Oviposition preferences in temperate grasshoppers: Conserved temperature requirements but contrasting responses to humidity across species

Oviposition preferences in temperate grasshoppers: Conserved temperature requirements but contrasting responses to humidity across species | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Oviposition preferences in temperate grasshoppers: Conserved temperature requirements but contrasting responses to humidity across species Michal Knapp, Tomáš Dvořák This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2924573/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 The right choice of an oviposition site is a crucial task for oviparous species without maternal care. In contrast to well investigated biotic factors, e.g., larval food preferences, parasitism, predation, and competition avoiding, abiotic factors affecting oviposition preferences in insects have been rarely investigated in comparative studies. To improve our current understanding of oviposition site selection in orthoptera, we investigated the influence of substrate temperature and moisture on oviposition behaviour for 14 temperate grasshopper species. Conspecific groups of adults were kept in arenas with simultaneous temperature and moisture gradients. For each ootheca produced during the experiment (1192 in total) we recorded its depth and local microclimatic conditions. Our results indicate that microclimatic oviposition preferences significantly differ among species, however, correlations between adult habitat preferences and microclimatic oviposition preferences were surprisingly weak. Even oligothermic species preferred substrate temperatures around 30°C and some xerothermic species preferred higher humidity. Hypothesized tendency to place oothecae closer to the ground within grass tussocks under hot and dry conditions was confirmed. It is possible that species evaluate microclimatic conditions for oviposition in the context of occupied habitat, i.e., in a relative rather than absolute manner. Biological sciences/Zoology/Animal behaviour Biological sciences/Ecology/Behavioural ecology Bare ground climate change ecophysiology egg laying tussock thermal niche Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Searching for the optimal environment that allows for the successful development of eggs and subsequent offspring survival is one of the crucial issues faced by every oviparous ectotherm species without a direct maternal care 1 . Most studies have focused on biotic factors associated with the optimal food availability for hatchlings or parasitism, predation, and competition avoiding 2 – 6 . Abiotic factors, such as moisture and temperature, have received significantly less attention 7 – 9 and comparative studies are largely missing. In addition, many studies are focused on reptile oviposition site selection (e.g. 10 – 12 ), and only limited information exist on insect species without direct maternal care, representing the most diverse and numerous group of ectotherms 13 , 14 . Especially with ongoing climate change and anthropogenic habitat degradation frequently affecting local moisture and temperature, understanding the role of microclimate on oviposition behaviour and preferences in insects has become an important research task for ecologists and entomologists 15 – 17 . Current theory on oviposition site selection in insects can be classified into two main hypotheses: 1) the environmental matching hypothesis postulates that oviposition preferences are associated with the overall xerothermophility (an affinity for dry and warm sites) of a species 7 , 13 , 18 , i.e., species inhabiting dry and warm habitats prefer to lay eggs in dry and warm microhabitats; 2) the insurance hypothesis postulates that all species prefer relatively humid substrate, independently of their habitat preferences, to avoid egg desiccation 9 , 19 , 20 . To test these hypotheses, multispecies comparative studies that uses standardized experimental designs are needed 13 , 21 . Unfortunately, the existing studies have mostly investigated a single species 9 , 18 , 20 , 22 or compared two species with contrasting habitat requirements 7 , 19 , 23 . For a few species, intraspecific differences between various populations have been also investigated 24 , 25 . Despite a potentially strong synergic effect, moisture and temperature have been rarely investigated in parallel 26 , 27 . Experimental designs that combine independent gradients of both variables are needed to distinguish between independent and interactive effects of local temperature and substrate humidity. Temperate grasshoppers from the subfamily Gomphocerinae represent an optimal model group for multispecies comparative studies. This subfamily contains a high number of morphologically uniform species with similar life histories 21 and, at the same time, particular species strongly differ in their habitat preferences 28 – 30 . Existing limited knowledge indicates that grasshoppers follow the environmental matching hypothesis rather than the insurance hypothesis, i.e., their microclimatic oviposition preferences are correlated with species habitat preferences 7 , 21 , 31 , 32 . However, a more systematic investigation is needed to provide a more nuanced and unambiguous conclusion. Within a suitable microhabitat, ovipositing females can further adjust their laying behaviour to meet optimal conditions for egg development 8 , 33 . For example, species using soil and grass tussocks for egg laying can manipulate oviposition depth (according to the vertical distribution plasticity hypothesis), as moisture commonly increases and temperature decreases with increasing depth 21 , 24 , 34 . In particular, it has been hypothesized that xerothermophilic grasshopper species lay eggs deeper into the substrate to avoid exposure to extreme temperatures and drought, whiles species inhabiting cold and moist habitats prefer shallow oviposition or grass tussocks to increase developmental temperature or to escape egg flooding 7 , 21 . However, for orthopterans, there is still only very limited evidence on inter- and intraspecific variation in this trait, as well as the overall variability in condition-dependent oviposition depth 21 , 24 . This study investigated the oviposition preferences of 14 Central European gomphocerid grasshopper species using laboratory experiments that combined independent moisture and temperature gradients. The specific goals were to: 1) identify moisture, temperature, and depth preferences for egg-laying of all investigated species; 2) evaluate the oviposition preference hypotheses by comparing data from this study with adult habitat preferences investigated in our previous study (xerothermophility indices; 30 ); 3) assess whether grasshoppers modify their oviposition behaviour (laying depth) according to local microclimatic conditions. Materials and methods Specimen collection Grasshoppers used for the laboratory experiment were collected between June 15 th and July 15 th , 2018, when most of Central European grasshopper species reach adulthood. All collecting sites were located within the Czech Republic. More specifically, xerothermophilic species were obtained from karst areas of Český kras and steppe habitats in České středohoří, hygro- and psychrophilic Omocestus viridulus were collected from Brdy Mountains, and the rest of the species were collected within the capital city of Prague. All sampled sites represented typical habitats for each species 35 . Grasshoppers were visually checked, and only freshly emerged adults (with a soft cuticle and deflated abdomen) or subadults were chosen for the laboratory experiment. This approach reduced a potential age effect on oviposition preferences 21 . Finally, fourteen grasshopper species with contrasting habitat requirements were included in the laboratory experiment (Table 1). Experimental design The laboratory experiment was performed in large cages (100 × 50 × 30 cm) made of 4 mm thick sheets of polycarbonate (bottom) and plastic mesh (walls). The bottom was divided lengthwise into four 100 × 12.5 c m sized compartments, and each compartment was filled 5 cm thick substrate layer consisting of 1:1 peat and sand mixture. During the testing stage we repeatedly measured water content of substrate samples originating from various areas of large cages (measured gravimetrically). Our substrate ensured even distribution of moisture within each compartment as confirmed by a pilot experiment (testing stage). Based on various watering regimes tested during the testing phase we developed the appropriate final experimental setting. Compartments (moisture treatments) within a cage differed in their moisture levels as follows: 1) dry (approximately 5% moisture, measured gravimetrically), 2) slightly wet (20%), 3) wet (35%) or 4) soaked (50%). The dry compartment did not get any extra water during the experiment. The slightly wet compartment was watered every other day with 200 ml, which resulted in transient wetness of the upper soil layer. The wet compartment was watered with 400 ml every other day to maintain permanent wetness across the whole soil profile. The soaked compartment received 600 ml every other day to reach fully saturated soil.. . A line of three 53 W halogen bulbs arranged 10 cm above the soil level was used to create a temperature gradient perpendicular to moisture gradient (Figure 1). The function describing the relation of soil-surface temperature and the distance from the heat source was computed based on measurements performed across the cage length in 5 cm intervals, separately for each moisture compartment. Maximal surface temperature (42 °C) was reached directly under the heat source in the dry compartment. In the other compartments, maximal temperature gradually decreased with increasing moisture level (32 °C in the soaked compartment). Moreover, there was no other light source in the breeding room and cages were visually isolated from each other. Grasshopper species differ in their preferred oviposition substrate, some species preferring bare ground, others place eggs (oothecae) into grass roots or grass tussocks, and some species use both strategies 21 . Therefore, four grass tussocks (ca. 