Integrated Multi-Trait Analysis of Photosynthetic Carbon Assimilation Pathways and Adaptive Patterns in Dendrobium Species

Integrated Multi-Trait Analysis of Photosynthetic Carbon Assimilation Pathways and Adaptive Patterns in Dendrobium Species | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Integrated Multi-Trait Analysis of Photosynthetic Carbon Assimilation Pathways and Adaptive Patterns in Dendrobium Species Zhe Yang, Lingzhi Wei, Lihui Peng, Zhenhai Deng, Yujing Wei, Zhongchen Xiong, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8304845/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract To analyze the diversity of photosynthetic carbon assimilation pathways and ecological adaptation characteristics in Dendrobium species, this study selected nine native Chinese Dendrobium species. A comprehensive approach was employed, integrating diurnal dynamics of net CO 2 exchange, carbon isotope ratios (δ 13 C), diurnal titratable acid accumulation (ΔH + ), leaf anatomical structure, and mesophyll succulent index (Sm). The results indicate: that Dendrobium parishii , Dendrobium aphyllum , and Dendrobium anosmum maintain positive net CO 2 exchange at night, with δ 13 C values exceeding − 16‰ and high nocturnal acid accumulation. Combined with their substantial leaf thickness (LT) and Sm, these species are classified as typical CAM plants; Dendrobium cariniferum , Dendrobium gibsonii , and Dendrobium hancockii exhibited no positive CO 2 exchange throughout the night, with δ 13 C values below − 26‰ and low ΔH + levels, demonstrating physiological characteristics consistent with the C 3 pathway; Additionally, Dendrobium linawianum , Dendrobium moschatum , and Dendrobium crystallinum exhibited weak nocturnal CO 2 exchange and moderate to low acid accumulation. However, their δ 13 C values remained within the C 3 range, indicating they are not stable CAM types but may exhibit plastic expression under specific environmental conditions, displaying characteristics of intermediate C 3 /CAM photosynthetic plants. The LT and SM exhibited partial overlap between C 3 /CAM and strictly C 3 species, highlighting the limitations of using morphological traits as sole diagnostic criteria. Correlation analyses further revealed that although LT was significantly associated with Nighttime Net CO 2 Exchange (NNEE) and δ 13 C, the Sm displayed only weak correlations with key physiological indicators. In contrast, NNEE, δ 13 C, and ΔH + were strongly aligned with one another, underscoring that structural characteristics can serve only as supplementary references, while reliable classification must depend on the coordinated variation of physiological metrics. Furthermore, the photosynthetic types of the examined Dendrobium species closely corresponded to moisture availability and light conditions in their native habitats, reflecting the ecological plasticity of their carbon-assimilation strategies. In summary, Dendrobium species exhibit remarkable diversity and environmental plasticity in their photosynthetic carbon assimilation strategies. This study, through multi-indicator integrated assessment, not only clarifies the photosynthetic types of different species but also provides important references for identifying photosynthetic carbon assimilation pathways and cultivating Dendrobium species. Dendrobium species carbon assimilation pathways crassulacean acid metabolism ecological adaptability leaf anatomy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Photosynthesis is the fundamental metabolic process through which plants convert light energy into chemical energy (Blankenship 2021 ; Nelson and Yocum 2006 ). Throughout evolution, plants have developed several distinct pathways for photosynthetic carbon assimilation, including the C 3 , C 4 and crassulacean acid metabolism (CAM) pathways, to adapt to diverse ecological environments (Ehleringer and Monson 1993 ; Sage 2004 ). These pathways differ significantly in their in carbon-fixation efficiency, water-use characteristics and ecological adaptability. The C 3 pathway, being the most ancient and widespread form and predominates in temperate and humid regions; however, C 3 plants experience pronounced photorespiration under high temperatures, drought, and intense light. In contrast, C 4 pathway evolved as an adaptation to such stress conditions. This pathway relies on Kranz anatomy, a specialized leaf structure and a spatial CO 2 -concentrating mechanism that suppresses photorespiration, allowing plants to maintain efficient carbon fixation in hot and high-light habitats (Sage and Zhu 2011 ). CAM represents another specialized adaptation that shifts CO 2 exchange to the night, storing it as organic acids. By closing their stomata during the day and relying on nocturnally accumulated malate for CO 2 release, CAM