samples were subsequently purified using the Monarch PCR & DNA Cleanup Kit (New England Biolabs) according to the manufacturer's instructions. DNA concentration of purified samples was determined using Qubit HS dsDNA kit (Thermo Fisher Scientific) and a TECAN Infinite F200 Pro (Tecan Life Sciences) after which all samples that had successfully amplified, were prepared for Sanger sequencing by Eurofins Genomics (Germany). Species identification using barcoding The obtained COI sequence for each animal was used for species identification using BLAST’s blastn function 60 . An identity percentage above 98% and a report from at least two different accession numbers was considered reliable species identification. For sequences with an identity percentage lower than 98%, the best result was used, manually checked, included and noted (Appendix 2). For the taxonomic analysis, one reference sequence from the BOLDsystems database was included in the analysis for each identified species, with a preference for sequences from samples collected in subarctic regions. Every sample was manually curated by comparing recorded taxonomy to literature of local arthropod diversity 18 under a principle of conservative taxonomic assignments. Subsequently, all assigned taxonomies were compared to images of the sequenced animal 18 . In 49 cases, manual curation revealed obvious mismatches between images and recorded taxonomy e.g. wrong taxonomic order and these samples were removed from further analysis. All sequences obtained were aligned using the MEGA software 61 and the ClustalW algorithm with a gap opening quality of 15.00 and a gap extension penalty set at 6.66. Sequences that did not allow for alignment on account of short sequence length were removed from the analysis. All aligned sequences were shortened to consist of only a 410 bp region. Consensus sequences were generated from the aligned sequences obtained in this study using BioEdit (0.6). All phylogenetic trees were generated from aligned consensus sequences using MEGA software and the Neighbour-joining tree function with 1000 bootstrap iterations. Modelling exposure to microclimatic temperatures exceeding thermal thresholds To investigate how the tested species may be affected by projected increases in diurnal temperatures, we compared species-specific CT max estimates to measured and expected future microclimatic temperatures in the period 19 July to 23 Aug 2023. First, we estimated hourly air temperatures (2 m height) at the sampling location with the ‘NicheMapR’ package v3.2.1 62 (Figure S2). As no reliable projections of future microclimates exists yet we first calculated the differences in air temperatures between current (2000-2021) and projected future conditions (2081-2100; SSP370 scenario) (see Figures S1-2 for details). This difference, ΔT air ,was added to the measures obtained from our TOMST loggers to describe the current microclimate (T micro_current ). We obtained estimates of hourly future microclimate temperatures using the formula: T micro_future = T micro_current + a ∙e ( b ∙ΔTair) , where a and b are coefficients based on the exponential relationship between air and microclimate temperatures (different for day and nighttime; Figures S2-3). Statistical analysis Following species identification, outliers (for CT max and CT min ) within each species were identified and removed if having a median absolute deviation (MAD) of 3 or more, removing 53 samples 63 . This was done to remove individuals with extreme low CT max and high CT min suggesting that they were harmed in the process of catching them and / or getting them into the test tube (all analyses was also performed without removing outliers and conclusions remained the same). Thermal scope was calculated for species with a measure of both CT max and CT min as the absolute difference between medians of bootstrap resampled data from each species using 10,000 iterations. Kruskal-Wallis tests were performed to compare medians within groups to determine potential differences. In case of significant differences, a post hoc Dunn’s test was performed to compare groups. To investigate the presence of phylogenetic signals in each trait (CT max , CT min and thermal scope), Pagel’s Lambda was determined using the phytools R package running 1000 simulations 64,65 . Correlations between thermal tolerance traits were corrected for phylogenetic signals by performing Phylogenetic Generalized Least Squares (PGLS) models in the nlme R package 66 . Declarations Acknowledgements: We thank Frederik Kjær Nielsen, Christian Dupont Danielsen and Andreas Mølgaard Andersen for technical assistance in the laboratory, Elma Huremovic for assistance in literature research and Simon Bahrndorff for constructive comments on earlier versions of the manuscript and for supporting access to field facilities in Narsarsuaq, Greenland. Author contributions: Jonas Bruhn Wesseltoft: Conceptualization, Methodology, Data Curation, Formal Analysis, Visualization, Writing – Original Draft. Michael Ørsted: Formal Analysis, Funding Acquisition, Writing – Review and Editing, Supervision. Nadieh de Jonge: Methodology, Formal Analysis, Writing – Review and Editing. Michael M. Hansen: Writing – Review and Editing. Toke Thomas Høye: Resources, Writing – Review and Editing. Torsten Nygaard Kristensen: Conceptualization, Methodology, Resources, Project Administration, Funding Acquisition, Writing – Original Draft, Supervision. Funding: The work was funded by the European co-funded Partnership BiodivClim-191 ASICS (0156-00024B to TNK), and by the Novo Nordisk Foundation (grant NNF23OC0082599 to MØ). Laboratory facilities in Narsarsuaq, Greenland, was supported by the Greenland Integrated Observing System (GIOS) (the Danish National Fund for Research Infrastructure (NUFI)). 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J., Devey, D., Wilkinson, T. & Papadopulos, A. S. T. Field-based species identification of closely-related plants using real-time nanopore sequencing. Sci. Rep. 7 , 8345 (2017). Hansen, J., Topp-Jørgensen, E. and Christensen, T. R. (eds.). Zackenberg Ecological Research Operations 21st Annual Report, 2015. Aarhus University, DCE – Danish Centre for Environment and Energy. 96 pp. (2017). Bawa, S. A., Gregg, P. C., Del Soccoro, A. P., Miller, C. & Andrew, N. R. Estimating the differences in critical thermal maximum and metabolic rate of Helicoverpa punctigera (Wallengren) (Lepidoptera: Noctuidae) across life stages. PeerJ 9 , e12479 (2021). Anthony, S. E., Buddle, C. M., Høye, T. T. & Sinclair, B. J. Thermal limits of summer-collected Pardosa wolf spiders (Araneae: Lycosidae) from the Yukon Territory (Canada) and Greenland. Polar. Biol. 42 , 2055–2064 (2019). Chidawanyika, F. & Terblanche, J. S. Rapid thermal responses and thermal tolerance in adult codling moth Cydia pomonella (Lepidoptera: Tortricidae). J. Insect Physiol. 57 , 108–117 (2011). Hanna, C. J. & Cobb, V. A. Critical thermal maximum of the green lynx spider, Peucetia viridans (Araneae, Oxyopidae). J. Arachnol. 35 , 193–196 (2007). Weaving, H., Terblanche, J. S. & English, S. How plastic are upper thermal limits? A comparative study in tsetse (family: Glossinidae) and wider Diptera. J. Therm. Biol. 118 , 103745 (2023). Lighton, J. R. B. Hot hypoxic flies: Whole-organism interactions between hypoxic and thermal stressors in Drosophila melanogaster. J. Therm. Biol. 32 , 134–143 (2007). Folk, D. G., Hoekstra, L. A. & Gilchrist, G. W. Critical thermal maxima in knockdown-selected Drosophila : are thermal endpoints correlated? J. Exp. Biol. 210 , 2649–2656 (2007). Leclerc, M. A. J., Guivarc’h, L., Lazzari, C. R. & Pincebourde, S. Thermal tolerance of two Diptera that pollinate thermogenic plants. J. Therm. Biol. 109 , 103339 (2022). Jones, K. K. et al. The critical thermal maximum of diving beetles (Coleoptera: Dytiscidae): a comparison of subterranean and surface-dwelling species. Curr. Res. Insect Sci. 1 , 100019 (2021). Käfer, H., Kovac, H. & Stabentheiner, A. Resting metabolism and critical thermal maxima of vespine wasps (Vespula sp.). J. Insect Physiol. 