10 × 10 cm ) were planted in each compartment on both sides of the heat source at a distance of 12 and 35 cm (see the cage setup in Figure 1). Twenty females and 15 males per species were released in each cage and each species had its own cage (i.e., 14 cages were used in total). Similar population densities were used in various laboratory studies on Central European gomphocerinae grasshoppers 32,36,37 . However, natural population densities are probably lower. Cages were maintained until the death of the last female, typically for two or three months. Grasshoppers were provided with food in the form of a mixture of grasses placed in water-filled plastic tubes. Several tubes were dispersed across each cage and grasses were changed every three days or when depleted. Cages were kept at a L16 : D8 photoperiod and ambient temperature fluctuating between 26 ± 2 °C during the light period and 17 ± 2 °C during the dark period. Ambient humidity ranged 40 – 60 %. All investigated species are strictly day-active 21 and thus it is expected that oviposition took place solely during the daytime. After the death of the last female, each cage was carefully inspected for oothecae. For each ootheca, the substrate (bare ground vs. tussock), the compartment moisture level (dry, slightly wet, wet, soaked), the vertical position relative to the soil surface (the depth in the soil, or the height above the ground in the case of an oviposition on grasses), and the distance from the heat source (to calculate temperature) were recorded. The vertical position of ootheca was measured as the position of its center with a precision of± 0.5 cm. Data analysis Phylogenetic data To address phylogenetic relations between investigated species, we constructed a phylogenetic tree following methods and data of the most recently published phylogeny 38 and added additional COI sequence of Stenobothrus crassipes , which was obtained from the BOLD database (39; BOLD: AAD9833). The final phylogenetic tree is included in supplementary materials (Figure S4). Moisture and temperature indices To describe species oviposition preferences, we assessed the most preferred level (mode) and mean of moisture (dry, slightly wet, wet or soaked), the mean laying depth (vertical position according to the soil surface), and the mean preferred soil surface temperature. To test the existence of specific preferences for moisture, chi-squared tests were used for each species separately. Our null hypothesis assumed the random distribution among moisture compartments, i.e., 25% probability for each compartment. Significant differences between species oviposition temperature and vertical distribution were tested with a One-way ANOVA. Only species producing more than 10 oothecae per substrate type were included in our analyses. To investigate the relationship between the index of xerothermophility (developed in our previous study; Table 1; for details see 30 ) and species-specific microclimatic preferences for moisture, temperature and vertical distribution of oothecae, several phylogenetic linear mixed-effect models were fitted. In all models, species identity was included as a random effect. Separate models were used for all investigated response variables and substrate type combinations, i.e., bare ground-laid and tussock-laid oothecae were analysed independently. To account for phylogenetic relations, we used “Almer” extension for “lme4” package 40 . For analyses of the vertical distribution of oothecae, the contribution of each species had to be weighted by the ratio of bare ground-laid or tussock-laid oothecae to decrease the importance of species as a contributing factor (as cases of rare oothecae in non-preferred substrate type could be problematic). Finally, we investigated the effects of microclimatic conditions on the vertical distribution of oothecae using phylogenetic linear mixed-effect models in which the vertical position of ootheca was the response variable. Moisture, temperature, and their interaction were used as fixed effects. Species identity was included as a random effect. Two separate models were fitted for bare ground-laid and tussock-laid oothecae. All analyses were performed in R 3.5.2 41 . Results Oviposition preferences A total of 1192 oothecae were produced by 280 females from 14 grasshopper species investigated in this study (Table 1). All species showed a non-random oviposition microhabitat selection with respect to moisture (Figure 2), with Stenobothrus nigromaculatus being the closest to a random distribution of oothecae (χ 2 = 10.7, df = 3, p = 0.013). The other extreme was represented by Omocestus haemorrhoidalis , which laid only 10 oothecae exclusively in the wet compartment. Three species preferred soaked substrate, six species preferred wet substrate and five species preferred slightly wet or dry substrate (Table S1). Overall, across all investigated species, wet substrate is the most preferred (contained 36% of all oothecae), followed by soaked (25%), slightly wet (22%) and dry substrate (17%). The strongest preference for moist substrate was recorded for Pseudochorthippus montanus , and the driest substrate was selected by Chorthippus vagans and Stenobothrus crassipes. The highest average temperature for soil substrate-laid oothecae was recorded for Chorthippus albomarginatus (33.2 °C), and the lowest for Chorthippus mollis (29.2 °C). Among tussocks-laid oothecae, the highest average temperature was observed for Omocestus viridulus (33.3 °C), and the lowest for Pseudochorthippus parallelus (29.6 °C). The preferred soil temperatures for oviposition are summarised in Table S1 and visualised in Figure 3. Thermal preferences significantly differed between some species (bare ground-laid oothecae: F = 11.43, df = 9, p < 0.001; tussocks-laid oothecae: F = 14.47, df = 10, p < 0.001), however, there was also a large group of species with very similar thermal preferences (Table S1). Vertical positions of oothecae ranged from 4 cm below ground to 5 cm above the soil surface (Table S1; Figure S1). The deepest ootheca was buried by Chorthippus vagans , but the lowest mean depth was observed for Euchorthippus pulvinatus (2.37 cm below ground). The species with the highest mean laying location was Omocestus viridulus with a mean of 1.34 cm above the soil surface . There were significant differences in the vertical distribution of oothecae between the great majority of investigated species (bare ground-laid oothecae: F = 20.3, df = 9, p < 0.001; tussocks-laid oothecae: F = 36.58, df = 10, p < 0.001; Table S1). Oviposition site selection hypotheses Contrary to our expectation, and the environmental matching hypothesis, there was no significant relationship between species-specific oviposition preferences (preferred moisture level, substrate temperature, and partially the vertical distribution of oothecae) and the index of xerothermophility for both bare ground-laid and tussock-laid oothecae, with the exception of the vertical distribution of tussocks-laid oothecae (Table 2). Highly xerothermophilic species (based on habitat requirements of adults published in 30 ) placed their oothecae significantly closer to the ground within grass tussock than less xerothermophilic species. Interestingly, the significance of this relationship was not confirmed for bare ground laying species despite the noticeable regression slope (Figure 4). Our results for moisture strongly support the insurance hypothesis, as wet substrate was, in general, the most preferred substrate and the fewest number of oothecae were placed in the dry compartment across investigated species (Figure2). In general, the vertical distribution of oothecae at the interspecific level was significantly affected by moisture level and partially by temperature in accordance with the vertical distribution plasticity hypothesis. Oothecae were placed shallower in bare ground or higher on tussocks with increasing local moisture (Figures S2 and S3). Oothecae were placed significantly lower on tussocks and tend to be placed deeper in bare ground with increasing local temperature (Table 3, Figure 5, S3). There was no significant interactive effect of local moisture and temperature on vertical distribution of oothecae. Note that the above-described results represent overall patterns (across species) and patterns for particular species can be slightly different (for details see Figures 5, S2 and S3). Discussion All investigated orthopteran species showed a non-random choice of the moisture level, surface temperature, and vertical position when ovipositing. Interestingly, there was no relationship between preferred moisture or temperature during oviposition and species xerothermophility based on species habitat preferences. Instead, generally higher moisture and a relatively narrow range of temperature were preferred across species. Nevertheless, there was a significant relationship between species xerothermophility and vertical distribution of oothecae laid in tussocks. Furthermore, at the intraspecific level, oothecae were placed shallower in bare ground on higher on tussocks with increasing local moisture and partially also temperature. In general, our results indicate that orthopterans optimise oviposition depth and even xeric orthopteran species prefer substrates with higher moisture to protect oothecae from desiccation. Moisture is the most relevant factor determining successful egg development in insects 14 . Many species need to absorb water before embryogenesis begins, and eggs have to continuously resist desiccation until hatching occurs 21 . Almost all the investigated species laid at least some eggs into each moisture level (compartment), which suggests a relatively relaxed moisture preferences across orthopterans. Even in the most drought preferring species, Chorthippus vagans and Stenobothrus crassipes , females only slightly preferred the dry substrate over slightly wet or wet substrates, and some oothecae were laid in soaked soil. In general, wet soil was preferred the most and dry soil the least. Our findings provide support for the insurance hypothesis, which predicts a general tendency to oviposit into substrates with high moisture levels independently of the habitat preferred by adults 