plants minimize water loss and can survive in extremely arid environments (Winter and Smith 1996 ; Borland et al. 2009 ). Importantly, the C 3 , C 4 and CAM pathways are not entirely isolated from one another. C 3 /C 4 intermediate species are present in several plant groups. These intermediate forms reflect the intrinsic plasticity of photosynthetic carbon assimilation and provide valuable model systems for investigating how plants adjust their carbon-fixation strategies in response to environmental variations. The long-term evolution and distribution of these pathways have been influenced by changes in atmospheric CO 2 concentration, temperature and water availability. Since the Cenozoic era, global aridification, coupled with declining atmospheric CO 2 levels is believed to have promoted multiple independent origins of both C 4 and CAM photosynthesis (Sage et al. 2011 ; Winter and Holtum 2014 ). The interplay of these biochemical mechanisms, along with their varying modes of spatio-temporal CO 2 concentration, has resulted in a diverse array of adaptive carbon assimilation strategies. This diversity forms the theoretical foundation for understanding the ecological drivers of photosynthetic evolution. In the crassulacean acid metabolism (CAM) pathway, plants exhibit distinctive adaptive advantages. Studies exhibit that the water-use efficiency of typical CAM species can be up to six times greater than that of C 3 plants and three times greater than that of C 4 plants (Nobel 1996 ). This highly efficient water-saving strategy enables CAM plants to maintain a competitive edge in arid and semi-arid environments. Additionally, CAM species demonstrate significant tolerance to intense light and high temperatures, which collectively define their ecological roles in dry and semi-dry ecosystems (Silvera et al. 2010 ). CAM is characterized by a unique CO 2 fixation mechanism and operates through four distinct phases. During Phase I, which occurs at night, stomata open and CO 2 is fixed by phosphoenolpyruvate carboxylase to form malate, which is stored in vacuoles. Phase II occurs around dawn, when stomata are partially open, allowing both C 3 and C 4 carbon fixation to occur simultaneously. Phase III takes place during the day, when stomata remain closed and malate is decarboxylated, releasing CO 2 for the Calvin cycle through the action of Rubisco. Finally, Phase IV occurs in the late afternoon, when stomata reopen briefly and CO 2 is fixed directly via the C 3 pathway (Osmond 1978 ; Ting 1985 ). CAM expression can be further categorized into obligate CAM, which is constitutively expressed throughout the plant’s life cycle, facultative CAM, which is induced under drought or other stress conditions, CAM cycling, which involves nocturnal acid accumulation without net CO 2 exchange, and CAM idling, which occurs under extreme drought when stomata remain nearly completely closed and carbon recycling is maintained internally (Winter and Holtum 2014 ). This diversity reflects the high degree of plasticity with which plants adapt to their environment. Research on CAM and the broader spectrum of carbon assimilation pathways is therefore essential for understanding plant stress physiology and offers new strategies for crop genetic improvement. Insights into the water-saving and heat-tolerant traits of CAM provide a foundation for breeding drought-resistant and water-efficient crops. Moreover, the carbon-fixation characteristics of CAM plants are enhancing for refining global carbon-cycle models and improving predictions of ecosystem function in the context of climate change. Dendrobium is one of the largest genera within the Orchidaceae family, encompassing over 1,500 species that are widely distributed across tropical and subtropical regions of Asia (Hou et al. 2017 ). Species within this genus hold significant ecological and economic value. Many Dendrobium species are integral components of forest ecosystems and serve as important medicinal and horticultural resources. Their survival status and physiological adaptability are directly related to biodiversity conservation and sustainable utilization (Cheng et al. 2019 ). Recently, the diversity of photosynthetic carbon assimilation pathways and their environmental regulation have emerged as a major focus in plant physiological ecology (Sage et al. 2012 ). Prior studies have demonstrated that the Orchidaceae family exhibits a wide range of photosynthetic types, spanning from typical C 3 species to strong CAM species (Silvera et al. 2010 ). Evidence of plastic carbon assimilation in Dendrobium suggests that some species may modify their carbon-acquisition strategies in response to abiotic stress (Silvera et al. 2009 ; Zhang et al. 2020 ). This physiological plasticity may represent one of the key mechanisms that