Phenotypic variability and thermal adaptation in social spider mites: Insights into speciation and local adaptation

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In this study, we compared thermal life history traits among three closely related social spider mites: Stigmaeopsis sabelisi , S. miscanthi high-aggression (HG) form, and their common ancestral group, S. miscanthi mild-aggression (ML) form. We investigated the minimum temperature thresholds for development by measuring the days required for egg hatching under five constant temperature conditions (15°C, 20°C, 25°C, 30°C, 32°C) and estimating the thresholds using linear and nonlinear regression models. Additionally, we assessed their reproductive diapause attributes. Our results revealed that the minimum development thresholds were slightly lower in S. sabelisi from colder regions compared to S. miscanthi HG form and S. miscanthi ML form distributed in warmer and subtropical regions. Notably, high-temperature stress negatively affected development only in S. sabelisi , suggesting local adaptation. Reproductive diapause attributes also varied: reproductive diapause was induced under short-day conditions in S. sabelisi , whereas the other two species lacked such diapause. Moreover, phenotypic variation in the number of days required for egg hatching was highest in S. miscanthi ML form, suggesting retained ancestral variability that may have facilitated subsequent divergence. These findings support the hypothesis that populations from colder environments exhibit lower thermal thresholds and more intense diapause than those from warmer environments, and also provide insights into the mechanisms driving local adaptation and speciation in the social spider mites. Geographic variation local adaptation lower developmental threshold thermal adaptation speciation social spider mites Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Arthropods, including mites and ticks, are ectothermic organisms whose body temperature and, consequently, physiological processes closely follow ambient environmental conditions. As a result, their development, reproduction, and population dynamics are strongly influenced by thermal environments (Taylor 1981 ). Among thermal traits, the minimum and maximum temperature thresholds for development are often considered as indicators of local adaptation (Honek 2013 ). Another key trait associated with thermal adaptation is diapause, a common strategy in arthropods that facilitates survival during adverse conditions, particularly low winter temperatures (Tougeron 2019 ; Denlinger 2023 ). It is generally hypothesised that populations from colder regions exhibit lower minimum temperature thresholds for development and more intense diapause attributes than those from warmer regions, allowing them to maximise the use of limited thermal resources and enhance overwintering success (Honek 2013 ). While this pattern is well documented for diapause (Masaki 1961 ; Schmidt et al. 2005 ; Posledovich et al. 2015 ), empirical support for similar geographic trends in the thermal thresholds for development remains inconsistent; some taxa conform to the pattern, while others deviate from it (Campbell et al. 1974 ; Tauber et al. 1987 ; Honek 2013 ; Kipyatkov and Lopatina 2013 ), suggesting that the patterns of geographical/latitudinal variation in thermal thresholds for development would differ among taxa. Stigmaeopsis sabelisi and Stigmaeopsis miscanthi high aggression (HG) form are social spider mites infesting the Chinese silver grass, Miscanthus sinensis . Spider mites produce silken threads, and those of the genus Stigmaeopsis use the threads to construct woven nests, where they live in groups (Saito and Sato 2024 ). Within the nests, individuals exhibit a variety of cooperative behaviours among nest mates, including faeces management to maintain nest hygiene (Sato et al. 2003 ; Sato and Saito 2006 , 2008 ), removal of exuviae, other debris and bacteria scattered inside the nests (Kanazawa et al. 2011 ), and collective defence against predators which invade the woven nests (Saitō 1986 ; Yano et al. 2011 ). Because of these cooperative behaviours, Stigmaeopsis mites are recognised as social spider mites (Schausberger et al. 2021 ; Saito and Sato 2024 ). Among Stigmaeopsis mites, species infesting the Chinese silver grass, known as Stigmaeopsis miscanthi species group, exhibit a unique social behaviour: male-killing, in which males eliminate rival males to establish harems (Saitō 1990 ). Notably, the frequency of male-killing differs between species: being lower in S. sabelisi and higher in S. miscanthi HG form (Saito 1995 ; Saito and Sahara 1999 ). Previously, S. sabelisi was referred to as the low-aggression (LW) form of S. miscanthi , as the two were once considered conspecific variants differing in male aggression levels. However, subsequent studies have revealed morphological differences (Saito 1995 ), reproductive isolation (Sato et al. 2000a , b , 2015 , 2018 ) and distinct molecular phylogenetic relationships (Ito and Fukuda 2009 ; Sakagami et al. 2009 ; Sakamoto et al. 2017 ; Sato et al. 2019 ), supporting their classification as separate species (Saito et al. 2018 ). These two species differ in their geographic distributions; S. sabelisi is found in colder regions of Japan and Korea, whereas S. miscanthi HG form is primarily distributed in warmer areas of Japan (Saito and Sahara 1999 ; Sato et al. 2019 ). Corresponding to this habitat difference, differences in diapause attributes have been reported between them (Saito et al. 2002 ). In both species, short-day lengths with lower temperatures during the developmental period (from egg to adult emergence) induce reproductive diapause in females, and long-day lengths with higher temperatures terminate the diapause, allowing females to initiate oviposition. S. sabelisi females require a significantly longer period to terminate reproductive diapause and initiate oviposition compared to those of S. miscanthi HG form (Saito et al. 2002 ). Regarding thermal responses in development, the two species exhibit similar minimum temperature thresholds for development ( S. sabelisi : 11.7°C, S. miscanthi HG form: 11.8°C) (Saito et al. 2013 ). Developmental speed, however, is significantly slower in S. sabelisi at any temperature in the range 10°C to 25°C, resulting in a lower intrinsic rate of natural increase ( r m ) compared to S. miscanthi HG form (Saito et al. 2013 ). Recently, populations exhibiting intermediate levels of male aggression, referred to as S. miscanthi mild-aggression (ML) form, have been identified in subtropical regions of Japan. S. miscanthi ML form also exhibits an intermediate male weapon morphology and is reproductively isolated from S. sabelisi and S. miscanthi HG form (Sato et al. 2013 , 2019 ). Phylogenetic analysis based on mitochondrial cytochrome c oxidase subunit I (mt COI) sequences, including populations from Japan, Korea and Taiwan, revealed that both S. sabelisi and S. miscanthi HG form were derived from a group with mild male aggression, including S. miscanthi ML form, approximately 20,000–40,000 years before present (BP) and 5,494 − 10,988 years BP, respectively (Sato et al. 2019 ). These findings suggest that S. sabelisi and S. miscanthi HG form emerged from a common ancestral group distributed in the subtropical regions, including the S. miscanthi ML form, through subsequent adaptation to colder and warmer environments, respectively. If this evolutionary scenario is correct, then investigating the thermal responses in development and diapause attributes of the S. miscanthi ML form is essential for understanding the ecological and evolutionary processes underlying speciation in this group. This study has two primary aims: (1) to test the hypothesis that populations from colder regions exhibit lower minimum temperature thresholds for development and more pronounced reproductive diapause than those from warmer and subtropical regions, and (2) to gain deeper insight into the speciation process associated with local adaptation in the social spider mites. To address these aims, we compared thermal life history traits: the minimum temperature thresholds for development and reproductive diapause, among S. sabelisi , S. miscanthi HG form, and S. miscanthi ML form. For the minimum temperature thresholds for development, we investigated the number of days required for egg hatching under five constant temperature conditions (15°C, 20°C, 25°C, 30°C and 32°C), and estimated the thresholds using linear and non-linear regressions. We also calculated the coefficient of variation in developmental time to assess phenotypic variability, which reflects intra- and inter-lineage diversity and offers insights into evolutionary potential (Gamelon et al. 2025 ). In addition, we investigated reproductive diapause induction under short-day conditions based on previous findings (Saito et al. 2013 ). Collectively, these data offer a comprehensive perspective on how thermal adaptation may contribute to ecological divergence and speciation in this group. Materials and Methods The minimum temperature threshold for development Mites We used S. miscanthi ML form collected from Ishigaki Island (latitude and longitude: 24.522789°N, 124.283347°E, Altitude: 30.1 m; Okinawa Prefecture, Japan) in July 2022, S. miscanthi HG form collected from Naha City (latitude and longitude: 26.212532°N, 127.71723°E, altitude: 40.1 m; Okinawa