9 , 20 , 42 . In contrast, we did not find evidence supporting the environmental matching hypothesis, which predicts a correlation between species habitat preferences (xerothermophility) and oviposition preferences 7 , 18 . It seems that species do not detect moisture in an absolute manner, but only relatively in the context of occupied habitat. For example, the xerothermophilic species Chorthippus mollis strongly preferred soaked substrate in the experiment, despite that this species almost never comes into contact with soaked soil in nature. Omocestus viridulus , which usually occurs in humid habitats, preferred tussocks within only slightly wet soil in our experiment. This can be an adaptation to spring flooding, which are common in its preferred habitats 21 , 35 . The embryonic development rate is closely related to ambient temperature and the sum of degree days during a season can be a limiting factor for many temperate insects 32 , 43 . On the other hand, lethal temperature thresholds are usually very close to the thermal optima and a fine equilibrium between these two factors has to be reached 14 , 44 . Our data show that optima of all investigated species occurred between 29 and 34°C, which fits well with previously published single-species data 21 , 25 , 31 , 45 . The relatively narrow range of preferred oviposition temperatures across species originating from variable habitats is quite surprising and seems to be quite conservative within the Gomphocerinae subfamily. In contrast to findings of Schnebel & Grossfield (1986), we observed no tendency of xerothermophilic species to prefer higher surface temperature for oviposition. However, Schnebel & Grossfield (1986) studied species (fruit flies) along a latitudinal gradient ranging from arctic to tropic regions, not species from a single region. Again, a possible explanation of our findings can be based on individual life strategies of different species. For example, despite its high xerothermophility, Chorthippus mollis preferred the lowest oviposition temperature out of all investigated species. This species has a relatively long embryonic development and the choice of relatively cold and moist microhabitats within hot and dry steppes, where it live, can protect developing immobile embryos from potentially damaging temperatures at the start of summer when other species already hatched 32 , 35 . It has been hypothesized that xerothermophilic species should lay eggs deeper into the ground or lower within tussocks to avoid damaging hot and dry conditions 7 , 21 , 46 than oligothermic species. Our results partly supported this hypothesis, as tussocks-laid oothecae were placed closer to ground with increasing xerothermophility of species, and there was a similar tendency in bare ground-laid oothecae. Nevertheless, using more species, especially more hygro- and psychrophilic ones, in a future study will allow for a better evaluation of this pattern. Additional support for this relationship was provided at the intraspecific level, as oothecae were laid deeper in dry and hot conditions in the great majority of investigated species (following the vertical distribution plasticity hypothesis). A similar finding was previously reported for the grasshopper Romalea microptera in which females laid eggs shallower into the substrate when exposed to higher levels of moisture 24 . Only a limited number of studies have investigated the effects of microclimate on oviposition behaviour compared to the wider range of published works that explore the effects of biotic factors, e.g., optimal food, parasitism, or competition avoidance 5 , 6 . Abiotic factors are directly modified by ongoing climate change and anthropogenic habitat alterations, which increases the relevance of such studies for nature conservation 16 , 47 . Moreover, eggs are an immobile life stage unable to escape from suboptimal conditions and frequently the most vulnerable to extreme temperatures 32 , 48 . This highlights the importance of studies investigating microhabitat oviposition preferences in insects. Especially valuable are studies employing a comparative framework, i.e., investigating a high number of species using a standardized experimental setting. Unfortunately, such studies are rare with the exception of a historical study conducted for Drosophila flies 13 . Declarations Acknowledgements: We are grateful to the Nature Conservation Agency of the Czech Republic for permitting experiments with endangered species Stenobothrus eurasius. Many thanks go to Jakub Prokop for financial and technical support, Ondřej Balvín for help with the phylogenetic analysis, Ezequiel González for insightful comments and David N. Awde for language corrections. 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BARCODING: bold: The Barcode of Life Data System (http://www.barcodinglife.org): BARCODING. Mol. Ecol. Notes 7 , 355–364 (2007). Bolstad, G. H. et al. genetic constraints predict evolutionary divergence in Dalechampia blossoms. Philos. Trans. R. Soc. B Biol. Sci. 369 , 20130255 (2014). R Core Team. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/. (2020). Marrone, P. G. & Stinner, R. E. Effects of soil moisture and texture on oviposition preference of the bean leaf beetle, Cerotoma trifurcata (Forster) (Coleoptera: Coccinellidae). Environ. Entomol. 12 , 426–428 (1983). Sibly, R. & Monk, K. A Theory of Grasshopper Life Cycles. Oikos 48 , 186–194 (1987). Feder, M. E., Blair, N. & Figueras, H. Oviposition site selection: unresponsiveness ofDrosophilato cues of potential thermal stress. Anim. Behav. 53 , 585–588 (1997). Fielding, D. J. Developmental time of Melanoplus sanguinipes (Orthoptera: Acrididae) at high latitudes. Environ. Entomol. 33 , 1513–1522 (2004). Gustin, R. D. Effect of Two Moisture and Population Levels on Oviposition of the Western Corn Rootworm123. Environ. Entomol. 8 , 406–407 (1979). Fartmann, T., Brüggeshemke, J., Poniatowski, D. & Löffler, F. Summer drought affects abundance of grassland grasshoppers differently along an elevation gradient. Ecol. Entomol. 47 , 778–790 (2022). Knapp, M. & Nedvěd, O. Gender and Timing during Ontogeny Matter: Effects of a Temporary High Temperature on Survival, Body Size and Colouration in Harmonia axyridis. PLoS ONE 8 , e74984 (2013). Tables Table 1: Overview of investigated species. Number of laid oothecae per species and their associations with grass tussocks is stated. Also the indices of xerothermophility obtained from our previous study are presented here. Species Total amount of oothecae Tussocks-associated oothecae (%) Index of xerothermophility Euchorthippuspulvinatus 42 0 0.896 Chorthpippusalbomarginatus 51 2 0.336 Chorthippusbiguttulus 160 0 0.518 Chorthippusdorsatus 145 100 0.375 Chorthippusmollis 54 4 0.900 Chorthippusvagans 92 12 0.960 Omocestushaemorrhoidalis 10 40 0.799 Omocestusviridulus 189 100 0.109 Pseudochorthippusmontanus 41 0 0.021 Pseudochorthippusparallelus 41 32 0.291 Stenobothrus crassipes 80 51 0.780 Stenobothruseurasius 26 88 1.000 Stenobothruslineatus 183 100 0.777 Stenobothrusnigromaculatus 79 100 0.952 Table 2: Effects of species xerothermophility on oviposition preferences. Results of phyllogenetic mixed-effects models investigating the relationship between the index of xerothermophility (independent variable) and species oviposition preferences for moisture, temperature, and ootheca vertical position (separate model was fitted for each response variable and substrate type). Dependent variable Substrate type Chi-sq d.f. p-value Moisture Bare ground 0.0068 1 0.9344 Tussock 1.8825 1 0.17 Temperature Bare ground 0.658 1 0.4173 Tussock 0.7725 1 0.3953 Vertical position Bare ground 3.0814 1 0.0792 Tussock 5.5023 1 0.019 Table 3: Results of the phyllogenetic mixed-effect model testing whether there was plasticity of laying depth based on changing moisture and temperature. Substrate type Independent variable Chi-sq d.f. p Bare ground moisture 18.02 3 <0.001 temperature 3.26 1 0.0706 moisture : temperature 2.6 3 0.4586 Tussock moisture 27.61 3 <0.001 temperature 6.09 1 0.0136 moisture : temperature 6.25 3 0.1 Additional Declarations There is NO Competing Interest. Supplementary Files rawdata.csv Raw dataset SupplementarytableS1.xlsx Basic descriptive statistics Supplementaryfigures.docx 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-2924573","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":201093502,"identity":"ddbba7ea-cfad-42c3-b12e-d44579679931","order_by":0,"name":"Michal Knapp","email":"","orcid":"","institution":"Faculty of Environmental Sciences, Czech University of Life Sciences Prague","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Michal","middleName":"","lastName":"Knapp","suffix":""},{"id":201093503,"identity":"e9485567-9235-41a4-9015-e0fe256cb961","order_by":1,"name":"Tomáš Dvořák","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYBACNnYg8YCBgYeBvYFILfzMQCIBpIXnAJFaJJshWhgYJBKI1GJwmP3ih4SKOhl+yecPH/7cYRfNwH78AQEtPMUSCWcO80jOzjE2kDyTnNvAk4DfPqCWBInEtgM8Brdz2CQM25hzGyQYDuDVYn+YJ/lH4r86Hvubx5//SGyrB2phbCDkl2MSiQ3MPAYSDGYMB9sOA7Uw49UBchibRcKxwzwSZ3KMJRvbjue28aQR0HK8/fGNDzV19vztxx9+/NlWndtPKMSAkWiAymcjoB4I2AmZOQpGwSgYBSMeAAA790Yv+i+p7QAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-9182-4333","institution":"Faculty of Environmental Sciences, Czech University of Life Sciences Prague","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Tomáš","middleName":"","lastName":"Dvořák","suffix":""}],"badges":[],"createdAt":"2023-05-11 21:22:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2924573/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2924573/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":37249775,"identity":"282eda1d-6604-4a15-8dbd-020d90517070","added_by":"auto","created_at":"2023-05-19 16:05:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":28164,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme of our experimental cages (view from above).\u003c/strong\u003e Background colours indicate local surface temperature, green squares represent grass tussocks, and crossed circles indicate positions of 53W halogen bulbs (placed 10 cm above the surface). Substrate humidity decreased from top to bottom.