Temperature Tolerance and Thermal Environment of European Seed Bugs

Heteroptera, or true bugs populate many climate zones, coping with different environmental conditions. The aim of this study was the evaluation of their thermal limits and derived traits, as well as climatological parameters which might influence their distribution. We assessed the thermal limits (critical thermal maxima, CTmax, and minima, CTmin) of eight seed bug species (Lygaeidae, Pyrrhocoridae) distributed over four Köppen–Geiger climate classification types (KCC), approximately 6° of latitude, and four European countries (Austria, Italy, Croatia, Bulgaria). In test tubes, a temperature ramp was driven down to −5 °C for CTmin and up to 50 °C for CTmax (0.25 °C/min) until the bugs’ voluntary, coordinated movement stopped. In contrast to CTmin, CTmax depended significantly on KCC, species, and body mass. CTmax showed high correlation with bioclimatic parameters such as annual mean temperature and mean maximum temperature of warmest month (BIO5), as well as three parameters representing temperature variability. CTmin correlated with mean annual temperature, mean minimum temperature of coldest month (BIO6), and two parameters representing variability. Although the derived trait cold tolerance (TC = BIO6 − CTmin) depended on several bioclimatic variables, heat tolerance (TH = CTmax − BIO5) showed no correlation. Seed bugs seem to have potential for further range shifts in the face of global warming.


Introduction
Heteroptera, or true bugs, are a widely distributed, highly diverse insect taxon with approximately 45,300 [1] species worldwide. They populate every climate zone from arctic to desert [2][3][4], showing great ability to cope with a variety of environmental factors. This leads to a substantial potential for invasiveness, and indeed many bug species are known to be highly invasive. For example, Halyomorpha halys, an Asian stink bug invading Europe and North America, made it to the top four "most wanted" species list in the USA, inflicting economic damage of USD 37 million to tree fruit production in 2010 [5]. However, although some species thrive over wide latitudinal ranges, other closely related species are more restricted in their distribution range (e.g., Orsillus depressus: Southern and Central Europe, northwards until Scandinavia; O. maculatus: Southern Europe, around the Mediterranean and Black Sea) [6]. Species such as Oxycarenus lavaterae are known to have spread from their Mediterranean These bug species are distributed over wide areas of Europe, some restricted to the southern and central parts, and some spread to regions north of the polar circle [43]. All physiological measurements were performed on adult individuals at the laboratory in Austria. The animals were transported to the laboratory by ourselves or were delivered via express mail within 3 to 5 days. The bugs were sampled randomly at the respective sample sites. Sex of the individuals was not determined. All individuals were similar in their external characteristics (habitus, size, body mass). Therefore, we assume that we mapped a natural gender relationship of the respective populations. Individuals that were noticeably behaviorally impaired (possibly by transport) were excluded from the evaluation. This as well as different numbers of sampled individuals resulted in an inconsistent number (n) per species/sample site/experiment. All experiments were conducted immediately after the individuals arrived in our laboratory. All bug sampling, transportation, and experiments took place in concurrence with national and EU regulations.

Critical Thermal Maximum (CTmax)
We used a similar experimental setup as in Section 2.2, without the shaking apparatus activated. Stimulation was not necessary, as the insects were always active at higher temperatures. Up to 10  Table 2. Species, sample location and Köppen-Geiger climate classification (KCC), mean body mass with SD, critical thermal minima and maxima (CT min , CT max ) with SD, thermal tolerance breadth (TTB), and heat and cold tolerance (TH, TC) of the true bugs assessed.

Species
Location -KCC

Critical Thermal Minimum (CT min )
We placed the true bugs (see Table 2 for n) in individual measurement chambers (acrylic glass test tubes, about 3 to 10 ml, depending on size, which were mounted in a self-constructed shaking device. The device was submerged into a temperature-controlled water bath (JULABO F33, JULABO Labortechnik GmbH, Seelbach, Germany). Up to nine individuals of the same species were tested per trial run. An additional empty tube contained a thermocouple connected to a data logger (ALMEMO 2690, Ahlborn GmbH, Holzkirchen, Germany), which recorded the exact temperature the animals were exposed to in one second intervals. After 10 min of habituation at 15 • C, we drove a temperature ramp at a slope of −0.25 • C/min down to −5 • C. After 5 min at this temperature, we warmed the chambers at a rate of 2 • C/min until 15 • C was reached again. The measurement chambers were shaken forcefully by the shaking device once a minute for 1 s for the entire experiment (approximately 100 min). The experiments were recorded via video camera (Sony HDR-CX730E, Sony Europe Limited, Vienna, Austria) for later evaluation of the animals' behavior. To determine the lower threshold of activity, the last appearance of voluntary movement of antennae or legs at a shaking event was determined. The temperature at the next shaking event was specified as the CT min (according to [44]). All evaluated individuals survived the experiment and regained full mobility.