Prefecture, Japan) in July 2022 and S. sabelisi collected from Mt. Amagi (latitude and longitude: 34.8836111°N, 139.0713889°E, altitude: 491.6 m; Shizuoka Prefecture, Japan) in June 2022. More than 20 females were used to establish each laboratory culture, and the mites were reared on the host plant leaves, M. sinensis placed on wet cotton in plastic trays in an air-conditioned room at 25°C, with a relative humidity of 60–80% and a 15:9 h light:dark photoperiod. Experiment We introduced a female collected from the laboratory culture onto a detached M. sinensis leaf (1 × 2 cm) placed on wet cotton in an insect breeding dish (5.0 cm diameter, 1.5 cm high; SPL Life Sciences, Gyeonggi-do, Korea). We allowed the female to construct a nest on the leaf and lay eggs in it for 24 h. When the female laid more than one egg, we crushed the extra eggs with a needle to make one egg per nest. We removed the female from the nest and observed the development of the egg every 24 h. We recorded the number of days required for egg hatching to occur. We carried out these manipulations for three species/forms ( S. miscanthi ML form, S. miscanthi HG form and S. sabelisi ) under five different temperature conditions (15°C, 20°C, 25°C, 30°C and 32°C) using incubators, where other conditions were the same (50–70% relative humidity and 15:9 h light:dark photoperiod). The number of replicates was approximately 20 per species per treatment. As some eggs died during the experiments, we excluded them from the analysis, resulting in a final number of replications ranging from 12 to 21 per species per treatment. Statistical analysis For the statistical analyses, we used the software R ver.4.0.2 (R Core Team 2024 ). We analysed the number of days required for egg hatching with species/form, temperature and the interaction using a generalised linear model, where a gamma distribution was applied as the error distribution (gamma GLM), since the data was significantly deviated from the normal distribution (Shapiro-Wilk normality test, W = 0.741, P < 0.001). Then, we performed a Tukey post-hoc test to examine pairwise differences among species using the package emmeans (Lenth 2024 ). We analysed the phenotypic variability by calculating the coefficient of variation (standard deviation divided by the mean) of the number of days required for egg hatching for each temperature and species/form. We compared the coefficient of variation among species/forms at each temperature using an asymptotic test for the equality of coefficients of variance as implemented in the package cvequality (Feltz and Miller 1996 ; Marwick and Krishnamoorthy 2019 ). We adjusted the P -values for multiple testing using the Benjamini–Hochberg (BH) method (Benjamini and Hochberg 1995 ). For the estimation of the minimum temperature thresholds for development, we performed a linear regression analysis on the developmental speed (1 divided by days to hatch) and temperature in each species/form, as a previous study on S. sabelisi and S. miscanthi HG form used the linear regression for the estimation (Saito et al. 2013 ). We also performed a non-linear regression analysis based on the Brière model (Briere et al. 1999 ), which takes into account both the minimum and maximum temperature thresholds, as a previous study examined them at the temperature range from 10°C to 25°C, whereas this study also included 30°C and 32°C, where hatching may be delayed due to high temperature disturbance. The fitted model was: $$\:Developmental\:speed\:\left(T\right)=a\times\:T\times\:(T-{T}_{min})\sqrt{{T}_{max}-T}$$ where \(\:T\) is temperature, \(\:{T}_{min}\) and \(\:{T}_{max}\) are the minimum and maximum temperature thresholds for development, and \(\:a\) is constant. For the model fitting, we used nonlinear least squares, and we placed constraints that \(\:{T}_{min}\) and \(\:{T}_{max}\) should be in the range from 0 to 20 and from 20 to 40, respectively. Reproductive diapause Mites We used S. miscanthi ML form collected from Ishigaki Island (latitude and longitude: 24.5213078°N, 124.2832838°E, Altitude: 33.5 m; Okinawa Prefecture, Japan) in July 2024, S. miscanthi HG form collected from Naha City (latitude and longitude: 26.212532°N, 127.71723°E, altitude: 40.1 m; Okinawa Prefecture, Japan) in July 2022 and S. sabelisi collected from Mt. Unzen (latitude and longitude: 32.75686°N, 130.275699°E, altitude: 878.5 m; Nagasaki Prefecture, Japan) in June 2024. More than 20 females were used to establish each laboratory culture, and the mites were reared on the host plant leaves, M. sinensis placed on wet cotton in plastic trays in an air-conditioned room at 25°C, with a relative humidity of 60–80% and a 15:9 h light:dark photoperiod. Experiment The experimental procedure followed Saito et al. ( 2002 ), which examined diapause attributes of S. sabelisi and S. miscanthi HG form. Although physiological indicators such as changes in body colouration were not observed in the previous study, it clearly showed that S. sabelisi from colder regions required significantly more time to initiate oviposition than the S. miscanthi HG form from warmer regions, when females that had developed under short-day conditions were transferred to long-day conditions. This delay in oviposition timing under a shift from short to long photoperiods thus serves as a reliable proxy for evaluating reproductive diapause in S. miscanthi species group. We introduced 10–20 fertilised females collected from the laboratory culture onto a detached M. sinensis leaf (1 × 4 cm) placed on wet cotton in an insect breeding dish (5.0 cm diameter, 1.5 cm high; SPL Life Sciences, Gyeonggi-do, Korea). We allowed the females to lay eggs for approximately 10 days, removed the females, and reared the eggs until they developed into the teleiochrysalis stage under short-day conditions (9:15 h light:dark photoperiod) to induce diapause. As controls, we reared the eggs under long-day conditions (15:9 h light:dark photoperiod). We collected teleiochrysalis females from detached leaves and introduced them onto new detached leaves together with males. One day after adult emergence, we introduced the females individually onto new detached M. sinensis leaves and left them under long-day conditions (15:9 h light:dark photoperiod) to terminate diapause. We observed the survival of females and whether they initiated oviposition every day. We carried out these manipulations under the conditions of 18°C and a relative humidity of 50–70%. The number of replicates was 20–22 per treatment and per species/form. Statistical analysis For the statistical analyses, we used the software R v.4.0.2 (R Core Team 2024 ). We analysed the timing of oviposition initiation using non-parametric survival analysis: a Kaplan-Meier curve and a log-rank test in the package survival (Therneau 2019 ). We compared the timing of oviposition initiation between females which had developed under short-day conditions (treatment) and those which had developed under long-day conditions (control) in each species/form. We adjusted the P -values for multiple testing using the BH method (Benjamini and Hochberg 1995 ). We did not use a semi-parametric survival analysis: Cox proportional hazards regression model, as the assumption of proportional hazards for the fit of the Cox regression model was not satisfied in our dataset. Results The minimum temperature threshold for development The curve of the number of days required for egg hatching along temperature was significantly different among species (gamma GLM, species × temperature, F 2,239 = 9.296, P < 0.001). Naturally, the number of days decreased as the temperature increased, although the number of days at 32°C was longer than that at 30°C in S. sabelisi (Fig. 1 ). S. sabelisi required significantly longer days for egg hatching than the other two species (Tukey post-hoc test; S. sabelisi vs. S. miscanthi HG form: P < 0.001, S. sabelisi vs. S. miscanthi ML form: P < 0.001). A significant difference in the number of days was not found between S. miscanthi HG form and S. miscanthi ML form (Tukey post-hoc test, S. miscanthi HG form vs. S. miscanthi ML form: P = 0.185; Fig. 1 ). The coefficient of variation appeared higher in S. miscanthi ML form than in the other two species/forms across five different temperatures (Fig. 2 ); however, significant differences were observed only at 20°C(asymptotic test with adjustment by BH method, 15°C: P = 0.486, 20°C: P = 0.012, 25°C: P = 0.414, 30°C: P = 0.385, 32°C: P = 0.385). Linear regressions for development rate and temperature successfully converged for each species/form ( S. sabelisi : F 1,77 = 675.8, P < 0.001; S. miscanthi HG form: F 1,84 = 1,420, P < 0.001; S. miscanthi ML form: F 1,78 = 732.3, P < 0.001; Table 1 a; Fig. 3 a). The regression lines estimated the minimum temperature threshold for development to be 10.16°C for S. sabelisi , 10.33°C for S. miscanthi HG form and 10.77°C for S. miscanthi ML form. Nonlinear regressions for development rate and temperature based on the Brière model (Briere et al. 1999 ) were performed successfully for each species/form (Table 1 b; Fig. 3 b). In the nonlinear regressions, the minimum temperature threshold for development was estimated to be 9.25°C for S. sabelisi , 9.63°C for S. miscanthi HG form and 10.16°C for S. miscanthi ML form (Table 1 b). Reproductive diapause The timing of oviposition initiation was significantly different between treatment and control in S. sabelisi (log-rank test with