58 , 679–689 (2012). Oyen, K. J., Giri, S. & Dillon, M. E. Altitudinal variation in bumble bee (Bombus) critical thermal limits. J. Therm. Biol. 59 , 52–57 (2016). Sørensen, J. G., Loeschcke, V. & Kristensen, T. N. Cellular damage as induced by high temperature is dependent on rate of temperature change – investigating consequences of ramping rates on molecular and organismal phenotypes in Drosophila melanogaster Meigen 1830. J. Exp. Biol. (2012). Overgaard, J., Kristensen, T. N. & Sørensen, J. G. Validity of thermal ramping assays used to assess thermal tolerance in arthropods. PLoS One 7 , e32758 (2012). Sandblom, E. et al. Physiological constraints to climate warming in fish follow principles of plastic floors and concrete ceilings. Nat. Commun. 7 , 11447 (2016). Overland, J. E. Rare events in the Arctic. Clim. Change 168 , 27 (2021). Fischer, E. M., Sippel, S. & Knutti, R. Increasing probability of record-shattering climate extremes. Nat. Clim. Chang. 11 , 689–695 (2021). Walsh, J. E. et al. Extreme weather and climate events in northern areas: A review. Earth Sci. Rev. 209 , 103324 (2020). Conti, M., Johnson, B., Kraft, T. & Ye, A. A comparison between ground-dwelling spider preferences for thermal control and vegetation reveals different microhabitat selection. CEC Research 7 , 1 (2023). Vives‐Ingla, M. et al. Interspecific differences in microhabitat use expose insects to contrasting thermal mortality. Ecol. Monogr. 93 , (2023). Freckleton, R. P. & Jetz, W. Space versus phylogeny: disentangling phylogenetic and spatial signals in comparative data. Proc. R. Soc. Lond. B. Biol. Sci. 276 , 21–30 (2009). Deutsch, C. A. et al. Impacts of climate warming on terrestrial ectotherms across latitude. Proc. Natl. Acad. Sci. U. S. A. 105 , 6668–6672 (2008). Clusella-Trullas, S., Garcia, R. A., Terblanche, J. S. & Hoffmann, A. A. How useful are thermal vulnerability indices? Trends Ecol. Evol. 36 , 1000–1010 (2021). Koltz, A. M., Schmidt, N. M. & Høye, T. T. Differential arthropod responses to warming are altering the structure of Arctic communities. R. Soc. Open Sci. 5 , 171503 (2018). Eggleton, P. The state of the world’s insects. Annu. Rev. Environ. Resour. 45 , 61–82 (2020). Gubler, D. J. Dengue and dengue hemorrhagic fever. Clin. Microbiol. Rev. 11 , 480–96 (1998). NG, J. C. K. & Perry, K. L. Transmission of plant viruses by aphid vectors. Mol. Plant Pathol. 5 , 505–511 (2004). Noer, N. K. et al. Rapid Adjustments in Thermal Tolerance and the Metabolome to Daily Environmental Changes – A Field Study on the Arctic Seed Bug Nysius groenlandicus . Front. Physiol. 13 , (2022). Folmer, O., Black, M., Hoeh, W., Lutz, R. & Vrijenhoek, R. DNA primers for amplication of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Biol. Biotechnol. 3 , 294–299 (1994). Elbrecht, V. et al. Validation of COI metabarcoding primers for terrestrial arthropods. PeerJ 7 , e7745 (2019). Camacho, C. et al. BLAST+: architecture and applications. BMC Bioinform. 10 , 421 (2009). Tamura, K., Stecher, G. & Kumar, S. MEGA11: Molecular evolutionary genetics analysis Version 11. Mol. Biol. Evol. 38 , 3022–3027 (2021). Kearney, M. R. & Porter, W. P. NicheMapR – an R package for biophysical modelling: the ectotherm and Dynamic Energy Budget models. Ecography 43 , 85–96 (2020). Leys, C., Ley, C., Klein, O., Bernard, P. & Licata, L. Detecting outliers: Do not use standard deviation around the mean, use absolute deviation around the median. J. Exp. Soc. Psychol. 49 , 764–766 (2013). Revell, L. J. phytools 2.0: an updated R ecosystem for phylogenetic comparative methods (and other things). PeerJ 12 , e16505 (2024). Pagel, M. Inferring the historical patterns of biological evolution. Nature 401 , 877–884 (1999). Pinheiro, J., Bates, D. & R Core Team. nlme: Linear and nonlinear mixed effects models. https://CRAN.R-project.org/package=nlme (2024). Additional Declarations There is NO Competing Interest. Supplementary Files SupplementalFigures.docx Supplementary Figures Appendix1.docx Species Catalogue Appendix2.xlsx Dataset 1 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5640068","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":391891118,"identity":"c5123266-386d-468f-b42b-2101900939cd","order_by":0,"name":"Jonas Wesseltoft","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArUlEQVRIiWNgGAWjYBACxnYGxscMBlAezwFitDQzMBuTpoWBmYFNGs4hSgtzM49ZdUGBDQN/+xkDhjdniHIYj9ntGQZpDBJncgwY59wgSgvvtts8BocZGA7kGDDzfCBSSzFIi/z5NyRoYQZpMbgBsoU4h/F/lgb6hcfwxrOCg3OI8b5he1vi54I/NnJy55M3PnhzjBgtDRCaB0QcIEIDA4M8UapGwSgYBaNgZAMARakyeLSxbnIAAAAASUVORK5CYII=","orcid":"https://orcid.org/0009-0006-8368-8972","institution":"Aalborg University","correspondingAuthor":true,"prefix":"","firstName":"Jonas","middleName":"","lastName":"Wesseltoft","suffix":""},{"id":391891119,"identity":"6bba96b5-dedf-49f0-a1c7-271d09b39dce","order_by":1,"name":"Michael Ørsted","email":"","orcid":"https://orcid.org/0000-0001-8222-8399","institution":"Aarhus University","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Ørsted","suffix":""},{"id":391891120,"identity":"77c5ccf9-c537-42ab-8381-71393fdf0b4f","order_by":2,"name":"Nadieh Jonge","email":"","orcid":"","institution":"Department of Chemistry and Bioscience, Aalborg University","correspondingAuthor":false,"prefix":"","firstName":"Nadieh","middleName":"","lastName":"Jonge","suffix":""},{"id":391891121,"identity":"a181f648-479f-488f-83ad-678ea9c6964e","order_by":3,"name":"Michael Hansen","email":"","orcid":"","institution":"Aarhus University","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Hansen","suffix":""},{"id":391891122,"identity":"2df6b537-a3f9-479a-8b35-a5ff1074d877","order_by":4,"name":"Toke Høye","email":"","orcid":"https://orcid.org/0000-0001-5387-3284","institution":"Aarhus University","correspondingAuthor":false,"prefix":"","firstName":"Toke","middleName":"","lastName":"Høye","suffix":""},{"id":391891123,"identity":"3cfcb596-9fc4-4bf1-ad14-6a6e4056b965","order_by":5,"name":"Torsten Kristensen","email":"","orcid":"","institution":"Section of Genetics, Ecology and Evolution, Department of Bioscience, Aarhus University","correspondingAuthor":false,"prefix":"","firstName":"Torsten","middleName":"","lastName":"Kristensen","suffix":""}],"badges":[],"createdAt":"2024-12-13 18:25:55","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5640068/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5640068/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":72138072,"identity":"f06aa385-ca13-4705-8481-b2978e13536a","added_by":"auto","created_at":"2024-12-23 06:08:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":256229,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eNeighbour-joining Phylogenetic tree of all collected species based on the sequenced COI region. Clockwise around the perimeter the symbols indicate taxonomic order as follows: Diptera, Neuroptera, Lepidoptera, Coleoptera, Chilopoda, Hemiptera, Hymenoptera, Trombidiformes, Trichoptera, Araneae and Pectinida. Species names in parenthesis indicate phylogenetic placement outside taxonomic order. Silhouettes from phylopic.org.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5640068/v1/f8c701c5c713df68c53bd856.png"},{"id":72137597,"identity":"c8ee2d7e-020d-4f70-b420-5a69ec52670a","added_by":"auto","created_at":"2024-12-23 06:00:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":239516,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eBoxplot of A) CT\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e, B) CT\u003c/em\u003e\u003csub\u003e\u003cem\u003emin\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e and C) thermal scope measures for the different orders. Each black circle represents the median of a given species within the indicated order. Orders with shared letter denominators show no significant differences between them as per the post-hoc Dunns test.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5640068/v1/04fd243fadbbf9d497b5b522.png"},{"id":72137592,"identity":"25b6bfc4-d73f-499d-9e4c-19c12b301b59","added_by":"auto","created_at":"2024-12-23 06:00:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":450967,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eNeighbour-joining Phylogenetic trees for A) CT\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e, B) \u003c/em\u003eCT\u003csub\u003emin\u003c/sub\u003e\u003cem\u003e and C) thermal scope at species resolution level. Colours at branch tips indicate the median thermal limit or scope of the given species while colours of node branches represent mean of immediately connected branches.