Critical Thermal Maximum (CT max )
We used a similar experimental setup as in Section 2.2, without the shaking apparatus activated. Stimulation was not necessary, as the insects were always active at higher temperatures. Up to 10 measurement chambers were used for the bugs; the thermocouple for measuring the temperature was placed in an additional similar chamber, near the animals. Following a standardized method (e.g., [45][46][47]), the experiments started at a temperature of 25 • C. After 10 min of habituation, we drove a temperature ramp up to 50 • C at a rate of 0.25 • C/min. Again, the animals' behavior was recorded via video camera for later evaluation. The cease of coordinated movement marked the so-called knockdown point the experimental ambient temperature measured at this time was described as CT max ( [40][41][42]). As we did not abort the experiment after the first individual reached CT max but finished the temperature ramp, no individuals survived the CT max experiments.

Thermal Tolerance Breadth (TTB), Heat Tolerance (TH), Cold Tolerance (TC)
Thermal tolerance breadth (TTB) is the temperature range in which the bugs were able to exhibit voluntary coordinated movement. It was calculated as TTB = CT max − CT min . The heat tolerance (TH) and cold tolerance (TC) were calculated from the upper and lower thermal limits and bioclimatic variables: TH = CT max − BIO5 (maximum temperature of the warmest month), and TC = BIO6 − CT min (BIO6 = minimum temperature of the coldest month), respectively. Positive values for TH and TC show the bugs' ability to endure the occurring ambient temperatures. Negative values indicate that they will have to avoid heat or cold damage by other means. TTB, TH, and TC were calculated from mean CT min and CT max values per species and sample location.

Climate Data, Bioclimatic Variables, and Data Analysis
The climate regions of our study animals were detailed from the Köppen-Geiger climate classification system (KCC, Figure 1) updated by Kottek et al. [48]. The animal data were correlated with eight bioclimatic variables (BIO1 to BIO7; BIO12) extracted from the WorldClim dataset (

Testing for a Phylogenetic Signal
A detailed phylogenetic cladogram was not available for our sampled bug species. To model phylogenetic relations between the species, we generated a branching tree using an established taxonomy ( [6,43], Table 1), which is preferable to ignoring taxonomic relationship entirely [50]. For the analyses regarding the phylogenetic signal, we built a phylogenetic tree based on recent studies of Heteroptera: Pentatomomorpha [51] using Mesquite software 3.6 [52]. The taxonomic positions of relevant subfamilies in this study were taken as template for the eight species (19 populations) assessed in our study, where polytomies were randomly resolved into a series of dichotomies. Due to missing exact relationships between species, we set branch length to 1 ( [53]; see Figure S1). To incorporate phylogenetic signals (i.e., related species that have similar traits) for all temperature parameters, species, and body mass, we calculated Pagel's λ [54] using R 3.5.1 [55]. The Pagel's λ ranges between 0 (no phylogenetic signal) and 1 (strong phylogenetic signal) and was determined using the pgls function in the caper package [56].
A multifactorial ANOVA with mass as covariate revealed a correlation of CT min with KCC, but not with species or body mass ( Table 3). The hotter the climate, the lower the tolerance against low temperatures was observed. General linear model (GLM) statistics revealed significant influence of bioclimatic parameters on CT min (p < 0.0001). It was directly related to the mean diurnal temperature range (BIO2), isothermality (BIO3), and minimum temperatures (BIO6) (Table 3, Figure 2, Figure S4).
By contrast, for CT max , we found highly significant relationships with all three parameters: KCC, species, and body mass. Species had the highest F-quotient, but also KCC showed significant influence (Table 3). A generalized linear model (GLM) for CT max showed highly significant correlations both for the entire model as well as for every single bioclimatic parameter. Temperature seasonality (BIO4) and minimum temperature of coldest month (BIO6) seemed to have a lesser but still significant effect. For details, see Table 3. Figure 2 shows the correlations of CT min and CT max with latitude and climatic parameters BIO1, and BIO4 to BIO7.

