Global warming disrupts a thermogenesis-driven pollination mutualism

Global warming disrupts a thermogenesis-driven pollination mutualism | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Global warming disrupts a thermogenesis-driven pollination mutualism Yibo Luo, Yuan-Jun Yu, Yan Luo, Wufan Zhang, Xiang Ding, Xiqiang Song This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7182269/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Global warming threatens plant-pollinator mutualisms, but how microclimates modu-late this impact remains poorly understood. Using the thermogenic Alocasia odora and its specialized Colocasiomyia pollinators, we show floral thermogenesis—driven by male floral alternative oxidase (AOX) upregulation—creates vertically partitioned thermal microclimates. Field observations and manipulative experiments revealed that these thermal gradients guide flies via push-pull dynamics: flies are pushed toward female flowers for daytime pollination while pulled toward warm male parts for nighttime thermoregulation. Crucially, warming amplifies system vulnerability: inflo-rescence production declines 5.4% per 1°C increase, fruit set drops from 89% (cool seasons) to 68% (hot seasons), but pollinator declines are species-specific (C. alocasiae: 92.4% summer loss vs. C. xenalocasiae: 31.9%). While thermogenesis en-ables precision pollination, it converts microclimates into thermal traps under warm-ing due to directional niche plasticity, traits that buffered cold stress in evolutionary history cannot mitigate novel heat extremes in future. Biological sciences/Ecology/Behavioural ecology Biological sciences/Ecology/Climate-change ecology Biological sciences/Ecology/Community ecology Biological sciences/Ecology/Ecological networks Biological sciences/Ecology/Conservation biology Thermogenesis Microclimate Pollination Mutualism Climate Change Araceae Alternative Oxidase Figures Figure 1 Figure 2 Figure 3 Figure 4 Main Global warming profoundly affects all levels of biological organization—from individual growth and developmental rates 1 to population dynamics 2 and community structure 3 . These changes occur across a wide range of spatial scales, from within organisms to entire ecosystems 4,5 . However, the warming effects observed at broad (e.g., regional to global) scales do not necessarily reflect the actual thermal conditions experienced by organisms at micro-scales 6 . For example, flowers 7 , leaf surfaces 8 , and tree canopies 9 often form "microclimates" that differ significantly from the ambient environment, and many critical biotic interactions take place within these microscale habitats 10 . Yet, quantitative research into how broad-scale climate warming alters micro-scale or microclimates and thereby influences species interactions remains scarce 11 . This knowledge gap hampers our ability to accurately assess the ecological consequences of climate change. Among diverse species interactions, mutualistic relationships—particularly plant-pollinator relationships—may be highly sensitive to temperature changes 12 . This sensitivity stems from positive feedback dynamics, a process where an initial change triggers responses that amplify the original effect, creating a self-reinforcing cycle. Classical ecological theory, as proposed by Robert May, suggests that mutualisms are inherently prone to instability owing to these positive feedbacks 13 . This gives rise to a concerning prediction: if global warming leads to reduced floral abundance, pollinators may suffer from resource scarcity and population decline, which in turn further reduces future plant reproductive success through pollination process, initiating a "lose-lose" spiral of mutual collapse 14 . However, this assumes floral temperatures passively track ambient warming—ignoring flowers’ active microclimate regulation via reflectance, evaporative cooling, and circadian rhythms 15-17 . In fact, some plant lineages have evolved specialized metabolic mechanisms to actively modulate thermal environments 18 . A striking example is floral thermogenesis—the metabolic production of heat through alternative oxidase (AOX) and uncoupling proteins (UCPs), which elevates inflorescence temperatures by converting respiratory energy into thermal output 19-21 . Floral thermogenesis is an ancient trait that has independently evolved multiple times across at least 14 plant families 22,23 . Notably, thermogenesis is restricted to reproductive organs (e.g., inflorescences), suggesting its direct role in reproduction or pollination processes 24 . Several ecological hypotheses posited that floral heat production enhances plant–pollinator interactions. It may do so by (1) accelerating the release and diffusion of volatile compounds that attract pollinators 25-27 , (2) providing thermal rewards in cold environments, 28,29 or (3) generating temperature gradients that guide pollinators into the floral structure 30 . Crucially, it is unknown if thermogenesis decouples floral temperatures from ambient warming, and whether such decoupling buffers or amplifies climate impacts on mutualisms. Theoretically, two contrasting outcomes are possible. Firstly, a buffering effect: because thermogenic flowers already maintain elevated temperatures, they may be relatively insensitive to additional warming, thus offering a form of "pre-adaptation" that buffers plant–pollinator interactions 31,32 . Alternatively, an amplifying effect may occur: prolonged high temperatures could inhibit thermogenic metabolism or induce transpiration-based cooling 33 , thereby reducing floral attractiveness and impairing plant reproductive success. Thermogenic plant–pollinator systems thus provide a natural experimental model for studying climate change impacts on micro-scale ecosystems, and may serve as early warning indicators for broader ecosystem vulnerabilities through scaling up the temperature effects to the different biological organization levels. Alocasia odora , a typical thermogenic species in the Araceae family, exhibits clearly partitioned spadix structures, with thermogenic appendix and male flowers at the upper part and sterile male and female flowers at the lower part 34 . Previous studies indicate unique behavioral patterns among its specialized pollinators, flies of the genus Colocasiomyia (Drosophilidae): Colocasiomyia alocasiae preferentially oviposits in the upper male flower part, while Colocasiomyia xenalocasiae selects the lower female flower part 35 . Notably, Alocasia odora primarily depends on these Colocasiomyia flies for pollination despite visits from various insects, forming highly specialized mutualistic interactions 36 . Despite observed spatiotemporal variation in fly aggregation 37 , the roles of thermogenesis and climate variation in mediating these behaviors and mutualism stability are unquantified. In this study, we investigate a year-round flowering population of Alocasia odora in the tropics to examine the interactions between climate variation, floral thermogenesis, and pollination mutualisms. Our specific objectives are: (1) Characterize thermogenesis patterns/mechanisms in Alocasia inflorescences; (2) Clarify the ecological function of thermogenesis in mediating pollinator behavior; (3) Evaluate thermogenic buffering vs. amplification of climate impacts using seasonal variation as a warming proxy. Buffering is defined as maintained pollination success/fitness across temperature gradients; amplification as synergistic declines indicating destabilization. Results AOX-mediated thermogenesis creates vertically partitioned microclimates Temperature measurements revealed distinct thermogenic patterns across floral parts. During the female stage, the male flowers and appendixes reached a peak temperature of 30°C at noon, significantly higher than the ambient temperature of 25°C. As the ambient temperature decreased after noon, the temperature of the whole flower also declined. At night, the minimum temperature of the male flowers was 22°C, while the ambient temperature dropped to 17°C. In contrast, non-thermogenic female flowers showed minimal temperature elevation (≈1°C above ambient at night; Fig. 2a). Transcriptomic analysis identified molecular drivers of this thermogenesis. De novo assembly yielded 85,898 unigenes (N50=1.85 kb), with homology searches revealing three AOX ( AOX1a , AOX1b , AOX4 ) and three UCP ( UCP1 , UCP5 , UCPB ) homologs (Fig. 2b). Expression profiling demonstrated significant upregulation of AOX1b in male flowers during the female stage (2626 FPKM vs. <172 FPKM in other tissues; p 0.05). Temperature-driven push-pull dynamics guide precision pollination Flies exhibited temperature-dependent relocation within inflorescences. Each inflorescence hosted 10–30 flies that migrated vertically in response to thermal gradients: (1) At dawn/dusk (≈23°C), flies aggregated on the warmer upper (thermogenic) part (Fig. 2c); (2) At noon (>25°C in upper part), they moved to the cooler lower (non-thermogenic) part; (3) At night (ambient ≈17°C), flies returned to the upper part (maintained >20°C). Olfactory cues did not drive this behavior: Total VOC emissions were low during crepuscular/nocturnal periods (0.5 × 10 9 peak area; non-attractive) and high at noon (1–3 × 10 9 ; P <0.05), yet flies avoided the upper part during peak scent emission. This confirms temperature—not scent—as the primary driver of vertical movement. The activity of Colocasiomyia flies was significantly influenced by artificial manipulation of inflorescence temperatures. At noon, the male (upper) part of the inflorescence generates heat, causing the flies to primarily reside in the cooler lower (female) part. When we reduced the overall temperature of the inflorescence to nighttime levels (17–19°C), an average of 51% of the flies moved to the upper part within 30 minutes. Conversely, when we artificially heated the lower part to simulate higher temperatures (over 25°C) while keeping the upper part cool (under 25°C), approximately 85% of the flies departed the warmer lower part and relocated to the cooler upper part (Fig. 2d). Light conditions influenced fly behavior exclusively during the day. Shading the inflorescence to reduce light intensity caused about 17% of the flies to move from the lower part to the upper part. In contrast, increasing light intensity at night had little effect on fly movement. Light experiments suggests that light intensity alone may not sufficiently influence the flies' vertical movement during the night (Fig. 2e). Thermogenesis converts microclimates into thermal traps under warming The flowering peak of A. odora during the low-temperature phase (November to March), with an average of 0.61 ± 0.11 inflorescences per plant per month (mean ± SD). In contrast, the high-temperature phase (April to October) exhibited a substantial reduction in inflorescence numbers, averaging 0.30 ± 0.20 inflorescences per plant per month (Fig. 3b). Similarly, the fruit set rate was notably higher during the low-temperature phase, reaching 89.19 ± 7.83%, but declined to 67.92 ± 15.72% during the high-temperature phase. The abundance of Colocasiomyia flies showed significant seasonal patterns (Fig 3c). During the low-temperature phase, both C. alocasiae and C. xenalocasiae were present in higher numbers. Specifically, C. alocasiae averaged 688.59 ± 454.22 individuals based on 40 inflorescences, corresponding to 17.21 ± 11.36 flies