\u0026nbsp; Species names in parentheses indicate phylogenetic placement outside taxonomic order. Species marked with a star indicate species recorded in the larval stage. Number by nodes are bootstrap values.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5640068/v1/ebcfcde3c4f0019e745f99b2.png"},{"id":72138075,"identity":"b2ce8623-e4b4-406a-a1df-652b4ded220f","added_by":"auto","created_at":"2024-12-23 06:08:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":250863,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eCorrelation analyses of thermal limits. Each dot represents the median of a species in an order designated by the colour of the dot. The solid line represents a linear model not accounting for phylogeny, while the dashed line represents a Phylogenetic Generalized Least Squares (PGLS) model.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5640068/v1/794c56426f6982d2eee10632.png"},{"id":72137602,"identity":"c3288ffb-e11b-4751-8305-4a83eb1cc7e2","added_by":"auto","created_at":"2024-12-23 06:00:24","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":246805,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eExample of exposure of three study species (Calliphora uralensis, Spilogona arctica, and Nebria rufescens) to microclimatic temperatures exceeding their thermal tolerance limits (grey horizontal lines). Solid lines represent air temperature (blue) and microclimate temperatures under current conditions (maroon: hourly average TOMST logger data) and projected future conditions (red, see methods and Figures S1-S3) in the period 30 July to 2 August 2023. During these four days, exposure above CT\u003c/em\u003e\u003csub\u003e\u003cem\u003emax \u003c/em\u003e\u003c/sub\u003e\u003cem\u003eis highlighted under current and future conditions (maroon and red areas, respectively), while Nebria rufescens is only exposed to T\u0026gt;CT\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e under future microclimatic conditions (yellow area). The duration of\u003c/em\u003e \u003cem\u003eT\u0026gt;CT\u003c/em\u003e\u003csub\u003e\u003cem\u003emax \u003c/em\u003e\u003c/sub\u003e\u003cem\u003efor Calliphora uralensis is shown as an example highlighting that not only are some species going to experience more days with temperatures above CT\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e, but the duration of exposure will also lengthen (from 7 to 10 hours). The sum of exposure to temperatures above CT\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e for the exemplary four days is shown under current and future conditions.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-5640068/v1/3133bdcea86c429b51e470ae.png"},{"id":72138923,"identity":"a3d295ed-9ba7-4273-8029-03421e1fecd1","added_by":"auto","created_at":"2024-12-23 06:16:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2135237,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5640068/v1/19d567c9-d986-40b0-8161-5e3405b86182.pdf"},{"id":72137594,"identity":"378c163a-5d16-421e-a62c-0fb92d22a7ff","added_by":"auto","created_at":"2024-12-23 06:00:23","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":818222,"visible":true,"origin":"","legend":"Supplementary Figures","description":"","filename":"SupplementalFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-5640068/v1/ba50e02e16c58a0e9c56ee75.docx"},{"id":72138082,"identity":"b6b1423e-260d-4167-a2a0-900e5a0a53f7","added_by":"auto","created_at":"2024-12-23 06:08:25","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":77046819,"visible":true,"origin":"","legend":"Species Catalogue","description":"","filename":"Appendix1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5640068/v1/8a154b6418821be7852c7e09.docx"},{"id":72137598,"identity":"43c26bae-ebab-4510-b837-39086c3849ac","added_by":"auto","created_at":"2024-12-23 06:00:24","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":114828,"visible":true,"origin":"","legend":"Dataset 1","description":"","filename":"Appendix2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5640068/v1/f83340b02e2f608dce25da02.xlsx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Heat tolerance and thermal scope are evolutionarily constrained in Greenlandic terrestrial arthropods","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAs the earth continues to warm, knowing the thermal tolerance limits of organisms is critical for predicting how ecosystems will respond and be affected\u003csup\u003e1\u0026ndash;4\u003c/sup\u003e. With increasing mean temperatures and more frequent extreme weather events, many species will experience temperatures exceeding their optimal or even permissible temperature range\u003csup\u003e1,5,6\u003c/sup\u003e. This can result in heat (or cold) stress, which has marked negative impacts on multiple life-history traits and can ultimately be lethal\u003csup\u003e7\u0026ndash;9\u003c/sup\u003e. When populations and species experience climatic stress, they can either migrate, adapt through evolution\u003csup\u003e5,10\u003c/sup\u003e, or via plasticity in physiological, behavioural and/or morphological traits\u003csup\u003e5,11,12\u003c/sup\u003e. However, recent findings have suggested that while animals, and ectotherms in particular, can adapt to colder environments through evolution and plasticity, their capacity to increase heat tolerance through evolutionary adaptation or plasticity is limited\u003csup\u003e10,13\u0026ndash;15\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe current warming rate in arctic regions is almost four times higher than the global mean\u003csup\u003e16\u003c/sup\u003e making it of outmost importance to investigate the effects of climate change on thermal biology of arthropods in this region\u003csup\u003e17\u003c/sup\u003e. Further a low species diversity, in arctic and sub-artic regions makes it a relatively simple system to investigate\u003csup\u003e18\u003c/sup\u003e. Globally, knowledge of thermal tolerances across species and genera of arthropods, is far from complete\u003csup\u003e19,20\u003c/sup\u003e, and there has recently been a call for more focus on thermal limits and adaptations to climate stress of especially polar arthropods\u003csup\u003e17,21,22\u003c/sup\u003e. Historically, work on thermal biology of arthropods from high latitudes and altitudes has focused on their ability to cope with low temperatures\u003csup\u003e17,22\u0026ndash;24\u003c/sup\u003e and only recently have studies on responses to high temperature stress been performed\u003csup\u003e21,25,26\u003c/sup\u003e. Efforts to reconcile the fragmented knowledge of thermal tolerances across species and latitudes into centralized databases clearly illustrate the lack of data, as less than 0.0003% of described terrestrial arthropod species have estimates of thermal stress tolerance levels in the database GLOBTHERM\u003csup\u003e20\u003c/sup\u003e. Thus, more data on more species is urgently needed to expand our understanding of consequences of climate changes globally.\u003c/p\u003e\n\u003cp\u003eIn this study performed in Southern Greenland, we adopt a novel catch-test-and-sequence methodology, enabling simultaneous determination of thermal limits using ramping assays and species identification utilizing cytochrome c oxidase I (COI) barcoding. We present heat and / or cold tolerance data on 93 species of terrestrial Greenlandic insects, spiders and collembolans covering ~8 % of the described terrestrial Greenlandic arthropod species. Our results reveal marked inter-specific differences in thermal tolerances and a clear phylogenetic signal for both upper thermal limits and thermal scope suggesting evolutionary constraints. These results provide evidence that rapid climate changes in Greenland will likely have strong impact on the future distribution and abundance of arthropods in this region.