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2924573/v1/4e5c1702aec533a3a389f8ff.png"},{"id":37250211,"identity":"2e1f962a-7a47-4869-b39a-1814a38dc133","added_by":"auto","created_at":"2023-05-19 16:13:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":40563,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpecies-specific distribution of oothecae in relation to humidity.\u003c/strong\u003e Data for all species significantly differed from a random distribution (0.25 : 0.25 : 0.25 : 0.25).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2924573/v1/27c4ffc6366ba4e4a5df78b9.png"},{"id":37249779,"identity":"cc207db7-dbf4-4f07-b53a-ae5c8546ae67","added_by":"auto","created_at":"2023-05-19 16:05:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":52603,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpecies specific distribution of oothecae in relation to temperature.\u003c/strong\u003e Bare ground-laid and tussocks-laid oothecae are distinguished by colours (see the legend).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2924573/v1/28384b0a2a555f85d9ac1471.png"},{"id":37249777,"identity":"667b541d-1d47-44cc-a143-922498e39514","added_by":"auto","created_at":"2023-05-19 16:05:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":38281,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRelationship between vertical distribution of oothecae and species index of xerothermophility.\u003c/strong\u003e Each dot represents mean vertical position of oothecae laid by one species (error bars represent ± SD). Bare ground-laid (dark grey) and tussocks-laid (light grey) oothecae are shown separately. Dot size represents species’ affinity to the given substrate type. The full line represents the phyllogeneticaly corrected significant relationship between the vertical distribution of oothecae and the index of xerothermophility for tussocks-laid oothecae. The dashed line represents the same but only marginally significant relationship for bare ground-laid oothecae.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2924573/v1/a2191143cc87c9ec3c5778eb.png"},{"id":37249782,"identity":"5f997fab-f2ed-4f07-a611-0e6b042528a0","added_by":"auto","created_at":"2023-05-19 16:05:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":159036,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of temperature on vertical position of oothecae.\u003c/strong\u003e Separate regression lines are shown for each species. Left shows tussock-laid oothecae and right shows bare ground-laid oothecae. The overall relationship (across species) between temperature and vertical position of oothecae was significant for tussock-laid oothecae and marginally significant for bare ground-laid oothecae.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2924573/v1/8e8dc268462d6bfa98711933.png"},{"id":45574129,"identity":"62e867d2-f27f-4f39-92f7-915c0f7a3d94","added_by":"auto","created_at":"2023-10-31 17:14:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":774989,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2924573/v1/5ce37956-d857-40c1-89ce-4edff35fd1ec.pdf"},{"id":37250212,"identity":"e710b61f-3284-409c-b53f-30dea5346213","added_by":"auto","created_at":"2023-05-19 16:13:47","extension":"csv","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":86443,"visible":true,"origin":"","legend":"Raw dataset","description":"","filename":"rawdata.csv","url":"https://assets-eu.researchsquare.com/files/rs-2924573/v1/c51ad483b6f024d33a6e28c4.csv"},{"id":37250829,"identity":"d6fba02f-2e96-4995-a23c-d5e1d9611b97","added_by":"auto","created_at":"2023-05-19 16:21:47","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":15590,"visible":true,"origin":"","legend":"\u003cp\u003eBasic descriptive statistics\u003c/p\u003e","description":"","filename":"SupplementarytableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-2924573/v1/d61eba47b4245dcdc6a89393.xlsx"},{"id":37250213,"identity":"c0e88f95-1976-4a63-97e2-227a4d117719","added_by":"auto","created_at":"2023-05-19 16:13:47","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":318749,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-2924573/v1/121f013aac32dbca06215938.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Oviposition preferences in temperate grasshoppers: Conserved temperature requirements but contrasting responses to humidity across species","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSearching for the optimal environment that allows for the successful development of eggs and subsequent offspring survival is one of the crucial issues faced by every oviparous ectotherm species without a direct maternal care \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Most studies have focused on biotic factors associated with the optimal food availability for hatchlings or parasitism, predation, and competition avoiding \u003csup\u003e\u003cspan additionalcitationids=\"CR3 CR4 CR5\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Abiotic factors, such as moisture and temperature, have received significantly less attention \u003csup\u003e\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e and comparative studies are largely missing. In addition, many studies are focused on reptile oviposition site selection (e.g. \u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e), and only limited information exist on insect species without direct maternal care, representing the most diverse and numerous group of ectotherms \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Especially with ongoing climate change and anthropogenic habitat degradation frequently affecting local moisture and temperature, understanding the role of microclimate on oviposition behaviour and preferences in insects has become an important research task for ecologists and entomologists \u003csup\u003e\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCurrent theory on oviposition site selection in insects can be classified into two main hypotheses: 1) the environmental matching hypothesis postulates that oviposition preferences are associated with the overall xerothermophility (an affinity for dry and warm sites) of a species \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, i.e., species inhabiting dry and warm habitats prefer to lay eggs in dry and warm microhabitats; 2) the insurance hypothesis postulates that all species prefer relatively humid substrate, independently of their habitat preferences, to avoid egg desiccation \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. To test these hypotheses, multispecies comparative studies that uses standardized experimental designs are needed \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Unfortunately, the existing studies have mostly investigated a single species \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e or compared two species with contrasting habitat requirements \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. For a few species, intraspecific differences between various populations have been also investigated \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Despite a potentially strong synergic effect, moisture and temperature have been rarely investigated in parallel \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Experimental designs that combine independent gradients of both variables are needed to distinguish between independent and interactive effects of local temperature and substrate humidity.\u003c/p\u003e \u003cp\u003eTemperate grasshoppers from the subfamily Gomphocerinae represent an optimal model group for multispecies comparative studies. This subfamily contains a high number of morphologically uniform species with similar life histories \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e and, at the same time, particular species strongly differ in their habitat preferences \u003csup\u003e\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Existing limited knowledge indicates that grasshoppers follow the environmental matching hypothesis rather than the insurance hypothesis, i.e., their microclimatic oviposition preferences are correlated with species habitat preferences \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. However, a more systematic investigation is needed to provide a more nuanced and unambiguous conclusion.\u003c/p\u003e \u003cp\u003eWithin a suitable microhabitat, ovipositing females can further adjust their laying behaviour to meet optimal conditions for egg development \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. For example, species using soil and grass tussocks for egg laying can manipulate oviposition depth (according to the vertical distribution plasticity hypothesis), as moisture commonly increases and temperature decreases with increasing depth \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. In particular, it has been hypothesized that xerothermophilic grasshopper species lay eggs deeper into the substrate to avoid exposure to extreme temperatures and drought, whiles species inhabiting cold and moist habitats prefer shallow oviposition or grass tussocks to increase developmental temperature or to escape egg flooding \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. However, for orthopterans, there is still only very limited evidence on inter- and intraspecific variation in this trait, as well as the overall variability in condition-dependent oviposition depth \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThis study investigated the oviposition preferences of 14 Central European gomphocerid grasshopper species using laboratory experiments that combined independent moisture and temperature gradients. The specific goals were to: 1) identify moisture, temperature, and depth preferences for egg-laying of all investigated species; 2) evaluate the oviposition preference hypotheses by comparing data from this study with adult habitat preferences investigated in our previous study (xerothermophility indices; \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e); 3) assess whether grasshoppers modify their oviposition behaviour (laying depth) according to local microclimatic conditions.