Testing for Phylogenetic Signal
A strong phylogenetic signal was detected in body mass (Table 5), which generally can be explained by small mass differences between several species (compare Figure S2). We found no significant phylogenetic signal in the tested physiological parameters using Pagel's λ for critical thermal minimum (CT min ), temperature breath (TTB), heat tolerance (TH), and cold tolerance (TC) (λ = 0 with distinct values for hypothesis and null hypothesis). For the critical thermal maximum (CT max ), a phylogenetic signal was indicated (Pagel's λ = 1, p (λ=1) = 1) but the null hypothesis of no signal showed also a certain probability (p (λ=0) = 0.235).   Table 2 and Table S2.  Table 2 and Table S2.  Table 2 and Table S2.  Table 2 and Table S2.  Table 5. Statistical analysis of phylogenetic signal in fresh body mass, CTmin, CTmax, TTB (thermal tolerance breadth), TC (cold tolerance), and TH (heat tolerance). Estimation of phylogenetic signals for single traits with Pagel's λ: p(λ=0) states the significance level of "no phylogenetic signal", and P(λ=1) the significance level of "a strong phylogenetic signal. In the case of body mass, for example, p(λ=0) < 0.001 means that the hypothesis of no phylogenetic signal was discarded, and P(λ=1) = 1 means that the hypothesis of a strong phylogenetic signal was confirmed (i.e., is not discarded).

Discussion
True bugs are an abundant and successful insect group, capable of populating a wide variety of habitats. The species covered in this paper are closely related, of the families Lygaeidae and Pyrrhocoridae, and colonized four Köppen-Geiger climate regions over a latitudinal range of almost 6°.
Upper thermal limits in the tested bug species varied stronger (SD = 2.5 °C, range = 39.4-49.5 °C; total mean CTmax = 45.3 °C) than lower limits (SD = 1.4 °C, range = −4.6-2.1 °C; mean CTmin = −3.3 °C) (p <0.001, F-test; see Table 2). Regarding the upper limits, this concurs with the findings of Kellermann et al. ( [57], on fruit flies). However, the ability to withstand high temperatures (CTmax) declines with an improved tolerance to low temperatures (CTmin) in dung beetles [19]. In our seed bugs, CTmax correlated significantly with all studied climatic parameters (Table 3), except BIO7 (a derived bioclimatic parameter: BIO7 = BIO5 − BIO6), despite the moderate variation in latitude (6°) and thus climate variability. This finding demonstrates that climate has a strong impact on seed bug heat tolerance. Only temperature seasonality (BIO4) and minimum temperature of the coldest month (BIO6) had a  Table 5. Statistical analysis of phylogenetic signal in fresh body mass, CT min , CT max , TTB (thermal tolerance breadth), TC (cold tolerance), and TH (heat tolerance). Estimation of phylogenetic signals for single traits with Pagel's λ: p (λ=0) states the significance level of "no phylogenetic signal", and P (λ=1) the significance level of "a strong phylogenetic signal. In the case of body mass, for example, p (λ=0) < 0.001 means that the hypothesis of no phylogenetic signal was discarded, and P (λ=1) = 1 means that the hypothesis of a strong phylogenetic signal was confirmed (i.e., is not discarded).