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eCatch-test-and-sequence method describes thermal limits for ~8% of Greenlandic terrestrial arthropods \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCOI barcode sequencing of the tested arthropods allowed us to obtain high resolution taxonomic information on 552 of the 701 collected animals (~79%) across 11 taxonomic orders (Table 1 \u0026amp; Figure 1). A total of 99 species were described in this study comprising 8.25 % of the described terrestrial arthropod fauna in Greenland\u003csup\u003e18\u003c/sup\u003e. CT\u003csub\u003emax\u003c/sub\u003e and / or CT\u003csub\u003emin\u003c/sub\u003e estimates were obtained for 93 of the recorded species. For six species we could not obtain measures of thermal tolerance either because they were physically too big to test in the vials (\u003cem\u003eApis mellifera\u003c/em\u003e and \u003cem\u003eVespula rufa\u003c/em\u003e) or water entered the vials drowning the individuals during testing. The highest degree of species coverage was observed in the least diverse orders where between 50-100% of the described Greenlandic terrestrial arthropods were included in our study while the most diverse orders had a coverage between 8% and 15% (Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTable 1: Coverage of thermal tolerances and species across taxonomic orders. All reference to Greenlandic (GL) arthropods is according to \u003c/em\u003e\u003cem\u003eB\u0026ouml;cher et. al.\u003csup\u003e18\u003c/sup\u003e\u003c/em\u003e\u003cem\u003e. % indicates percentage of described GL species found in the current study. Orders above 100% of described GL species are on account of lower taxonomic resolution (e.g. Lamyctes sp.). \u003c/em\u003e\u003c/p\u003e\n\u003ctable border=\"1\" width=\"650\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"151\"\u003e\n\u003cp\u003e\u003cstrong\u003eOrder\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003e\u003cstrong\u003e# of described GL \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003especies\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"97\"\u003e\n\u003cp\u003e\u003cstrong\u003e# and % of GL species recorded in current study \u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"100\"\u003e\n\u003cp\u003e\u003cstrong\u003e# of species with CT\u003csub\u003emax\u003c/sub\u003e\u003c/strong\u003e\u003cstrong\u003e measures obtained in current study\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e\u003cstrong\u003e# species with CT\u003csub\u003emin\u003c/sub\u003e\u003c/strong\u003e\u003cstrong\u003e measures obtained in current study\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e\u003cstrong\u003e# of species where thermal scope could be calculated\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"151\"\u003e\n\u003cp\u003e\u003cstrong\u003eAraneae\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003e75\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"97\"\u003e\n\u003cp\u003e11 (15%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"100\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"151\"\u003e\n\u003cp\u003e\u003cstrong\u003eChilopoda\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"97\"\u003e\n\u003cp\u003e2 (200%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"100\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"151\"\u003e\n\u003cp\u003e\u003cstrong\u003eColeoptera\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003e36 (native)\u003c/p\u003e\n\u003cp\u003e36\u003c/p\u003e\n\u003cp\u003e(introduced)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"97\"\u003e\n\u003cp\u003e9 (25%) (native)\u003c/p\u003e\n\u003cp\u003e0 (introduced)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"100\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"151\"\u003e\n\u003cp\u003e\u003cstrong\u003eDiptera\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003e~370\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"97\"\u003e\n\u003cp\u003e36 (10%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"100\"\u003e\n\u003cp\u003e28\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"151\"\u003e\n\u003cp\u003e\u003cstrong\u003eEntomobryomorpha\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"97\"\u003e\n\u003cp\u003e1 (50%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"100\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"151\"\u003e\n\u003cp\u003e\u003cstrong\u003eHemiptera\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003e41\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"97\"\u003e\n\u003cp\u003e11 (27%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"100\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"151\"\u003e\n\u003cp\u003e\u003cstrong\u003eHymenoptera\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003e~ 200\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"97\"\u003e\n\u003cp\u003e16 (8%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"100\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"151\"\u003e\n\u003cp\u003e\u003cstrong\u003eLepidoptera\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003e58\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"97\"\u003e\n\u003cp\u003e9 (16%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"100\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"151\"\u003e\n\u003cp\u003e\u003cstrong\u003eNeuroptera\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"97\"\u003e\n\u003cp\u003e2 (100%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"100\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"151\"\u003e\n\u003cp\u003e\u003cstrong\u003eTrichoptera\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"97\"\u003e\n\u003cp\u003e2 (25%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"100\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"151\"\u003e\n\u003cp\u003e\u003cstrong\u003eTrombidiformes\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"97\"\u003e\n\u003cp\u003e2 (100%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"100\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"151\"\u003e\n\u003cp\u003e\u003cstrong\u003eTOTAL\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003e830\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"97\"\u003e\n\u003cp\u003e101 (12%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"100\"\u003e\n\u003cp\u003e71\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e56\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e33\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHeat and cold tolerance vary drastically within and among orders\u003c/p\u003e\n\u003cp\u003eWe then tested the inter-order differences of critical thermal limits and thermal scope and found that the median values of CT\u003csub\u003emax\u003c/sub\u003e for each species differed significantly across taxonomic orders (Kruskal-Wallis, \u0026Chi;\u003csup\u003e2\u003c/sup\u003e = 35.558, df = 8, p \u0026lt; 0.001) (Figure 2A). The same was seen for thermal scope, which was also significantly different across orders (\u0026Chi;\u003csup\u003e2\u003c/sup\u003e = 15.354, df = 6, p = 0.0177) (Figure 2C). However, differences across orders were not observed for CT\u003csub\u003emin\u003c/sub\u003e (\u0026Chi;\u003csup\u003e2\u003c/sup\u003e = 12.969, df = 9, p = 0.164) (Figure 2B).\u003c/p\u003e\n\u003cp\u003eFor CT\u003csub\u003emax\u003c/sub\u003e, Araneae ranked as having the highest median values across orders, while the lowest was observed for Diptera. A post-hoc pairwise Dunns tests revealed that Araneae and Lepidoptera had significantly higher median CT\u003csub\u003emax\u003c/sub\u003e compared to species of Diptera (p = \u0026lt;0.001 and 0.0028). Other orders did not differ in CT\u003csub\u003emax\u003c/sub\u003e (Figure 2A). For thermal scope, Araneae and Diptera had the highest and lowest median values, respectively and only these two were found to be significantly different (p = 0.0021) (Figure 2C).