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003ch2\u003eSpecimen collection\u003c/h2\u003e\n\u003cp\u003eGrasshoppers used for the laboratory experiment were collected between June 15\u003csup\u003eth\u003c/sup\u003e and July 15\u003csup\u003eth\u003c/sup\u003e, 2018, when most of Central European grasshopper species reach adulthood. All collecting sites were located within the Czech Republic. More specifically, xerothermophilic species were obtained from karst areas of Česk\u0026yacute; kras and steppe habitats in Česk\u0026eacute; středohoř\u0026iacute;, hygro- and psychrophilic \u003cem\u003eOmocestus viridulus\u0026nbsp;\u003c/em\u003ewere collected from Brdy Mountains, and the rest of the species were collected within the capital city of Prague. All sampled sites represented typical habitats for each species \u003csup\u003e35\u003c/sup\u003e. Grasshoppers were visually checked, and only freshly emerged adults (with a soft cuticle and deflated abdomen) or subadults were chosen for the laboratory experiment. This approach reduced a potential age effect on oviposition preferences \u003csup\u003e21\u003c/sup\u003e. Finally, fourteen grasshopper species with contrasting habitat requirements were included in the laboratory experiment (Table 1).\u003c/p\u003e\n\u003ch2\u003eExperimental design\u003c/h2\u003e\n\u003cp\u003eThe laboratory experiment was performed in large cages (100 \u003cstrong\u003e\u0026times;\u0026nbsp;\u003c/strong\u003e50 \u003cstrong\u003e\u0026times;\u0026nbsp;\u003c/strong\u003e30 cm) made of 4 mm thick sheets of polycarbonate (bottom) and plastic mesh (walls). The bottom was divided lengthwise into four 100 \u003cstrong\u003e\u0026times;\u0026nbsp;\u003c/strong\u003e12.5\u003cstrong\u003e\u0026nbsp;c\u003c/strong\u003em sized compartments, and each compartment was filled 5 cm thick substrate layer consisting of 1:1 peat and sand mixture. During the testing stage we repeatedly measured water content of substrate samples originating from various areas of large cages (measured gravimetrically). Our substrate ensured even distribution of moisture within each compartment as confirmed by a pilot experiment (testing stage). Based on various watering regimes tested during the testing phase we developed the appropriate final experimental setting. Compartments (moisture treatments) within a cage differed in their moisture levels as follows: 1) dry (approximately 5% moisture, measured gravimetrically), 2) slightly wet (20%), 3) wet (35%) or 4) soaked (50%). The dry compartment did not get any extra water during the experiment. The slightly wet compartment was watered every other day with 200 ml, which resulted in transient wetness of the upper soil layer. The wet compartment was watered with 400 ml every other day to maintain permanent wetness across the whole soil profile. The soaked compartment received 600 ml every other day to reach fully saturated soil.. . A line of three 53 W halogen bulbs arranged 10 cm above the soil level was used to create a temperature gradient perpendicular to moisture gradient (Figure 1). The function describing the relation of soil-surface temperature and the distance from the heat source was computed based on measurements performed across the cage length in 5 cm intervals, separately for each moisture compartment. Maximal surface temperature (42 \u0026deg;C) was reached directly under the heat source in the dry compartment. In the other compartments, maximal temperature gradually decreased with increasing moisture level (32 \u0026deg;C in the soaked compartment). Moreover, there was no other light source in the breeding room and cages were visually isolated from each other.\u003c/p\u003e\n\u003cp\u003eGrasshopper species differ in their preferred oviposition substrate, some species preferring bare ground, others place eggs (oothecae) into grass roots or grass tussocks, and some species use both strategies\u0026nbsp;\u003csup\u003e21\u003c/sup\u003e. Therefore, four grass tussocks (ca. 10 \u003cstrong\u003e\u0026times; 10 cm\u003c/strong\u003e) were planted in each compartment on both sides of the heat source at a distance of 12 and 35 cm (see the cage setup in Figure 1).\u003c/p\u003e\n\u003cp\u003eTwenty females and 15 males per species were released in each cage and each species had its own cage (i.e., 14 cages were used in total). Similar population densities were used in various laboratory studies on Central European gomphocerinae grasshoppers \u003csup\u003e32,36,37\u003c/sup\u003e. However, natural population densities are probably lower. Cages were maintained until the death of the last female, typically for two or three months. Grasshoppers were provided with food in the form of a mixture of grasses placed in water-filled plastic tubes. Several tubes were dispersed across each cage and grasses were changed every three days or when depleted. Cages were kept at a L16 : D8 photoperiod and ambient temperature fluctuating between 26 \u0026plusmn; 2 \u0026deg;C during the light period and 17 \u0026plusmn; 2 \u0026deg;C during the dark period. Ambient humidity ranged 40 \u0026ndash; 60 %. All investigated species are strictly day-active \u003csup\u003e21\u003c/sup\u003e and thus it is expected that oviposition took place solely during the daytime.\u003c/p\u003e\n\u003cp\u003eAfter the death of the last female, each cage was carefully inspected for oothecae. For each ootheca, the substrate (bare ground vs. tussock), the compartment moisture level (dry, slightly wet, wet, soaked), the vertical position relative to the soil surface (the depth in the soil, or the height above the ground in the case of an oviposition on grasses), and the distance from the heat source (to calculate temperature) were recorded. The vertical position of ootheca was measured as the position of its center with a precision of\u0026plusmn; 0.5 cm. \u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eData analysis\u003c/h2\u003e\n\u003ch2\u003ePhylogenetic data\u003c/h2\u003e\n\u003cp\u003eTo address phylogenetic relations between investigated species, we constructed a phylogenetic tree following methods and data of the most recently published phylogeny\u0026nbsp;\u003csup\u003e38\u003c/sup\u003e and added additional COI sequence of \u003cem\u003eStenobothrus crassipes\u003c/em\u003e, which was obtained from the BOLD database\u0026nbsp;(39;\u0026nbsp;BOLD: AAD9833). The final phylogenetic tree is included in supplementary materials (Figure S4).\u003c/p\u003e\n\u003cp\u003eMoisture and temperature indices\u003c/p\u003e\n\u003cp\u003eTo describe species oviposition preferences, we assessed the most preferred level (mode) and mean of moisture (dry, slightly wet, wet or soaked), the mean laying depth (vertical position according to the soil surface), and the mean preferred soil surface temperature. To test the existence of specific preferences for moisture, chi-squared tests were used for each species separately. Our null hypothesis assumed the random distribution among moisture compartments, i.e., 25% probability for each compartment. Significant differences between species oviposition temperature and vertical distribution were tested with a One-way ANOVA. Only species producing more than 10 oothecae per substrate type were included in our analyses.\u003c/p\u003e\n\u003cp\u003eTo investigate the relationship between the index of xerothermophility (developed in our previous study; Table 1; for details see\u0026nbsp;\u003csup\u003e30\u003c/sup\u003e) and species-specific microclimatic preferences for moisture, temperature and vertical distribution of oothecae, several phylogenetic linear mixed-effect models were fitted. In all models, species identity was included as a random effect. Separate models were used for all investigated response variables and substrate type combinations, i.e., bare ground-laid and tussock-laid oothecae were analysed independently. To account for phylogenetic relations, we used \u0026ldquo;Almer\u0026rdquo; extension for \u0026ldquo;lme4\u0026rdquo; package\u0026nbsp;\u003csup\u003e40\u003c/sup\u003e. For analyses of the vertical distribution of oothecae, the contribution of each species had to be weighted by the ratio of bare ground-laid or tussock-laid oothecae to decrease the importance of species as a contributing factor (as cases of rare oothecae in non-preferred substrate type could be problematic).\u003c/p\u003e\n\u003cp\u003eFinally, we investigated the effects of microclimatic conditions on the vertical distribution of oothecae using phylogenetic linear mixed-effect models in which the vertical position of ootheca was the response variable. Moisture, temperature, and their interaction were used as fixed effects. Species identity was included as a random effect. Two separate models were fitted for bare ground-laid and tussock-laid oothecae. All analyses were performed in R 3.5.2\u0026nbsp;\u003csup\u003e41\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003eOviposition preferences\u003c/h2\u003e\n\u003cp\u003eA total of 1192 oothecae were produced by 280 females from 14 grasshopper species investigated in this study (Table 1). All species showed a non-random oviposition microhabitat selection with respect to moisture (Figure 2), with \u003cem\u003eStenobothrus nigromaculatus\u003c/em\u003e being the closest to a random distribution of oothecae (\u0026chi;\u003csup\u003e2\u003c/sup\u003e = 10.7, df = 3, p = 0.013). The other extreme was represented by \u003cem\u003eOmocestus haemorrhoidalis\u003c/em\u003e, which laid only 10 oothecae exclusively in the wet compartment. Three species preferred soaked substrate, six species preferred wet substrate and five species preferred slightly wet or dry substrate (Table S1). Overall, across all investigated species, wet substrate is the most preferred (contained 36% of all oothecae), followed by soaked (25%), slightly wet (22%) and dry substrate (17%). The strongest preference for moist substrate was recorded for \u003cem\u003ePseudochorthippus montanus\u003c/em\u003e, and the driest substrate was selected by \u003cem\u003eChorthippus vagans\u003c/em\u003e and \u003cem\u003eStenobothrus crassipes.