Lacking sex-specific temperature preferences of 9 coexisting temperate sepsid dung fly species (Diptera: Sepsidae)

Lacking sex-specific temperature preferences of 9 coexisting temperate sepsid dung fly species (Diptera: Sepsidae) | 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 Research Article Lacking sex-specific temperature preferences of 9 coexisting temperate sepsid dung fly species (Diptera: Sepsidae) Ramon Dallo, Martin Kapun, Wolf Blanckenhorn This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4252799/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 Many similar sepsid dung fly species coexist on European pastures, contradicting conventional wisdom of niche theory and competitive exclusion. We hypothesized that closely-related sepsid species on the same pasture in Switzerland avoid each other by having different spatio-temporal microhabitat niche preferences, thus enabling coexistence. A thermal racetrack experiment in the laboratory tested the thermal preferences of males and females of 9 coexisting temperate Sepsis dung fly species from Switzerland at two acclimation temperatures. The sepsid species investigated here showed no strong differences in thermal preferences. Flies of all species preferred to settle at cooler temperatures, and otherwise utilized the entire range (from 12°C to 30°C) offered for their activities. This was the case for both sexes, and also for both acclimation temperatures (18°C, 24°C). Our findings suggest that physiological thermal adaptation or acclimation is not an important mechanism by which adult sepsid flies avoid interspecific competition. Our experiment supports previous findings of widespread sepsid flies lacking local adaptation but high phenotypic plasticity, again highlighting the necessity of experimentally assessing putative biological mechanisms facilitating coexistence. Thermal preference Sepsidae niche theory competition competitive exclusion coexistence temperature thermal racetrack Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Temperature is a key environmental factor that directly influences the physiology of all living organisms by affecting biological processes from the molecular to the ecosystem levels (Gilbert and Miles 2017 , Hoffmann 2010 , Huey and Berrigan 2001 , Kellermann et al. 2012a,b). Temperature shapes the entire organismal life history, encompassing activity, development, reproduction and survival. In contrast to endotherms, ectotherms have only limited control over their own body temperature, so they are considerably affected by all environmental variation experienced (Angiletta et al. 2004, Angiletta 2009). By choosing niches with temperatures close to their preferred optimum, ectotherms can adjust their body temperature towards their preferred temperature. Thus thermoregulation is mainly achieved through behavioral plasticity (Stevenson 1985 ). Preferred temperatures might change depending on life stage (propagule, juvenile, adult, etc.) or activity (foraging, mating, egg-laying, etc.; Addeo et al. 2022 , Blanckenhorn et al. 2014 , MacLean et al. 2019a, Rajpurohit and Schmidt 2016 ). Nevertheless, every organism has a temperature range in which it can survive and reproduce, as delimited by their so-called thermal performance curve (Angilletta 2006 , 2009 , Berger et al. 2014 , Blanckenhorn et al. 2021a , Huey and Kingsolver 1989 , Huey and Stevenson 1979 ). Experiencing temperatures outside this range can in the worst-case lead to the death of the organism (Gilbert and Miles 2017 , Lutterschmidt and Hutchison 1997 ). Within this range every organism likely has a thermal optimum T opt that varies according to its precise physiological needs, a temperature it therefore should prefer given no further ecological constraints (Huey and Kingsolver 1989 , Huey and Stevenson 1979 , Gilbert and Miles 2017 ). Together with these (internal) physiological preferences, the (external) environmental temperature thus co-defines an organism’s ecological niche, and ultimately the species composition in a given habitat (Angilletta 2004 , Gilbert and Miles 2017 , Huey and Berrigan 2001 , Magnuson et al. 1979 ). Another important factor shaping species composition and coexistence is competition (Case and Gilpin 1974 , Gause 1934 ). Competition between different species occurs over various essential resources such as food, time, or (nesting) space (Crombie 1947 , de Camargo et al. 2016 , Tilman 1987 ). The competitive exclusion model (Armstrong and McGehee 1980 , den Boer 1986 , Gause 1934 , Hardin 1960 , Levin 1970 ) and ecological niche theory (Alley 1982 , Buckley er al. 2015, Case and Gilpin 1974 , Holt et al. 1994 , Kylafis and Loreau 2011 , MacArthur and Levins 1967 , Tilman et al. 1981 ) predict that when different species are competing for the same resources, a few dominant species will evolve to exclude all others. Moreover, closely related species compete more severely for the same resources (Cavender-Bares et al. 2009 , Silvertown et al. 2001 , Violle et al. 2011 , Webb et al. 2002 ) because they are ecologically more similar (Darwin 1859 ; the competition-relatedness hypothesis: Cahill et al. 2008 ). The predicted result overall should be a community composed of few species, and even fewer related species, in any particular habitat. Nonetheless, previous work documents an opposite pattern for sepsid dung flies (Diptera: Seaside; Rohner 2015 , Rohner et al. 2019 , Blanckenhorn et al. 2020 , 2021b). This group of flies plays an essential ecological role as primary degraders of rotting plant material and animal feces. Sepsid flies have a broad world-wide distribution across various biogeographic zones (Ang et al. 2013 , Pont and Meier 2002 ). In Europe multiple temperate species share the same ecological niche, livestock pastures and more generally grasslands, where eight or more closely related species may occur in sympatry in and around their breeding substrates, most commonly cow dung (Rohner 2015 , Rohner et al. 2014 , 2015 , 2019 ). It is argued that competitive exclusion does not hold if competing species have a means of evading each other (Gao et al. 2020 , Kronfeld-Schor and Dayan 2003 , Loreau and Ebenhoh 1994 , Schoener 1974 , Wiens 1986 ). The circadian or diurnal cycle of an organism can deflect interspecific competition, thus potentially facilitating coexistence via spatial and/or temporal niches (Corral et al. 2022 , Daan 1982 , Hagey et al. 2016 , Hood et al. 2021 , Kenagy 1973 , Richards 2002 , Schoener 1986 , Shorrocks et al. 1984 , Wiens 1986 ). As revealed by long-term repeated sampling in time and space, the abundance of various coexisting sepsid species in Switzerland depends on geography, altitude, the season, and even the time of day (Khelifa et al. 2019 , Rohner et al. 2014 , 2015 , 2019 ). One key factor potentially mediating this spatio-temporal abundance variation is temperature. Having different thermal preferences therefore might be a potential mechanism by which ecologically and phylogenetically similar sepsid species avoid each other. We here investigate whether widespread and often coexisting European sepsid species are differentiated in terms of thermal microhabitat preferences in accordance with their geographic, altitudinal, seasonal and/or diurnal distribution (Rohner et al. 2014 , 2015 , 2019 ). We generally hypothesize that flies experiencing higher average temperatures in nature should also prefer higher temperatures in the laboratory. We tested this by comparing the thermal preferences of 9 closely related black scavenger or dung fly species of the genus Sepsis native to Switzerland in a laboratory thermal racetrack, a classic approach in this field of climate research (e.g., Deal 1941 , Dillon et al. 2009 for a review, MacLean et al. 2019a, b, Yamamoto 1994 ). In doing so, we also considered well-established effects of acclimation temperature (MacLean et al. 2019a,b) as well as potential differences between the sexes relating to their different life histories and sexual roles (Addeo et al. 2022 , Rajpurohit and Schmidt 2016 ). Methods Fly populations Nine species of sepsid flies were used for our experiment: S. punctum , S. neocynipsea , S. cynipsea , S. duplicata , S. thoracica , S. orthocnemis , S. violacea , S. fulgens , S. flavimana (see Ang et al. 2013 , Pont and Meier 2002 ). Flies of all species were caught originally at Ziegelhütte in Schwamendingen, Zürich, Switzerland, in 2022 and subsequently bred individually to higher numbers as iso-female lines (i.e., the offspring of one field-caught female) for no more than three generations in the laboratory. All lines were held in 1.5-liter group containers with water, sugar and cow dung (see e.g. Puniamoorthy et al. 2012 for general rearing methods) in walk-in climate chambers at both 18°C and 24°C. Five to ten iso-female lines were later combined to create the mixed populations used in our experiment. Thermal gradient experiment Thermal preferences were tested with a self-constructed racetrack apparatus modelled after similar devices used in the past for small insects (see Dillon et al. 2009 , see supp. Fig. S0). The racetrack consisted of an aluminum base plate and a plastic top. The plastic top was shaped into two tracks (each track of 700 mm length x 10 mm width x 8 mm height) running along the base plate. A strip of white filter paper was placed between the baseplate and the plastic top to assure contrast and maintain humidity. At each end underneath the base plate a Peltier element (P&N Technology, Xiamen Fujian China) was positioned for either heating or cooling. Underneath each Peltier element a heat or cold sink (Fischer Electronic, Germany) lead off excess heating or cooling energy (the former into a replenishable ice container). Forty minutes before the first run of the day the power source of the Peltier elements was switched on to produce a constant linear temperature gradient from ca. 12℃ to 30℃ along which individual flies could align according to their preferences. All runs were conducted in a completely dark room at 21℃ (± 0.5℃). Groups of 4 flies of the same sex and acclimation treatment were aspirated into one track via a hole in the middle. Flies had been reared at either 18℃ or 24℃ for several generations (acclimation treatment). We thus had 36 treatment combinations (9 species * 2 sexes * 2 rearing temperatures), and flies from the same treatment combination were used in both tracks for any given run. Three thermometers (Analog devices, USA) recorded the temperature every 100 seconds at both ends of the racetrack and in the middle. Every 5 minutes an infrared camera took a picture from above that was also saved to an attached Raspberry pi 3B + computer (Raspberry Pi Foundation, UK). After 25 minutes, at which point the flies were typically resting, a run was stopped, and the last pictures were used for the final analysis. Four different