\u003c/p\u003e\n\u003cp\u003ePhylogenetic signals show evolutionary constraint of upper thermal limits in arctic arthropods\u003c/p\u003e\n\u003cp\u003eWe found a disparity between phylogenetic groups in CT\u003csub\u003emax\u003c/sub\u003e and thermal scope when these are presented with phylogenetic relations (Figure 3A). This corresponds with the results reported above showing that Araneae had the highest CT\u003csub\u003emax\u003c/sub\u003e with some species having individuals still being active at temperatures above 50˚C. In contrast Dipterans had the lowest CT\u003csub\u003emax\u003c/sub\u003e typically below 35˚C. The comparatively higher CT\u003csub\u003emax\u003c/sub\u003e for Lepidopterans seen in Figure 2, was also observed here across species (and life stages) (Figure 3A). For CT\u003csub\u003emin\u003c/sub\u003e, no apparent trend is observed (Figure 3B) which aligns with results showing no significant effect of order for CT\u003csub\u003emin\u003c/sub\u003e (Figure 2B). Finally, for thermal scope (Figure 3C) Araneae species have the highest values, while Lepidoptera have a lower measure of thermal scope, and the Dipterans the lowest values. To test for phylogenetic signal in each of these thermal limit measures, Pagel\u0026rsquo;s \u0026lambda; is calculated which reveals a strong and highly significant phylogenetic signal for thermal scope (\u0026lambda; = 0.99, p \u0026lt; 0.001), and CT\u003csub\u003emax \u003c/sub\u003e(\u0026lambda; = 0.49, p \u0026lt; 0.001) suggesting that closely related species have a more similar thermal scope and CT\u003csub\u003emax\u003c/sub\u003e than more distantly related species. In contrast, no phylogenetic signal was observed for CT\u003csub\u003emin\u003c/sub\u003e (\u0026lambda; \u0026lt; 0.001, p = 1).\u003c/p\u003e\n\u003cp\u003eIndependent evolution of upper and lower thermal limits\u003c/p\u003e\n\u003cp\u003eA Phylogenetic Generalized Least Squares model (PGLS) revealed that there was a clear positive association between CT\u003csub\u003emax\u003c/sub\u003e and thermal scope across all samples with a slope of 1.06 (SE = 0.09, t = 12.03, p \u0026lt; 0.001) (Figure 4A). CT\u003csub\u003emin\u003c/sub\u003e was also found to be strongly associated with thermal scope, however negatively given the nature of CT\u003csub\u003emin \u003c/sub\u003eas low values are evident of high cold tolerance, resulting in a slope of -0.89 (SE = 0.15, t = -5.77, p \u0026lt; 0.001) (Figure 4B). As opposed to CT\u003csub\u003emax\u003c/sub\u003e, this PGLS model for CT\u003csub\u003emin\u003c/sub\u003e and thermal scope differed from a linear model with a higher intercept for thermal scope (46.10 and 43.12), but otherwise similar coefficients with the linear model having a slope of -1.11 (SE = 0.26, t = -4.24, p = 0.001). However, no correlation was observed between CT\u003csub\u003emin\u003c/sub\u003e and CT\u003csub\u003emax\u003c/sub\u003e with a slope of -0.08 (SE = 0.01, t = -0.77, p = 0.44) (Figure 4C), indicating independent evolution of the two traits. Additionally, no significant relationships were observed between the medians of length of specimens and both CT\u003csub\u003emax\u003c/sub\u003e and CT\u003csub\u003emin\u003c/sub\u003e at species level (R\u003csup\u003e2 \u003c/sup\u003e\u0026lt; 0.001, p = 0.89 and (R\u003csup\u003e2\u0026nbsp; \u003c/sup\u003e= 0.010, p = 0.47) respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProjected future microclimatic temperatures may exceed upper thermal tolerance limits\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eComparing hourly microclimatic measurements from the summer of 2023 in Narsarsuaq with air temperatures in Narsarsuaq in the same period shows microclimatic temperatures far exceeding those measured in the air (Figure 5 and Figure S2). \u0026nbsp;Using the CT\u003csub\u003emax\u003c/sub\u003e estimates reported here, we show that during the entire summer period of 2023 only two species (\u003cem\u003eDelia platura \u003c/em\u003eand \u003cem\u003eCalliphora uralensis\u003c/em\u003e) are likely to have experienced temperatures above their CT\u003csub\u003emax\u003c/sub\u003e (Table S1). However, during this period, these stressful temperatures occurred regularly for the two species at 89% and 77% of days, respectively, in the investigated period.\u003c/p\u003e\n\u003cp\u003eInterestingly, when associating the CT\u003csub\u003emax\u003c/sub\u003e measures to the projected microclimatic temperatures (end-of-century SSP370 scenario), the number of species that will potentially experience temperatures above their upper thermal limit will increase dramatically to 17 (24% of species with CT\u003csub\u003emax\u003c/sub\u003e estimates in our study) and on average they will be exposed to stressful temperatures (defined as time spend exposed to temperatures above their estimated CT\u003csub\u003emax\u003c/sub\u003e) 35.77% of days across the investigated period (Table S1). \u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we investigated thermal tolerances of a wide range of terrestrial arthropods from Narsarsuaq in Southern Greenland. Our results 1) contribute to reducing the knowledge gap on thermal tolerance limits in the Greenlandic terrestrial arthropods, 2) highlight the power of incorporating molecular species identification in arthropod field surveys, 3) reveal evidence of evolutionary constraints in thermal tolerance traits in arctic arthropod species as seen for heat but not cold tolerance in species from other latitudes\u003csup\u003e10,14\u0026nbsp;\u003c/sup\u003eand 4) reveal that periodically a quarter of the investigated species will be exposed to temperatures above their CT\u003csub\u003emax\u003c/sub\u003e under expected future microclimatic conditions. With the present work, we have established an inventory of thermal tolerance measures of south Greenlandic arthropods and a resource with photos and size measurements of investigated species for use in future studies on Greenlandic arthropods.\u003c/p\u003e\n\u003cp\u003eBy combining rapid thermal tolerance measurements with COI barcoding, we enabled a high-throughput identification of 101 species and simultaneous measures of upper and lower thermal limits. Thus, this study provides a novel framework for rapid biodiversity and eco-physiological field assessments and offers new insights into the capacity of 93 Greenlandic arthropod species to withstand low and high temperatures. This constitutes a significant portion of the described Greenlandic terrestrial arthropod species diversity (~8%) and globally represents a large increase in the number of arthropod species where information on thermal tolerances is available\u003csup\u003e20\u003c/sup\u003e. Although molecular methods for species identification are routinely used\u003csup\u003e27\u0026ndash;29\u003c/sup\u003e, there is a large unutilized potential in incorporating barcoding approaches with phenotypic and physiological data for a wide range of species. This combination offers the possibility of covering much of the knowledge gap with relatively little effort, as in this case, where the field work was performed within a week. This is particularly important when working with limited resources or in areas with adverse working conditions or a short growing season as is the case in polar regions\u003csup\u003e30\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn this study, individual imaging of the collected specimen was used as a tool for curation of the taxonomic classification provided using the DNA barcoding. In fact, for 49 individuals it was determined based on the images that the taxonomic order provided by the DNA barcoding was erroneous. This illuminates an inherent weakness in blindly relying upon sequence data for species identification even when including a surface bleaching to avoid cross contamination. Interestingly, we observed an abundance (9 cases) of the parasitic wasp \u003cem\u003eIchneumon sarcitorius\u003c/em\u003e among the erroneous identifications. This could explain the wrong identification in some cases, as it is reasonable to suspect the investigated individuals could be carrying wasp DNA, which was not removed by the bleach treatment. This suggest that images can be a crucial part of species identification when performed on a scale as in the present study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe observed a clear and significant trend that members of the order\u0026nbsp;Araneae had both the highest CT\u003csub\u003emax\u003c/sub\u003e and the lowest CT\u003csub\u003emin\u003c/sub\u003e. Alongside Araneae, Lepidoptera were also found to have a high CT\u003csub\u003emax\u003c/sub\u003e, but this was mainly driven by the larval individuals from the\u0026nbsp;Noctuidae family which have previously been shown to have higher\u0026nbsp;CT\u003csub\u003emax\u003c/sub\u003e in the larval stage\u003csup\u003e31\u003c/sup\u003e. Similarly,\u0026nbsp;Bahrndorff et al.