per inflorescence. Similarly, C. xenalocasiae averaged 827.45 ± 518.96 individuals, equating to 20.69 ± 12.97 flies per inflorescence. In the high-temperature phase, the number of C. alocasiae flies markedly decreased, averaging 52.51 ± 147.28 individuals or 1.31 ± 3.68 flies per inflorescence. In some inflorescences, C. alocasiae was entirely absent. Conversely, C. xenalocasiae maintained relatively high numbers, averaging 564.62 ± 463.96 individuals or 14.12 ± 11.60 flies per inflorescence. Throughout one year, the sex ratios of both Colocasiomyia species remained relatively stable. C. alocasiae had an average female-to-male ratio of 1.13 ± 0.89, while C. xenalocasiae averaged 1.10 ± 0.28. Temperature was the dominant environmental driver of pollination metrics (Fig. 3a left). Correlations between pollination-related variables (inflorescences, fly numbers, fruit set rate) and the environmental variables humidity, light, and precipitation were low and non-significant (|r| 0.05), we focus exclusively on temperature in the following analyses. Temperature was negatively correlated with the number of inflorescences (r=-0.68, P<0.05). The fly number of C. alocasiae was negatively correlated with temperature (r=-0.56, P<0.05), while that of C. xenalocasiae was negatively correlated with both light intensity (r=-0.4, P<0.05) and temperature (r=-0.36, P0.39, P<0.05). In the cross-correlation analysis (Fig. 3a, right), temperature exhibited a significant negative correlation with the number of inflorescences at a lag of zero months, indicating an immediate effect of temperature on inflorescence numbers. Additionally, temperature was significantly negatively correlated with fruit set rate at a lag of one month. This suggests that the excessive temperature in the current month affects the fruit set rate in the following month. Furthermore, the number of inflorescences was significantly positively correlated with the number of C. alocasiae at a lag of zero months and with the number of C. xenalocasiae at a lag of two months. The number of C. alocasiae was significantly positively correlated with fruit set rate at a lag of one month, whereas the number of C. xenalocasiae showed a positive correlation with fruit set rate at a lag of zero months. Generalized Linear Mixed Models confirmed temperature as the key stressor. Temperature was the only significant factor affecting inflorescence numbers, exhibiting a negative effect (β = −1.03, P < 0.001). Within the temperature range of 25°C to 35°C, each 1°C increase corresponded to an average decrease of 5.37% in inflorescence numbers (Fig. 4a).Additionally, temperature had a significant negative effect on the abundance of C. alocasiae (β = −0.38, P < 0.01). In contrast, inflorescence numbers positively affected the abundance of both C. alocasiae (β = 6.50, P < 0.001; Fig. 4c) and C. xenalocasiae (β = 2.83, P < 0.001; Fig. 4b). Furthermore, temperature negatively influenced the fruit set rate (β = −0.224, P < 0.001). Conversely, the abundance of C. alocasiae and C. xenalocasiae both positively affected the fruit set rate (P < 0.01; Fig. 4d). Discussion AOX-Driven Thermogenesis Creates Spatiotemporally Segregated Microclimates Plant endogenous heat production (thermogenesis) is driven by two alternative respiratory pathways—AOX and UCP—that divert electron flow from ATP synthesis to heat 38,39 . Typically, these two pathways do not operate simultaneously in plants. For instance, species like Arum maculatum and Nelumbo nucifera rely primarily on the AOX pathway 20,40 . Our transcriptomic data showed that AOX was strongly up-regulated in the thermogenic male-flower part of Alocasia odora , whereas UCP transcripts were negligible. Specifically, we found that in the heat-producing region (the male flowers), AOX expression was significantly upregulated, whereas UCP expression was either absent or negligible. In contrast, the female flowers generated minimal heat, with neither AOX nor UCP exhibiting significant upregulation 41 . Consequently, the inflorescence of A. odora exhibited two distinct thermal microclimates along its vertical axis: a warmer upper thermogenic region (comprising the appendix and male flowers) and a cooler lower non-thermogenic region (including the sterile part and female flowers). In addition to heat production, the inflorescence displays spatial and temporal variations that further enhance its internal thermal heterogeneity. The spatial architecture and phenological progression of thermogenic inflorescences inherently necessitate pollinator mobility. Like other large-flowered thermogenic species (e.g., Amorphophallus titanum or Rafflesia arnoldii ), A. odora exhibits significant physical separation between male and female reproductive organs 42 . This spatial disjunction, spanning 15–20 cm in A. odora , physically constrains autonomous self-pollination and obligates pollen vectors to traverse between floral parts 43 . Concurrently, the temporal dimension of thermogenesis reinforces this requirement. Most thermogenic plants, including A. odora , exhibit protogyny—a sequential maturation where female receptivity precedes male anthesis 44,45 . This dichogamy creates a phenological mismatch that compels pollinators to visit pistillate florets during the initial flowering phase and later relocate to staminate florets. Thus, the spatiotemporal orchestration of thermogenesis not only reflects developmental constraints but also strategically facilitates directed pollinator movement. A Novel Pollination Mechanism Mediated By Temperature-Heterogeneous Microclimates At the diel scale, A. odora amplifies daytime heat but buffers the night-time chill, thereby narrowing overall temperature fluctuation within its inflorescence. Generally, insects have limited thermoregulatory capacity and consequently rely on microhabitat choice to maintain optimal body temperatures 46,47 . The behavioral adaptation is particularly pronounced in small-bodied insects (<2 mm), such as Colocasiomyia flies, whose ectothermic physiology renders them highly sensitive to ambient temperature fluctuations and enables precise navigation toward favorable thermal niches across millimeter-scale distances 48 . Within the confined yet thermally heterogeneous space of the A. odora inflorescence, minute pollinators must relocate to find the most suitable microenvironment for thermoregulation 49 . Specifically, our observations and behavioral experiments show that Colocasiomyia flies prefer temperatures of 20–25°C, consistent with those of other drosophilid flies 50 . The A. odora inflorescence, through thermogenesis, creates dynamic microclimates that fall within the flies’ preferred range (Fig. 2a). At midday, the upper inflorescence heats to 30°C—above the flies’ preferred threshold—driving flies downward to the cooler female part. Artificial manipulations confirmed temperature dominance: cooling the upper part prompted upward migration, while heating the lower part to 30°C caused near-total abandonment (Fig. 2d). Intense midday light also pushed flies downward; however, shading produced only minor upward movement—far less than that induced by temperature (Fig. 2e). Temperature-mediated pollinator repulsion has been reported in cycads, whose uniformly heated male cones expel thrips at midday, thereby enhancing pollen export to female cones 25,51 . However, this cycad mechanism operates stochastically, lacking spatial precision due to homogeneous cone heating that expels pollinators externally rather than directing intrafloral navigation. In contrast, the structurally distinct thermal microclimates within the spadix of A. odora guide heat-repelled pollinators to their preferred cooler regions instead of ejecting them entirely. At night the same thermogenic heat attracts Colocasiomyia flies. When ambient temperature falls below 20°C, the upper chamber stabilizes at 20–25°C, drawing flies back upward. During the transition from the female to the male phase the lower chamber gradually closes as the spathe contracts; flies that remain risk entrapment, yet we seldom observed corpses. Floral odours attract flies only at crepuscular periods, and nocturnal scent concentrations are negligible (Supplementary Fig. 1). Accordingly, we conclude that temperature, not scent, precisely drives the upward movement of flies at night. Thermal gradients dissipate rapidly in air, preventing temperature from serving as a long-range pollinator attractant; instead, it primarily enhances volatile emissions over distance 44 . For instance, mosquitoes rely on olfactory cues for long-range host detection, switching to thermal sensing only at close proximity 52 . In our study, Colocasiomyia flies detected subtle thermal differences between two closely adjacent but thermally distinct chambers . This proximity shortens the signal path and increases the efficacy of temperature as an attractant. Thus, the dual strategy—repelling flies at noon and attracting them at night—promotes the pollination efficiency and maintains a suitable feeding and nursery habitat for the flies. We therefore propose an unrecognised pollination mechanism in which contrasting intrafloral microclimates alternately repel and attract the same pollinators within a single day, ensuring precise pollen transfer while supplying reliable feeding and nursery sites. From Cold-Buffering to Heat-Amplification: Climate-Change Implications The thermogenic capacity of A. odora inflorescences partially buffers temperature fluctuations during cold periods (e.g., nights or winter). However, because these inflorescences generate heat but possess no effective cooling mechanism, they buffer against cold stress but not heat stress 53,54 . Floral thermogenesis may exacerbate ambient heat, particularly when ambient temperatures already exceed the preferred range of Colocasiomyia flies, thereby disrupting temperature-mediated pollination. Consequently, the A. odora pollination system may experience more severe impacts during hot seasons compared with non-thermogenic plant–pollinator systems. To assess the system’s resilience to temperature fluctuations and project future global-warming impacts, we conducted year-round monitoring of climatic and pollination-related variables. Seasonal monitoring revealed a significant negative correlation between temperature and inflorescence number, with no detectable lag effect—monthly temperatures directly influenced inflorescence production within the same month. This finding aligns with A. odora ’s floral development cycle, which requires less than one month from bud differentiation to anthesis 36 . Elevated temperatures typically suppress floral-bud differentiation, favoring vegetative over reproductive growth 55 . Accordingly, summer inflorescence numbers were substantially lower than winter numbers, suggesting that climate warming may exacerbate this disparity and could even eliminate summer inflorescences. While fruit-set rate is primarily determined by pollinator activity, our study also showed declining fruit-set rates with rising temperatures 56 . This pattern implies that reduced inflorescence numbers may indirectly diminish Colocasiomyia populations, further accelerating reproductive failure. Thus, temperature directly disrupts A. odora flowering and fruiting while indirectly impacting its obligate pollinators. Alocasia inflorescences maintain temperatures 5–10°C above ambient. Despite long-term