adjustment by BH method: χ 2 = 14.90, df = 1, P < 0.001; Fig. 4 ), hhowever such a significant difference was not detected in S. miscanthi HG form (log-rank test with adjustment by BH method: χ 2 = 0.40, df = 1, P = 0.75; Fig. 4 ) and S. miscanthi ML form (log-rank test with adjustment by BH method: χ 2 = 0.0, df = 1, P = 1.000; Fig. 4 ). Discussion The minimum temperature threshold for development estimated by linear regression was 10.16°C for S. sabelisi , 10.33°C for S. miscanthi HG form, and 10.77°C for S. miscanthi ML form. The threshold estimated by non-linear regression based on the Brière model (Briere et al. 1999 ) was 9.25°C, 9.63°C, and 10.16°C for the respective species/forms. Although these estimated values did not differ substantially among the three species/forms, they corresponded well to the climates of their geographic distributions, as S. sabelisi , S. miscanthi HG form and S. miscanthi ML form are distributed in colder, warmer and subtropical regions, respectively. Additionally, high-temperature stress was detected only in S. sabelisi , which is distributed in colder regions. A previous study examined the developmental speed of S. sabelisi and S. miscanthi HG form in the temperature range of 10 to 25°C, and observed a consistent increase in developmental speed with temperature for both species/forms (Saito et al. 2013 ). In contrast, this study extended the temperature range to include 30°C and 32°C. We observed that developmental speed increased from 15 to 30°C but declined from 30 to 32°C in S. sabelisi . Such a decrease was not observed in S. miscanthi HG form and S. miscanthi ML form, suggesting that the developmental response to temperature of these species/forms is finely tuned to their respective thermal environments. Similar patterns were observed for diapause induction. In our experiment, reproductive diapause was induced by short-day conditions at 18°C during development in S. sabelisi , but not in either S. miscanthi HG form or S. miscanthi ML form. A previous study reported reproductive diapause in both S. sabelisi and S. miscanthi HG form, using populations collected from regions around 33°N latitude (Kyushu and Shikoku, Japan) (Saito et al. 2002 ). In contrast, S. miscanthi HG form we used was collected from the region approximately 26°N latitude (Central Ryukyu, Japan), a considerably more southern location, and did not exhibit diapause under the same conditions. This discrepancy suggests geographic variation in diapause attributes within S. miscanthi HG form. Meanwhile, S. miscanthi ML form, collected from around 24°N latitude (South Ryukyu), did not exhibit diapause either. Given that S. miscanthi ML form is restricted to this southern region, it may generally lack reproductive diapause irrespective of population. Overall, these results indicate that the three species/forms possess life history traits—developmental responses to temperature and diapause attributes—that are well adapted to the climates of their geographic distributions. Although comparisons were made among closely related species/forms rather than populations within a species, our results on S. miscanthi species group supported the hypothesis that populations from the colder regions exhibit lower thermal thresholds in development and more intense diapause attributes than those from the warmer regions (Campbell et al. 1974 ; Tauber et al. 1987 ; Honek 2013 ; Kipyatkov and Lopatina 2013 ). To better understand inconsistencies across taxa, future studies should assess broader phylogenetic and ecological contexts. The results of this study provide a valuable contribution toward that goal. In this study, we examined not only average developmental responses to temperature but also phenotypic variability. Interestingly, the variation in the number of days required for egg hatching was more pronounced in S. miscanthi ML form than in the other two species/forms, indicating greater phenotypic variability in this lineage. As mentioned earlier, S. sabelisi and S. miscanthi HG form are estimated to have originated from populations with mild male aggression, including S. miscanthi ML form (Sato et al. 2019 ). The elevated variability observed in S. miscanthi ML form may reflect ancestral phenotypic and genetic diversity, which could have facilitated subsequent divergence and local adaptation in S. sabelisi and S. miscanthi HG form. A similar pattern is observed in the cichlid fishes of Lake Malawi, where ancestral morphological and ecological variation might have enabled rapid speciation via adaptive radiation (McGee et al. 2020 ). Such intra-population variability is often associated with broader environmental tolerance and greater evolutionary potential (Gamelon et al. 2025 ). Given that S. miscanthi ML form is distributed in subtropical regions with relatively stable, high-temperature environments and lacks diapause, it is plausible that relaxed thermal pressures in such environments may have allowed the persistence of developmental thermal trait variability. In contrast, the colder or more variable climates inhabited by S. sabelisi and S. miscanthi HG form may have imposed directional thermal selection, leading to more canalised traits, as reflected by their lower phenotypic variance and distinct thermal profiles. These three species/forms also differ in male aggression: it is high in S. miscanthi HG, low in S. sabelisi , and intermediate in S. miscanthi ML, as previously described. This variation has been interpreted through the concept of kin selection theory, which suggests that winter harshness influences the average relatedness among nestmates, thereby driving differences in male aggression across populations (Saito 1995 ; Saito and Sahara 1999 ; Sato et al. 2013 ). Male aggression is assessed by measuring the probability that one of two males placed in a nest will die within five days. As with thermal developmental traits, if male aggression shows greater variability in S. miscanthi ML form, for example, due to a mixture of high- and low-aggression individuals, then the observed “intermediate” aggression level may reflect underlying polymorphism rather than a stable intermediate phenotype. Therefore, examining variation in behavioural traits, in addition to life history traits, may provide further insight into the evolutionary mechanisms underlying both physiological and behavioural divergence. Declarations Data availability The datasets generated during and/or analysed during the current study are available from figshare (doi: 10.6084/m9.figshare.29287496; doi: 10.6084/m9.figshare.29287673) Acknowledgments We thank Mr. Shota Konaka, Mr. Naoki Matsumoto, Mr. Gomei Yoda, Mr. Taito Sano, Mr. Ryuto Uchiyama, Ms. Sayuka (Nagase) Nitta, Ms. Aina Yokoi, Ms. Ayana Tanino, Mr. Takamitsu Furukawa, Ms. Yuanhong Li, Mr. Keita Shindo, Ms. Mayu Tahara, Dr. Kazuharu Ohashi, and Prof. Kenji Miura for their valuable suggestions and support. This research was supported in part by the Suzuki Takahisa Memorial Grant, the University of Tsukuba (to YS), the Research Enhancement Project of Mountain Science Centre, the University of Tsukuba (to YS), and Nakatsuji Foresight Foundation (to YS). Author contributions YS and RY contributed to the study conception and design. 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Front Arachn Sci 3:1436082. https://doi.org/10.3389/frchs.2024.1436082 Saito Y, Sato Y, Chittenden AR et al (2018) Description of two new species of Stigmaeopsis , Banks 1917 (Acari, Tetranychidae) inhabiting Miscanthus grasses (Poaceae). Acarologia 58:414–429. https://doi.org/10.24349/acarologia/20184250 Sakagami T, Saito Y, Kongchuensin M, Sahara K (2009) Molecular phylogeny of Stigmaeopsis , with special reference to speciation through host plant shift. Ann Entomol Soc Am 102:360–366. https://doi.org/10.1603/008.102.0303 Sakamoto H, Matsuda T, Suzuki R et al (2017) Molecular identification of seven species of the genus Stigmaeopsis (Acari: Tetranychidae) and preliminary attempts to establish their phylogenetic relationship. Syst Appl Acarol 22:91–101. https://doi.org/10.11158/saa.22.1.10 Sato Y, Breeuwer JAJ, Egas M, Sabelis MW (2015) Incomplete premating and postmating reproductive barriers between two parapatric populations of a social spider mite. Exp Appl Acarol 65:277–291. https://doi.org/10.1007/s10493-015-9878-3 Sato Y, Egas M, Sabelis MW, Mochizuki A (2013) Male–male aggression peaks at intermediate relatedness in a social spider mite. Ecol Evol 3:2661–2669. https://doi.org/10.1002/ece3.661 Sato Y, Saito Y (2006) Nest sanitation in social spider mites: Interspecific differences in defecation behavior. Ethology 112:664–669. https://doi.org/10.1111/j.1439-0310.2005.01184.x Sato Y, Saito Y (2008) Evolutionary view of waste-management behavior using volatile chemical cues in social spider mites. J Ethol 26:267–272. https://doi.org/10.1007/s10164-007-0069-5 Sato Y, Saito Y, Mori K (2000a) Reproductive isolation between populations showing different aggression in a subsocial spider mite, Schizotetranychus miscanthi Saito (Acari: Tetranychidae). Appl Entomol Zool 35:605–610. https://doi.org/10.1303/aez.2000.605 Sato Y, Saito Y, Mori K (2000b) Patterns of reproductive isolation between two groups of Schizotetranychus miscanthi Saito (Acari: Tetranychidae) showing different male aggression traits. Appl Entomol Zool 35:611–618. https://doi.org/10.1303/aez.2000.611 Sato Y, Saito Y, Sakagami T (2003) Rules for nest sanitation in a social spider