Discussion
True bugs are an abundant and successful insect group, capable of populating a wide variety of habitats. The species covered in this paper are closely related, of the families Lygaeidae and Pyrrhocoridae, and colonized four Köppen-Geiger climate regions over a latitudinal range of almost 6 • .
Upper thermal limits in the tested bug species varied stronger (SD = 2.5 • C, range = 39.4-49.5 • C; total mean CT max = 45.3 • C) than lower limits (SD = 1.4 • C, range = −4.6-2.1 • C; mean CT min = −3.3 • C) (p < 0.001, F-test; see Table 2). Regarding the upper limits, this concurs with the findings of Kellermann et al. ([57], on fruit flies). However, the ability to withstand high temperatures (CT max ) declines with an improved tolerance to low temperatures (CT min ) in dung beetles [19]. In our seed bugs, CT max correlated significantly with all studied climatic parameters (Table 3), except BIO7 (a derived bioclimatic parameter: BIO7 = BIO5 − BIO6), despite the moderate variation in latitude (6 • ) and thus climate variability. This finding demonstrates that climate has a strong impact on seed bug heat tolerance. Only temperature seasonality (BIO4) and minimum temperature of the coldest month (BIO6) had a less pronounced (though significant) effect on CT max . Besides bioclimatic parameters, mass showed a strong positive effect on CT max (Table 3, Figure S3). This coincides with findings in beetles [58,59]. In other insects, however, results are not consistent. In ants, a positive correlation was reported [60,61] but also a decrease with mass [62,63]. In termites, a positive [64] or no correlation was reported [65].
Concerning the ability to tolerate cold, by contrast, the results are more diverse (Table 3). Although CT min is influenced by bioclimatic variables in general, only some bioclimatic variables seemed to have a direct effect, that is, the minimum temperature of the coldest month (BIO6) and associated parameters (mean diurnal range, BIO2; isothermality, BIO3). This seems plausible, because minimum environmental temperatures strongly determine survival during cold seasons, when behavioral avoidance is restricted, and thus the evolutionary drive to withstand them by physiological adaptation is high. If one accepts this interpretation, it seems intelligible that the annual mean temperature (BIO1) has only a weak effect on CT min . Mass had no effect on the CT min of seed bugs (Table 3, Figure S3). By contrast, in ants, a decrease with mass was reported [63]. In a seasonal comparison of two termite species, no effect of mass on CT min was reported [65]. Raschmanová et al. [66] observed a significant decrease of cold resistance with increasing body length in Collembola species inhabiting soil and subterranean habitats.
Responses to temperatures below the favorable temperature range (i.e., cold stress) often rely on different physiological mechanisms than those to heat stress. They are decoupled evolutionarily, and therefore may change differently in the course of the species' colonization of new areas [67]. Our findings support the hypothesis of decoupled thermal limits. Although cold tolerance (TC) correlated significantly with several bioclimatic parameters in a GLM analysis, heat tolerance (TH) did not (Table 4). This is in good accordance with a study of Addo-Bediako et al. [20], who found that upper thermal limits show little geographical variation, but the lower bounds of supercooling points and lower lethal temperatures do decline with latitude (and thus minimum temperature). In individual regression analyses (where other bioclimatic parameters were not included as covariates), cold tolerance (TC) depended on all tested bioclimatic parameters (Figure 3). On the one hand, it is plausible that with decreasing annual mean temperature (BIO1) and the minimum temperature of the coldest month (BIO6), the bugs adapted to endure lower temperatures (have a lower CT min ; Figure 2). On the other hand, the observed negative values of cold tolerance (TC) under cold conditions (minimum temperature of the coldest month, BIO6; Figure 3) would indicate that the bugs were below their safe thermal conditions. This suggests that they seek out more favorable microclimatic environments in good time, that is, at higher ambient temperatures where unhindered mobility is guaranteed. The temperature in these microclimatic environments may well be higher than indicated by the macroclimatic variables [68]. By contrast, heat tolerance TH, which represents the difference of CT max to the maximum temperature of the warmest month (BIO5), depended only on the annual mean temperature (BIO1) and the maximum temperature of the warmest month (BIO5) (Figure 3, Table S2). A decreasing heat tolerance with these bioclimatic variables (BIO1, BIO5) indicates a decrease of the upper thermal safety margin (compare Figure 4). It remains unclear, however, whether this occurs because of physiological restraints (i.e., inability for a higher CT max ) or a lack of need to increase CT max further (because the thermal safety range is already sufficient).
Both cold and heat tolerance (TC, TH) did not correlate with latitude ( Figure 3). This could have been due to the narrow latitudinal range of 6 • , as already mentioned above. Our results demonstrated that the investigation of variables directly affecting the studied organisms, instead of surrogate variables such as latitude or elevation, should increase the ability to understand the mechanisms driving animal distribution and biodiversity (see also [69]).