coexistence with thermogenic flowers, neither Colocasiomyia species shows local thermal adaptation 57,58 —as evidenced by significant negative correlations between fly abundance and temperature. Specifically, C. alocasiae (ovipositing in heated male florets) declines more sharply in summer than C. xenalocasiae (in non-thermogenic female parts), indicating that summer temperatures approach its thermal maximum 59 . Consequently, C. alocasiae larvae face severe heat stress in male florets, forcing a thermoregulation-fitness trade-off absent in C. xenalocasiae 46 . This contrast is compounded by host persistence differences: male inflorescences last ~7 days versus female infructescences’ 1–2 months. This extended developmental period buffers C. xenalocasiae against summer population declines: adults emerging during peak heat (July–August) originate from eggs laid during cooler conditions 1–2 months earlier, providing a continuous influx of new adults that mitigates what would otherwise be a severe seasonal low. We predict that global warming will disrupt the Alocasia - Colocasiomyia mutualism: shortened cold seasons diminish the cold-buffering utility of thermogenesis, while intensified summer heat converts male-part microclimates into thermal traps. This trajectory threatens C. alocasiae local extinction, collapsing fruit set and eroding pollination network diversity. Although our one-year dataset cannot fully resolve long-term climate responses, the observed temperature-driven declines indicate an ongoing destabilisation. Crucially, this paradox—where an evolutionarily adaptive microclimate trait amplifies climate vulnerability—reveals a broader risk for niche-constructed symbioses. The core mechanism involves directional niche plasticity, whereby traits like floral thermogenesis effectively buffer historical stressors (e.g., cold fluctuations) but fail to mitigate novel thermal extremes. Three interdependent factors, empirically demonstrated in our system, explain this vulnerability: (1) Active niche construction generating partner-dependent microhabitats, (2) Directional trait plasticity resisting historical stressors but not novel extremes (cold-buffering to heat-dissipation), and (3) Tight specialization tethering mutualists to engineered niches. Such systems risk accelerated degradation as warming converts adaptive traits into demographic liabilities, entangling partners in a co-declining feedback loop. Methods Study Sites and Species This study was conducted in Haikou, Hainan Province, China (19°32′–20°05′ N, 110°10′–110°41′ E), characterized by a tropical monsoon climate with cool, humid winters (November to April) and hot, rainy summers (May to October) 60 . Three populations of Alocasia odora were studied in three parks: Baishamen, Jinniuling, and Fengxiang, each with over 200 plants and spaced over 5 km apart. All experiments except the seasonal data collection were conducted from November to December, aligning with peak flowering. Alocasia odora is a common understory, hermaphroditic perennial herb with protogynous spadices in this region. Each individual plant comprises multiple ramets, each bearing a single inflorescence. The bloom of a single inflorescence lasts approximately 6–7 days. Flowering among ramets of the same individual is asynchronous 36 . As a result, an individual A. odora typically flowers continuously, and it can simultaneously produce both inflorescences and infructescences on different ramets (Fig. 1). This continuous flowering ensures that, within the populations studied, inflorescences remain in bloom throughout the entire year. The inflorescence of A. odora consists of four distinct parts along the spadix. The upper portion comprises the appendix and male (staminate) flowers, while the lower portion contains sterile male flowers and female (pistillate) flowers. The flowering process begins with the opening of the spathe and the maturation of female flowers in the lower part (the female stage). Concurrently, the upper part initiates heat production. After one to three days, during the night, the spathe encloses the lower part, signaling the onset of the male stage. The identification of two Colocasiomyia species—serving as pollinators—was based on established morphological criteria, primarily the examination of costal bristle patterns 61 . Inflorescence Thermogenesis and RNA-seq Temperature and Volatile Organic Compound Measurement We measured the temperatures of the appendix, male flowers, and female flowers of Alocasia odora throughout the entire flowering period of three inflorescence. A four-channel temperature detector (DT-3891G; Shenzhen Everbest Machinery Industry Co., Ltd., Shenzhen, China) was used, with ambient air temperature serving as a control (accuracy ±0.1°C). We recorded temperatures every three minutes, beginning at the female stage and continuing for three days. Simultaneously, volatile organic compounds (VOCs) were quantitatively sampled from the same three inflorescences using dynamic headspace collection. Air samples were drawn at 1 L/min through Porapak Q adsorbent tubes (ORBO-32, Supelco) for 4-hour intervals during critical periods: (1) first-day daytime (12:00–13:00), (2) first-day nighttime (23:00–24:00), and (3) second-day daytime (12:00–13:00). Ambient air samples (>5 m from inflorescences) served as background controls. Adsorbed VOCs were eluted with 200 μL dichloromethane and analyzed byAgilent 7890A GC system (Agilent Technologies, Santa Clara, CA, USA) coupled with 5975C mass spectrometry (MS). Total VOC emission was quantified by summing the peak areas of all detected compounds (60–450 m/z) after background subtraction. Generation of RNA Samples and Data Analysis Fresh tissue samples (approximately 1 cm³) were obtained from the female and male flower parts of three inflorescences at the bud stage and three inflorescences at the female stage, resulting in four groups and a total of 12 samples. Following collection, RNA was extracted, purified, and used to construct sequencing libraries. Paired-end RNA sequencing was then performed using the Illumina sequencing platform (Illumina, CA, USA) at Personal Biotechnology Co., Ltd (Shanghai, China). We used Trinity software to assemble the clean reads into transcripts, followed by de novo analysis without a reference genome. Gene function annotation was performed on the unigenes using databases including NR (NCBI non-redundant protein sequences), GO (Gene Ontology), KEGG (Kyoto Encyclopedia of Genes and Genomes), eggNOG (evolutionary genealogy of genes: Non-supervised Orthologous Groups), Swiss-Prot, and Pfam. Given their known crucial roles in plant thermogenesis, we specifically searched for genes identified as AOX (Alternative Oxidase) and UCP (Uncoupling Protein). Transcript abundance was quantified using RSEM software to calculate the fragments per kilobase of transcript per million fragments mapped (FPKM) values for each gene 62 . Movement Pattern of Colocasiomyia Flies Field Behavior Observation We monitored three simultaneously blooming inflorescences over four consecutive days, counting the number of Colocasiomyia flies on the upper and lower parts at hourly intervals. To facilitate observation, we removed part of the spathe covering the lower inflorescence, allowing flies to access this area even when the female spathe was closed during the male stage. Since the two Colocasiomyia species are indistinguishable during their activity on the inflorescences, we recorded only the total number of flies. Temperature and Light Manipulations in Fields To investigate how temperature and light affect the vertical movement of Colocasiomyia flies within the inflorescence, we manipulated the ambient temperature and light conditions in the field. Between 12:00 p.m. and 2:00 p.m., we cooled the male flower part by placing ice packs around the spathe. To increase the temperature, we manually warmed the spathe using gloved hands. During these manipulations, we monitored the temperatures of the male and female flower parts, the appendix, and the surrounding air. Heating or cooling was ceased once the desired temperature was reached, and each treatment was maintained for approximately 30 minutes. For light manipulation, we covered the inflorescence with a black cardboard box at noon to ensure darkness. In contrast, at night the daylight lamps were used to simulate midday light intensity. Before and after each temperature and light manipulation, we counted the number of flies on the upper and lower parts of the inflorescence to assess changes in their vertical distribution. Seasonal Data Collection Data collection spanned from May 2022 to May 2023 among three populations of A. odora . The observations conducted between 13:00 and 15:00 each time, and was performed one to four times per month in each population. Seasonal Environment We focused on four seasonal environmental variables: temperature, relative humidity, light intensity, and precipitation. Temperature and humidity were measured using a thermo-hygrometer (Model: DECEMTHM01, Zhejiang Delixi Network Technology Co., Ltd.), while light intensity was gauged using a lux meter (Model: SW6013, Guangzhou Shuwei Electronic Technology Co., Ltd.). During each field survey, measurements were systematically collected at five distinct locations within each Alocasia population. These readings were then averaged to create a composite dataset representing the microclimate of each population. Additionally, daily precipitation data for Haikou were obtained from the Haikou Meteorological Bureau website (http://www.hkqx.net/) corresponding to the survey dates. Number of Inflorescences and Fruit Set Rate Forty plants were randomly selected from each population to assess inflorescence flowering status. Inflorescences were classified as flowering if they were in the bud, female, or male stage; otherwise, they were classified as non-flowering. To determine the average number of flowering inflorescences per plant, the total number of flowering inflorescences observed was divided by the number of sampled plants. For example, if 20 flowering inflorescences were recorded among the 40 sampled plants, the average number of flowering inflorescences per plant would be 0.5 or 50%. During each field survey, five inflorescences of A. odora were randomly selected for laboratory observation. Female flowers on each inflorescence were counted to evaluate pollination success. The fruit set rate was calculated as the ratio of the number of developed and swollen female flowers (indicating successful pollination) to the total number of female flowers on each inflorescence. Number of Colocasiomyia Flies For each field survey, approximately 40 inflorescences were randomly selected to assess the abundance of Colocasiomyia flies. Identification of Colocasiomyia species and sexes based solely on morphology is challenging due to the morphological similarity between species and sexes. To address this, approximately 100 flies were randomly captured during each field survey. If individual inflorescences contained fewer than 100 flies, additional flies were captured from three to six other inflorescences to ensure a sufficient sample size while minimizing disturbance to the natural behavior of the flies in the whole population of A. odora . The captured flies were then transported to the laboratory for detailed examination under a stereomicroscope (Model: TS-30H, Beijing