orientations of the racetrack in the room were implemented by (1) rotating the racetrack situated on a rolling cart by 180 degrees, thus creating two different placements in the room, and (2) by switching the heating and cooling side for each placement in the room (see Fig. 1 ). Runs of each treatment combination were equally distributed in the morning or afternoon. Fly age was not tracked individually, but the adult flies were from 7 to 13 days old (post-emergence). A total of 2336 flies were used. All pictures were analyzed in ImageJ (Schneider et al. 2012 ). A custom python script (written by Martin Kapun) calculated a linear temperature gradient from the three thermometer readings, the linearity of which had been verified at the start of our study. The location coordinates (at the end of each 25 min run) of the black flies on white filter paper were determined and then converted to the corresponding temperature along the linear gradient (script by Martin Kapun). Statistical Analysis A generalized linear mixed model (GLMMs) with random factors (glmer) was used from the package “lme4” in R (Bates et al. 2015 , R Core Team 2021). Species, sex, acclimation temperature, and orientation were entered as fixed factors in a type III ANOVA using the “car” package (Fox and Weisberg 2019 ), initially including all relevant interactions, which were subsequently removed from the model bottom-up if non-significant. Group ID was the random factor. Additionally, analogous GLMMs with sex, acclimation temperature and orientation as fixed factors and group ID as a random factor were implemented for each species separately (see table S1 appendix). Some pairwise comparisons of species and/or acclimation temperatures were tested with Tukey’s HSD tests performed in the package “emmeans” (Lenth 2021). All plots were created using the package “ggplot2” (Wickham 2016 ). The effect of phylogeny (Su et al. 2016) was tested using a Bayesian multilevel model in the package “brms” (Bürkner 2021 ). For this analysis a new ranking variable was introduced. Every species received a unique rank, ranging from 1 (coldest) to 9 (hottest), according to their realized temperature niche in the wild derived from their geographic distribution from Pont and Meier ( 2002 ) and Rohner et al. ( 2015 , 2019 ). The analysis showed no significant effect of phylogeny on the results, therefore the original linear model without phylogeny was presented in the end. Results The frequency distribution of fly positions for all orientation combinations is given in Fig. 2 . Flies mostly settled at the cool end of the temperature gradient, although for orientation 3 the fly distribution is rather uniform across all temperatures, for unknown reasons. Given any external confounding factor, we would have expected orientations 1 & 4, as well as 2 & 3, to have yielded similar outcomes. Based on this result we performed two analyses: one including data for all four orientations, with orientation as a random blocking effect, and one conservative analysis using only orientations 1 & 4 (reported below). In the end, exclusion of orientations 2 & 3 only slightly changed the overall species and sex results. While we found significant temperature preference variation across the 9 species (p < 0.001, F(8, 269) = 5.4; see Table 1 , Fig. 3 ), we did not observe good correspondence with their natural temperature niches inferred from previous work, all of which admittedly are very similar (Rohner et al. 2015 , 2019 , Khelifa et al., 2019 ). Overall, all species preferred to rest at cooler temperatures, nevertheless using the whole temperature spectrum (Fig. 3 ). On average, S. cynipsea and S. punctum chose the warmest temperatures > 15°C, while S. flavimana and S. orthocnemis preferred the lowest temperatures around 13°C (Fig. 3 ). Table 1 ANOVA Output including all 4 orientations (top), or orientations 1 & 4 only (bottom). Anova of GLMM model with all orientations MS df den. df F P Species 55.27 8 554.61 4.24 < 0.001 Acclimation Temperature 157.97 1 554.61 12.13 < 0.001 Sex 17.04 1 554.67 1.31 0.253 Orientaion 2020.24 3 554.60 155.17 < 0.001 Species:Acclimation Temperature 50.58 8 554.61 3.88 < 0.001 Species:Sex 14.47 8 554.61 1.11 0.354 Anova of GLMM model with orientations 1 & 4 MS df den. df F P Species 34.16 8 269.27 5.40 < 0.001 Acclimation Temperature 43.96 1 269.31 6.95 0.009 Sex 11.33 1 269.36 1.79 0.182 Orientaion 0.01 1 269.24 0.00 0.968 Species:Acclimation Temperature 32.14 8 269.28 5.08 < 0.001 Species:Sex 15.74 8 269.32 2.49 0.013 The thermal preference of males and females did not systematically differ across species, but there was a significant interaction between sex and species indicating unsystematic sex differences across species (p = 0.013, F(8, 269) = 2.49; Fig. 5 , Table 1 ). Only for S. duplicata (p = 0.008, F(1, 27) = 8.13; Table S1 ) and S. fulgens (p = 0.029, F(1, 27) = 5.29; Table S1 ) was the main effect of sex significant, with males preferring higher temperatures than females (Fig. 5 ). Flies acclimated to the higher temperature (24℃) showed no preference for higher temperatures in general, although the acclimation effect was overall significant in the right direction (p = 0.009, F(1, 269) = 6.95; Table 1 ), as was the interaction between species and acclimation temperature (p < 0.001, F(8, 269) = 5.08; Table 1 ). This resulted from 3 species showing significant differences: S. flavimana (p < 0.001, F(1, 27) = 22.72; supp. Table S1 ) and S. violacea (p < 0.001, F(1, 27) = 18.48; Table S1 ) flies acclimated to the higher temperature (24℃) indeed preferred higher resting temperatures, while S. neocynipsea flies so raised (p = 0.024, F(1, 138) = 5.24, Table S1 ) preferred lower temperatures (Fig. 4 ). Discussion Many temperate species of the dung fly genus Sepsis coexist in high numbers in European pastoral habitats, contradicting conventional wisdom of competitive exclusion (Armstrong and McGehee 1980 , den Boer 1986 , Gause 1934 , Hardin 1960 , Levin 1970 ) and ecological niche theory (Alley 1982 , Buckley er al. 2015, Case and Gilpin 1974 , Holt et al. 1994 , Kylafis and Loreau 2011 , MacArthur and Levins 1967 , Rohner et al. 2019 , Tilman et al. 1981 ). We thus expected such coexistence of closely-related sepsid species on the same pasture in Switzerland to be avoided by preferring different spatio-temporal microhabitat niches (Blanckenhorn et al. 2020 , 2021a ). Temperature has a great effect on the behavior of any organism, especially of small ectotherms (Angiletta 2009), so we here specifically hypothesized and tested in the laboratory whether 9 coexisting Sepsis species have different thermal preferences. Thermal avoidance may occur along latitude or altitude, or over the season, in the extreme over the time course of a day (Rohner et al. 2015 , 2019 , Roy et al. 2018 ). A thermal racetrack experiment, by now a classic approach in thermal research of small organisms (Dillon et al. 2009 ), did not reveal very different thermal niches of the temperate sepsid species investigated, at least in the laboratory. Flies of all species preferred to settle (or rest) at cooler temperatures between 13°C and 15°C, but regularly utilized the entire range between 12°C and 30°C offered for their activity (see Fig. 3 ). This was the case for flies of both sexes, and also for both acclimation temperatures used here (18°C & 24°C). Based on previous studies (Pont and Meier 2002 , Rohner et al. 2015 , 2019 , Roy et al. 2018 , Khelifa et al. 2019 ) we had expected the thermal preferences of our 9 knowingly similar sepsid species co-occurring regularly in Swiss grasslands to roughly accord with their geographic distribution in nature. Our ranking was based on the following factors: 1) The distributional data in the latitudinal dimension: the more northern the range of a given species, the lower their assumed thermal preference (Pont and Meier 2002 , Roy et al. 2018 ). 2) The seasonal abundance of species in Switzerland: if a species is more abundant in summer, we assumed a higher temperature preference (Rohner et al. 2019 ). 3) The altitudinal distribution and abundance of species in Switzerland, assuming that species reaching higher altitudes also have a lower preferred temperatures (Blanckenhorn 1997 , Rohner et al. 2015 ). 4) As additional qualitative support of our ranking, we considered the distribution of the same sepsid species on the British Isles (Pont 1988 ; shared by Steven Crellin, Dipterists Forum Sepsid Recording Scheme). The integrated final relative qualitative ranking of all 9 species investigated here is presented in Figs. 3 – 5 , from the coolest predicted preference on the left to the warmest on the right. The species at both ends of the ranking with lowest and highest thermal preferences ( S. neocynipsea, S. punctum, S. fulgens and S. cynipsea ) are quite well supported by the literature (op. cit.); the other species in the mid-range however strongly overlap in their thermal preferences, so their ranking is based on minute differences and hence not particularly reliable. We did not find support for our overall ranking, nor were there many significant differences between particular species pairs (see supp. Table S1 ). For instance, S. cynipsea and S. punctum preferred significantly higher temperatures, in line with our expectations, and S. flavimana and S. orthocnemis preferred lower temperatures. For S. flavimana this was expected, but not for S. orthocnemis (see Fig. 3 ). From these results we conclude that S. cynipsea prefers the highest temperatures, followed by S. punctum , while S. orthocnemis and S . flavimana prefer the lowest. This might suggest that at least these species could avoid direct competition by avoiding each other through micro-habitat choices in time or space reflecting their differing thermal preferences. However, these differences remain slight, as there tremendous overlap in these flies’ thermal range (Fig. 3 ). Thus, avoidance of competition likely occurs merely for a small fraction of species sometimes, likely not resulting in a strong distributional signal or strongly differing thermal performance curves (Khelifa et al. 2019 , Rohner et al. 2015 , 2019 , Roy et al. 2018 ). Nevertheless, we cannot completely exclude the possibility that the physiological thermal preferences of some of these species contribute to adaptations for avoiding competition with similar related species. Given no clear inter-specific differences in thermal preferences found here, implying broadly overlapping thermal performance curves (cf. Khelifa et al. 2019 , MacLean et al. 2019a, b, Blanckenhorn et al. 2021a ) and thus also broadly overlapping geographical distributions of most Sepsis species, we must conclude that any observed differences between these sepsid species arise from unknown other, potentially random processes (genetic drift), thus confirming our previous studies