mite, Schizotetranychus miscanthi Saito (Acari: Tetranychidae). Ethology 109:713–724. https://doi.org/10.1046/j.1439-0310.2003.00905.x Sato Y, Sakamoto H, Gotoh T et al (2018) Patterns of reproductive isolation in a haplodiploid – strong postmating, prezygotic barriers among three forms of a social spider mite. J Evol Biol 31:866–881. https://doi.org/10.1111/jeb.13270 Sato Y, Tsuda Y, Sakamoto H et al (2019) Phylogeography of lethal male fighting in a social spider mite. Ecol Evol 9:1590–1602. https://doi.org/10.1002/ece3.4770 Schausberger P, Yano S, Sato Y (2021) Cooperative behaviors in group-living spider mites. Front Ecol Evol 9:745036. https://doi.org/10.3389/fevo.2021.745036 Schmidt PS, Matzkin L, Ippolito M, Eanes WF (2005) Geographic variation in diapause incidence, life-history traits, and climatic adaptation in Drosophila Melanogaster . Evolution 59:1721–1732. https://doi.org/10.1111/j.0014-3820.2005.tb01821.x Tauber CA, Tauber MJ, Nechols JR (1987) Thermal requirements for development in Chrysopa oculata : A geographically stable trait. Ecology 68:1479–1487. https://doi.org/10.2307/1939231 Taylor F (1981) Ecology and evolution of physiological time in insects. Am Nat 117:1–23. https://doi.org/10.1086/283683 Therneau TM (2019) Coxme: Mixed effects Cox models. R package version 22 – 14 https://CRANR-project.org/package=coxme Tougeron K (2019) Diapause research in insects: historical review and recent work perspectives. Entomol Exp Appl 167:27–36. https://doi.org/10.1111/eea.12753 Yano J, Saito Y, Chittenden AR, Sato Y (2011) Variation in counterattack effect against a phytoseiid predator between two forms of the social spider mite, Stigmaeopsis miscanthi . J Ethol 29:337–342. https://doi.org/10.1007/s10164-010-0265-6 Tables Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.docx Cite Share Download PDF Status: Published Journal Publication published 31 Jul, 2025 Read the published version in Experimental and Applied Acarology → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6875858","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":471430641,"identity":"80ffc8dd-1dcf-49bd-a92c-3c65d9132611","order_by":0,"name":"Ryu Yatabe","email":"","orcid":"","institution":"University of Tsukuba","correspondingAuthor":false,"prefix":"","firstName":"Ryu","middleName":"","lastName":"Yatabe","suffix":""},{"id":471430642,"identity":"a07aba32-a105-4495-9bc4-0e496b969ef9","order_by":1,"name":"Yukie Sato","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYFACHoYDIIofRCQUEK0lgYFBsgGkxYBILQwgLQZgu4jRYnD87MEDH3/Y5RmfX5344YEBgzy/2AECWs7kJRyckZBcbHbj7WYJoMMMZ85OIKDlBo/BYZ4E5sRtN85uAGlJMLhNjJY/CfWJm2ec3fyDeC0MCYcTN/D3biPOFskzOQYHe9KOJ864wbvNIsFAgrBf+I6fMf7ww6Y6sb//7OabPyps5PmlCWhBAAmwSglilYMA/wFSVI+CUTAKRsFIAgB3zUnWFbhIIgAAAABJRU5ErkJggg==","orcid":"","institution":"University of Tsukuba","correspondingAuthor":true,"prefix":"","firstName":"Yukie","middleName":"","lastName":"Sato","suffix":""}],"badges":[],"createdAt":"2025-06-12 02:53:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6875858/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6875858/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10493-025-01055-1","type":"published","date":"2025-07-31T16:13:08+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":84912821,"identity":"883655da-05ac-49c7-a00b-e688440b0b17","added_by":"auto","created_at":"2025-06-18 17:34:10","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":171152,"visible":true,"origin":"","legend":"\u003cp\u003eNumber of days required for egg hatching at five constant temperatures (15°C, 20°C, 25°C, 30°C, and 32°C) in \u003cem\u003eStigmaeopsis sabelisi\u003c/em\u003e, \u003cem\u003eS. miscanthi\u003c/em\u003e high aggression (HG) form and \u003cem\u003eS. miscanthi\u003c/em\u003e mild aggression (ML) form. Filled circles represent mean values, error bars indicate standard errors, and dashed lines show model predictions based on a generalised linear model with a gamma error distribution. Blue, red, and orange correspond to \u003cem\u003eS. sabelisi\u003c/em\u003e, \u003cem\u003eS. miscanthi\u003c/em\u003e HG form, and \u003cem\u003eS. miscanth\u003c/em\u003ei ML form, respectively.\u003c/p\u003e","description":"","filename":"figure11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6875858/v1/b2b81512620d3417aa92c18c.jpg"},{"id":84913901,"identity":"1f869c61-ee64-428a-af48-b8c9cdf3c3aa","added_by":"auto","created_at":"2025-06-18 17:42:10","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":174623,"visible":true,"origin":"","legend":"\u003cp\u003eCoefficients of variation in the number of days for egg hatching at five constant temperatures (15°C, 20°C, 25°C, 30°C, and 32°C) in \u003cem\u003eStigmaeopsis sabelisi\u003c/em\u003e, \u003cem\u003eS. miscanthi\u003c/em\u003e high aggression (HG) form and \u003cem\u003eS. miscanthi\u003c/em\u003e mild aggression (ML) form. Filled circles represent the coefficient of variation values, and dashed lines connect values within each species/form for visual clarity. Blue, red, and orange correspond to \u003cem\u003eS. sabelisi\u003c/em\u003e, \u003cem\u003eS. miscanthi\u003c/em\u003e HG form, and \u003cem\u003eS. miscanth\u003c/em\u003ei ML form, respectively.\u003c/p\u003e","description":"","filename":"figure12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6875858/v1/ffc89760238998605a33e492.jpg"},{"id":84912822,"identity":"a76bf0a2-5ea1-414b-a40a-607d5d5ed3ff","added_by":"auto","created_at":"2025-06-18 17:34:10","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":251411,"visible":true,"origin":"","legend":"\u003cp\u003eDevelopmental speed at five constant temperatures (15°C, 20°C, 25°C, 30°C, and 32°C) in \u003cem\u003eStigmaeopsis sabelisi\u003c/em\u003e, \u003cem\u003eS. miscanthi\u003c/em\u003e high aggression (HG) form and \u003cem\u003eS. miscanthi\u003c/em\u003emild aggression (ML) form, with fitted lines based on linear regressions (a) and non-linear regressions using the Brière model (b). Filled circles represent mean values, error bars indicate standard errors, and dashed lines show the fitted lines for each species/form based on linear regressions (a) and nonlinear regressions (b). Blue, red, and orange correspond to \u003cem\u003eS. sabelisi\u003c/em\u003e, \u003cem\u003eS. miscanthi\u003c/em\u003e HG form, and \u003cem\u003eS. miscanth\u003c/em\u003ei ML form, respectively.\u003c/p\u003e","description":"","filename":"figure13.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6875858/v1/c9bdf23f7329619f97941429.jpg"},{"id":84914137,"identity":"a6bc82f0-8a95-47e1-8847-9ff1fc7ad79e","added_by":"auto","created_at":"2025-06-18 17:50:10","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":214014,"visible":true,"origin":"","legend":"\u003cp\u003eOviposition patterns at 18°C under long-day conditions in \u003cem\u003eStigmaeopsis sabelisi\u003c/em\u003e, \u003cem\u003eS. miscanthi\u003c/em\u003e high aggression (HG) form and \u003cem\u003eS. miscanthi\u003c/em\u003e mild aggression (ML) form. Dashed lines represent oviposition patterns of females that developed at 18°C under short-day conditions (9:15 h light:dark photoperiod), while solid lines show oviposition patterns of females that developed at 18°C under long-day conditions (15:9 h light:dark photoperiod15:9 h light:dark photoperiod). Blue, red, and orange correspond to \u003cem\u003eS. sabelisi\u003c/em\u003e, \u003cem\u003eS. miscanthi\u003c/em\u003e HG form, and \u003cem\u003eS. miscanth\u003c/em\u003ei ML form, respectively.\u003c/p\u003e","description":"","filename":"figure14.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6875858/v1/3de2e9698f7a18e011026925.jpg"},{"id":88268492,"identity":"a2c3c3a1-a9cd-4eca-b3f0-0d973ed3b3ac","added_by":"auto","created_at":"2025-08-04 16:52:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1446743,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6875858/v1/61eb7d9a-c428-4fdc-b06c-d60903d33d17.pdf"},{"id":84912826,"identity":"a141ae59-ae74-4f32-8935-441d3fdf3399","added_by":"auto","created_at":"2025-06-18 17:34:10","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18035,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6875858/v1/9d52ebd3e82d945993646263.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Phenotypic variability and thermal adaptation in social spider mites: Insights into speciation and local adaptation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eArthropods, including mites and ticks, are ectothermic organisms whose body temperature and, consequently, physiological processes closely follow ambient environmental conditions. As a result, their development, reproduction, and population dynamics are strongly influenced by thermal environments (Taylor \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1981\u003c/span\u003e). Among thermal traits, the minimum and maximum temperature thresholds for development are often considered as indicators of local adaptation (Honek \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Another key trait associated with thermal adaptation is diapause, a common strategy in arthropods that facilitates survival during adverse conditions, particularly low winter temperatures (Tougeron \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Denlinger \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). It is generally hypothesised that populations from colder regions exhibit lower minimum temperature thresholds for development and more intense diapause attributes than those from warmer regions, allowing them to maximise the use of limited thermal resources and enhance overwintering success (Honek \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). While this pattern is well documented for diapause (Masaki \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1961\u003c/span\u003e; Schmidt et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Posledovich et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), empirical support for similar geographic trends in the thermal thresholds for development remains inconsistent; some taxa conform to the pattern, while others deviate from it (Campbell et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1974\u003c/span\u003e; Tauber et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Honek \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Kipyatkov and Lopatina \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), suggesting that the patterns of geographical/latitudinal variation in thermal thresholds for development would differ among taxa.