\u003c/em\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe highest average temperature for soil substrate-laid oothecae was recorded for \u003cem\u003eChorthippus albomarginatus\u003c/em\u003e (33.2 \u0026deg;C), and the lowest for \u003cem\u003eChorthippus mollis\u003c/em\u003e (29.2 \u0026deg;C). Among tussocks-laid oothecae, the highest average temperature was observed for \u003cem\u003eOmocestus viridulus\u003c/em\u003e (33.3 \u0026deg;C), and the lowest for \u003cem\u003ePseudochorthippus parallelus\u003c/em\u003e (29.6 \u0026deg;C). The preferred soil temperatures for oviposition are summarised in Table S1 and visualised in Figure 3. Thermal preferences significantly differed between some species (bare ground-laid oothecae: F = 11.43, df = 9, p \u0026lt; 0.001; tussocks-laid oothecae: F = 14.47, df = 10, p \u0026lt; 0.001), however, there was also a large group of species with very similar thermal preferences (Table S1).\u003c/p\u003e\n\u003cp\u003eVertical positions of oothecae ranged from 4 cm below ground to 5 cm above the soil surface (Table S1; Figure S1). The deepest ootheca was buried by \u003cem\u003eChorthippus vagans\u003c/em\u003e, but the lowest mean depth was observed for \u003cem\u003eEuchorthippus pulvinatus\u003c/em\u003e (2.37 cm below ground). The species with the highest mean laying location was \u003cem\u003eOmocestus viridulus\u003c/em\u003e with a mean of 1.34 cm above the soil surface\u003cem\u003e.\u003c/em\u003e There were significant differences in the vertical distribution of oothecae between the great majority of investigated species (bare ground-laid oothecae: F = 20.3, df = 9, p \u0026lt; 0.001; tussocks-laid oothecae: F = 36.58, df = 10, p \u0026lt; 0.001; Table S1).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eOviposition site selection hypotheses\u003c/h2\u003e\n\u003cp\u003eContrary to our expectation, and the environmental matching hypothesis, there was no significant relationship between species-specific oviposition preferences (preferred moisture level, substrate temperature, and partially the vertical distribution of oothecae) and the index of xerothermophility for both bare ground-laid and tussock-laid oothecae, with the exception of the vertical distribution of tussocks-laid oothecae (Table 2). Highly xerothermophilic species (based on habitat requirements of adults published in \u003csup\u003e30\u003c/sup\u003e) placed their oothecae significantly closer to the ground within grass tussock than less xerothermophilic species. Interestingly, the significance of this relationship was not confirmed for bare ground laying species despite the noticeable regression slope (Figure 4). Our results for moisture strongly support the insurance hypothesis, as wet substrate was, in general, the most preferred substrate and the fewest number of oothecae were placed in the dry compartment across investigated species (Figure2).\u003c/p\u003e\n\u003cp\u003eIn general, the vertical distribution of oothecae at the interspecific level was significantly affected by moisture level and partially by temperature in accordance with the vertical distribution plasticity hypothesis. Oothecae were placed shallower in bare ground or higher on tussocks with increasing local moisture (Figures S2 and S3). Oothecae were placed significantly lower on tussocks and tend to be placed deeper in bare ground with increasing local temperature (Table 3, Figure 5, S3). There was no significant interactive effect of local moisture and temperature on vertical distribution of oothecae. Note that the above-described results represent overall patterns (across species) and patterns for particular species can be slightly different (for details see Figures 5, S2 and S3).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAll investigated orthopteran species showed a non-random choice of the moisture level, surface temperature, and vertical position when ovipositing. Interestingly, there was no relationship between preferred moisture or temperature during oviposition and species xerothermophility based on species habitat preferences. Instead, generally higher moisture and a relatively narrow range of temperature were preferred across species. Nevertheless, there was a significant relationship between species xerothermophility and vertical distribution of oothecae laid in tussocks. Furthermore, at the intraspecific level, oothecae were placed shallower in bare ground on higher on tussocks with increasing local moisture and partially also temperature. In general, our results indicate that orthopterans optimise oviposition depth and even xeric orthopteran species prefer substrates with higher moisture to protect oothecae from desiccation.\u003c/p\u003e \u003cp\u003eMoisture is the most relevant factor determining successful egg development in insects \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Many species need to absorb water before embryogenesis begins, and eggs have to continuously resist desiccation until hatching occurs \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Almost all the investigated species laid at least some eggs into each moisture level (compartment), which suggests a relatively relaxed moisture preferences across orthopterans. Even in the most drought preferring species, \u003cem\u003eChorthippus vagans\u003c/em\u003e and \u003cem\u003eStenobothrus crassipes\u003c/em\u003e, females only slightly preferred the dry substrate over slightly wet or wet substrates, and some oothecae were laid in soaked soil. In general, wet soil was preferred the most and dry soil the least. Our findings provide support for the insurance hypothesis, which predicts a general tendency to oviposit into substrates with high moisture levels independently of the habitat preferred by adults \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. In contrast, we did not find evidence supporting the environmental matching hypothesis, which predicts a correlation between species habitat preferences (xerothermophility) and oviposition preferences \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. It seems that species do not detect moisture in an absolute manner, but only relatively in the context of occupied habitat. For example, the xerothermophilic species \u003cem\u003eChorthippus mollis\u003c/em\u003e strongly preferred soaked substrate in the experiment, despite that this species almost never comes into contact with soaked soil in nature. \u003cem\u003eOmocestus viridulus\u003c/em\u003e, which usually occurs in humid habitats, preferred tussocks within only slightly wet soil in our experiment. This can be an adaptation to spring flooding, which are common in its preferred habitats \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe embryonic development rate is closely related to ambient temperature and the sum of degree days during a season can be a limiting factor for many temperate insects \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. On the other hand, lethal temperature thresholds are usually very close to the thermal optima and a fine equilibrium between these two factors has to be reached \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Our data show that optima of all investigated species occurred between 29 and 34\u0026deg;C, which fits well with previously published single-species data \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. The relatively narrow range of preferred oviposition temperatures across species originating from variable habitats is quite surprising and seems to be quite conservative within the Gomphocerinae subfamily. In contrast to findings of Schnebel \u0026amp; Grossfield (1986), we observed no tendency of xerothermophilic species to prefer higher surface temperature for oviposition. However, Schnebel \u0026amp; Grossfield (1986) studied species (fruit flies) along a latitudinal gradient ranging from arctic to tropic regions, not species from a single region. Again, a possible explanation of our findings can be based on individual life strategies of different species. For example, despite its high xerothermophility, \u003cem\u003eChorthippus mollis\u003c/em\u003e preferred the lowest oviposition temperature out of all investigated species. This species has a relatively long embryonic development and the choice of relatively cold and moist microhabitats within hot and dry steppes, where it live, can protect developing immobile embryos from potentially damaging temperatures at the start of summer when other species already hatched \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIt has been hypothesized that xerothermophilic species should lay eggs deeper into the ground or lower within tussocks to avoid damaging hot and dry conditions \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e than oligothermic species. Our results partly supported this hypothesis, as tussocks-laid oothecae were placed closer to ground with increasing xerothermophility of species, and there was a similar tendency in bare ground-laid oothecae. Nevertheless, using more species, especially more hygro- and psychrophilic ones, in a future study will allow for a better evaluation of this pattern. Additional support for this relationship was provided at the intraspecific level, as oothecae were laid deeper in dry and hot conditions in the great majority of investigated species (following the vertical distribution plasticity hypothesis). A similar finding was previously reported for the grasshopper \u003cem\u003eRomalea microptera\u003c/em\u003e in which females laid eggs shallower into the substrate when exposed to higher levels of moisture \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOnly a limited number of studies have investigated the effects of microclimate on oviposition behaviour compared to the wider range of published works that explore the effects of biotic factors, e.g., optimal food, parasitism, or competition avoidance \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Abiotic factors are directly modified by ongoing climate change and anthropogenic habitat alterations, which increases the relevance of such studies for nature conservation \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Moreover, eggs are an immobile life stage unable to escape from suboptimal conditions and frequently the most vulnerable to extreme temperatures \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. This highlights the importance of studies investigating microhabitat oviposition preferences in insects. Especially valuable are studies employing a comparative framework, i.e., investigating a high number of species using a standardized experimental setting. Unfortunately, such studies are rare with the exception of a historical study conducted for \u003cem\u003eDrosophila\u003c/em\u003e flies \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to the Nature Conservation Agency of the Czech Republic for permitting experiments with endangered species \u003cem\u003eStenobothrus eurasius.