in this regard (Blanckenhorn et al. 2020 , 2021b, Kehlifa et al. 2019, Rohner et al. 2015 , 2019 , Roy et al., 2019, Zeender et al. 2019 ). That is, we ultimately reject our hypothesis that sepsids at the adult life stage avoid competition through thermal micro-niche differentiation mediated by different physiological preferences. Nevertheless, we hesitate to outright reject niche differentiation of European sepsid flies at a more general level. Previous research has shown that the evolution of differentiating spatio-temporal (micro)niches is a multifaceted and complex, but ultimately successful strategy to avoid competition (Corral et al. 2022 , Daan 1982 , Hagey et al. 2016 , Hood et al. 2021 , Kenagy 1973 , Richards 2002 , Schoener 1986 , Wiens 1986 ). Thus, there is still the possibility that any research misses the crucial factors or investigates the ‘wrong’ traits. The here discussed species all lay their eggs into cow (and other) dung (Laux et al. 2019 ), which the larvae consume and thus recycle to ultimately pupate in the ground underneath. Since dung can be limited not only in absolute abundance but also in space and time, intra- and inter-specific competition might very well be much stronger at the juvenile rather than the adult stage (Ezeakacha and Yee 2019 , Gurney and Nisbet 1985 , Khelifa et al. 2019 , Moll and Brown 2008 , Skidmore 1991 ). We thus might miss the crucial part of thermal competition between the species in the dung, though direct evidence for strong dung specialization of sepsid larvae is equally limited (Pont and Meier 2002 , Laux et al. 2019 ). It would therefore be interesting to perform a similar study with larvae, for which our racetrack set-up can be adapted. Crucial ecological factors to be considered could be the age of dung for egg laying, species composition in the dung pat, and others. Surprisingly, we found expected acclimation effects (MacLean et al. 2019a) of thermal preferences merely in S. flavimana and S. violacea , plus to a lesser extent in S. duplicata and S. thoracica (Fig. 4 ), with flies indeed preferring warmer resting temperatures when reared at warmer 24℃ rather than 18℃. The small S. flavimana is particularly active at cool temperatures late in the season in the highland areas, whereas the relatively large S. violacea and S. neocynipsea are particularly (or exclusively) active in early spring (Rohner et al. 2015 , 2019 ). All species had been held and bred for no more than three generations at these temperatures before being used in our experiment, as we wanted to minimize effects of laboratory adaptation (e.g., Berger et al. 2014 ). We therefore interpret all observed acclimation effects to reflect a plastic response rather than genetic adaptation, which often already results within generations (MacLean et al. 2019b ). So, we wonder why only about half the species here showed any signs of physiological acclimation, and S. neocynipsea even showed a significantly opposite effect (preference for warmer temperatures when reared at cooler 18℃, Fig. 4 ). S. flavimana and S. violacea might simply have shown faster acclimation than the other species, which could confer a fitness benefit. Another possible interpretation is that higher temperatures impose particularly strong selection on these two cold-loving species, potentially resulting in quick (laboratory) adaptation and evolution. By contrast, the opposite acclimation response of S. neocynipsea is intriguing. This sister species of S. cynipsea is rare in Europe (while common even in warm regions of North America), where it appears to be marginalized towards higher altitude habitats such as the Swiss Alps; it is thus considered cold-adapted (Giesen et al. 2017 , Rohner et al. 2015 , 2019 ). So, in contrast to the other species, S. neocynipsea seem to escape from high temperatures to prefer even lower temperatures when reared at high temperatures, instead of adapting its preference to match the surrounding temperature. Nevertheless, overall our results are in line with MacLean et al. (2019a), who found no clear change in the thermal optimum in response to acclimation temperatures in 10 Drosophila species. This indicates that widespread tropical and temperate flies, such as sepsids or Drosophila , show minimal plastic responses to changing temperatures, suggesting that their thermal preferences are quite robust to short-term temperature changes, and likely only start to change under long-term adaptation. Based on the results of MacLean et al. (2019a,b) and our study, we suggest that short-term plastic acclimation responses are not a common phenomenon in widespread species in general. With the exception of S. duplicata and S. fulgens we found no differences in thermal preferences between the sexes, as also found in other studies (Addeo et al. 2022 ). This confirms our implicit assumption that thermal performance (including preference) is mainly a physiological characteristic of a species affecting its general activity. However, Rajpurohit and Schmidt ( 2016 ) found thermal preferences to differ between the sexes. We might expect differing preferences of males and females for any sex-specific behavior, such as e.g. egg-laying. However, in our experiment all adult flies were older than one week, with access to plenty of food, water and the possibility to oviposit into dung in their holding containers, so no such differences were expected. S. fulgens and S. duplicata males preferred higher temperatures than females (Fig. 5 ). S. duplicata is also exceptional in that males of this species have no foreleg spines and instead show an elaborate courtship dance before mating (Puniamoorthy et al. 2009 ), similar to Drosophila melanogaster (cf. Addeo et al. 2022 , MacLean et al. 2019a, b, Rajpurohit and Schmidt 2016 ). In nature this species is also specialized on dryer dung, as females lay their eggs into the tunnels produced by dung beetles to reach the more humid, inner dung. However, in the end we cannot be certain about precisely what generated the sex differences in thermal preference in S. duplicata and S. fulgens but not the other species. We implemented four different orientations of the race track (Fig. 1 ) to control for essentially unknown but possible environmental (blocking) variables deriving from the room or other external sources (sounds, magnetic field, etc.), or from our experimental set-up itself. We changed the orientation of the racetrack in the room by 180 degrees, and also changed the cooling and heating Peltier elements for each orientation. Orientation had an ultimately unknown strong effect (Fig. 2 ), so we conclude that, in addition to temperature, there is some other external factor pulling the flies preferentially towards one side of the lab (Figs. 2 , 3 ). This result was confirmed in another project using our racetrack (Dallo et al. 2024). In this regard our findings also support the results of Strunov et al., 2023 , who conducted a thermal preference experiment in D. melanogaster using two different racetrack designs, one of which being highly similar to ours. They found a substantial difference in absolute values depending on the racetrack and experimental design, thus highlighting the need for careful experimental design in thermal preference behavioral studies. In the end, it is possible that resources, notably food (here mainly cattle dung), is so abundant in time and space (Crombie 1947 , de Camargo et al. 2016 , Tilman 1987 ) that competition among the various European sepsid species is only weak or not apparent at all most of the time, thus allowing coexistence. We need to continue studying more fully the extent and mechanisms of competition, species composition and ecological niche differentiation not only of sepsid dung flies, but also of other widespread species. Declarations FUNDING This project was funded by the Swiss National Foundation SNF grant no. 31003A-176055. CONFLICTS OF INTEREST None ETHICS APPROVAL Not necessary CONSENTS TO PARTICIPATE OR PUBLISH Not necessary DATA FILES Are supplied as supplement CODE Nothing beyond standard ANOVA statistics Acknowledgments We thank Marcel Freund of the University of Zurich who contributed to the design of the handcrafted racetrack apparatus. We are very grateful to Jeannine Roy who helped with fly maintenance, sampling and breeding of the flies. This project was funded by the Swiss National Foundation SNF grant no. 31003A-176055. Author Contributions All authors contributed to the conceptual development of the study/experiment. MK developed the apparatus. RD conducted the study as part of his PhD (data gathering; statistical analysis), with MK & WUB as supervisors. RD wrote the first draft; WUB & MK co-wrote. 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Biological Records Centre Institute of Terrestrial Ecology Puniamoorthy N, Ismail MRB, Tan DSH, Meier R (2009) From kissing to belly stridulation: comparative analysis reveals surprising diversity, rapid evolution, and much homoplasy in the mating behaviour of 27 species of sepsid flies (Diptera: Sepsidae). J Evol Biol 22:2146–2156 R Core Team (2017) R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing Rajpurohit S, Schmidt PS (2016) Measuring thermal behavior in smaller insects: A case study in Drosophila melanogaster demonstrates effects of sex, geographic origin, and rearing temperature on adult behavior. Fly 10:149–161 Richards SA (2002) Temporal partitioning and aggression among foragers: modeling the effects of stochasticity and individual state. Behav Ecol 13:427–438 Rohner PT (2015) An updated checklist of the Sepsidae (Diptera) of Switzerland, including the first record of Themira superba (Haliday, 1833). 