\u003c/p\u003e \u003cp\u003e \u003cem\u003eStigmaeopsis sabelisi\u003c/em\u003e and \u003cem\u003eStigmaeopsis miscanthi\u003c/em\u003e high aggression (HG) form are social spider mites infesting the Chinese silver grass, \u003cem\u003eMiscanthus sinensis\u003c/em\u003e. Spider mites produce silken threads, and those of the genus \u003cem\u003eStigmaeopsis\u003c/em\u003e use the threads to construct woven nests, where they live in groups (Saito and Sato \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Within the nests, individuals exhibit a variety of cooperative behaviours among nest mates, including faeces management to maintain nest hygiene (Sato et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Sato and Saito \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2006\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), removal of exuviae, other debris and bacteria scattered inside the nests (Kanazawa et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), and collective defence against predators which invade the woven nests (Saitō \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1986\u003c/span\u003e; Yano et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Because of these cooperative behaviours, \u003cem\u003eStigmaeopsis\u003c/em\u003e mites are recognised as social spider mites (Schausberger et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Saito and Sato \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Among \u003cem\u003eStigmaeopsis\u003c/em\u003e mites, species infesting the Chinese silver grass, known as \u003cem\u003eStigmaeopsis miscanthi\u003c/em\u003e species group, exhibit a unique social behaviour: male-killing, in which males eliminate rival males to establish harems (Saitō \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). Notably, the frequency of male-killing differs between species: being lower in \u003cem\u003eS. sabelisi\u003c/em\u003e and higher in \u003cem\u003eS. miscanthi\u003c/em\u003e HG form (Saito \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Saito and Sahara \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Previously, \u003cem\u003eS. sabelisi\u003c/em\u003e was referred to as the low-aggression (LW) form of \u003cem\u003eS. miscanthi\u003c/em\u003e, as the two were once considered conspecific variants differing in male aggression levels. However, subsequent studies have revealed morphological differences (Saito \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1995\u003c/span\u003e), reproductive isolation (Sato et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2000a\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003eb\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and distinct molecular phylogenetic relationships (Ito and Fukuda \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Sakagami et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Sakamoto et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Sato et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), supporting their classification as separate species (Saito et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThese two species differ in their geographic distributions; \u003cem\u003eS. sabelisi\u003c/em\u003e is found in colder regions of Japan and Korea, whereas \u003cem\u003eS. miscanthi\u003c/em\u003e HG form is primarily distributed in warmer areas of Japan (Saito and Sahara \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Sato et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Corresponding to this habitat difference, differences in diapause attributes have been reported between them (Saito et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). In both species, short-day lengths with lower temperatures during the developmental period (from egg to adult emergence) induce reproductive diapause in females, and long-day lengths with higher temperatures terminate the diapause, allowing females to initiate oviposition. \u003cem\u003eS. sabelisi\u003c/em\u003e females require a significantly longer period to terminate reproductive diapause and initiate oviposition compared to those of \u003cem\u003eS. miscanthi\u003c/em\u003e HG form (Saito et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Regarding thermal responses in development, the two species exhibit similar minimum temperature thresholds for development (\u003cem\u003eS. sabelisi\u003c/em\u003e: 11.7\u0026deg;C, \u003cem\u003eS. miscanthi\u003c/em\u003e HG form: 11.8\u0026deg;C) (Saito et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Developmental speed, however, is significantly slower in \u003cem\u003eS. sabelisi\u003c/em\u003e at any temperature in the range 10\u0026deg;C to 25\u0026deg;C, resulting in a lower intrinsic rate of natural increase (\u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e) compared to \u003cem\u003eS. miscanthi\u003c/em\u003e HG form (Saito et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRecently, populations exhibiting intermediate levels of male aggression, referred to as \u003cem\u003eS. miscanthi\u003c/em\u003e mild-aggression (ML) form, have been identified in subtropical regions of Japan. \u003cem\u003eS. miscanthi\u003c/em\u003e ML form also exhibits an intermediate male weapon morphology and is reproductively isolated from \u003cem\u003eS. sabelisi\u003c/em\u003e and \u003cem\u003eS. miscanthi\u003c/em\u003e HG form (Sato et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Phylogenetic analysis based on mitochondrial cytochrome c oxidase subunit I (mt COI) sequences, including populations from Japan, Korea and Taiwan, revealed that both \u003cem\u003eS. sabelisi\u003c/em\u003e and \u003cem\u003eS. miscanthi\u003c/em\u003e HG form were derived from a group with mild male aggression, including \u003cem\u003eS. miscanthi\u003c/em\u003e ML form, approximately 20,000\u0026ndash;40,000 years before present (BP) and 5,494\u0026thinsp;\u0026minus;\u0026thinsp;10,988 years BP, respectively (Sato et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). These findings suggest that \u003cem\u003eS. sabelisi\u003c/em\u003e and \u003cem\u003eS. miscanthi\u003c/em\u003e HG form emerged from a common ancestral group distributed in the subtropical regions, including the \u003cem\u003eS. miscanthi\u003c/em\u003e ML form, through subsequent adaptation to colder and warmer environments, respectively. If this evolutionary scenario is correct, then investigating the thermal responses in development and diapause attributes of the \u003cem\u003eS. miscanthi\u003c/em\u003e ML form is essential for understanding the ecological and evolutionary processes underlying speciation in this group.\u003c/p\u003e \u003cp\u003eThis study has two primary aims: (1) to test the hypothesis that populations from colder regions exhibit lower minimum temperature thresholds for development and more pronounced reproductive diapause than those from warmer and subtropical regions, and (2) to gain deeper insight into the speciation process associated with local adaptation in the social spider mites. To address these aims, we compared thermal life history traits: the minimum temperature thresholds for development and reproductive diapause, among \u003cem\u003eS. sabelisi\u003c/em\u003e, \u003cem\u003eS. miscanthi\u003c/em\u003e HG form, and \u003cem\u003eS. miscanthi\u003c/em\u003e ML form. For the minimum temperature thresholds for development, we investigated the number of days required for egg hatching under five constant temperature conditions (15\u0026deg;C, 20\u0026deg;C, 25\u0026deg;C, 30\u0026deg;C and 32\u0026deg;C), and estimated the thresholds using linear and non-linear regressions. We also calculated the coefficient of variation in developmental time to assess phenotypic variability, which reflects intra- and inter-lineage diversity and offers insights into evolutionary potential (Gamelon et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In addition, we investigated reproductive diapause induction under short-day conditions based on previous findings (Saito et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Collectively, these data offer a comprehensive perspective on how thermal adaptation may contribute to ecological divergence and speciation in this group.