Paidiwei Instrument Co., Ltd.). Species and sex were identified based on distinct morphological characteristics. The average species and sex ratios calculated from these samples were then used to estimate the overall population structure within the 40 randomly selected inflorescences. Statistical analyses We analyzed the relationships between environmental variables (temperature, light intensity, relative humidity, and precipitation) and population variables (inflorescence numbers, fruit set rates, and number of Colocasiomyia flies) using pairwise and cross-correlation methods. Cross-correlation analysis allowed us to assess how the effect of one variable on another might change over time, accounting for possible time lags. A lag of zero indicates the strongest correlation within one month, while a lag of one suggests a delayed effect by one month. We established four Generalized Linear Mixed Models (GLMMs) using the Number of Inflorescences, Fruit Set Rate, and Number of C. alocasiae or C. xenalocasiae as dependent variables, with other factors as independent variables and location information as a random effect. Based on the characteristics of each dependent variable, we selected either a Beta regression model or a zero-inflated negative binomial regression model for fitting. Independent variables that did not significantly impact the dependent variables were removed, and we compared the Akaike Information Criterion (AIC) values of the models before and after modification. The simpler model with a lower AIC value was chosen as the final model. All analyses were performed using R version 4.4.1 (R Core Team 2023), and graphs were generated with GraphPad Prism 9.0 (GraphPad Software Inc.). References Dell, A. I., Pawar, S. & Savage, V. M. Systematic variation in the temperature dependence of physiological and ecological traits. 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RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC bioinformatics 12 , 1-16 (2011). Additional Declarations There is NO Competing Interest. Supplementary Files GA.png Graphical abstract . Thermal regulation of Colocasiomyia flies' behavior by A. odora inflorescences and the impact of global warming on their population size. During the female stage (12:00), midday heat "pushes" flies to the lower part of the inflorescence. In the male stage (24:00), nighttime warmth "pulls" flies to the upper, with the female part closed. The projected effect of global warming (red line) on A. odora and Colocasiomyia fly population size across seasons, showing a marked reduction compared to current conditions (black line). SF1.png Supplementary Fig. 1: Dynamic differences in inflorescences scent concentration. Total peak area of inflorescences headspace volatiles in GC-MS at different times. Different letters indicate significant differences at P < 0.05. 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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-7182269","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":499832507,"identity":"4e1e334b-6856-4dbc-95da-5b1a63bf4f8a","order_by":0,"name":"Yibo Luo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAu0lEQVRIiWNgGAWjYBACPiBmZqiQSCBeCxtYyxmStTC2MZCiRSL5mHThPIs8/vbjDxh+1DDImxPWkpYmPXObRLHEmRwDxp5jDIY7GwhqyTGT5t0mkbiBIYeBgbeBIcHgAFFa5gC18D9/wPiXeC0NQC0SCQbMxNnC8yzZesYxoF9uvDE4LHNMwnADIS387MkHbxfU1OXx96c/fPimxkaeoC1AwCIBYwEVS+BRiADMH4hSNgpGwSgYBSMXAACf8jcqiPUsuwAAAABJRU5ErkJggg==","orcid":"","institution":"State Key Laboratory of Plant Diversity and Prominent Crops, Institute of Botany, Chinese Academy of Sciences","correspondingAuthor":true,"prefix":"","firstName":"Yibo","middleName":"","lastName":"Luo","suffix":""},{"id":499832508,"identity":"7142f036-c717-4ed5-b5ae-c8e463638aef","order_by":1,"name":"Yuan-Jun Yu","email":"","orcid":"https://orcid.org/0000-0002-8786-8296","institution":"State Key Laboratory of Plant Diversity and Prominent Crops, Institute of Botany, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yuan-Jun","middleName":"","lastName":"Yu","suffix":""},{"id":499832509,"identity":"de7bd792-1dcc-4014-9f0b-89f703856da7","order_by":2,"name":"Yan Luo","email":"","orcid":"","institution":"State Key Laboratory of Plant Diversity and Prominent Crops, Xishuangbanna Tropi-cal Botanical Garden, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Luo","suffix":""},{"id":499832510,"identity":"5532a920-da1a-4e63-9fdc-5eaa4169fbda","order_by":3,"name":"Wufan Zhang","email":"","orcid":"","institution":"State Key Laboratory of Resource Insects, Institute of Apicultural Research, Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Wufan","middleName":"","lastName":"Zhang","suffix":""},{"id":499832511,"identity":"89a57c9b-ba70-4657-b4f8-03afeda8b34c","order_by":4,"name":"Xiang Ding","email":"","orcid":"","institution":"Key Laboratory of Genetics and Germplasm Innovation of Tropical Special Forest Trees and Ornamental Plants, Ministry of Education, School of Tropical Agriculture and Forestry, Hainan University","correspondingAuthor":false,"prefix":"","firstName":"Xiang","middleName":"","lastName":"Ding","suffix":""},{"id":499832512,"identity":"63ed0246-23b1-45e5-9bb4-2cdca57f857e","order_by":5,"name":"Xiqiang Song","email":"","orcid":"","institution":"Key Laboratory of Genetics and Germplasm Innovation of Tropical Special Forest Trees and Ornamental Plants, Ministry of Education, School of Tropical Agriculture and Forestry, Hainan University","correspondingAuthor":false,"prefix":"","firstName":"Xiqiang","middleName":"","lastName":"Song","suffix":""}],"badges":[],"createdAt":"2025-07-22 04:01:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7182269/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7182269/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89058071,"identity":"e536d740-88c3-414e-b4c2-45b804d3ac1e","added_by":"auto","created_at":"2025-08-14 08:51:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2576198,"visible":true,"origin":"","legend":"\u003cp\u003eContinuous flowering phenology of an \u003cem\u003eAlocasia odora \u003c/em\u003eindividual, illustrating the simultaneous presence of a floral bud (a), an anthesis-stage inflorescence (b), multiple developing infructescences (c), and a senesced fruit (d).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7182269/v1/cf2a6b0cf0a1de37080a5601.png"},{"id":89058070,"identity":"7d8151b5-90f4-4af0-8513-035a95d6b33c","added_by":"auto","created_at":"2025-08-14 08:51:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":472524,"visible":true,"origin":"","legend":"\u003cp\u003eThermogenesis of the \u003cem\u003eA. odora \u003c/em\u003einflorescence and activity of \u003cem\u003eColocasiomyia\u003c/em\u003e flies. (a) The left panel shows changes over time in temperatures of different parts of the spadix and the ambient temperature; the right panel illustrates the corresponding positions on the inflorescence. (b) FPKM expression levels of thermogenesis-related genes in female and male flowers during the bud stage and female (pistillate) flowering stage: F−, female flowers at bud stage; F+, female flowers at female stage; M−, male flowers at bud stage; M+, male flowers at female stage. (c) Distribution of \u003cem\u003eColocasiomyia\u003c/em\u003e flies on the inflorescence at the same times as in (a). Proportions of \u003cem\u003eColocasiomyia\u003c/em\u003e flies on the upper and lower parts of the inflorescence under controlled conditions at different temperatures (d) and light conditions (e). * indicates P \u0026lt; 0.05; ** indicates P \u0026lt; 0.01; ns indicates no significant difference (P \u0026gt; 0.05).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7182269/v1/9622f40aef8e5d17fe858a4e.png"},{"id":89058074,"identity":"03ccd215-dd1e-4711-a946-ed44dc7eb1ab","added_by":"auto","created_at":"2025-08-14 08:51:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":575148,"visible":true,"origin":"","legend":"\u003cp\u003e(a) The lower left displays correlations between environmental and population variables, while the upper right shows cross-correlations between these factors. Black numbers represent correlation values, and white numbers indicate relative lag times in months, * denotes P \u0026lt; 0.05. (b) Seasonal temperature fluctuations alongside flowering and fruit set dynamics of \u003cem\u003eA. odora\u003c/em\u003e populations. (c) Seasonal temperature fluctuations with population dynamics of \u003cem\u003eC. alocasiae\u003c/em\u003e and \u003cem\u003eC. xenalocasiae\u003c/em\u003e on an average of 30 inflorescences.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7182269/v1/85cc924e3e0ede64756b14fd.png"},{"id":89059284,"identity":"7add7618-2ad9-4f50-a2dc-1072df0276a6","added_by":"auto","created_at":"2025-08-14 08:59:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":382549,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Relationship between temperature and the number of inflorescences. Relationship between the number of inflorescences and (b) C. xenalocasiae and (c) C. alocasiae. (d) Relationship between C. xenalocasiae and fruit set rate. Points represent individual survey data, while the fitted lines and shaded areas (95% confidence intervals) show predictions from the GLMM statistical model.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7182269/v1/e8351ed5dd2d2c76f8445bd8.png"},{"id":91730936,"identity":"bc8fb7d5-dce0-4d18-8141-0451559dfc8d","added_by":"auto","created_at":"2025-09-19 16:04:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6942135,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7182269/v1/a212d15c-e05b-40b3-991a-3082e56d81a8.pdf"},{"id":89058073,"identity":"9da1fc5d-4054-4963-85da-51fc90e8684e","added_by":"auto","created_at":"2025-08-14 08:51:59","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":260445,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical abstract . \u003c/strong\u003eThermal regulation of \u003cem\u003eColocasiomyia\u003c/em\u003e flies' behavior by \u003cem\u003eA. odora \u003c/em\u003einflorescences and the impact of global warming on their population size. During the female stage (12:00), midday heat \"pushes\" flies to the lower part of the inflorescence. In the male stage (24:00), nighttime warmth \"pulls\" flies to the upper, with the female part closed. The projected effect of global warming (red line) on \u003cem\u003eA. odora \u003c/em\u003eand \u003cem\u003eColocasiomyia\u003c/em\u003e fly population size across seasons, showing a marked reduction compared to current conditions (black line).