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Nat Methods 9:671–675 Schoener TW (1974) Resource Partitioning in Ecological Communities: Research on how similar species divide resources helps reveal the natural regulation of species diversity. Science 185:27–39 Schoener TW (1986) Resource partitioning. Community ecology pattern and process Shorrocks B, Rosewell J, Edwards K, Atkinson W (1984) Interspecific competition is not a major organizing force in many insect communities. Nature 310:310–312 Silvertown J, Dodd M, Gowing D (2001) Phylogeny and the niche structure of meadow plant communities: Phylogenetic origins of niche structure . J Ecol 89:428–435 Skidmore P (1991) Insects of the British cow-dung community. Field Studies Council Stevenson RD (1985) The Relative Importance of Behavioral and Physiological Adjustments Controlling Body Temperature in Terrestrial Ectotherms. Am Nat 126:362–386 Strunov A, Schoenherr C, Kapun M (2023) Wolbachia has subtle effects on thermal preference in highly inbred Drosophila melanogaster which vary with life stage and environmental conditions. Sci Rep 13:13792. doi.org/10.1038/s41598-023-40781-7 Tilman D, Mattson M, Langer S (1981) Competition and nutrient kinetics along a temperature gradient: An experimental test of a mechanistic approach to niche theory1: Nutrient competition. Limnol Oceanogr 26:1020–1033 Tilman D (1987) The Importance of the Mechanisms of Interspecific Competition. Am Nat 129:769–774 Violle C, Nemergut DR, Pu Z, Jiang L (2011) Phylogenetic limiting similarity and competitive exclusion: Phylogenetic relatedness and competition. Ecol Lett 14:782–787 Webb CO, Ackerly DD, McPeek MA, Donoghue MJ (2002) Phylogenies and Community Ecology. Annu Rev Ecol Syst 33:475–505 Wickham H (2016) ggplot2: Elegant Graphics for Data Analysis. Springer-, New York Wiens JA (1986) Overview: the importance of spatial and temporal scale in ecological investigations. Community Ecol 145–153 Yamamoto AH (1994) Temperature preference of Drosophila immigrans and D. virilis: Intra- and inter-population genetic variation. Jpn J Genet 69:67–76 Zeender V et al (2019) Comparative reproductive dormancy differentiation in European black scavenger flies (Diptera: Sepsidae). Oecologia 189:905–917 Supplementary Files DalloEtAl2024ComparativeSepsidTemperaturePrefSupplement.docx DataracetrackcomparativeallSepsisspp.xlsx 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-4252799","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":311040700,"identity":"64902db9-56bd-494d-8177-ba278561553f","order_by":0,"name":"Ramon Dallo","email":"","orcid":"","institution":"Universität Zürich: Universitat Zurich","correspondingAuthor":false,"prefix":"","firstName":"Ramon","middleName":"","lastName":"Dallo","suffix":""},{"id":311040701,"identity":"aed50b93-30b9-41d9-a866-246273a78901","order_by":1,"name":"Martin Kapun","email":"","orcid":"","institution":"Naturhistorisches Museum Wien","correspondingAuthor":false,"prefix":"","firstName":"Martin","middleName":"","lastName":"Kapun","suffix":""},{"id":311040702,"identity":"e6d4760a-8a87-479a-b17a-2539abb62a13","order_by":2,"name":"Wolf Blanckenhorn","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEklEQVRIie3QsUrEMBjA8YRAuvT2lBt8he84uHpY6atcOKijuDkmFNLFB1D0ITr2NkuhXfIAFZeTwk0OJ463+NnJIefh5pA/hJSmP5KGEJ/vX8bUnqpzfOCEUIVzoNW4AOMrV1TfUyV+kLD+MxEr8iuBTmtFK3EWB60cbqrkOo4GPYSkSWPCdlsXsTUSK2abu6yZP9hsuXmU+RwJWyoeg4MsellsqRG07AMznZgG4FWaKRIOzyEXboK7GJEiKQ4jeamLA5LwFJFlz1s2kp4ahkQcI+n3v0gr1qXN1niwDMDKPHqCK4CGL1wkKvJafVTJZdm1s8+JSQC67m3/fnuRQpfvXGRs5bhJHOzY9z6fz+c71Re532XKFeSPJgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-0713-3944","institution":"University of Zurich","correspondingAuthor":true,"prefix":"","firstName":"Wolf","middleName":"","lastName":"Blanckenhorn","suffix":""}],"badges":[],"createdAt":"2024-04-11 13:37:59","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4252799/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4252799/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59052000,"identity":"0a6a3998-5c51-4734-8817-d219baec877f","added_by":"auto","created_at":"2024-06-25 20:10:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":8845,"visible":true,"origin":"","legend":"\u003cp\u003eThe experimental set-up in four orientations (1-4) relative to the room. The black line represents the aluminium racetrack (2 separate tracks 700mm long) with its cold (12℃) and hot (30℃) ends. The blue square represents the space on which the rod with the attached IR camera stood. The space between the camera and the racetrack is 740mm. The square (410 x 1000mm) illustrates the baseplate of the whole apparatus on which all other elements stood. The baseplate was attached to a cart (grey area). On the cart and underneath the baseplate the computer screen and two power sources were placed, while the white area was overhanging the cart.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4252799/v1/2206f821b8bba87c503cd889.png"},{"id":59052004,"identity":"37b69d3a-5216-4c78-b9ae-b5067ede9be2","added_by":"auto","created_at":"2024-06-25 20:10:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":37375,"visible":true,"origin":"","legend":"\u003cp\u003eFrequency distribution of all flies across all treatments for the four racetrack orientations (cf. Fig. 1). Orientations 1 \u0026amp; 4 (red \u0026amp; purple) show similar fly distributions across temperature with higher abundance at the cool end. Orientation 3 (green) shows a rather uniform pattern with more evenly distributed flies and the highest abundance at the warm end. Orientation 2 (blue) is in between, again showing the highest abundance at the cool end (similar to 1 \u0026amp; 4), but also a second maximum at the warm end.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4252799/v1/9f50b93935f3fdf428e31f2e.png"},{"id":59052005,"identity":"7dc592af-3189-46bb-9903-fe1fa5c40630","added_by":"auto","created_at":"2024-06-25 20:10:43","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":226036,"visible":true,"origin":"","legend":"\u003cp\u003eViolin plots of the thermal preference of 9 Swiss Sepsis species from Zürich. Mean preferred temperature and standard deviation are illustrated by black lines. The species are ordered by their overall mean temperature niches inferred from natural distribution data (coolest left to warmest right): Pont and Meier 2002, Rohner et al. 2015, 2019, Khelifa et al., 2019). There is merely weak correspondence.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4252799/v1/26342d7a78394d74e0337352.jpeg"},{"id":59052001,"identity":"e7e2668b-63cd-4311-971b-3095c35b6ffe","added_by":"auto","created_at":"2024-06-25 20:10:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":90669,"visible":true,"origin":"","legend":"\u003cp\u003eThermal preference of 9 Sepsis from Zürich, Switzerland at two different acclimation temperatures. On average warmer acclimation temperatures lead to preference for warmer temperature, but not for all species. Species are ordered by their overall mean temperature niches inferred from natural distribution data (coolest left to warmest right): Pont and Meier 2002, Rohner et al. 2015, 2019, Khelifa et al., 2019).\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4252799/v1/b911e244ce1f15295b7fa226.png"},{"id":59052002,"identity":"940ef4e6-46e6-47fc-a777-eb0d987e5303","added_by":"auto","created_at":"2024-06-25 20:10:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":89640,"visible":true,"origin":"","legend":"\u003cp\u003eSex-specific thermal preferences of 9 Sepsis from Ziegelhütte, Zürich, Switzerland. Expect for S. duplicata and S. fulgens, for which males preferred higher temperatures than females, we found no significant differences between sexes. Species are ordered by their overall mean temperature niches inferred from natural distribution data (coolest left to warmest right): Pont and Meier 2002, Rohner et al. 2015, 2019, Khelifa et al., 2019).\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4252799/v1/8e8a72865cc56b6c2bc5f9f8.png"},{"id":62844652,"identity":"b964b03e-c503-42ca-84d9-022aef014efa","added_by":"auto","created_at":"2024-08-20 07:12:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1183692,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4252799/v1/634ac9f9-0c65-43a1-9a28-5a6119536905.pdf"},{"id":59052006,"identity":"b56d8a8f-2fb7-44df-ae4a-3e8b0df9c314","added_by":"auto","created_at":"2024-06-25 20:10:43","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":8230341,"visible":true,"origin":"","legend":"","description":"","filename":"DalloEtAl2024ComparativeSepsidTemperaturePrefSupplement.docx","url":"https://assets-eu.researchsquare.com/files/rs-4252799/v1/da065d82d8c48fa0fb693aaa.docx"},{"id":59052003,"identity":"f0b78541-5320-4842-a018-30e54e03aa5c","added_by":"auto","created_at":"2024-06-25 20:10:43","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":169871,"visible":true,"origin":"","legend":"","description":"","filename":"DataracetrackcomparativeallSepsisspp.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4252799/v1/bf9efb6f0734e7534d82a94c.xlsx"}],"financialInterests":"","formattedTitle":"Lacking sex-specific temperature preferences of 9 coexisting temperate sepsid dung fly species (Diptera: Sepsidae)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTemperature is a key environmental factor that directly influences the physiology of all living organisms by affecting biological processes from the molecular to the ecosystem levels (Gilbert and Miles \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Hoffmann \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, Huey and Berrigan \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2001\u003c/span\u003e, Kellermann et al. 2012a,b). Temperature shapes the entire organismal life history, encompassing activity, development, reproduction and survival. In contrast to endotherms, ectotherms have only limited control over their own body temperature, so they are considerably affected by all environmental variation experienced (Angiletta et al. 2004, Angiletta 2009). By choosing niches with temperatures close to their preferred optimum, ectotherms can adjust their body temperature towards their preferred temperature. Thus thermoregulation is mainly achieved through behavioral plasticity (Stevenson \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e1985\u003c/span\u003e). Preferred temperatures might change depending on life stage (propagule, juvenile, adult, etc.) or activity (foraging, mating, egg-laying, etc.; Addeo et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Blanckenhorn et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, MacLean et al. 2019a, Rajpurohit and Schmidt \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNevertheless, every organism has a temperature range in which it can survive and reproduce, as delimited by their so-called thermal performance curve (Angilletta \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2006\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, Berger et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Blanckenhorn et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e, Huey and Kingsolver \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1989\u003c/span\u003e, Huey and Stevenson \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1979\u003c/span\u003e). Experiencing temperatures outside this range can in the worst-case lead to the death of the organism (Gilbert and Miles \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Lutterschmidt and Hutchison \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Within this range every organism likely has a thermal optimum T\u003csub\u003eopt\u003c/sub\u003e that varies according to its precise physiological needs, a temperature it therefore should prefer given no further ecological constraints (Huey and Kingsolver \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1989\u003c/span\u003e, Huey and Stevenson \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1979\u003c/span\u003e, Gilbert and Miles \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Together with these (internal) physiological preferences, the (external) environmental temperature thus co-defines an organism\u0026rsquo;s ecological niche, and ultimately the species composition in a given habitat (Angilletta \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2004\u003c/span\u003e, Gilbert and Miles \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Huey and Berrigan \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2001\u003c/span\u003e, Magnuson et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e1979\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAnother important factor shaping species composition and coexistence is competition (Case and Gilpin \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1974\u003c/span\u003e, Gause \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1934\u003c/span\u003e). Competition between different species occurs over various essential resources such as food, time, or (nesting) space (Crombie \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1947\u003c/span\u003e, de Camargo et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Tilman \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). The competitive exclusion model (Armstrong and McGehee \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1980\u003c/span\u003e, den Boer \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1986\u003c/span\u003e, Gause \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1934\u003c/span\u003e, Hardin \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1960\u003c/span\u003e, Levin \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1970\u003c/span\u003e) and ecological niche theory (Alley \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1982\u003c/span\u003e, Buckley er al. 2015, Case and Gilpin \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1974\u003c/span\u003e, Holt et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1994\u003c/span\u003e, Kylafis and Loreau \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, MacArthur and Levins \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1967\u003c/span\u003e, Tilman et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e1981\u003c/span\u003e) predict that when different species are competing for the same resources, a few dominant species will evolve to exclude all others. Moreover, closely related species compete more severely for the same resources (Cavender-Bares et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, Silvertown et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2001\u003c/span\u003e, Violle et al. \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Webb et al. \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) because they are ecologically more similar (Darwin \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1859\u003c/span\u003e; the competition-relatedness hypothesis: Cahill et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The predicted result overall should be a community composed of few species, and even fewer related species, in any particular habitat.\u003c/p\u003e \u003cp\u003eNonetheless, previous work documents an opposite pattern for sepsid dung flies (Diptera: Seaside; Rohner \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Rohner et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Blanckenhorn et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, 2021b). This group of flies plays an essential ecological role as primary degraders of rotting plant material and animal feces. Sepsid flies have a broad world-wide distribution across various biogeographic zones (Ang et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Pont and Meier \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). In Europe multiple temperate species share the same ecological niche, livestock pastures and more generally grasslands, where eight or more closely related species may occur in sympatry in and around their breeding substrates, most commonly cow dung (Rohner \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Rohner et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt is argued that competitive exclusion does not hold if competing species have a means of evading each other (Gao et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Kronfeld-Schor and Dayan \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2003\u003c/span\u003e, Loreau and Ebenhoh \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1994\u003c/span\u003e, Schoener \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e1974\u003c/span\u003e, Wiens \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). The circadian or diurnal cycle of an organism can deflect interspecific competition, thus potentially facilitating coexistence via spatial and/or temporal niches (Corral et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Daan \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1982\u003c/span\u003e, Hagey et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Hood et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Kenagy \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1973\u003c/span\u003e, Richards \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2002\u003c/span\u003e, Schoener \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e1986\u003c/span\u003e, Shorrocks et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e1984\u003c/span\u003e, Wiens \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). As revealed by long-term repeated sampling in time and space, the abundance of various coexisting sepsid species in Switzerland depends on geography, altitude, the season, and even the time of day (Khelifa et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Rohner et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). One key factor potentially mediating this spatio-temporal abundance variation is temperature. Having different thermal preferences therefore might be a potential mechanism by which ecologically and phylogenetically similar sepsid species avoid each other.\u003c/p\u003e \u003cp\u003eWe here investigate whether widespread and often coexisting European sepsid species are differentiated in terms of thermal microhabitat preferences in accordance with their geographic, altitudinal, seasonal and/or diurnal distribution (Rohner et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). We generally hypothesize that flies experiencing higher average temperatures in nature should also prefer higher temperatures in the laboratory. We tested this by comparing the thermal preferences of 9 closely related black scavenger or dung fly species of the genus \u003cem\u003eSepsis\u003c/em\u003e native to Switzerland in a laboratory thermal racetrack, a classic approach in this field of climate research (e.g., Deal \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1941\u003c/span\u003e, Dillon et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e for a review, MacLean et al. 2019a, b, Yamamoto \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). In doing so, we also considered well-established effects of acclimation temperature (MacLean et al. 2019a,b) as well as potential differences between the sexes relating to their different life histories and sexual roles (Addeo et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Rajpurohit and Schmidt \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eFly populations\u003c/h2\u003e \u003cp\u003eNine species of sepsid flies were used for our experiment: \u003cem\u003eS. punctum\u003c/em\u003e, \u003cem\u003eS. neocynipsea\u003c/em\u003e, \u003cem\u003eS. cynipsea\u003c/em\u003e, \u003cem\u003eS. duplicata\u003c/em\u003e, \u003cem\u003eS. thoracica\u003c/em\u003e, \u003cem\u003eS. orthocnemis\u003c/em\u003e, \u003cem\u003eS. violacea\u003c/em\u003e, \u003cem\u003eS. fulgens\u003c/em\u003e, \u003cem\u003eS. flavimana\u003c/em\u003e (see Ang et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Pont and Meier \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Flies of all species were caught originally at Ziegelh\u0026uuml;tte in Schwamendingen, Z\u0026uuml;rich, Switzerland, in 2022 and subsequently bred individually to higher numbers as iso-female lines (i.e., the offspring of one field-caught female) for no more than three generations in the laboratory. All lines were held in 1.5-liter group containers with water, sugar and cow dung (see e.g. Puniamoorthy et al. 2012 for general rearing methods) in walk-in climate chambers at both 18\u0026deg;C and 24\u0026deg;C. Five to ten iso-female lines were later combined to create the mixed populations used in our experiment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eThermal gradient experiment\u003c/h2\u003e \u003cp\u003eThermal preferences were tested with a self-constructed racetrack apparatus modelled after similar devices used in the past for small insects (see Dillon et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, see supp. Fig. S0). The racetrack consisted of an aluminum base plate and a plastic top. The plastic top was shaped into two tracks (each track of 700 mm length x 10 mm width x 8 mm height) running along the base plate. A strip of white filter paper was placed between the baseplate and the plastic top to assure contrast and maintain humidity. At each end underneath the base plate a Peltier element (P\u0026amp;N Technology, Xiamen Fujian China) was positioned for either heating or cooling. Underneath each Peltier element a heat or cold sink (Fischer Electronic, Germany) lead off excess heating or cooling energy (the former into a replenishable ice container).\u003c/p\u003e \u003cp\u003eForty minutes before the first run of the day the power source of the Peltier elements was switched on to produce a constant linear temperature gradient from ca. 12℃ to 30℃ along which individual flies could align according to their preferences. All runs were conducted in a completely dark room at 21℃ (\u0026plusmn;\u0026thinsp;0.5℃). Groups of 4 flies of the same sex and acclimation treatment were aspirated into one track via a hole in the middle. Flies had been reared at either 18℃ or 24℃ for several generations (acclimation treatment). We thus had 36 treatment combinations (9 species * 2 sexes * 2 rearing temperatures), and flies from the same treatment combination were used in both tracks for any given run.