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eThe minimum temperature threshold for development\u003c/h2\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003eMites\u003c/h2\u003e \u003cp\u003eWe used \u003cem\u003eS. miscanthi\u003c/em\u003e ML form collected from Ishigaki Island (latitude and longitude: 24.522789\u0026deg;N, 124.283347\u0026deg;E, Altitude: 30.1 m; Okinawa Prefecture, Japan) in July 2022, \u003cem\u003eS. miscanthi\u003c/em\u003e HG form collected from Naha City (latitude and longitude: 26.212532\u0026deg;N, 127.71723\u0026deg;E, altitude: 40.1 m; Okinawa Prefecture, Japan) in July 2022 and \u003cem\u003eS. sabelisi\u003c/em\u003e collected from Mt. Amagi (latitude and longitude: 34.8836111\u0026deg;N, 139.0713889\u0026deg;E, altitude: 491.6 m; Shizuoka Prefecture, Japan) in June 2022. More than 20 females were used to establish each laboratory culture, and the mites were reared on the host plant leaves, \u003cem\u003eM. sinensis\u003c/em\u003e placed on wet cotton in plastic trays in an air-conditioned room at 25\u0026deg;C, with a relative humidity of 60\u0026ndash;80% and a 15:9 h light:dark photoperiod.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eExperiment\u003c/h3\u003e\n\u003cp\u003eWe introduced a female collected from the laboratory culture onto a detached \u003cem\u003eM. sinensis\u003c/em\u003e leaf (1 \u0026times; 2 cm) placed on wet cotton in an insect breeding dish (5.0 cm diameter, 1.5 cm high; SPL Life Sciences, Gyeonggi-do, Korea). We allowed the female to construct a nest on the leaf and lay eggs in it for 24 h. When the female laid more than one egg, we crushed the extra eggs with a needle to make one egg per nest. We removed the female from the nest and observed the development of the egg every 24 h. We recorded the number of days required for egg hatching to occur. We carried out these manipulations for three species/forms (\u003cem\u003eS. miscanthi\u003c/em\u003e ML form, \u003cem\u003eS. miscanthi\u003c/em\u003e HG form and \u003cem\u003eS. sabelisi\u003c/em\u003e) under five different temperature conditions (15\u0026deg;C, 20\u0026deg;C, 25\u0026deg;C, 30\u0026deg;C and 32\u0026deg;C) using incubators, where other conditions were the same (50\u0026ndash;70% relative humidity and 15:9 h light:dark photoperiod). The number of replicates was approximately 20 per species per treatment. As some eggs died during the experiments, we excluded them from the analysis, resulting in a final number of replications ranging from 12 to 21 per species per treatment.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eFor the statistical analyses, we used the software R ver.4.0.2 (R Core Team \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). We analysed the number of days required for egg hatching with species/form, temperature and the interaction using a generalised linear model, where a gamma distribution was applied as the error distribution (gamma GLM), since the data was significantly deviated from the normal distribution (Shapiro-Wilk normality test, \u003cem\u003eW\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.741, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Then, we performed a Tukey post-hoc test to examine pairwise differences among species using the package \u003cem\u003eemmeans\u003c/em\u003e (Lenth \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe analysed the phenotypic variability by calculating the coefficient of variation (standard deviation divided by the mean) of the number of days required for egg hatching for each temperature and species/form. We compared the coefficient of variation among species/forms at each temperature using an asymptotic test for the equality of coefficients of variance as implemented in the package \u003cem\u003ecvequality\u003c/em\u003e (Feltz and Miller \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Marwick and Krishnamoorthy \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). We adjusted the \u003cem\u003eP\u003c/em\u003e-values for multiple testing using the Benjamini\u0026ndash;Hochberg (BH) method (Benjamini and Hochberg \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1995\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor the estimation of the minimum temperature thresholds for development, we performed a linear regression analysis on the developmental speed (1 divided by days to hatch) and temperature in each species/form, as a previous study on \u003cem\u003eS. sabelisi\u003c/em\u003e and \u003cem\u003eS. miscanthi\u003c/em\u003e HG form used the linear regression for the estimation (Saito et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). We also performed a non-linear regression analysis based on the Bri\u0026egrave;re model (Briere et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), which takes into account both the minimum and maximum temperature thresholds, as a previous study examined them at the temperature range from 10\u0026deg;C to 25\u0026deg;C, whereas this study also included 30\u0026deg;C and 32\u0026deg;C, where hatching may be delayed due to high temperature disturbance. The fitted model was:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:Developmental\\:speed\\:\\left(T\\right)=a\\times\\:T\\times\\:(T-{T}_{min})\\sqrt{{T}_{max}-T}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:T\\)\u003c/span\u003e\u003c/span\u003e is temperature, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{T}_{min}\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{T}_{max}\\)\u003c/span\u003e\u003c/span\u003e are the minimum and maximum temperature thresholds for development, and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:a\\)\u003c/span\u003e\u003c/span\u003e is constant. For the model fitting, we used nonlinear least squares, and we placed constraints that \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{T}_{min}\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{T}_{max}\\)\u003c/span\u003e\u003c/span\u003e should be in the range from 0 to 20 and from 20 to 40, respectively.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eReproductive diapause\u003c/h3\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMites\u003c/h2\u003e \u003cp\u003eWe used \u003cem\u003eS. miscanthi\u003c/em\u003e ML form collected from Ishigaki Island (latitude and longitude: 24.5213078\u0026deg;N, 124.2832838\u0026deg;E, Altitude: 33.5 m; Okinawa Prefecture, Japan) in July 2024, \u003cem\u003eS. miscanthi\u003c/em\u003e HG form collected from Naha City (latitude and longitude: 26.212532\u0026deg;N, 127.71723\u0026deg;E, altitude: 40.1 m; Okinawa Prefecture, Japan) in July 2022 and \u003cem\u003eS. sabelisi\u003c/em\u003e collected from Mt. Unzen (latitude and longitude: 32.75686\u0026deg;N, 130.275699\u0026deg;E, altitude: 878.5 m; Nagasaki Prefecture, Japan) in June 2024. More than 20 females were used to establish each laboratory culture, and the mites were reared on the host plant leaves, \u003cem\u003eM. sinensis\u003c/em\u003e placed on wet cotton in plastic trays in an air-conditioned room at 25\u0026deg;C, with a relative humidity of 60\u0026ndash;80% and a 15:9 h light:dark photoperiod.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExperiment\u003c/h3\u003e\n\u003cp\u003eThe experimental procedure followed Saito et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), which examined diapause attributes of \u003cem\u003eS. sabelisi\u003c/em\u003e and \u003cem\u003eS. miscanthi\u003c/em\u003e HG form. Although physiological indicators such as changes in body colouration were not observed in the previous study, it clearly showed that \u003cem\u003eS. sabelisi\u003c/em\u003e from colder regions required significantly more time to initiate oviposition than the \u003cem\u003eS. miscanthi\u003c/em\u003e HG form from warmer regions, when females that had developed under short-day conditions were transferred to long-day conditions. This delay in oviposition timing under a shift from short to long photoperiods thus serves as a reliable proxy for evaluating reproductive diapause in \u003cem\u003eS. miscanthi\u003c/em\u003e species group.\u003c/p\u003e \u003cp\u003eWe introduced 10\u0026ndash;20 fertilised females collected from the laboratory culture onto a detached \u003cem\u003eM. sinensis\u003c/em\u003e leaf (1 \u0026times; 4 cm) placed on wet cotton in an insect breeding dish (5.0 cm diameter, 1.5 cm high; SPL Life Sciences, Gyeonggi-do, Korea). We allowed the females to lay eggs for approximately 10 days, removed the females, and reared the eggs until they developed into the teleiochrysalis stage under short-day conditions (9:15 h light:dark photoperiod) to induce diapause. As controls, we reared the eggs under long-day conditions (15:9 h light:dark photoperiod). We collected teleiochrysalis females from detached leaves and introduced them onto new detached leaves together with males. One day after adult emergence, we introduced the females individually onto new detached \u003cem\u003eM. sinensis\u003c/em\u003e leaves and left them under long-day conditions (15:9 h light:dark photoperiod) to terminate diapause. We observed the survival of females and whether they initiated oviposition every day. We carried out these manipulations under the conditions of 18\u0026deg;C and a relative humidity of 50\u0026ndash;70%. The number of replicates was 20\u0026ndash;22 per treatment and per species/form.