\u003c/p\u003e","description":"","filename":"GA.png","url":"https://assets-eu.researchsquare.com/files/rs-7182269/v1/4fd91f057cbadce814eaab59.png"},{"id":89058069,"identity":"3bc9e997-e532-4359-a58f-235accb732c6","added_by":"auto","created_at":"2025-08-14 08:51:59","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":65977,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Fig. 1: \u003c/strong\u003eDynamic differences in inflorescences scent concentration. Total peak area of inflorescences headspace volatiles in GC-MS at different times. Different letters indicate significant differences at P \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"SF1.png","url":"https://assets-eu.researchsquare.com/files/rs-7182269/v1/7792650fb662a11e71097edd.png"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Global warming disrupts a thermogenesis-driven pollination mutualism","fulltext":[{"header":"Main","content":"\u003cp\u003eGlobal warming profoundly affects all levels of biological organization\u0026mdash;from individual growth and developmental rates\u003csup\u003e1\u003c/sup\u003e to population dynamics\u003csup\u003e2\u003c/sup\u003e and community structure\u003csup\u003e3\u003c/sup\u003e. These changes occur across a wide range of spatial scales, from within organisms to entire ecosystems\u003csup\u003e4,5\u003c/sup\u003e. However, the warming effects observed at broad (e.g., regional to global) scales do not necessarily reflect the actual thermal conditions experienced by organisms at micro-scales\u003csup\u003e6\u003c/sup\u003e. For example, flowers\u003csup\u003e7\u003c/sup\u003e, leaf surfaces\u003csup\u003e8\u003c/sup\u003e, and tree canopies\u003csup\u003e9\u003c/sup\u003e often form \u0026quot;microclimates\u0026quot; that differ significantly from the ambient environment, and many critical biotic interactions take place within these microscale habitats\u003csup\u003e10\u003c/sup\u003e. Yet, quantitative research into how broad-scale climate warming alters micro-scale or microclimates and thereby influences species interactions remains scarce\u003csup\u003e11\u003c/sup\u003e. This knowledge gap hampers our ability to accurately assess the ecological consequences of climate change.\u003c/p\u003e\n\u003cp\u003eAmong diverse species interactions, mutualistic relationships\u0026mdash;particularly plant-pollinator relationships\u0026mdash;may be highly sensitive to temperature changes\u003csup\u003e12\u003c/sup\u003e. This sensitivity stems from positive feedback dynamics, a process where an initial change triggers responses that amplify the original effect, creating a self-reinforcing cycle. Classical ecological theory, as proposed by Robert May, suggests that mutualisms are inherently prone to instability owing to these positive feedbacks\u003csup\u003e13\u003c/sup\u003e. This gives rise to a concerning prediction: if global warming leads to reduced floral abundance, pollinators may suffer from resource scarcity and population decline, which in turn further reduces future plant reproductive success through pollination process, initiating a \u0026quot;lose-lose\u0026quot; spiral of mutual collapse\u003csup\u003e14\u003c/sup\u003e. However, this assumes floral temperatures passively track ambient warming\u0026mdash;ignoring flowers\u0026rsquo; active microclimate regulation via reflectance, evaporative cooling, and circadian rhythms\u003csup\u003e15-17\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn fact, some plant lineages have evolved specialized metabolic mechanisms to actively modulate thermal environments\u003csup\u003e18\u003c/sup\u003e. A striking example is floral thermogenesis\u0026mdash;the metabolic production of heat through alternative oxidase (AOX) and uncoupling proteins (UCPs), which elevates inflorescence temperatures by converting respiratory energy into thermal output\u003csup\u003e19-21\u003c/sup\u003e. Floral thermogenesis is an ancient trait that has independently evolved multiple times across at least 14 plant families\u003csup\u003e22,23\u003c/sup\u003e. Notably, thermogenesis is restricted to reproductive organs (e.g., inflorescences), suggesting its direct role in reproduction or pollination processes\u003csup\u003e24\u003c/sup\u003e. Several ecological hypotheses posited that floral heat production enhances plant\u0026ndash;pollinator interactions. It may do so by (1) accelerating the release and diffusion of volatile compounds that attract pollinators\u003csup\u003e25-27\u003c/sup\u003e, (2) providing thermal rewards in cold environments,\u003csup\u003e28,29\u003c/sup\u003e or (3) generating temperature gradients that guide pollinators into the floral structure\u003csup\u003e30\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eCrucially, it is unknown if thermogenesis decouples floral temperatures from ambient warming, and whether such decoupling buffers or amplifies climate impacts on mutualisms. Theoretically, two contrasting outcomes are possible. Firstly, a buffering effect: because thermogenic flowers already maintain elevated temperatures, they may be relatively insensitive to additional warming, thus offering a form of \u0026quot;pre-adaptation\u0026quot; that buffers plant\u0026ndash;pollinator interactions\u003csup\u003e31,32\u003c/sup\u003e. Alternatively, an amplifying effect may occur: prolonged high temperatures could inhibit thermogenic metabolism or induce transpiration-based cooling\u003csup\u003e33\u003c/sup\u003e, thereby reducing floral attractiveness and impairing plant reproductive success. Thermogenic plant\u0026ndash;pollinator systems thus provide a natural experimental model for studying climate change impacts on micro-scale ecosystems, and may serve as early warning indicators for broader ecosystem vulnerabilities through scaling up the temperature effects to the different biological organization levels.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAlocasia odora\u003c/em\u003e, a typical thermogenic species in the Araceae family, exhibits clearly partitioned spadix structures, with thermogenic appendix and male flowers at the upper part and sterile male and female flowers at the lower part\u003csup\u003e34\u003c/sup\u003e. Previous studies indicate unique behavioral patterns among its specialized pollinators, flies of the genus \u003cem\u003eColocasiomyia\u0026nbsp;\u003c/em\u003e(Drosophilidae): \u003cem\u003eColocasiomyia alocasiae\u003c/em\u003e preferentially oviposits in the upper male flower part, while\u003cem\u003e\u0026nbsp;Colocasiomyia xenalocasiae\u0026nbsp;\u003c/em\u003eselects the lower female flower part\u003csup\u003e35\u003c/sup\u003e. Notably, \u003cem\u003eAlocasia odora\u003c/em\u003e primarily depends on these \u003cem\u003eColocasiomyia\u003c/em\u003e flies for pollination despite visits from various insects, forming highly specialized mutualistic interactions\u003csup\u003e36\u003c/sup\u003e. Despite observed spatiotemporal variation in fly aggregation\u003csup\u003e37\u003c/sup\u003e, the roles of thermogenesis and climate variation in mediating these behaviors and mutualism stability are unquantified.\u003c/p\u003e\n\u003cp\u003eIn this study, we investigate a year-round flowering population of \u003cem\u003eAlocasia odora\u003c/em\u003e in the tropics to examine the interactions between climate variation, floral thermogenesis, and pollination mutualisms. Our specific objectives are: (1) Characterize thermogenesis patterns/mechanisms in \u003cem\u003eAlocasia\u003c/em\u003e inflorescences; (2) Clarify the ecological function of thermogenesis in mediating pollinator behavior; (3) Evaluate thermogenic buffering vs. amplification of climate impacts using seasonal variation as a warming proxy. Buffering is defined as maintained pollination success/fitness across temperature gradients; amplification as synergistic declines indicating destabilization.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eAOX-mediated thermogenesis creates vertically partitioned microclimates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTemperature measurements revealed distinct thermogenic patterns across floral parts. During the female stage, the male flowers and appendixes reached a peak temperature of 30\u0026deg;C at noon, significantly higher than the ambient temperature of 25\u0026deg;C. As the ambient temperature decreased after noon, the temperature of the whole flower also declined. At night, the minimum temperature of the male flowers was 22\u0026deg;C, while the ambient temperature dropped to 17\u0026deg;C. In contrast, non-thermogenic female flowers showed minimal temperature elevation (\u0026asymp;1\u0026deg;C above ambient at night; Fig. 2a).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Transcriptomic analysis identified molecular drivers of this thermogenesis. \u003cem\u003eDe novo\u003c/em\u003e assembly yielded 85,898 unigenes (N50=1.85 kb), with homology searches revealing three AOX (\u003cem\u003eAOX1a\u003c/em\u003e, \u003cem\u003eAOX1b\u003c/em\u003e, \u003cem\u003eAOX4\u003c/em\u003e) and three \u003cem\u003eUCP\u003c/em\u003e (\u003cem\u003eUCP1\u003c/em\u003e, \u003cem\u003eUCP5\u003c/em\u003e, \u003cem\u003eUCPB\u003c/em\u003e) homologs (Fig. 2b). Expression profiling demonstrated significant upregulation of \u003cem\u003eAOX1b\u003c/em\u003e in male flowers during the female stage (2626 FPKM vs. \u0026lt;172 FPKM in other tissues; p \u0026lt; 0.001). Other genes (\u003cem\u003eAOX1a\u003c/em\u003e, \u003cem\u003eAOX4\u003c/em\u003e, \u003cem\u003eUCP1\u003c/em\u003e, \u003cem\u003eUCP5\u003c/em\u003e, \u003cem\u003eUCPB\u003c/em\u003e) showed non-significant elevation in thermogenic tissues (p \u0026gt; 0.05).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTemperature-driven push-pull dynamics guide precision pollination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFlies exhibited temperature-dependent relocation within inflorescences. Each inflorescence hosted 10\u0026ndash;30 flies that migrated vertically in response to thermal gradients: (1) At dawn/dusk (\u0026asymp;23\u0026deg;C), flies aggregated on the warmer upper (thermogenic) part (Fig. 2c); (2) At noon (\u0026gt;25\u0026deg;C in upper part), they moved to the cooler lower (non-thermogenic) part; (3) At night (ambient \u0026asymp;17\u0026deg;C), flies returned to the upper part (maintained \u0026gt;20\u0026deg;C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOlfactory cues did not drive this behavior: Total VOC emissions were low during crepuscular/nocturnal periods (0.5 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e peak area; non-attractive) and high at noon (1\u0026ndash;3 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e; \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05), yet flies avoided the upper part during peak scent emission. This confirms temperature\u0026mdash;not scent\u0026mdash;as the primary driver of vertical movement.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe activity of \u003cem\u003eColocasiomyia\u003c/em\u003e flies was significantly influenced by artificial manipulation of inflorescence temperatures. At noon, the male (upper) part of the inflorescence generates heat, causing the flies to primarily reside in the cooler lower (female) part. When we reduced the overall temperature of the inflorescence to nighttime levels (17\u0026ndash;19\u0026deg;C), an average of 51% of the flies moved to the upper part within 30 minutes. Conversely, when we artificially heated the lower part to simulate higher temperatures (over 25\u0026deg;C) while keeping the upper part cool (under 25\u0026deg;C), approximately 85% of the flies departed the warmer lower part and relocated to the cooler upper part (Fig. 2d).