\u003c/p\u003e \u003cp\u003eThree thermometers (Analog devices, USA) recorded the temperature every 100 seconds at both ends of the racetrack and in the middle. Every 5 minutes an infrared camera took a picture from above that was also saved to an attached Raspberry pi 3B\u0026thinsp;+\u0026thinsp;computer (Raspberry Pi Foundation, UK). After 25 minutes, at which point the flies were typically resting, a run was stopped, and the last pictures were used for the final analysis. Four different orientations of the racetrack in the room were implemented by (1) rotating the racetrack situated on a rolling cart by 180 degrees, thus creating two different placements in the room, and (2) by switching the heating and cooling side for each placement in the room (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Runs of each treatment combination were equally distributed in the morning or afternoon. Fly age was not tracked individually, but the adult flies were from 7 to 13 days old (post-emergence). A total of 2336 flies were used.\u003c/p\u003e \u003cp\u003eAll pictures were analyzed in ImageJ (Schneider et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). A custom python script (written by Martin Kapun) calculated a linear temperature gradient from the three thermometer readings, the linearity of which had been verified at the start of our study. The location coordinates (at the end of each 25 min run) of the black flies on white filter paper were determined and then converted to the corresponding temperature along the linear gradient (script by Martin Kapun).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eA generalized linear mixed model (GLMMs) with random factors (glmer) was used from the package \u0026ldquo;lme4\u0026rdquo; in R (Bates et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, R Core Team 2021). Species, sex, acclimation temperature, and orientation were entered as fixed factors in a type III ANOVA using the \u0026ldquo;car\u0026rdquo; package (Fox and Weisberg \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), initially including all relevant interactions, which were subsequently removed from the model bottom-up if non-significant. Group ID was the random factor. Additionally, analogous GLMMs with sex, acclimation temperature and orientation as fixed factors and group ID as a random factor were implemented for each species separately (see table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e appendix). Some pairwise comparisons of species and/or acclimation temperatures were tested with Tukey\u0026rsquo;s HSD tests performed in the package \u0026ldquo;emmeans\u0026rdquo; (Lenth 2021). All plots were created using the package \u0026ldquo;ggplot2\u0026rdquo; (Wickham \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe effect of phylogeny (Su et al. 2016) was tested using a Bayesian multilevel model in the package \u0026ldquo;brms\u0026rdquo; (B\u0026uuml;rkner \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). For this analysis a new ranking variable was introduced. Every species received a unique rank, ranging from 1 (coldest) to 9 (hottest), according to their realized temperature niche in the wild derived from their geographic distribution from Pont and Meier (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) and Rohner et al. (\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The analysis showed no significant effect of phylogeny on the results, therefore the original linear model without phylogeny was presented in the end.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThe frequency distribution of fly positions for all orientation combinations is given in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. Flies mostly settled at the cool end of the temperature gradient, although for orientation 3 the fly distribution is rather uniform across all temperatures, for unknown reasons. Given any external confounding factor, we would have expected orientations 1 \u0026amp; 4, as well as 2 \u0026amp; 3, to have yielded similar outcomes. Based on this result we performed two analyses: one including data for all four orientations, with orientation as a random blocking effect, and one conservative analysis using only orientations 1 \u0026amp; 4 (reported below). In the end, exclusion of orientations 2 \u0026amp; 3 only slightly changed the overall species and sex results.\u003c/p\u003e\n\u003cp\u003eWhile we found significant temperature preference variation across the 9 species (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, F(8, 269)\u0026thinsp;=\u0026thinsp;5.4; see Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e), we did not observe good correspondence with their natural temperature niches inferred from previous work, all of which admittedly are very similar (Rohner et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e, Khelifa et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). Overall, all species preferred to rest at cooler temperatures, nevertheless using the whole temperature spectrum (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). On average, \u003cem\u003eS. cynipsea\u003c/em\u003e and \u003cem\u003eS. punctum\u003c/em\u003e chose the warmest temperatures\u0026thinsp;\u0026gt;\u0026thinsp;15\u0026deg;C, while \u003cem\u003eS. flavimana\u003c/em\u003e and \u003cem\u003eS. orthocnemis\u003c/em\u003e preferred the lowest temperatures around 13\u0026deg;C (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eANOVA Output including all 4 orientations (top), or orientations 1 \u0026amp; 4 only (bottom).\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"6\"\u003e\n \u003cp\u003eAnova of GLMM model with all orientations\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eMS\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003edf\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eden. df\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eF\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSpecies\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e55.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e554.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAcclimation Temperature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e157.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e554.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e554.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.253\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOrientaion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2020.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e554.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e155.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSpecies:Acclimation Temperature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e554.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSpecies:Sex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e554.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.354\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"6\"\u003e\n \u003cp\u003e\u003cstrong\u003eAnova of GLMM model with orientations 1 \u0026amp; 4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003edf\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eden. df\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eF\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSpecies\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e269.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAcclimation Temperature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e43.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e269.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.009\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e269.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.182\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOrientaion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e269.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.968\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSpecies:Acclimation Temperature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e269.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSpecies:Sex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e269.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.013\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"char\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003eThe thermal preference of males and females did not systematically differ across species, but there was a significant interaction between sex and species indicating unsystematic sex differences across species (p\u0026thinsp;=\u0026thinsp;0.013, F(8, 269)\u0026thinsp;=\u0026thinsp;2.49; Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Only for \u003cem\u003eS. duplicata\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.008, F(1, 27)\u0026thinsp;=\u0026thinsp;8.13; Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e) and \u003cem\u003eS. fulgens\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.029, F(1, 27)\u0026thinsp;=\u0026thinsp;5.29; Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e) was the main effect of sex significant, with males preferring higher temperatures than females (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eFlies acclimated to the higher temperature (24℃) showed no preference for higher temperatures in general, although the acclimation effect was overall significant in the right direction (p\u0026thinsp;=\u0026thinsp;0.009, F(1, 269)\u0026thinsp;=\u0026thinsp;6.95; Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e), as was the interaction between species and acclimation temperature (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, F(8, 269)\u0026thinsp;=\u0026thinsp;5.08; Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). This resulted from 3 species showing significant differences: \u003cem\u003eS. flavimana\u003c/em\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, F(1, 27)\u0026thinsp;=\u0026thinsp;22.72; supp. Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e) and \u003cem\u003eS. violacea\u003c/em\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, F(1, 27)\u0026thinsp;=\u0026thinsp;18.48; Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e) flies acclimated to the higher temperature (24℃) indeed preferred higher resting temperatures, while \u003cem\u003eS. neocynipsea\u003c/em\u003e flies so raised (p\u0026thinsp;=\u0026thinsp;0.024, F(1, 138)\u0026thinsp;=\u0026thinsp;5.24, Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e) preferred lower temperatures (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMany temperate species of the dung fly genus \u003cem\u003eSepsis\u003c/em\u003e coexist in high numbers in European pastoral habitats, contradicting conventional wisdom of competitive exclusion (Armstrong and McGehee \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1980\u003c/span\u003e, den Boer \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1986\u003c/span\u003e, Gause \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1934\u003c/span\u003e, Hardin \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1960\u003c/span\u003e, Levin \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1970\u003c/span\u003e) and ecological niche theory (Alley \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1982\u003c/span\u003e, Buckley er al. 2015, Case and Gilpin \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1974\u003c/span\u003e, Holt et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1994\u003c/span\u003e, Kylafis and Loreau \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, MacArthur and Levins \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1967\u003c/span\u003e, Rohner et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Tilman et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e1981\u003c/span\u003e). We thus expected such coexistence of closely-related sepsid species on the same pasture in Switzerland to be avoided by preferring different spatio-temporal microhabitat niches (Blanckenhorn et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e). Temperature has a great effect on the behavior of any organism, especially of small ectotherms (Angiletta 2009), so we here specifically hypothesized and tested in the laboratory whether 9 coexisting \u003cem\u003eSepsis\u003c/em\u003e species have different thermal preferences. Thermal avoidance may occur along latitude or altitude, or over the season, in the extreme over the time course of a day (Rohner et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Roy et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). A thermal racetrack experiment, by now a classic approach in thermal research of small organisms (Dillon et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), did not reveal very different thermal niches of the temperate sepsid species investigated, at least in the laboratory. Flies of all species preferred to settle (or rest) at cooler temperatures between 13\u0026deg;C and 15\u0026deg;C, but regularly utilized the entire range between 12\u0026deg;C and 30\u0026deg;C offered for their activity (see Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This was the case for flies of both sexes, and also for both acclimation temperatures used here (18\u0026deg;C \u0026amp; 24\u0026deg;C).\u003c/p\u003e \u003cp\u003eBased on previous studies (Pont and Meier \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2002\u003c/span\u003e, Rohner et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Roy et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Khelifa et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) we had expected the thermal preferences of our 9 knowingly similar sepsid species co-occurring regularly in Swiss grasslands to roughly accord with their geographic distribution in nature. Our ranking was based on the following factors: \u003cb\u003e1)\u003c/b\u003e The distributional data in the latitudinal dimension: the more northern the range of a given species, the lower their assumed thermal preference (Pont and Meier \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2002\u003c/span\u003e, Roy et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). \u003cb\u003e2)\u003c/b\u003e The seasonal abundance of species in Switzerland: if a species is more abundant in summer, we assumed a higher temperature preference (Rohner et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). \u003cb\u003e3)\u003c/b\u003e The altitudinal distribution and abundance of species in Switzerland, assuming that species reaching higher altitudes also have a lower preferred temperatures (Blanckenhorn \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1997\u003c/span\u003e, Rohner et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). \u003cb\u003e4)\u003c/b\u003e As additional qualitative support of our ranking, we considered the distribution of the same sepsid species on the British Isles (Pont \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; shared by Steven Crellin, Dipterists Forum Sepsid Recording Scheme). The integrated final relative qualitative ranking of all 9 species investigated here is presented in Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e, from the coolest predicted preference on the left to the warmest on the right. The species at both ends of the ranking with lowest and highest thermal preferences (\u003cem\u003eS. neocynipsea, S. punctum, S. fulgens\u003c/em\u003e and \u003cem\u003eS. cynipsea\u003c/em\u003e) are quite well supported by the literature (op. cit.); the other species in the mid-range however strongly overlap in their thermal preferences, so their ranking is based on minute differences and hence not particularly reliable.\u003c/p\u003e \u003cp\u003eWe did not find support for our overall ranking, nor were there many significant differences between particular species pairs (see supp. Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). For instance, \u003cem\u003eS. cynipsea\u003c/em\u003e and \u003cem\u003eS. punctum\u003c/em\u003e preferred significantly higher temperatures, in line with our expectations, and \u003cem\u003eS. flavimana\u003c/em\u003e and \u003cem\u003eS. orthocnemis\u003c/em\u003e preferred lower temperatures. For \u003cem\u003eS. flavimana\u003c/em\u003e this was expected, but not for \u003cem\u003eS. orthocnemis\u003c/em\u003e (see Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). From these results we conclude that \u003cem\u003eS. cynipsea\u003c/em\u003e prefers the highest temperatures, followed by \u003cem\u003eS. punctum\u003c/em\u003e, while \u003cem\u003eS. orthocnemis\u003c/em\u003e and \u003cem\u003eS\u003c/em\u003e. \u003cem\u003eflavimana\u003c/em\u003e prefer the lowest. This might suggest that at least these species could avoid direct competition by avoiding each other through micro-habitat choices in time or space reflecting their differing thermal preferences. However, these differences remain slight, as there tremendous overlap in these flies\u0026rsquo; thermal range (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Thus, avoidance of competition likely occurs merely for a small fraction of species sometimes, likely not resulting in a strong distributional signal or strongly differing thermal performance curves (Khelifa et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Rohner et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Roy et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Nevertheless, we cannot completely exclude the possibility that the physiological thermal preferences of some of these species contribute to adaptations for avoiding competition with similar related species. Given no clear inter-specific differences in thermal preferences found here, implying broadly overlapping thermal performance curves (cf. Khelifa et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, MacLean et al. 2019a, b, Blanckenhorn et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e) and thus also broadly overlapping geographical distributions of most \u003cem\u003eSepsis\u003c/em\u003e species, we must conclude that any observed differences between these sepsid species arise from unknown other, potentially random processes (genetic drift), thus confirming our previous studies in this regard (Blanckenhorn et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, 2021b, Kehlifa et al. 2019, Rohner et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Roy et al., 2019, Zeender et al. \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). That is, we ultimately reject our hypothesis that sepsids at the adult life stage avoid competition through thermal micro-niche differentiation mediated by different physiological preferences.\u003c/p\u003e \u003cp\u003eNevertheless, we hesitate to outright reject niche differentiation of European sepsid flies at a more general level. Previous research has shown that the evolution of differentiating spatio-temporal (micro)niches is a multifaceted and complex, but ultimately successful strategy to avoid competition (Corral et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Daan \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1982\u003c/span\u003e, Hagey et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Hood et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Kenagy \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1973\u003c/span\u003e, Richards \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2002\u003c/span\u003e, Schoener \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e1986\u003c/span\u003e, Wiens \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). Thus, there is still the possibility that any research misses the crucial factors or investigates the \u0026lsquo;wrong\u0026rsquo; traits. The here discussed species all lay their eggs into cow (and other) dung (Laux et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), which the larvae consume and thus recycle to ultimately pupate in the ground underneath. Since dung can be limited not only in absolute abundance but also in space and time, intra- and inter-specific competition might very well be much stronger at the juvenile rather than the adult stage (Ezeakacha and Yee \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Gurney and Nisbet \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1985\u003c/span\u003e, Khelifa et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Moll and Brown \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Skidmore \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). We thus might miss the crucial part of thermal competition between the species in the dung, though direct evidence for strong dung specialization of sepsid larvae is equally limited (Pont and Meier \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2002\u003c/span\u003e, Laux et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). It would therefore be interesting to perform a similar study with larvae, for which our racetrack set-up can be adapted. Crucial ecological factors to be considered could be the age of dung for egg laying, species composition in the dung pat, and others.