\u003csup\u003e21\u003c/sup\u003e reported a CT\u003csub\u003emax\u003c/sub\u003e value for adult \u003cem\u003eEurois occulta\u003c/em\u003e which was 3˚C lower than reported here, while presenting comparable values for several other species across different orders also tested here including \u003cem\u003eNysius groenlandicus, Psammotettix lividellus\u003c/em\u003e and \u003cem\u003eNabis flavimarginatus\u003c/em\u003e\u003csup\u003e21\u003c/sup\u003e. For the \u003cem\u003ePardosa\u003c/em\u003e genera, our estimates of CT\u003csub\u003emax\u003c/sub\u003e were higher and lower for CT\u003csub\u003emin\u003c/sub\u003e compared to findings in Anthony et al., 2019\u003csup\u003e32\u003c/sup\u003e.\u0026nbsp;Lepidoptera and Araneae typically have high upper thermal limits with estimates in the literature in the range 45 \u0026ndash; 50˚C\u003csup\u003e31,33\u003c/sup\u003e and 42 - 49˚C\u003csup\u003e32,34\u003c/sup\u003e respectively. Our results for these taxa were in alignment with these findings and generally\u0026nbsp;upper thermal limits across the other taxonomic orders corresponds to what has previously been reported in the literature. A general finding in our study is that Diptera has low and variable heat tolerance with CT\u003csub\u003emax\u003c/sub\u003e values in the range 22 \u0026ndash; 43˚C. This aligns with literature findings\u003csup\u003e7,21,35\u0026ndash;38\u003c/sup\u003e highlighting the large intra-order variability and the importance of high taxonomic resolution in thermal tolerance studies. Likewise, Coleoptera and Hymenoptera have been reported to have CT\u003csub\u003emax\u003c/sub\u003e between 38 and 45˚C\u003csup\u003e39\u0026ndash;41\u003c/sup\u003e, aligning with our findings. In comparing and interpretating CT\u003csub\u003emax\u003c/sub\u003e and CT\u003csub\u003emin\u003c/sub\u003e values across studies, it is however important that conditions including ramping rates, start temperature and pretest rearing temperatures can have marked impact on the estimates\u003csup\u003e42,43\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eEstimates of upper thermal limits obtained in this study were shown to be significantly dependent upon the phylogenetic relations across the tested species (Figure 3A), meaning that closely related species were more likely to have similar measures of upper thermal tolerance than distantly related species. This suggests that the upper thermal tolerance of the arctic arthropods investigated here show little capacity to adapt to higher temperatures through evolutionary adaptation. While this have been shown for other species of arthropods\u003csup\u003e13,14,44\u003c/sup\u003e, it has not previously been shown across arctic terrestrial arthropods, further highlighting the knowledge gap in these rapidly changing sensitive environments. Importantly, thermal scope of the tested arthropods (Figure 3C), similarly exhibited a strong phylogenetic signal suggesting that this trait is also phylogenetically constrained. Arctic and subarctic (and polar in general) climates vary dramatically on a diurnal and seasonal basis (Figure S1A and S2) and with climate change, we expect to see increases in the frequency of climatic extremes\u0026nbsp;and more unpredictable thermal fluctuations\u003csup\u003e45\u0026ndash;47\u003c/sup\u003e. Our data suggest that the ability to evolutionary adapt to high temperature fluctuations is limited, which could have serious implications for the abundance and distribution of the future Greenlandic\u0026nbsp;terrestrial arthropods.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe find that both upper thermal tolerance and thermal scope is conserved across species of the same order despite quite different modes of life as seen in the spiders with the brush and herb-living mesh-weavers (\u003cem\u003eDictyna major\u003c/em\u003e and \u003cem\u003eEmblyna borealis)\u0026nbsp;\u003c/em\u003eand the ground-dwelling wolf spiders (\u003cem\u003ePardosa groenlandica\u003c/em\u003e and \u003cem\u003ePardosa furcifera)\u003c/em\u003e\u003cem\u003e\u003csup\u003e18\u003c/sup\u003e\u003c/em\u003e. Microhabitat temperatures in their habitats are expected to differ between the two groups\u003csup\u003e48,49\u003c/sup\u003e but despite this, they all exhibit exceptionally high thermal tolerances (Figure 4A and 4C). Our results and literature findings thus support that the phylogenetic constraints observed here are likely not caused by shared environmental history and evolutionary pressures as suggested to be a causative agent of phylogenetic signals by Freckleton and Jetz\u003csup\u003e50\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFinally, by projecting future diurnal microclimatic temperatures according to climate predictions, we highlight the issue of increased potential exposure to stressful warm temperatures. A quarter of the tested species reported here will potentially experience future microclimatic temperatures exceeding their upper critical thermal limit. This finding is contrary to that of\u0026nbsp;Deutsch et. al.\u003csup\u003e51\u003c/sup\u003e, who argued that terrestrial ectotherms at temperate latitudes were far from their critical limits and thus not susceptible to being negatively affected by global warming as opposed to tropical species who had a smaller thermal safety margin and thus would be more negatively affected. However, as also pointed out by others\u003csup\u003e5,52\u003c/sup\u003e, the use of such measures of thermal safety margins can be problematic as they often rely on air temperature, which, as shown here, is not representative of the micro-environment experienced by small arthropods. Although behavioural thermoregulation and plasticity may protect arthropods against future warmer and more variable temperatures, our finding suggests that warming macro- and micro-climates in Arctic and sub-Arctic regions will have profound effects on the ecology, behaviour and distribution of the arthropod community\u003csup\u003e53\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eWhile individual studies reporting thermal limits for a wide range of arthropod species is essential to expand the limited knowledge base, it is also crucial to coordinate a joint effort to conglomerate all this information into large, united databases, as information is currently scattered and hard to assess for researchers and practitioners. Thus, to enable more powerful analysis that illuminate trends, not just locally, but globally and over a longer time scale, it is essential to make data as comparable and easily accessible as possible. The data and knowledge provided here are highly valuable for future studies aiming at predicting the potential changes in diversity, abundance and distribution of arthropods with a changing climate and how this will impact on complex ecological networks.\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eChanges in abundance and distribution of arctic terrestrial arthropods are important to understand as they play crucial roles in ecosystems, including pollination, nutrient cycling (decomposition), seed dispersal\u003csup\u003e54\u003c/sup\u003e, and functioning as disease vectors for both plants and animals, including humans\u003csup\u003e55,56\u003c/sup\u003e. Our study provides important insights into the thermal tolerances of a large proportion of Greenlandic arthropods and reveals stark phylogenetic signals for both CT\u003csub\u003emax\u003c/sub\u003e and thermal scope. The observed phylogenetic signal suggesting evolutionary constraints for these measures along with our results showing that a quarter of the investigated species will likely be exposed microclimates above their upper thermal limits for extended time periods in the future, suggest that climate changes in arctic regions will have profound impacts on arthropod diversity, abundance and distribution.