\u0026nbsp;\u003c/em\u003eMany thanks go to Jakub Prokop for financial and technical support, Ondřej Balv\u0026iacute;n for help with the phylogenetic analysis, Ezequiel Gonz\u0026aacute;lez for insightful comments and David N. Awde for language corrections. This study was also partly supported by the Technology Agency of the Czech Republic (grant number SS02030018 \u0026ndash; DivLand).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRefsnider, J. M. \u0026amp; Janzen, F. J. Putting Eggs in One Basket: Ecological and Evolutionary Hypotheses for Variation in Oviposition-Site Choice. \u003cem\u003eAnnu. Rev. Ecol. Evol. Syst.\u003c/em\u003e \u003cstrong\u003e41\u003c/strong\u003e, 39\u0026ndash;57 (2010).\u003c/li\u003e\n\u003cli\u003eMitchell, R. The Evolution of Oviposition Tactics in the Bean Weevil, Callosobruchus maculatus (F.). \u003cem\u003eEcology\u003c/em\u003e \u003cstrong\u003e56\u003c/strong\u003e, 696\u0026ndash;702 (1975).\u003c/li\u003e\n\u003cli\u003eJaenike, J. On Optimal Oviposition Behaviour in Phytophagous Insect. \u003cem\u003eTheor. Popul. Biol.\u003c/em\u003e 350\u0026ndash;356 (1978).\u003c/li\u003e\n\u003cli\u003eBernays, E. \u0026amp; Graham, M. 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Entomol.\u003c/em\u003e \u003cstrong\u003e33\u003c/strong\u003e, 1513\u0026ndash;1522 (2004).\u003c/li\u003e\n\u003cli\u003eGustin, R. D. Effect of Two Moisture and Population Levels on Oviposition of the Western Corn Rootworm123. \u003cem\u003eEnviron. Entomol.\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 406\u0026ndash;407 (1979).\u003c/li\u003e\n\u003cli\u003eFartmann, T., Br\u0026uuml;ggeshemke, J., Poniatowski, D. \u0026amp; L\u0026ouml;ffler, F. Summer drought affects abundance of grassland grasshoppers differently along an elevation gradient. \u003cem\u003eEcol. Entomol.\u003c/em\u003e \u003cstrong\u003e47\u003c/strong\u003e, 778\u0026ndash;790 (2022).\u003c/li\u003e\n\u003cli\u003eKnapp, M. \u0026amp; Nedvěd, O. Gender and Timing during Ontogeny Matter: Effects of a Temporary High Temperature on Survival, Body Size and Colouration in Harmonia axyridis. \u003cem\u003ePLoS ONE\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, e74984 (2013).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1: Overview of investigated species.\u0026nbsp;\u003c/strong\u003eNumber of laid oothecae per species and their associations with grass tussocks is stated. Also the indices of xerothermophility obtained from our previous study are presented here.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.46938775510204%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eSpecies\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.489795918367346%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal amount of oothecae\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.489795918367346%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eTussocks-associated oothecae (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.551020408163264%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eIndex of xerothermophility\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.46938775510204%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eEuchorthippuspulvinatus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.489795918367346%\" valign=\"top\"\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.489795918367346%\" valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.551020408163264%\" valign=\"top\"\u003e\n \u003cp\u003e0.896\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.46938775510204%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eChorthpippusalbomarginatus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.489795918367346%\" valign=\"top\"\u003e\n \u003cp\u003e51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.489795918367346%\" valign=\"top\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.551020408163264%\" valign=\"top\"\u003e\n \u003cp\u003e0.336\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.46938775510204%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eChorthippusbiguttulus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.489795918367346%\" valign=\"top\"\u003e\n \u003cp\u003e160\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.489795918367346%\" valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.551020408163264%\" valign=\"top\"\u003e\n \u003cp\u003e0.518\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.46938775510204%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eChorthippusdorsatus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.489795918367346%\" valign=\"top\"\u003e\n \u003cp\u003e145\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.489795918367346%\" valign=\"top\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.551020408163264%\" valign=\"top\"\u003e\n \u003cp\u003e0.375\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.46938775510204%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eChorthippusmollis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.489795918367346%\" valign=\"top\"\u003e\n \u003cp\u003e54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.489795918367346%\" valign=\"top\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.551020408163264%\" valign=\"top\"\u003e\n \u003cp\u003e0.900\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.46938775510204%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eChorthippusvagans\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.489795918367346%\" valign=\"top\"\u003e\n \u003cp\u003e92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.489795918367346%\" valign=\"top\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.551020408163264%\" valign=\"top\"\u003e\n \u003cp\u003e0.960\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.46938775510204%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eOmocestushaemorrhoidalis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.489795918367346%\" valign=\"top\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.489795918367346%\" valign=\"top\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.551020408163264%\" valign=\"top\"\u003e\n \u003cp\u003e0.799\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.46938775510204%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eOmocestusviridulus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.489795918367346%\" valign=\"top\"\u003e\n \u003cp\u003e189\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.489795918367346%\" valign=\"top\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.551020408163264%\" valign=\"top\"\u003e\n \u003cp\u003e0.109\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.46938775510204%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ePseudochorthippusmontanus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.489795918367346%\" valign=\"top\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.489795918367346%\" valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.551020408163264%\" valign=\"top\"\u003e\n \u003cp\u003e0.021\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.46938775510204%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ePseudochorthippusparallelus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.489795918367346%\" valign=\"top\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.489795918367346%\" valign=\"top\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.551020408163264%\" valign=\"top\"\u003e\n \u003cp\u003e0.291\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.46938775510204%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eStenobothrus crassipes\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.489795918367346%\" valign=\"top\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.489795918367346%\" valign=\"top\"\u003e\n \u003cp\u003e51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.551020408163264%\" valign=\"top\"\u003e\n \u003cp\u003e0.780\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.46938775510204%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eStenobothruseurasius\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.489795918367346%\" valign=\"top\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.489795918367346%\" valign=\"top\"\u003e\n \u003cp\u003e88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.551020408163264%\" valign=\"top\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.46938775510204%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eStenobothruslineatus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.489795918367346%\" valign=\"top\"\u003e\n \u003cp\u003e183\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.489795918367346%\" valign=\"top\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.551020408163264%\" valign=\"top\"\u003e\n \u003cp\u003e0.777\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.46938775510204%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eStenobothrusnigromaculatus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.489795918367346%\" valign=\"top\"\u003e\n \u003cp\u003e79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.489795918367346%\" valign=\"top\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.551020408163264%\" valign=\"top\"\u003e\n \u003cp\u003e0.952\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2: Effects of species xerothermophility on oviposition preferences.\u003c/strong\u003e Results of phyllogenetic mixed-effects models investigating the relationship between the index of xerothermophility (independent variable) and species oviposition preferences for moisture, temperature, and ootheca vertical position (separate model was fitted for each response variable and substrate type).