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLight conditions influenced fly behavior exclusively during the day. Shading the inflorescence to reduce light intensity caused about 17% of the flies to move from the lower part to the upper part. In contrast, increasing light intensity at night had little effect on fly movement. Light experiments suggests that light intensity alone may not sufficiently influence the flies\u0026apos; vertical movement during the night (Fig. 2e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThermogenesis converts microclimates into thermal traps under warming\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe flowering peak of \u003cem\u003eA. odora\u003c/em\u003e during the low-temperature phase (November to March), with an average of 0.61 \u0026plusmn; 0.11 inflorescences per plant per month (mean \u0026plusmn; SD). In contrast, the high-temperature phase (April to October) exhibited a substantial reduction in inflorescence numbers, averaging 0.30 \u0026plusmn; 0.20 inflorescences per plant per month (Fig. 3b). Similarly, the fruit set rate was notably higher during the low-temperature phase, reaching 89.19 \u0026plusmn; 7.83%, but declined to 67.92 \u0026plusmn; 15.72% during the high-temperature phase.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe abundance of \u003cem\u003eColocasiomyia\u003c/em\u003e flies showed significant seasonal patterns (Fig 3c). During the low-temperature phase, both\u003cem\u003e\u0026nbsp;C. alocasiae\u003c/em\u003e and \u003cem\u003eC. xenalocasiae\u0026nbsp;\u003c/em\u003ewere present in higher numbers. Specifically, \u003cem\u003eC. alocasiae\u003c/em\u003e averaged 688.59 \u0026plusmn; 454.22 individuals based on 40 inflorescences, corresponding to 17.21 \u0026plusmn; 11.36 flies per inflorescence. Similarly, \u003cem\u003eC. xenalocasiae\u003c/em\u003e averaged 827.45 \u0026plusmn; 518.96 individuals, equating to 20.69 \u0026plusmn; 12.97 flies per inflorescence. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the high-temperature phase, the number of\u003cem\u003e\u0026nbsp;C. alocasiae\u0026nbsp;\u003c/em\u003eflies markedly decreased, averaging 52.51 \u0026plusmn; 147.28 individuals or 1.31 \u0026plusmn; 3.68 flies per inflorescence. In some inflorescences, \u003cem\u003eC. alocasiae\u003c/em\u003e was entirely absent. Conversely, \u003cem\u003eC. xenalocasiae\u0026nbsp;\u003c/em\u003emaintained relatively high numbers, averaging 564.62 \u0026plusmn; 463.96 individuals or 14.12 \u0026plusmn; 11.60 flies per inflorescence. Throughout one year, the sex ratios of both \u003cem\u003eColocasiomyia\u003c/em\u003e species remained relatively stable. \u003cem\u003eC. alocasiae\u0026nbsp;\u003c/em\u003ehad an average female-to-male ratio of 1.13 \u0026plusmn; 0.89, while \u003cem\u003eC. xenalocasiae\u0026nbsp;\u003c/em\u003eaveraged 1.10 \u0026plusmn; 0.28.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTemperature was the dominant environmental driver of pollination metrics (Fig. 3a left). Correlations between pollination-related variables (inflorescences, fly numbers, fruit set rate) and the environmental variables humidity, light, and precipitation were low and non-significant (|r| \u0026lt; 0.5, P \u0026gt; 0.05), we focus exclusively on temperature in the following analyses. Temperature was negatively correlated with the number of inflorescences (r=-0.68, P\u0026lt;0.05). The fly number of \u003cem\u003eC. alocasiae\u003c/em\u003e was negatively correlated with temperature (r=-0.56, P\u0026lt;0.05), while that of \u003cem\u003eC. xenalocasiae\u003c/em\u003e was negatively correlated with both light intensity (r=-0.4, P\u0026lt;0.05) and temperature (r=-0.36, P\u0026lt;0.05). The number of inflorescences and fruit set rate were significantly positively correlated with \u003cem\u003eColocasiomyia\u003c/em\u003e fly\u0026rsquo;s numbers (r\u0026gt;0.39, P\u0026lt;0.05).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the cross-correlation analysis (Fig. 3a, right), temperature exhibited a significant negative correlation with the number of inflorescences at a lag of zero months, indicating an immediate effect of temperature on inflorescence numbers. Additionally, temperature was significantly negatively correlated with fruit set rate at a lag of one month. This suggests that the excessive temperature in the current month affects the fruit set rate in the following month. Furthermore, the number of inflorescences was significantly positively correlated with the number of\u003cem\u003e\u0026nbsp;C. alocasiae\u0026nbsp;\u003c/em\u003eat a lag of zero months and with the number of \u003cem\u003eC. xenalocasiae\u0026nbsp;\u003c/em\u003eat a lag of two months. The number of \u003cem\u003eC. alocasiae\u0026nbsp;\u003c/em\u003ewas significantly positively correlated with fruit set rate at a lag of one month, whereas the number of \u003cem\u003eC. xenalocasiae\u003c/em\u003e showed a positive correlation with fruit set rate at a lag of zero months.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGeneralized Linear Mixed Models confirmed temperature as the key stressor. Temperature was the only significant factor affecting inflorescence numbers, exhibiting a negative effect (\u0026beta; = \u0026minus;1.03, P \u0026lt; 0.001). Within the temperature range of 25\u0026deg;C to 35\u0026deg;C, each 1\u0026deg;C increase corresponded to an average decrease of 5.37% in inflorescence numbers (Fig. 4a).Additionally, temperature had a significant negative effect on the abundance of \u003cem\u003eC. alocasiae\u0026nbsp;\u003c/em\u003e(\u0026beta; = \u0026minus;0.38, P \u0026lt; 0.01). In contrast, inflorescence numbers positively affected the abundance of both \u003cem\u003eC. alocasiae\u0026nbsp;\u003c/em\u003e(\u0026beta; = 6.50, P \u0026lt; 0.001; Fig. 4c) and \u003cem\u003eC. xenalocasiae\u003c/em\u003e (\u0026beta; = 2.83, P \u0026lt; 0.001; Fig. 4b). Furthermore, temperature negatively influenced the fruit set rate (\u0026beta; = \u0026minus;0.224, P \u0026lt; 0.001). Conversely, the abundance of\u003cem\u003e\u0026nbsp;C. alocasiae\u0026nbsp;\u003c/em\u003eand \u003cem\u003eC. xenalocasiae\u0026nbsp;\u003c/em\u003eboth positively affected the fruit set rate (P \u0026lt; 0.01; Fig. 4d).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cstrong\u003eAOX-Driven Thermogenesis Creates Spatiotemporally Segregated Microclimates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlant endogenous heat production (thermogenesis) is driven by two alternative respiratory pathways\u0026mdash;AOX and UCP\u0026mdash;that divert electron flow from ATP synthesis to heat\u003csup\u003e38,39\u003c/sup\u003e. Typically, these two pathways do not operate simultaneously in plants. For instance, species like \u003cem\u003eArum maculatum\u003c/em\u003e and \u003cem\u003eNelumbo nucifera\u003c/em\u003e rely primarily on the AOX pathway\u003csup\u003e20,40\u003c/sup\u003e.\u0026nbsp;Our transcriptomic data showed that AOX was strongly up-regulated in the thermogenic male-flower part of \u003cem\u003eAlocasia odora\u003c/em\u003e, whereas UCP transcripts were negligible. Specifically, we found that in the heat-producing region (the male flowers), AOX expression was significantly upregulated, whereas UCP expression was either absent or negligible. In contrast, the female flowers generated minimal heat, with neither AOX nor UCP exhibiting significant upregulation\u003csup\u003e41\u003c/sup\u003e.\u0026nbsp;Consequently, the inflorescence of \u003cem\u003eA. odora\u003c/em\u003e exhibited two distinct thermal microclimates along its vertical axis: a warmer upper thermogenic region (comprising the appendix and male flowers) and a cooler lower non-thermogenic region (including the sterile part and female flowers). In addition to heat production, the inflorescence displays spatial and temporal variations that further enhance its internal thermal heterogeneity.\u003c/p\u003e\n\u003cp\u003eThe spatial architecture and phenological progression of thermogenic inflorescences inherently necessitate pollinator mobility. Like other large-flowered thermogenic species (e.g., \u003cem\u003eAmorphophallus titanum\u003c/em\u003e or \u003cem\u003eRafflesia arnoldii\u003c/em\u003e), \u003cem\u003eA. odora\u003c/em\u003e exhibits significant physical separation between male and female reproductive organs\u003csup\u003e42\u003c/sup\u003e. This spatial disjunction, spanning 15\u0026ndash;20 cm in \u003cem\u003eA. odora\u003c/em\u003e, physically constrains autonomous self-pollination and obligates pollen vectors to traverse between floral parts\u003cstrong\u003e\u003csup\u003e43\u003c/sup\u003e\u003c/strong\u003e. Concurrently, the temporal dimension of thermogenesis reinforces this requirement. Most thermogenic plants, including \u003cem\u003eA. odora\u003c/em\u003e, exhibit protogyny\u0026mdash;a sequential maturation where female receptivity precedes male anthesis\u003csup\u003e44,45\u003c/sup\u003e. This dichogamy creates a phenological mismatch that compels pollinators to visit pistillate florets during the initial flowering phase and later relocate to staminate florets. Thus, the spatiotemporal orchestration of thermogenesis not only reflects developmental constraints but also strategically facilitates directed pollinator movement.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA Novel Pollination Mechanism Mediated By Temperature-Heterogeneous Microclimates\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the diel scale, \u003cem\u003eA. odora\u003c/em\u003e amplifies daytime heat but buffers the night-time chill, thereby narrowing overall temperature fluctuation within its inflorescence. Generally, insects have limited thermoregulatory capacity and consequently rely on microhabitat choice to maintain optimal body temperatures\u003csup\u003e46,47\u003c/sup\u003e. The behavioral adaptation is particularly pronounced in small-bodied insects (\u0026lt;2 mm), such as \u003cem\u003eColocasiomyia\u003c/em\u003e flies, whose ectothermic physiology renders them highly sensitive to ambient temperature fluctuations and enables precise navigation toward favorable thermal niches across millimeter-scale distances\u003csup\u003e48\u003c/sup\u003e. Within the confined yet thermally heterogeneous space of the \u003cem\u003eA. odora\u003c/em\u003e inflorescence, minute pollinators must relocate to find the most suitable microenvironment for thermoregulation\u003csup\u003e49\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eSpecifically, our observations and behavioral experiments show that \u003cem\u003eColocasiomyia\u003c/em\u003e flies prefer temperatures of 20\u0026ndash;25\u0026deg;C, consistent with those of other drosophilid flies\u003csup\u003e50\u003c/sup\u003e. The \u003cem\u003eA. odora\u003c/em\u003e inflorescence, through thermogenesis, creates dynamic microclimates that fall within the flies\u0026rsquo; preferred range (Fig. 2a). At midday, the upper inflorescence heats to 30\u0026deg;C\u0026mdash;above the flies\u0026rsquo; preferred threshold\u0026mdash;driving flies downward to the cooler female part. Artificial manipulations confirmed temperature dominance: cooling the upper part prompted upward migration, while heating the lower part to 30\u0026deg;C caused near-total abandonment (Fig. 2d). Intense midday light also pushed flies downward; however, shading produced only minor upward movement\u0026mdash;far less than that induced by temperature (Fig. 2e). Temperature-mediated pollinator repulsion has been reported in cycads, whose uniformly heated male cones expel thrips at midday, thereby enhancing pollen export to female cones\u003csup\u003e25,51\u003c/sup\u003e. However, this cycad mechanism operates stochastically, lacking spatial precision due to homogeneous cone heating that expels pollinators externally rather than directing intrafloral navigation. In contrast, the structurally distinct thermal microclimates within the spadix of \u003cem\u003eA. odora\u003c/em\u003e guide heat-repelled pollinators to their preferred cooler regions instead of ejecting them entirely.