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eSample collection\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTerrestrial arthropods (insects, arachnids and collembola) were collected in Narsarsuaq, Southern Greenland (61.160\u0026deg;N, 45.424\u0026deg;W). This region is characterized by cool temperatures, long winters and short summers (average -7 \u0026deg;C in January and +11˚C in July, Figure S1A). The area has recently experienced rapidly increasing temperatures with mean annual temperature anomalies exceeding +1˚C on average (range -1.7 to +4.4˚C) since 2000 compared to the 1979-2023 baseline (Figure S1B). Importantly, summers in the area are very thermally variable with maximum temperatures at ground level exceeding 30˚C\u003csup\u003e57\u003c/sup\u003e. Ambient temperature was measured 7.5 cm above ground using TMS-4 loggers (TOMST) at 5 min resolution in two locations in Narsarsuaq (Figure S2). The study site is a heath-like, grass- and shrub covered area, where arthropods were collected from grasses and bushes using a sweep net or a pooter (a flexible tube used to catch animals directly from leaves or the ground). All individuals used in the experiment were caught in a 200 x 200 m area less than 100 m from the laboratory facilities. Animals were collected in the summer season from the 12\u003csup\u003eth\u003c/sup\u003e of August until the 17\u003csup\u003eth\u003c/sup\u003e of August 2023. All animals were collected during the day between 09:00 and 19:00, and all days throughout the study period showed similar temperature profiles (Figure S2). A subset of the collected arthropods was chosen based on morphological novelty (aiming at having as many different species as possible tested) and transferred to individual 5 mL glass vials with plastic screw-lids within 30 minutes after being collected. The vials with arthropods were kept outside of the laboratory in the shade for maximum one hour before being tested for either heat or cold tolerance. Animals were collected on six separate days constituting six batches. In each batch, between 80 and 138 individuals were tested. In total, 701 individuals were tested for either heat or cold stress tolerance. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCritical Thermal Limits\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVials with arthropods were placed randomly in a rack before being submerged in a circulating water bath for Critical Thermal maximum (CT\u003csub\u003emax\u003c/sub\u003e)or Critical Thermal minimum (CT\u003csub\u003emin\u003c/sub\u003e) assessments. Liquid in the water baths was kept at 20˚C until vials with animals had been submerged, whereafter the temperature was increased or lowered by 0.1˚C per minute to assess CT\u003csub\u003emax\u003c/sub\u003e or CT\u003csub\u003emin\u003c/sub\u003e, respectively. The temperature of onset of coma, i.e. at which the last movement was observed in response to agitation (facilitated by softly knocking on the vials with a metal rod), was recorded as the thermal tolerance limit to heat or cold stress for that individual\u003csup\u003e43\u003c/sup\u003e. Immediately following the critical thermal limit test, each individual was transferred to a 1.5 mL Eppendorf tube before being stored at -20˚C until imaging and DNA extraction (to be used for species identification based on DNA barcoding). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImaging\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImaging was performed using an Olympus SZX10 stereo microscope, and Olympus DP74 colour camera (Olympus Corporation). Animals were thawed in small batches of between 1 and 10, before being placed on a 1% agarose plate facilitating proper positioning of the animals. Each animal was pictured from multiple angles, including ventral, dorsal and profile in addition to identifying markers, i.e. wing webbings or cornicles. Additionally, each animal was measured from the tip of the head to the end of the abdomen and a micro-ruler was imaged at the same magnification for scale. Following imaging, each animal was returned to the Eppendorf tube and kept at 5˚C until DNA extraction was performed on the following day. Representative images of each observed species can be found in Appendix 1 while images of all individuals can be accessed at Dryad depository (https://doi.org/10.5061/dryad.1g1jwsv6w). \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA extraction\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll specimens were washed twice in 200 uL 2.5% bleach solution (Thermo Fisher Scientific) and twice in 800 uL UV treated ELGA water to minimize cross contamination from sample collection and imaging. Total genomic DNA was extracted from all individuals using the DNeasy Blood and Tissue kit (Qiagen) according to the manufacturer\u0026apos;s instructions for extraction of DNA from insects. Agilent Tapestation 2200 was used to run Genomic DNA Screentape (Agilent Technologies) to assess DNA quality. Following DNA extraction and quality control, samples were stored at -80˚C.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePCR, cleanup and COI barcode sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSamples of extracted DNA from all tested animals were diluted 1:10 in 2x UV treated nuclease free water. All samples were amplified using either the\u0026nbsp;HCO1490/LCO2198 primer pair\u003csup\u003e58\u003c/sup\u003e or the BF2/BR3 primer pair\u003csup\u003e59\u003c/sup\u003e (Appendix 2). For the former primer pair, a single touchdown PCR reaction was performed for each sample in a reaction volume of 25 uL (1X PCRBIO Ultramix (PCR Biosystems), 400 nM of each primer and 2 uL template DNA). The following thermocycler settings were used for the touchdown PCR: Initial denaturation at 95˚C for 2 minutes followed by 16 cycles of: 95˚C for 30 sec, 62˚C (-1 \u0026deg;C each cycle) for 1 minute, 72˚C for 1 minute, followed by 25 cycles of: 95˚C for 30 sec, 46˚C for 1 minute, 72˚C for 1 minute followed by a final extension at 72˚C for 5 minutes. For the latter primer pair, a single PCR was performed for each sample in a reaction volume of 25 uL (1X PCRBIO Ultramix (PCR Biosystems), 400 nM of each primer and 2 uL template DNA). The following thermocycler settings were used for the PCR: Initial denaturation at 95˚C for 2 minutes followed by 25 cycles of: 95˚C for 30 sec, 44˚C for 1 minute, 72˚C for 45 seconds, followed by a final extension at 72˚C for 5 minutes.\u003c/p\u003e\n\u003cp\u003eAll samples were subsequently purified using the Monarch PCR \u0026amp; DNA Cleanup Kit (New England Biolabs) according to the manufacturer\u0026apos;s instructions. DNA concentration of purified samples was determined using Qubit HS dsDNA kit (Thermo Fisher Scientific) and a TECAN Infinite F200 Pro (Tecan Life Sciences) after which all samples that had successfully amplified, were prepared for Sanger sequencing by Eurofins Genomics (Germany).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpecies identification using barcoding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe obtained COI sequence for each animal was used for species identification using BLAST\u0026rsquo;s blastn function\u003csup\u003e60\u003c/sup\u003e. \u0026nbsp;An identity percentage above 98% and a report from at least two different accession numbers was considered reliable species identification. For sequences with an identity percentage lower than 98%, the best result was used, manually checked, included and noted (Appendix 2). For the taxonomic analysis, one reference sequence from the BOLDsystems database was included in the analysis for each identified species, with a preference for sequences from samples collected in subarctic regions. Every sample was manually curated by comparing recorded taxonomy to literature of local arthropod diversity\u003csup\u003e18\u003c/sup\u003e under a principle of conservative taxonomic assignments. Subsequently, all assigned taxonomies were compared to images of the sequenced animal\u003csup\u003e18\u003c/sup\u003e. In 49 cases, manual curation revealed obvious mismatches between images and recorded taxonomy e.g. wrong taxonomic order and these samples were removed from further analysis. All sequences obtained were aligned using the MEGA software\u003csup\u003e61\u003c/sup\u003e and the ClustalW algorithm with a gap opening quality of 15.00 and a gap extension penalty set at 6.66. Sequences that did not allow for alignment on account of short sequence length were removed from the analysis. All aligned sequences were shortened to consist of only a 410 bp region. Consensus sequences were generated from the aligned sequences obtained in this study using BioEdit (0.6). All phylogenetic trees were generated from aligned consensus sequences using MEGA software and the Neighbour-joining tree function with 1000 bootstrap iterations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eModelling exposure to microclimatic temperatures exceeding thermal thresholds\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate how the tested species may be affected by projected increases in diurnal temperatures, we compared species-specific CT\u003csub\u003emax\u003c/sub\u003e estimates to measured and expected future microclimatic temperatures in the period 19 July to 23 Aug 2023. First, we estimated hourly air temperatures (2 m height) at the sampling location with the \u0026lsquo;NicheMapR\u0026rsquo; package v3.2.1\u003csup\u003e62\u003c/sup\u003e (Figure S2). As no reliable projections of future microclimates exists yet we first calculated the differences in air temperatures between current (2000-2021) and projected future conditions (2081-2100; SSP370 scenario) (see Figures S1-2 for details). This difference, \u0026Delta;T\u003csub\u003eair\u003c/sub\u003e,was added to the measures obtained from our TOMST loggers to describe the current microclimate (T\u003csub\u003emicro_current\u003c/sub\u003e). We obtained estimates of hourly future microclimate temperatures using the formula: T\u003csub\u003emicro_future\u0026nbsp;\u003c/sub\u003e= T\u003csub\u003emicro_current\u0026nbsp;\u003c/sub\u003e+ \u003cem\u003ea\u003c/em\u003e∙e\u003csup\u003e(\u003cem\u003eb\u003c/em\u003e∙\u0026Delta;Tair)\u003c/sup\u003e, where \u003cem\u003ea\u0026nbsp;\u003c/em\u003eand \u003cem\u003eb\u003c/em\u003e are coefficients based on the exponential relationship between air and microclimate temperatures (different for day and nighttime; Figures S2-3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing species identification, outliers (for CT\u003csub\u003emax\u003c/sub\u003e and CT\u003csub\u003emin\u003c/sub\u003e) within each species were identified and removed if having a median absolute deviation (MAD) of 3 or more, removing 53 samples\u003csup\u003e63\u003c/sup\u003e. This was done to remove individuals with extreme low CT\u003csub\u003emax\u003c/sub\u003e and high CT\u003csub\u003emin\u0026nbsp;\u003c/sub\u003esuggesting that they were harmed in the process of catching them and / or getting them into the test tube (all analyses was also performed without removing outliers and conclusions remained the same). Thermal scope was calculated for species with a measure of both CT\u003csub\u003emax\u003c/sub\u003e and CT\u003csub\u003emin\u003c/sub\u003e as the absolute difference between medians of bootstrap resampled data from each species using 10,000 iterations. Kruskal-Wallis tests were performed to compare medians within groups to determine potential differences. In case of significant differences, a post hoc Dunn\u0026rsquo;s test was performed to compare groups. To investigate the presence of phylogenetic signals in each trait (CT\u003csub\u003emax\u003c/sub\u003e, CT\u003csub\u003emin\u003c/sub\u003e and thermal scope), Pagel\u0026rsquo;s Lambda was determined using the phytools R package running 1000 simulations\u003csup\u003e64,65\u003c/sup\u003e.\u0026nbsp;Correlations between thermal tolerance traits were corrected for phylogenetic signals by performing Phylogenetic Generalized Least Squares (PGLS) models in the nlme R package\u003csup\u003e66\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003eWe thank Frederik Kj\u0026aelig;r Nielsen, Christian Dupont Danielsen and Andreas M\u0026oslash;lgaard Andersen for technical assistance in the laboratory, Elma Huremovic for assistance in literature research and Simon Bahrndorff for constructive comments on earlier versions of the manuscript and for supporting access to field facilities in Narsarsuaq, Greenland.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJonas Bruhn Wesseltoft:\u003c/strong\u003e Conceptualization, Methodology, Data Curation, Formal Analysis, Visualization, Writing \u0026ndash; Original Draft.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMichael \u0026Oslash;rsted:\u003c/strong\u003e Formal Analysis, Funding Acquisition, Writing \u0026ndash; Review and Editing, Supervision.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNadieh de Jonge:\u003c/strong\u003e Methodology, Formal Analysis, Writing \u0026ndash; Review and Editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMichael M. Hansen:\u0026nbsp;\u003c/strong\u003eWriting \u0026ndash; Review and Editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eToke Thomas H\u0026oslash;ye:\u0026nbsp;\u003c/strong\u003eResources, Writing \u0026ndash; Review and Editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTorsten Nygaard Kristensen:\u003c/strong\u003e Conceptualization, Methodology, Resources, Project Administration, Funding Acquisition, Writing \u0026ndash; Original Draft, Supervision.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThe work was funded by the European co-funded Partnership BiodivClim-191 ASICS (0156-00024B to TNK), and by the Novo Nordisk Foundation (grant NNF23OC0082599 to M\u0026Oslash;). Laboratory facilities in Narsarsuaq, Greenland, was supported by the Greenland Integrated Observing System (GIOS) (the Danish National Fund for Research Infrastructure (NUFI)).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLicenses:\u003c/strong\u003e All animals were collected in accordance with regulations from the government of Greenland under the non-exclusive license no. G23-006 for utilization of Greenland genetic resources as granted to Aalborg University for use between 01/05-2023 until 31/09-2023.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJ\u0026oslash;rgensen, L. B., \u0026Oslash;rsted, M., Malte, H., Wang, T. \u0026amp; Overgaard, J. Extreme escalation of heat failure rates in ectotherms with global warming. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e611\u003c/strong\u003e, 93\u0026ndash;98 (2022).\u003c/li\u003e\n\u003cli\u003eAnt\u0026atilde;o, L. 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For arctic ectotherms in particular, thermal tolerance limits and adaptive potential are mostly unknown. Such knowledge is urgently needed to predict climate change impacts on future distributions of biodiversity in these rapidly changing environments. Here, we provide new data on upper and lower thermal limits of 93 Greenlandic species of insects, arachnids, and collembolans identified using barcode sequencing representing ~8% of described terrestrial Greenlandic arthropod species. We found pronounced differences in heat and cold tolerance among species and a strong phylogenetic signal for both heat tolerance and thermal scope (difference between upper and lower thermal limits), suggesting that terrestrial Greenlandic arthropods are evolutionarily constrained in their capacity to cope with increasing and more variable future temperatures. Further, with projected future increases in microclimatic temperatures induced by climate change, we reveal a marked increase in the number of species that will experience potentially stressful temperatures for prolonged periods of time. Together, our results suggest that climate change will likely result in substantial changes in distributions and abundances of Greenlandic terrestrial arthropods. ","manuscriptTitle":"Heat tolerance and thermal scope are evolutionarily constrained in Greenlandic terrestrial arthropods","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-23 06:00:19","doi":"10.21203/rs.3.rs-5640068/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d96e9a71-0559-46b6-83ec-0f37bf34e869","owner":[],"postedDate":"December 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":41772546,"name":"Biological sciences/Ecology/Ecophysiology"},{"id":41772547,"name":"Biological sciences/Biological techniques/High-throughput screening"},{"id":41772548,"name":"Biological sciences/Ecology/Evolutionary ecology"}],"tags":[],"updatedAt":"2025-01-06T21:30:17+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-23 06:00:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5640068","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5640068","identity":"rs-5640068","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}