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eFor the statistical analyses, we used the software R v.4.0.2 (R Core Team \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). We analysed the timing of oviposition initiation using non-parametric survival analysis: a Kaplan-Meier curve and a log-rank test in the package \u003cem\u003esurvival\u003c/em\u003e (Therneau \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). We compared the timing of oviposition initiation between females which had developed under short-day conditions (treatment) and those which had developed under long-day conditions (control) in each species/form. We adjusted the \u003cem\u003eP\u003c/em\u003e-values for multiple testing using the BH method (Benjamini and Hochberg \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). We did not use a semi-parametric survival analysis: Cox proportional hazards regression model, as the assumption of proportional hazards for the fit of the Cox regression model was not satisfied in our dataset.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eThe minimum temperature threshold for development\u003c/h2\u003e\n \u003cp\u003eThe curve of the number of days required for egg hatching along temperature was significantly different among species (gamma GLM, species \u0026times; temperature, \u003cem\u003eF\u003c/em\u003e\u003csub\u003e2,239\u003c/sub\u003e = 9.296, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Naturally, the number of days decreased as the temperature increased, although the number of days at 32\u0026deg;C was longer than that at 30\u0026deg;C in \u003cem\u003eS. sabelisi\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). \u003cem\u003eS. sabelisi\u003c/em\u003e required significantly longer days for egg hatching than the other two species (Tukey post-hoc test; \u003cem\u003eS. sabelisi\u003c/em\u003e vs. \u003cem\u003eS. miscanthi\u003c/em\u003e HG form: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, \u003cem\u003eS. sabelisi\u003c/em\u003e vs. \u003cem\u003eS. miscanthi\u003c/em\u003e ML form: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). A significant difference in the number of days was not found between \u003cem\u003eS. miscanthi\u003c/em\u003e HG form and \u003cem\u003eS. miscanthi\u003c/em\u003e ML form (Tukey post-hoc test, \u003cem\u003eS. miscanthi\u003c/em\u003e HG form vs. \u003cem\u003eS. miscanthi\u003c/em\u003e ML form: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.185; Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eThe coefficient of variation appeared higher in \u003cem\u003eS. miscanthi\u003c/em\u003e ML form than in the other two species/forms across five different temperatures (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e); however, significant differences were observed only at 20\u0026deg;C(asymptotic test with adjustment by BH method, 15\u0026deg;C: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.486, 20\u0026deg;C: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.012, 25\u0026deg;C: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.414, 30\u0026deg;C: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.385, 32\u0026deg;C: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.385).\u003c/p\u003e\n \u003cp\u003eLinear regressions for development rate and temperature successfully converged for each species/form (\u003cem\u003eS. sabelisi\u003c/em\u003e: \u003cem\u003eF\u003c/em\u003e\u003csub\u003e1,77\u003c/sub\u003e = 675.8, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; \u003cem\u003eS. miscanthi\u003c/em\u003e HG form: \u003cem\u003eF\u003c/em\u003e\u003csub\u003e1,84\u003c/sub\u003e = 1,420, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; \u003cem\u003eS. miscanthi\u003c/em\u003e ML form: \u003cem\u003eF\u003c/em\u003e\u003csub\u003e1,78\u003c/sub\u003e = 732.3, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea; Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea). The regression lines estimated the minimum temperature threshold for development to be 10.16\u0026deg;C for \u003cem\u003eS. sabelisi\u003c/em\u003e, 10.33\u0026deg;C for \u003cem\u003eS. miscanthi\u003c/em\u003e HG form and 10.77\u0026deg;C for \u003cem\u003eS. miscanthi\u003c/em\u003e ML form.\u003c/p\u003e\n \u003cp\u003eNonlinear regressions for development rate and temperature based on the Bri\u0026egrave;re model (Briere et al. \u003cspan class=\"CitationRef\"\u003e1999\u003c/span\u003e) were performed successfully for each species/form (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb; Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb). In the nonlinear regressions, the minimum temperature threshold for development was estimated to be 9.25\u0026deg;C for \u003cem\u003eS. sabelisi\u003c/em\u003e, 9.63\u0026deg;C for \u003cem\u003eS. miscanthi\u003c/em\u003e HG form and 10.16\u0026deg;C for \u003cem\u003eS. miscanthi\u003c/em\u003e ML form (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eReproductive diapause\u003c/h2\u003e\n \u003cp\u003eThe timing of oviposition initiation was significantly different between treatment and control in \u003cem\u003eS. sabelisi\u003c/em\u003e (log-rank test with adjustment by BH method: \u003cem\u003e\u0026chi;\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;14.90, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e), hhowever such a significant difference was not detected in \u003cem\u003eS. miscanthi\u003c/em\u003e HG form (log-rank test with adjustment by BH method: \u003cem\u003e\u0026chi;\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.40, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.75; Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e) and \u003cem\u003eS. miscanthi\u003c/em\u003e ML form (log-rank test with adjustment by BH method: \u003cem\u003e\u0026chi;\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.0, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.000; Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe minimum temperature threshold for development estimated by linear regression was 10.16\u0026deg;C for \u003cem\u003eS. sabelisi\u003c/em\u003e, 10.33\u0026deg;C for \u003cem\u003eS. miscanthi\u003c/em\u003e HG form, and 10.77\u0026deg;C for \u003cem\u003eS. miscanthi\u003c/em\u003e ML form. The threshold estimated by non-linear regression based on the Bri\u0026egrave;re model (Briere et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) was 9.25\u0026deg;C, 9.63\u0026deg;C, and 10.16\u0026deg;C for the respective species/forms. Although these estimated values did not differ substantially among the three species/forms, they corresponded well to the climates of their geographic distributions, as \u003cem\u003eS. sabelisi\u003c/em\u003e, \u003cem\u003eS. miscanthi\u003c/em\u003e HG form and \u003cem\u003eS. miscanthi\u003c/em\u003e ML form are distributed in colder, warmer and subtropical regions, respectively. Additionally, high-temperature stress was detected only in \u003cem\u003eS. sabelisi\u003c/em\u003e, which is distributed in colder regions. A previous study examined the developmental speed of \u003cem\u003eS. sabelisi\u003c/em\u003e and \u003cem\u003eS. miscanthi\u003c/em\u003e HG form in the temperature range of 10 to 25\u0026deg;C, and observed a consistent increase in developmental speed with temperature for both species/forms (Saito et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In contrast, this study extended the temperature range to include 30\u0026deg;C and 32\u0026deg;C. We observed that developmental speed increased from 15 to 30\u0026deg;C but declined from 30 to 32\u0026deg;C in \u003cem\u003eS. sabelisi\u003c/em\u003e. Such a decrease was not observed in \u003cem\u003eS. miscanthi\u003c/em\u003e HG form and \u003cem\u003eS. miscanthi\u003c/em\u003e ML form, suggesting that the developmental response to temperature of these species/forms is finely tuned to their respective thermal environments.\u003c/p\u003e \u003cp\u003eSimilar patterns were observed for diapause induction. In our experiment, reproductive diapause was induced by short-day conditions at 18\u0026deg;C during development in \u003cem\u003eS. sabelisi\u003c/em\u003e, but not in either \u003cem\u003eS. miscanthi\u003c/em\u003e HG form or \u003cem\u003eS. miscanthi\u003c/em\u003e ML form. A previous study reported reproductive diapause in both \u003cem\u003eS. sabelisi\u003c/em\u003e and \u003cem\u003eS. miscanthi\u003c/em\u003e HG form, using populations collected from regions around 33\u0026deg;N latitude (Kyushu and Shikoku, Japan) (Saito et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). In contrast, \u003cem\u003eS. miscanthi\u003c/em\u003e HG form we used was collected from the region approximately 26\u0026deg;N latitude (Central Ryukyu, Japan), a considerably more southern location, and did not exhibit diapause under the same conditions. This discrepancy suggests geographic variation in diapause attributes within \u003cem\u003eS. miscanthi\u003c/em\u003e HG form. Meanwhile, \u003cem\u003eS. miscanthi\u003c/em\u003e ML form, collected from around 24\u0026deg;N latitude (South Ryukyu), did not exhibit diapause either. Given that \u003cem\u003eS. miscanthi\u003c/em\u003e ML form is restricted to this southern region, it may generally lack reproductive diapause irrespective of population.