\u003c/p\u003e\n\u003cp\u003eAt night the same thermogenic heat attracts \u003cem\u003eColocasiomyia\u003c/em\u003e flies. When ambient temperature falls below 20\u0026deg;C, the upper chamber stabilizes at 20\u0026ndash;25\u0026deg;C, drawing flies back upward. During the transition from the female to the male phase the lower chamber gradually closes as the spathe contracts; flies that remain risk entrapment, yet we seldom observed corpses. Floral odours attract flies only at crepuscular periods, and nocturnal scent concentrations are negligible (Supplementary Fig. 1). Accordingly, we conclude that temperature, not scent, precisely drives the upward movement of flies at night. Thermal gradients dissipate rapidly in air, preventing temperature from serving as a long-range pollinator attractant; instead, it primarily enhances volatile emissions over distance\u003csup\u003e44\u003c/sup\u003e. For instance, mosquitoes rely on olfactory cues for long-range host detection, switching to thermal sensing only at close proximity\u003csup\u003e52\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn our study, \u003cem\u003eColocasiomyia\u003c/em\u003e flies detected subtle thermal differences between \u003cstrong\u003etwo closely adjacent but thermally distinct\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003echambers\u003cstrong\u003e. \u003cstrong\u003eThis proximity shortens the signal path and increases the efficacy of temperature as an attractant.\u003c/strong\u003e\u0026nbsp;\u003c/strong\u003eThus, the dual strategy\u0026mdash;repelling flies at noon and attracting them at night\u0026mdash;promotes the pollination efficiency and maintains a suitable feeding and nursery habitat for the flies. We therefore propose an unrecognised pollination mechanism in which contrasting intrafloral microclimates alternately repel and attract the same pollinators within a single day, ensuring precise pollen transfer while supplying reliable feeding and nursery sites.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFrom Cold-Buffering to Heat-Amplification: Climate-Change Implications\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe thermogenic capacity of \u003cem\u003eA. odora\u003c/em\u003e inflorescences partially buffers temperature fluctuations during cold periods (e.g., nights or winter). However, because these inflorescences generate heat but possess no effective cooling mechanism, they buffer against cold stress but not heat stress\u003csup\u003e53,54\u003c/sup\u003e. Floral thermogenesis may exacerbate ambient heat, particularly when ambient temperatures already exceed the preferred range of \u003cem\u003eColocasiomyia\u003c/em\u003e flies, thereby disrupting temperature-mediated pollination. Consequently, the \u003cem\u003eA. odora\u003c/em\u003e pollination system may experience more severe impacts during hot seasons compared with non-thermogenic plant\u0026ndash;pollinator systems. To assess the system\u0026rsquo;s resilience to temperature fluctuations and project future global-warming impacts, we conducted year-round monitoring of climatic and pollination-related variables.\u003c/p\u003e\n\u003cp\u003eSeasonal monitoring revealed a significant negative correlation between temperature and inflorescence number, with no detectable lag effect\u0026mdash;monthly temperatures directly influenced inflorescence production within the same month. This finding aligns with \u003cem\u003eA. odora\u003c/em\u003e\u0026rsquo;s floral development cycle, which requires less than one month from bud differentiation to anthesis\u003csup\u003e36\u003c/sup\u003e. Elevated temperatures typically suppress floral-bud differentiation, favoring vegetative over reproductive growth\u003csup\u003e55\u003c/sup\u003e. Accordingly, summer inflorescence numbers were substantially lower than winter numbers, suggesting that climate warming may exacerbate this disparity and could even eliminate summer inflorescences. While fruit-set rate is primarily determined by pollinator activity, our study also showed declining fruit-set rates with rising temperatures\u003csup\u003e56\u003c/sup\u003e. This pattern implies that reduced inflorescence numbers may indirectly diminish \u003cem\u003eColocasiomyia\u003c/em\u003e populations, further accelerating reproductive failure. Thus, temperature directly disrupts \u003cem\u003eA. odora\u003c/em\u003e flowering and fruiting while indirectly impacting its obligate pollinators.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAlocasia inflorescences maintain temperatures 5\u0026ndash;10\u0026deg;C above ambient. Despite long-term coexistence with thermogenic flowers, neither Colocasiomyia species shows local thermal adaptation\u003c/em\u003e\u003csup\u003e57,58\u003c/sup\u003e\u0026mdash;as evidenced by significant negative correlations between fly abundance and temperature. Specifically,\u003cem\u003e\u0026nbsp;C. alocasiae\u0026nbsp;\u003c/em\u003e(ovipositing in heated male florets) declines more sharply in summer than\u003cem\u003e\u0026nbsp;C. xenalocasiae\u0026nbsp;\u003c/em\u003e(in non-thermogenic female parts), indicating that summer temperatures approach its thermal maximum\u003csup\u003e59\u003c/sup\u003e. Consequently, \u003cem\u003eC. alocasiae\u003c/em\u003e larvae face severe heat stress in male florets, forcing a thermoregulation-fitness trade-off absent in \u003cem\u003eC. xenalocasiae\u003c/em\u003e\u003csup\u003e46\u003c/sup\u003e. This contrast is compounded by host persistence differences: male inflorescences last ~7 days versus female infructescences\u0026rsquo; 1\u0026ndash;2 months. This extended developmental period buffers \u003cem\u003eC. xenalocasiae\u003c/em\u003e against summer population declines: adults emerging during peak heat (July\u0026ndash;August) originate from eggs laid during cooler conditions 1\u0026ndash;2 months earlier, providing a continuous influx of new adults that mitigates what would otherwise be a severe seasonal low.\u003c/p\u003e\n\u003cp\u003eWe predict that global warming will disrupt the \u003cem\u003eAlocasia\u003c/em\u003e-\u003cem\u003eColocasiomyia\u003c/em\u003e mutualism: shortened cold seasons diminish\u0026nbsp;the cold-buffering utility of thermogenesis,\u0026nbsp;while intensified summer heat converts male-part microclimates into thermal traps. This trajectory threatens \u003cem\u003eC. alocasiae\u0026nbsp;\u003c/em\u003elocal extinction, collapsing fruit set and eroding pollination network diversity. Although our one-year dataset cannot fully resolve long-term climate responses, the observed temperature-driven declines indicate an ongoing destabilisation. Crucially, this paradox\u0026mdash;where an evolutionarily adaptive microclimate trait amplifies climate vulnerability\u0026mdash;reveals a broader risk for niche-constructed symbioses. The core mechanism involves directional niche plasticity, whereby traits like floral thermogenesis effectively buffer historical stressors (e.g., cold fluctuations) but fail to mitigate novel thermal extremes. Three interdependent factors, empirically demonstrated in our system, explain this vulnerability: (1) Active niche construction generating partner-dependent microhabitats, (2) Directional trait plasticity resisting historical stressors but not novel extremes (cold-buffering to heat-dissipation), and (3) Tight specialization tethering mutualists to engineered niches.\u0026nbsp;Such systems risk accelerated degradation as warming converts adaptive traits into demographic liabilities, entangling partners in a co-declining feedback loop.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eStudy Sites and Species\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted in Haikou, Hainan Province, China (19\u0026deg;32\u0026prime;\u0026ndash;20\u0026deg;05\u0026prime; N, 110\u0026deg;10\u0026prime;\u0026ndash;110\u0026deg;41\u0026prime; E), characterized by a tropical monsoon climate with cool, humid winters (November to April) and hot, rainy summers (May to October)\u003csup\u003e60\u003c/sup\u003e. Three populations of \u003cem\u003eAlocasia odora\u003c/em\u003e were studied in three parks: Baishamen, Jinniuling, and Fengxiang, each with over 200 plants and spaced over 5 km apart. All experiments except the seasonal data collection were conducted from November to December, aligning with peak flowering.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAlocasia odora\u003c/em\u003e is a common understory, hermaphroditic perennial herb with protogynous spadices in this region. Each individual plant comprises multiple ramets, each bearing a single inflorescence. The bloom of a single inflorescence lasts approximately 6\u0026ndash;7 days. Flowering among ramets of the same individual is asynchronous\u003csup\u003e36\u003c/sup\u003e. As a result, an individual \u003cem\u003eA. odora\u003c/em\u003e typically flowers continuously, and it can simultaneously produce both inflorescences and infructescences on different ramets (Fig. 1). This continuous flowering ensures that, within the populations studied, inflorescences remain in bloom throughout the entire year. The inflorescence of \u003cem\u003eA. odora\u003c/em\u003e consists of four distinct parts along the spadix. The upper portion comprises the appendix and male (staminate) flowers, while the lower portion contains sterile male flowers and female (pistillate) flowers. The flowering process begins with the opening of the spathe and the maturation of female flowers in the lower part (the female stage). Concurrently, the upper part initiates heat production. After one to three days, during the night, the spathe encloses the lower part, signaling the onset of the male stage. The identification of two \u003cem\u003eColocasiomyia \u003c/em\u003especies\u0026mdash;serving as pollinators\u0026mdash;was based on established morphological criteria, primarily the examination of costal bristle patterns\u003csup\u003e61\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInflorescence Thermogenesis and RNA-seq\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTemperature and Volatile Organic Compound Measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe measured the temperatures of the appendix, male flowers, and female flowers of \u003cem\u003eAlocasia odora \u003c/em\u003ethroughout the entire flowering period of three inflorescence. A four-channel temperature detector (DT-3891G; Shenzhen Everbest Machinery Industry Co., Ltd., Shenzhen, China) was used, with ambient air temperature serving as a control (accuracy \u0026plusmn;0.1\u0026deg;C). We recorded temperatures every three minutes, beginning at the female stage and continuing for three days.