It has to be kept in mind, however, that our analysis does not include all ecological factors relevant for survival of unfavorable conditions. Mean minimum temperatures of the coldest month (BIO6) in Austria and Bulgaria were lower than the species' CT min , resulting in negative values of cold tolerance (TC) (Figure 4). Here, the overwintering individuals have to seek out microhabitats that provide a more benign microclimate, wherein they can endure cold periods [68,70,71]. However, if endured winter temperatures sink under the lethal temperatures of the bug species, colonization of such regions is not possible. This might explain the absence of, for instance, the Mediterranean Orsillus maculatus in colder regions of Europe, whereas Orsillus depressus thrives in these regions [43].
Heat tolerance (TH) in all species exceeded mean maximum temperatures of the warmest month (BIO5) in all locations (Table 2; by up to 25.9 • C in Oxycarenus lavaterae in Graz/AT), which coincides with the findings of Sunday et al. [24] in several ectotherms. As all individuals were sampled during the cold season (winter), an immediate acclimation to high temperatures is unlikely. However, as bioclimatic parameters were the climatological normal from 1970 to 2000, absolute maximum temperatures were likely to be higher at some times, but not to the extent that they make permanent populations impossible.
The climate variability hypothesis states that a positive relationship exists between the breadth of thermal tolerance (TTB, the degree of eurythermy) and the level of climatic variability experienced by taxa with increasing latitude, especially in terrestrial ectotherms [16,72]. However, on the basis of our results, we could not make a clear decision because there was no correlation with BIO7 (BIO5 − BIO6), the bioclimatic variable we assumed to characterize best the climatic variability, but there was a weak correlation with BIO4 (temperature seasonality) ( Table 4). One reason for the ambiguity could be the small latitudinal range covered in this study and the resulting small climatic variability.
The presence of a phylogenetic signal, that is, when related species share similar thermal responses, may indicate constraints of their thermal niches, which dictate the environments in which they can persist [73], or it may indicate similar selection pressures and similar environmental effects [73][74][75]. In our assessed seed bug species, kinship seems to have played a lesser, or at least no unambiguous, role in the adaptation to high environmental temperatures, and no role in low temperature adaptation (Table 5). This concurs with the findings of Teets and Hahn [76] on CT min of Drosophila. Concerning CT max , it concurs with findings of Ayrinhac et al. [77] and Hoffmann et al. [78] regarding the upper thermal limits in Drosophila, of Terblanche et al. [79] in Glossina pallidipes, of Vorhees et al. [39] in Culex tarsalis, and of Hamblin et al. [80] in a variety of bee species. In other words, our results suggest that despite phylogenetic relatedness, the seed bugs differ in their CT max because of adaptation to differing thermal environments, indicated by the comprehensive significant effects of bioclimatic variables on CT max (Figure 2, Tables 3 and 4).
In the face of global warming and the current "Mediterranaziation" of the Heteroptera fauna [81,82] in regions of previously unsuitable climatological (i.e., "too cold") conditions, we suggest that seed bugs will not experience troubles to disperse towards the north. Temperature-induced longer (reproductive) seasons and shortened diapause may result in an increase of generations per year or/and increased overwintering success [83][84][85]. Although winter mortality may vary depending on parameters such as gender, microhabitat choice, as well as size and coloration [86][87][88], temperature seems to be the main factor. That which might hinder the true bugs' dispersion could be the minimum environmental temperatures, which still can reach quite low values in Europe despite higher annual temperatures. Yukawa et al. [89] suggested the northward range expansion of Nezara viridula in Japan to be due to global warming, because the monthly mean temperature for January in the newly invaded areas exceeded the limit temperature of 5 • C, below which winter mortality increases by 15%-16.5% per 1 • C decrease in mean winter temperature [87,90,91]. Concerning the ability to invade colder areas in reaction to global temperature increase, we suggest a similar dispersion capacity of European seed bugs than predicted for Nezara viridula in Kyushu in Southern Japan [89].

Conclusions
Physiological traits such as lower and upper thermal limits play an important role in seed bug survival, reproduction, dispersion, and colonization under given environmental conditions. We suggest that their wide thermal breath and adaptive capacity have promoted their prosperity and dispersion. In this respect, there seems to be ample potential in the bugs for thriving and further proliferation, as well as invasion to new, hitherto unexploited areas of settlement, especially in the face of global warming.

Supplementary Materials:
The following are available online at http://www.mdpi.com/2075-4450/11/3/197/s1, Figure S1: Theoretical cladogram. Figure S2: Correlation of CT min and CT max with fresh body mass. Figure S3: Correlation of physiological parameters with bioclimatic variables. Table S1: Species, sample date, sample location, KCC, and bioclimatic parameters of the true bugs assessed. Table S2 Acknowledgments: We greatly appreciate the extensive help of Thomas Frieß (Ökoteam -Institut für Tierökologie und Naturraumplanung OG, Graz) in the identification of certain bug species, as well as for his valuable hints on distribution and ecology. Furthermore, we want to thank Iacopo Petrocelli for his help in acquiring bug samples, and Christine Malej and Oliver Olipitz for their help in data evaluation. We thank the reviewers for their particularly helpful comments in improving the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.