\u003c/p\u003e \u003cp\u003eOverall, these results indicate that the three species/forms possess life history traits\u0026mdash;developmental responses to temperature and diapause attributes\u0026mdash;that are well adapted to the climates of their geographic distributions. Although comparisons were made among closely related species/forms rather than populations within a species, our results on \u003cem\u003eS. miscanthi\u003c/em\u003e species group supported the hypothesis that populations from the colder regions exhibit lower thermal thresholds in development and more intense diapause attributes than those from the warmer regions (Campbell et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1974\u003c/span\u003e; Tauber et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Honek \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Kipyatkov and Lopatina \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). To better understand inconsistencies across taxa, future studies should assess broader phylogenetic and ecological contexts. The results of this study provide a valuable contribution toward that goal.\u003c/p\u003e \u003cp\u003eIn this study, we examined not only average developmental responses to temperature but also phenotypic variability. Interestingly, the variation in the number of days required for egg hatching was more pronounced in \u003cem\u003eS. miscanthi\u003c/em\u003e ML form than in the other two species/forms, indicating greater phenotypic variability in this lineage. As mentioned earlier, \u003cem\u003eS. sabelisi\u003c/em\u003e and \u003cem\u003eS. miscanthi\u003c/em\u003e HG form are estimated to have originated from populations with mild male aggression, including \u003cem\u003eS. miscanthi\u003c/em\u003e ML form (Sato et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The elevated variability observed in \u003cem\u003eS. miscanthi\u003c/em\u003e ML form may reflect ancestral phenotypic and genetic diversity, which could have facilitated subsequent divergence and local adaptation in \u003cem\u003eS. sabelisi\u003c/em\u003e and \u003cem\u003eS. miscanthi\u003c/em\u003e HG form. A similar pattern is observed in the cichlid fishes of Lake Malawi, where ancestral morphological and ecological variation might have enabled rapid speciation via adaptive radiation (McGee et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Such intra-population variability is often associated with broader environmental tolerance and greater evolutionary potential (Gamelon et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Given that \u003cem\u003eS. miscanthi\u003c/em\u003e ML form is distributed in subtropical regions with relatively stable, high-temperature environments and lacks diapause, it is plausible that relaxed thermal pressures in such environments may have allowed the persistence of developmental thermal trait variability. In contrast, the colder or more variable climates inhabited by \u003cem\u003eS. sabelisi\u003c/em\u003e and \u003cem\u003eS. miscanthi\u003c/em\u003e HG form may have imposed directional thermal selection, leading to more canalised traits, as reflected by their lower phenotypic variance and distinct thermal profiles.\u003c/p\u003e \u003cp\u003eThese three species/forms also differ in male aggression: it is high in \u003cem\u003eS. miscanthi\u003c/em\u003e HG, low in \u003cem\u003eS. sabelisi\u003c/em\u003e, and intermediate in \u003cem\u003eS. miscanthi\u003c/em\u003e ML, as previously described. This variation has been interpreted through the concept of kin selection theory, which suggests that winter harshness influences the average relatedness among nestmates, thereby driving differences in male aggression across populations (Saito \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Saito and Sahara \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Sato et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Male aggression is assessed by measuring the probability that one of two males placed in a nest will die within five days. As with thermal developmental traits, if male aggression shows greater variability in \u003cem\u003eS. miscanthi\u003c/em\u003e ML form, for example, due to a mixture of high- and low-aggression individuals, then the observed \u0026ldquo;intermediate\u0026rdquo; aggression level may reflect underlying polymorphism rather than a stable intermediate phenotype. Therefore, examining variation in behavioural traits, in addition to life history traits, may provide further insight into the evolutionary mechanisms underlying both physiological and behavioural divergence.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from \u003cem\u003efigshare\u003c/em\u003e (doi: 10.6084/m9.figshare.29287496; doi: 10.6084/m9.figshare.29287673)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Mr. Shota Konaka, Mr. Naoki Matsumoto, Mr. Gomei Yoda, Mr. Taito Sano, Mr. Ryuto Uchiyama, Ms. Sayuka (Nagase) Nitta, Ms. Aina Yokoi, Ms. Ayana Tanino, Mr. Takamitsu Furukawa, Ms. Yuanhong Li, Mr. Keita Shindo, Ms. Mayu Tahara, Dr. Kazuharu Ohashi, and Prof. Kenji Miura for their valuable suggestions and support. This research was supported in part by the Suzuki Takahisa Memorial Grant, the University of Tsukuba (to YS),\u0026nbsp;the Research Enhancement Project of Mountain Science Centre, the University of Tsukuba (to YS), and Nakatsuji Foresight Foundation (to YS).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYS and RY contributed to the study conception and design. RY conducted experiments, YS and RY analysed data, and wrote the first draft of the manuscript, read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e:\u0026nbsp;The authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e: This research was supported in part by the Suzuki Takahisa Memorial Grant, the University of Tsukuba (to YS), the Research Enhancement Project of Mountain Science Centre, the University of Tsukuba (to YS), and Nakatsuji Foresight Foundation (to YS).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBenjamini Y, Hochberg Y (1995) Controlling the false discovery rate: A practical and powerful approach to multiple testing. 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J Ethol 29:337\u0026ndash;342. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10164-010-0265-6\u003c/span\u003e\u003cspan address=\"10.1007/s10164-010-0265-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":true,"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":"Geographic variation, local adaptation, lower developmental threshold, thermal adaptation, speciation, social spider mites","lastPublishedDoi":"10.21203/rs.3.rs-6875858/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6875858/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThermal adaptation plays a crucial role in shaping the development, reproduction and population dynamics of ectothermic organisms. In this study, we compared thermal life history traits among three closely related social spider mites: \u003cem\u003eStigmaeopsis sabelisi\u003c/em\u003e, \u003cem\u003eS. miscanthi\u003c/em\u003e high-aggression (HG) form, and their common ancestral group, \u003cem\u003eS. miscanthi\u003c/em\u003e mild-aggression (ML) form. We investigated the minimum temperature thresholds for development by measuring the days required for egg hatching under five constant temperature conditions (15\u0026deg;C, 20\u0026deg;C, 25\u0026deg;C, 30\u0026deg;C, 32\u0026deg;C) and estimating the thresholds using linear and nonlinear regression models. Additionally, we assessed their reproductive diapause attributes. Our results revealed that the minimum development thresholds were slightly lower in \u003cem\u003eS. sabelisi\u003c/em\u003e from colder regions compared to \u003cem\u003eS. miscanthi\u003c/em\u003e HG form and \u003cem\u003eS. miscanthi\u003c/em\u003e ML form distributed in warmer and subtropical regions. Notably, high-temperature stress negatively affected development only in \u003cem\u003eS. sabelisi\u003c/em\u003e, suggesting local adaptation. Reproductive diapause attributes also varied: reproductive diapause was induced under short-day conditions in \u003cem\u003eS. sabelisi\u003c/em\u003e, whereas the other two species lacked such diapause. Moreover, phenotypic variation in the number of days required for egg hatching was highest in \u003cem\u003eS. miscanthi\u003c/em\u003e ML form, suggesting retained ancestral variability that may have facilitated subsequent divergence. These findings support the hypothesis that populations from colder environments exhibit lower thermal thresholds and more intense diapause than those from warmer environments, and also provide insights into the mechanisms driving local adaptation and speciation in the social spider mites.\u003c/p\u003e","manuscriptTitle":"Phenotypic variability and thermal adaptation in social spider mites: Insights into speciation and local adaptation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-18 17:34:05","doi":"10.21203/rs.3.rs-6875858/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":"0e769f05-c5e1-4adf-b207-83214e44262b","owner":[],"postedDate":"June 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-04T16:48:31+00:00","versionOfRecord":{"articleIdentity":"rs-6875858","link":"https://doi.org/10.1007/s10493-025-01055-1","journal":{"identity":"experimental-and-applied-acarology","isVorOnly":false,"title":"Experimental and Applied Acarology"},"publishedOn":"2025-07-31 16:13:08","publishedOnDateReadable":"July 31st, 2025"},"versionCreatedAt":"2025-06-18 17:34:05","video":"","vorDoi":"10.1007/s10493-025-01055-1","vorDoiUrl":"https://doi.org/10.1007/s10493-025-01055-1","workflowStages":[]},"version":"v1","identity":"rs-6875858","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6875858","identity":"rs-6875858","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}