\u003c/p\u003e\n\u003cp\u003eSimultaneously, volatile organic compounds (VOCs) were quantitatively sampled from the same three inflorescences using dynamic headspace collection. Air samples were drawn at 1 L/min through Porapak Q adsorbent tubes (ORBO-32, Supelco) for 4-hour intervals during critical periods: (1) first-day daytime (12:00\u0026ndash;13:00), (2) first-day nighttime (23:00\u0026ndash;24:00), and (3) second-day daytime (12:00\u0026ndash;13:00). Ambient air samples (\u0026gt;5 m from inflorescences) served as background controls. Adsorbed VOCs were eluted with 200 \u0026mu;L dichloromethane and analyzed byAgilent 7890A GC system (Agilent Technologies, Santa Clara, CA, USA) coupled with 5975C mass spectrometry (MS). Total VOC emission was quantified by summing the peak areas of all detected compounds (60\u0026ndash;450 m/z) after background subtraction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneration of RNA Samples and Data Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFresh tissue samples (approximately 1 cm\u0026sup3;) were obtained from the female and male flower parts of three inflorescences at the bud stage and three inflorescences at the female stage, resulting in four groups and a total of 12 samples. Following collection, RNA was extracted, purified, and used to construct sequencing libraries. Paired-end RNA sequencing was then performed using the Illumina sequencing platform (Illumina, CA, USA) at Personal Biotechnology Co., Ltd (Shanghai, China).\u003c/p\u003e\n\u003cp\u003eWe used Trinity software to assemble the clean reads into transcripts, followed by de novo analysis without a reference genome. Gene function annotation was performed on the unigenes using databases including NR (NCBI non-redundant protein sequences), GO (Gene Ontology), KEGG (Kyoto Encyclopedia of Genes and Genomes), eggNOG (evolutionary genealogy of genes: Non-supervised Orthologous Groups), Swiss-Prot, and Pfam. Given their known crucial roles in plant thermogenesis, we specifically searched for genes identified as AOX (Alternative Oxidase) and UCP (Uncoupling Protein). Transcript abundance was quantified using RSEM software to calculate the fragments per kilobase of transcript per million fragments mapped (FPKM) values for each gene\u003csup\u003e62\u003c/sup\u003e. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMovement Pattern of \u003cem\u003eColocasiomyia \u003c/em\u003eFlies \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eField Behavior Observation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe monitored three simultaneously blooming inflorescences over four consecutive days, counting the number of \u003cem\u003eColocasiomyia\u003c/em\u003e flies on the upper and lower parts at hourly intervals. To facilitate observation, we removed part of the spathe covering the lower inflorescence, allowing flies to access this area even when the female spathe was closed during the male stage. Since the two \u003cem\u003eColocasiomyia\u003c/em\u003e species are indistinguishable during their activity on the inflorescences, we recorded only the total number of flies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTemperature and Light Manipulations in Fields\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate how temperature and light affect the vertical movement of \u003cem\u003eColocasiomyia\u003c/em\u003e flies within the inflorescence, we manipulated the ambient temperature and light conditions in the field. Between 12:00 p.m. and 2:00 p.m., we cooled the male flower part by placing ice packs around the spathe. To increase the temperature, we manually warmed the spathe using gloved hands. During these manipulations, we monitored the temperatures of the male and female flower parts, the appendix, and the surrounding air. Heating or cooling was ceased once the desired temperature was reached, and each treatment was maintained for approximately 30 minutes.\u003c/p\u003e\n\u003cp\u003eFor light manipulation, we covered the inflorescence with a black cardboard box at noon to ensure darkness. In contrast, at night the daylight lamps were used to simulate midday light intensity. Before and after each temperature and light manipulation, we counted the number of flies on the upper and lower parts of the inflorescence to assess changes in their vertical distribution.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSeasonal Data Collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData collection spanned from May 2022 to May 2023 among three populations of \u003cem\u003eA. odora\u003c/em\u003e. The observations conducted between 13:00 and 15:00 each time, and was performed one to four times per month in each population. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSeasonal Environment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe focused on four seasonal environmental variables: temperature, relative humidity, light intensity, and precipitation. Temperature and humidity were measured using a thermo-hygrometer (Model: DECEMTHM01, Zhejiang Delixi Network Technology Co., Ltd.), while light intensity was gauged using a lux meter (Model: SW6013, Guangzhou Shuwei Electronic Technology Co., Ltd.). During each field survey, measurements were systematically collected at five distinct locations within each \u003cem\u003eAlocasia\u003c/em\u003e population. These readings were then averaged to create a composite dataset representing the microclimate of each population. Additionally, daily precipitation data for Haikou were obtained from the Haikou Meteorological Bureau website (http://www.hkqx.net/) corresponding to the survey dates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNumber of Inflorescences and Fruit Set Rate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eForty plants were randomly selected from each population to assess inflorescence flowering status. Inflorescences were classified as flowering if they were in the bud, female, or male stage; otherwise, they were classified as non-flowering. To determine the average number of flowering inflorescences per plant, the total number of flowering inflorescences observed was divided by the number of sampled plants. For example, if 20 flowering inflorescences were recorded among the 40 sampled plants, the average number of flowering inflorescences per plant would be 0.5 or 50%.\u003c/p\u003e\n\u003cp\u003eDuring each field survey, five inflorescences of \u003cem\u003eA. odora \u003c/em\u003ewere randomly selected for laboratory observation. Female flowers on each inflorescence were counted to evaluate pollination success. The fruit set rate was calculated as the ratio of the number of developed and swollen female flowers (indicating successful pollination) to the total number of female flowers on each inflorescence.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNumber of \u003cem\u003eColocasiomyia\u003c/em\u003e Flies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor each field survey, approximately 40 inflorescences were randomly selected to assess the abundance of \u003cem\u003eColocasiomyia\u003c/em\u003e flies. Identification of \u003cem\u003eColocasiomyia\u003c/em\u003e species and sexes based solely on morphology is challenging due to the morphological similarity between species and sexes. To address this, approximately 100 flies were randomly captured during each field survey. If individual inflorescences contained fewer than 100 flies, additional flies were captured from three to six other inflorescences to ensure a sufficient sample size while minimizing disturbance to the natural behavior of the flies in the whole population of \u003cem\u003eA. odora\u003c/em\u003e. The captured flies were then transported to the laboratory for detailed examination under a stereomicroscope (Model: TS-30H, Beijing Paidiwei Instrument Co., Ltd.). Species and sex were identified based on distinct morphological characteristics. The average species and sex ratios calculated from these samples were then used to estimate the overall population structure within the 40 randomly selected inflorescences.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe analyzed the relationships between environmental variables (temperature, light intensity, relative humidity, and precipitation) and population variables (inflorescence numbers, fruit set rates, and number of \u003cem\u003eColocasiomyia \u003c/em\u003eflies) using pairwise and cross-correlation methods. Cross-correlation analysis allowed us to assess how the effect of one variable on another might change over time, accounting for possible time lags. A lag of zero indicates the strongest correlation within one month, while a lag of one suggests a delayed effect by one month. \u003c/p\u003e\n\u003cp\u003eWe established four Generalized Linear Mixed Models (GLMMs) using the Number of Inflorescences, Fruit Set Rate, and Number of \u003cem\u003eC. alocasiae\u003c/em\u003e or \u003cem\u003eC. xenalocasiae\u003c/em\u003e as dependent variables, with other factors as independent variables and location information as a random effect. Based on the characteristics of each dependent variable, we selected either a Beta regression model or a zero-inflated negative binomial regression model for fitting. Independent variables that did not significantly impact the dependent variables were removed, and we compared the Akaike Information Criterion (AIC) values of the models before and after modification. 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RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. \u003cem\u003eBMC bioinformatics\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 1-16 (2011).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Thermogenesis, Microclimate, Pollination Mutualism, Climate Change, Araceae, Alternative Oxidase","lastPublishedDoi":"10.21203/rs.3.rs-7182269/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7182269/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Global warming threatens plant-pollinator mutualisms, but how microclimates modu-late this impact remains poorly understood. Using the thermogenic Alocasia odora and its specialized Colocasiomyia pollinators, we show floral thermogenesis—driven by male floral alternative oxidase (AOX) upregulation—creates vertically partitioned thermal microclimates. Field observations and manipulative experiments revealed that these thermal gradients guide flies via push-pull dynamics: flies are pushed toward female flowers for daytime pollination while pulled toward warm male parts for nighttime thermoregulation. Crucially, warming amplifies system vulnerability: inflo-rescence production declines 5.4% per 1°C increase, fruit set drops from 89% (cool seasons) to 68% (hot seasons), but pollinator declines are species-specific (C. alocasiae: 92.4% summer loss vs. C. xenalocasiae: 31.9%). While thermogenesis en-ables precision pollination, it converts microclimates into thermal traps under warm-ing due to directional niche plasticity, traits that buffered cold stress in evolutionary history cannot mitigate novel heat extremes in future.","manuscriptTitle":"Global warming disrupts a thermogenesis-driven pollination mutualism","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-14 08:51:55","doi":"10.21203/rs.3.rs-7182269/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":"d56731dd-c5b6-4a79-89e4-0d11a2db17d7","owner":[],"postedDate":"August 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":53091274,"name":"Biological sciences/Ecology/Behavioural ecology"},{"id":53091275,"name":"Biological sciences/Ecology/Climate-change ecology"},{"id":53091276,"name":"Biological sciences/Ecology/Community ecology"},{"id":53091277,"name":"Biological sciences/Ecology/Ecological networks"},{"id":53091278,"name":"Biological sciences/Ecology/Conservation biology"}],"tags":[],"updatedAt":"2025-09-19T15:56:14+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-14 08:51:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7182269","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7182269","identity":"rs-7182269","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}