\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"31.29139072847682%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eDependent variable\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.66887417218543%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSubstrate type\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.894039735099337%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eChi-sq\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.596026490066226%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ed.f.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.549668874172184%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ep-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"31.29139072847682%\" valign=\"top\"\u003e\n \u003cp\u003eMoisture\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.66887417218543%\" valign=\"top\"\u003e\n \u003cp\u003eBare ground\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.894039735099337%\" valign=\"top\"\u003e\n \u003cp\u003e0.0068\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.596026490066226%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.549668874172184%\" valign=\"top\"\u003e\n \u003cp\u003e0.9344\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"31.29139072847682%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.66887417218543%\" valign=\"top\"\u003e\n \u003cp\u003eTussock\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.894039735099337%\" valign=\"top\"\u003e\n \u003cp\u003e1.8825\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.596026490066226%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.549668874172184%\" valign=\"top\"\u003e\n \u003cp\u003e0.17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"31.29139072847682%\" valign=\"top\"\u003e\n \u003cp\u003eTemperature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.66887417218543%\" valign=\"top\"\u003e\n \u003cp\u003eBare ground\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.894039735099337%\" valign=\"top\"\u003e\n \u003cp\u003e0.658\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.596026490066226%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.549668874172184%\" valign=\"top\"\u003e\n \u003cp\u003e0.4173\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"31.29139072847682%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.66887417218543%\" valign=\"top\"\u003e\n \u003cp\u003eTussock\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.894039735099337%\" valign=\"top\"\u003e\n \u003cp\u003e0.7725\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.596026490066226%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.549668874172184%\" valign=\"top\"\u003e\n \u003cp\u003e0.3953\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"31.29139072847682%\" valign=\"top\"\u003e\n \u003cp\u003eVertical position\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.66887417218543%\" valign=\"top\"\u003e\n \u003cp\u003eBare ground\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.894039735099337%\" valign=\"top\"\u003e\n \u003cp\u003e3.0814\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.596026490066226%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.549668874172184%\" valign=\"top\"\u003e\n \u003cp\u003e0.0792\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"31.29139072847682%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.66887417218543%\" valign=\"top\"\u003e\n \u003cp\u003eTussock\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.894039735099337%\" valign=\"top\"\u003e\n \u003cp\u003e5.5023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.596026490066226%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.549668874172184%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.019\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3:\u003c/strong\u003e Results of the phyllogenetic mixed-effect model testing whether there was plasticity of laying depth based on changing moisture and temperature.\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.479338842975206%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSubstrate type\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.388429752066116%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eIndependent variable\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.867768595041323%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eChi-sq\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.578512396694215%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ed.f.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.68595041322314%\" valign=\"top\"\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 width=\"22.479338842975206%\" valign=\"top\"\u003e\n \u003cp\u003eBare ground\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.388429752066116%\" valign=\"top\"\u003e\n \u003cp\u003emoisture\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.867768595041323%\" valign=\"top\"\u003e\n \u003cp\u003e18.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.578512396694215%\" valign=\"top\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.68595041322314%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.479338842975206%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.388429752066116%\" valign=\"top\"\u003e\n \u003cp\u003etemperature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.867768595041323%\" valign=\"top\"\u003e\n \u003cp\u003e3.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.578512396694215%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.68595041322314%\" valign=\"top\"\u003e\n \u003cp\u003e0.0706\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.479338842975206%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.388429752066116%\" valign=\"top\"\u003e\n \u003cp\u003emoisture : temperature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.867768595041323%\" valign=\"top\"\u003e\n \u003cp\u003e2.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.578512396694215%\" valign=\"top\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.68595041322314%\" valign=\"top\"\u003e\n \u003cp\u003e0.4586\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.479338842975206%\" valign=\"top\"\u003e\n \u003cp\u003eTussock\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.388429752066116%\" valign=\"top\"\u003e\n \u003cp\u003emoisture\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.867768595041323%\" valign=\"top\"\u003e\n \u003cp\u003e27.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.578512396694215%\" valign=\"top\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.68595041322314%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.479338842975206%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.388429752066116%\" valign=\"top\"\u003e\n \u003cp\u003etemperature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.867768595041323%\" valign=\"top\"\u003e\n \u003cp\u003e6.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.578512396694215%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.68595041322314%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0136\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.479338842975206%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.388429752066116%\" valign=\"top\"\u003e\n \u003cp\u003emoisture : temperature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.867768595041323%\" valign=\"top\"\u003e\n \u003cp\u003e6.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.578512396694215%\" valign=\"top\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.68595041322314%\" valign=\"top\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\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":"Bare ground, climate change, ecophysiology, egg laying, tussock, thermal niche","lastPublishedDoi":"10.21203/rs.3.rs-2924573/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2924573/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe right choice of an oviposition site is a crucial task for oviparous species without maternal care. In contrast to well investigated biotic factors, e.g., larval food preferences, parasitism, predation, and competition avoiding, abiotic factors affecting oviposition preferences in insects have been rarely investigated in comparative studies. To improve our current understanding of oviposition site selection in orthoptera, we investigated the influence of substrate temperature and moisture on oviposition behaviour for 14 temperate grasshopper species. Conspecific groups of adults were kept in arenas with simultaneous temperature and moisture gradients. For each ootheca produced during the experiment (1192 in total) we recorded its depth and local microclimatic conditions. Our results indicate that microclimatic oviposition preferences significantly differ among species, however, correlations between adult habitat preferences and microclimatic oviposition preferences were surprisingly weak. Even oligothermic species preferred substrate temperatures around 30\u0026deg;C and some xerothermic species preferred higher humidity. Hypothesized tendency to place oothecae closer to the ground within grass tussocks under hot and dry conditions was confirmed. It is possible that species evaluate microclimatic conditions for oviposition in the context of occupied habitat, i.e., in a relative rather than absolute manner.\u003c/p\u003e","manuscriptTitle":"Oviposition preferences in temperate grasshoppers: Conserved temperature requirements but contrasting responses to humidity across species","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-05-19 16:05:42","doi":"10.21203/rs.3.rs-2924573/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":"395c62ab-986b-402d-bb0b-884a2b2b7648","owner":[],"postedDate":"May 19th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":21564514,"name":"Biological sciences/Zoology/Animal behaviour"},{"id":21564515,"name":"Biological sciences/Ecology/Behavioural ecology"}],"tags":[],"updatedAt":"2023-10-31T17:05:58+00:00","versionOfRecord":[],"versionCreatedAt":"2023-05-19 16:05:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2924573","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2924573","identity":"rs-2924573","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}