\u003c/p\u003e \u003cp\u003eSurprisingly, we found expected acclimation effects (MacLean et al. 2019a) of thermal preferences merely in \u003cem\u003eS. flavimana\u003c/em\u003e and \u003cem\u003eS. violacea\u003c/em\u003e, plus to a lesser extent in \u003cem\u003eS. duplicata and S. thoracica\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), with flies indeed preferring warmer resting temperatures when reared at warmer 24℃ rather than 18℃. The small \u003cem\u003eS. flavimana\u003c/em\u003e is particularly active at cool temperatures late in the season in the highland areas, whereas the relatively large \u003cem\u003eS. violacea\u003c/em\u003e and \u003cem\u003eS. neocynipsea\u003c/em\u003e are particularly (or exclusively) active in early spring (Rohner et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). All species had been held and bred for no more than three generations at these temperatures before being used in our experiment, as we wanted to minimize effects of laboratory adaptation (e.g., Berger et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). We therefore interpret all observed acclimation effects to reflect a plastic response rather than genetic adaptation, which often already results within generations (MacLean et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e). So, we wonder why only about half the species here showed any signs of physiological acclimation, and \u003cem\u003eS. neocynipsea\u003c/em\u003e even showed a significantly opposite effect (preference for warmer temperatures when reared at cooler 18℃, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). \u003cem\u003eS. flavimana\u003c/em\u003e and \u003cem\u003eS. violacea\u003c/em\u003e might simply have shown faster acclimation than the other species, which could confer a fitness benefit. Another possible interpretation is that higher temperatures impose particularly strong selection on these two cold-loving species, potentially resulting in quick (laboratory) adaptation and evolution. By contrast, the opposite acclimation response of \u003cem\u003eS. neocynipsea\u003c/em\u003e is intriguing. This sister species of \u003cem\u003eS. cynipsea\u003c/em\u003e is rare in Europe (while common even in warm regions of North America), where it appears to be marginalized towards higher altitude habitats such as the Swiss Alps; it is thus considered cold-adapted (Giesen et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Rohner et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). So, in contrast to the other species, \u003cem\u003eS. neocynipsea\u003c/em\u003e seem to escape from high temperatures to prefer even lower temperatures when reared at high temperatures, instead of adapting its preference to match the surrounding temperature.\u003c/p\u003e \u003cp\u003eNevertheless, overall our results are in line with MacLean et al. (2019a), who found no clear change in the thermal optimum in response to acclimation temperatures in 10 \u003cem\u003eDrosophila\u003c/em\u003e species. This indicates that widespread tropical and temperate flies, such as sepsids or \u003cem\u003eDrosophila\u003c/em\u003e, show minimal plastic responses to changing temperatures, suggesting that their thermal preferences are quite robust to short-term temperature changes, and likely only start to change under long-term adaptation. Based on the results of MacLean et al. (2019a,b) and our study, we suggest that short-term plastic acclimation responses are not a common phenomenon in widespread species in general.\u003c/p\u003e \u003cp\u003eWith the exception of \u003cem\u003eS. duplicata\u003c/em\u003e and \u003cem\u003eS. fulgens\u003c/em\u003e we found no differences in thermal preferences between the sexes, as also found in other studies (Addeo et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This confirms our implicit assumption that thermal performance (including preference) is mainly a physiological characteristic of a species affecting its general activity. However, Rajpurohit and Schmidt (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) found thermal preferences to differ between the sexes. We might expect differing preferences of males and females for any sex-specific behavior, such as e.g. egg-laying. However, in our experiment all adult flies were older than one week, with access to plenty of food, water and the possibility to oviposit into dung in their holding containers, so no such differences were expected. \u003cem\u003eS. fulgens\u003c/em\u003e and \u003cem\u003eS. duplicata\u003c/em\u003e males preferred higher temperatures than females (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e). \u003cem\u003eS. duplicata\u003c/em\u003e is also exceptional in that males of this species have no foreleg spines and instead show an elaborate courtship dance before mating (Puniamoorthy et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), similar to \u003cem\u003eDrosophila melanogaster\u003c/em\u003e (cf. Addeo et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, MacLean et al. 2019a, b, Rajpurohit and Schmidt \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In nature this species is also specialized on dryer dung, as females lay their eggs into the tunnels produced by dung beetles to reach the more humid, inner dung. However, in the end we cannot be certain about precisely what generated the sex differences in thermal preference in \u003cem\u003eS. duplicata\u003c/em\u003e and \u003cem\u003eS. fulgens\u003c/em\u003e but not the other species.\u003c/p\u003e \u003cp\u003eWe implemented four different orientations of the race track (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) to control for essentially unknown but possible environmental (blocking) variables deriving from the room or other external sources (sounds, magnetic field, etc.), or from our experimental set-up itself. We changed the orientation of the racetrack in the room by 180 degrees, and also changed the cooling and heating Peltier elements for each orientation. Orientation had an ultimately unknown strong effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), so we conclude that, in addition to temperature, there is some other external factor pulling the flies preferentially towards one side of the lab (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This result was confirmed in another project using our racetrack (Dallo et al. 2024). In this regard our findings also support the results of Strunov et al., \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, who conducted a thermal preference experiment in \u003cem\u003eD. melanogaster\u003c/em\u003e using two different racetrack designs, one of which being highly similar to ours. They found a substantial difference in absolute values depending on the racetrack and experimental design, thus highlighting the need for careful experimental design in thermal preference behavioral studies.\u003c/p\u003e \u003cp\u003eIn the end, it is possible that resources, notably food (here mainly cattle dung), is so abundant in time and space (Crombie \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1947\u003c/span\u003e, de Camargo et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Tilman \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e1987\u003c/span\u003e) that competition among the various European sepsid species is only weak or not apparent at all most of the time, thus allowing coexistence. We need to continue studying more fully the extent and mechanisms of competition, species composition and ecological niche differentiation not only of sepsid dung flies, but also of other widespread species.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project was funded by the Swiss National Foundation SNF grant no. 31003A-176055.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICTS OF INTEREST\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eETHICS APPROVAL\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot necessary\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONSENTS TO PARTICIPATE OR PUBLISH\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot necessary\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA FILES\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAre supplied as supplement\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCODE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNothing beyond standard ANOVA statistics\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Marcel Freund of the University of Zurich who contributed to the design of the handcrafted racetrack apparatus. We are very grateful to Jeannine Roy who helped with fly maintenance, sampling and breeding of the flies. This project was funded by the Swiss National Foundation SNF grant no. 31003A-176055.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the conceptual development of the study/experiment.\u003c/p\u003e\n\u003cp\u003eMK developed the apparatus.\u003c/p\u003e\n\u003cp\u003eRD conducted the study as part of his PhD (data gathering; statistical analysis), with MK \u0026amp; WUB as supervisors.\u003c/p\u003e\n\u003cp\u003eRD wrote the first draft; WUB \u0026amp; MK co-wrote.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAddeo NF et al (2022) Impact of age, size, and sex on adult black soldier fly [ \u003cem\u003eHermetia illucens\u003c/em\u003e L. (Diptera: Stratiomyidae)] thermal preference. 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Oecologia 189:905\u0026ndash;917\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"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},"keywords":"Thermal preference, Sepsidae, niche theory, competition, competitive exclusion, coexistence, temperature, thermal racetrack","lastPublishedDoi":"10.21203/rs.3.rs-4252799/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4252799/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMany similar sepsid dung fly species coexist on European pastures, contradicting conventional wisdom of niche theory and competitive exclusion. We hypothesized that closely-related sepsid species on the same pasture in Switzerland avoid each other by having different spatio-temporal microhabitat niche preferences, thus enabling coexistence. A thermal racetrack experiment in the laboratory tested the thermal preferences of males and females of 9 coexisting temperate \u003cem\u003eSepsis\u003c/em\u003e dung fly species from Switzerland at two acclimation temperatures. The sepsid species investigated here showed no strong differences in thermal preferences. Flies of all species preferred to settle at cooler temperatures, and otherwise utilized the entire range (from 12\u0026deg;C to 30\u0026deg;C) offered for their activities. This was the case for both sexes, and also for both acclimation temperatures (18\u0026deg;C, 24\u0026deg;C). Our findings suggest that physiological thermal adaptation or acclimation is not an important mechanism by which adult sepsid flies avoid interspecific competition. Our experiment supports previous findings of widespread sepsid flies lacking local adaptation but high phenotypic plasticity, again highlighting the necessity of experimentally assessing putative biological mechanisms facilitating coexistence.\u003c/p\u003e","manuscriptTitle":"Lacking sex-specific temperature preferences of 9 coexisting temperate sepsid dung fly species (Diptera: Sepsidae)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-25 20:10:37","doi":"10.21203/rs.3.rs-4252799/v1","editorialEvents":[{"type":"communityComments","content":0}],"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":"59c68c9a-919d-4e78-b2d6-2fe8a954f017","owner":[],"postedDate":"June 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-20T07:04:35+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-25 20:10:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4252799","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4252799","identity":"rs-4252799","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}