enable Dendrobium species to thrive in diverse habitats. Given the extensive distribution and varied ecological contexts of the genus, it is probable that a rich variety of photosynthetic types exists, particularly within the transitional range between CAM and C 3 pathways. Nonetheless, systematic research on photosynthetic carbon assimilation pathways in Dendrobium remains limited. Most existing studies concentrate on a few economically significant species, and comprehensive comparisons that integrate multiple indicators are still lacking. To address these issues, this study investigates nine native Chinese Dendrobium species, including D. parishii , D. aphyllum , D. ellipsophyllum , D. cariniferum , D. gibsonii , D. hancockii , D. linawianum , D. moschatum and D. crystallinum. The photosynthetic carbon assimilation pathways of these species have not been previously examined. The innovation of this study lies in the application of a comprehensive analytical framework that integrates five categories of indicators: net CO 2 exchange, δ 13 C values, titratable acid accumulation (ΔH + ), leaf anatomical traits and the mesophyll succulent index. This framework facilitates a systematic assessment of C 3 , CAM and intermediate C 3/ CAM photosynthetic types, revealing potential photosynthetic plasticity. Measurements of net CO 2 exchange enable the characterization of diel gas-exchange patterns (van Tongerlo et al. 2021 ). Changes in titratable acidity serve as an important indicator of carbon metabolism and reflect the magnitude of nocturnal CO 2 fixation and daytime decarboxylation (Cushman and Borland 2002 ). Carbon isotope ratios distinguish among C 3 , C 4 , and CAM pathways, providing long-term information about water-use efficiency (Farquhar et al. 1982 ). Leaf anatomical traits are closely linked to photosynthetic pathways; for instance, C 4 plants typically possess Kranz anatomy, while CAM plants often exhibit thick leaves with well-developed water-storage tissue (Orsenigo et al. 1997 ). The mesophyll succulent index offers a quantitative measure of xeromorphic structure, reflecting the degree of tissue water storage. Higher succulence is commonly associated with typical CAM species and other plants adapted to arid habitats (Herrera 2009 ). By integrating physiological, biochemical and anatomical indicators, this study aims to accurately classify the photosynthetic pathways of the nine species and identify potential intermediate types between C 3 and CAM. The findings provide insights into the environmental adaptation mechanisms of Dendrobium , offering a scientific basis for taxonomy, conservation and cultivation management. This is particularly pertinent in the context of climate change and the sustainable utilization of germplasm resources. Methods Study site and plant materials The experiment was conducted in 2023 at the Guangxi Institute of Botany, located in the Yanshan District of Guilin City, Guangxi, China (25°01′ N, 110°17′ E). The study site is situated at an elevation of approximately 180 m and is characterized by a subtropical monsoon climate. The region experiences a mean annual temperature of 18.8°C, with January being the coldest month, averaging 8.3°C, and July being the hottest month, averaging 28.3°C. Annual precipitation ranges from 1,900 to 2,000 mm, with over 70% occurring between April and August. The mean annual relative humidity is 78%, and the annual sunshine duration is approximately 1,500 h. Nine Dendrobium species were introduced from Guangxi and Yunnan and cultivated in the germplasm nursery of the Guangxi Institute of Botany. The species included D. parishii , D. aphyllum , D. anosmum , D. cariniferum , D. gibsonii , D. hancockii , D. linawianum , D. moschatum , and D. crystallinum , as identified by Zhongchen Xiong of the Guangxi Institute of Botany. Detailed information regarding the plant materials is provided in Table 1 . The plants were grown in plastic pots (height: 18 cm; inner diameter: 16 cm) filled with a substrate composed of tree bark, coconut fiber, and perlite in a ratio of 1:1:1. All test plants were three-year-old mature individuals maintained under mild drought stress. Shade nets with a transmittance of 30% were installed on the greenhouse roof, allowing the plants to grow under natural light conditions.Net CO 2 exchange measurements were conducted in early October 2023, and leaf samples were subsequently collected for analyses of carbon isotope ratios (δ 13 C), titratable acidity (ΔH + ), leaf anatomical structure, and mesophyll succulent index. Table 1 Experimental materials Species Determiner Determining Institution Site of Introduction D. parishii Zhongchen Xiong Guangxi Institute of Botany Baoshan, Yunnan D. aphyllum Zhongchen Xiong Guangxi Institute of Botany Pu’er, Yunnan D. anosmum Zhongchen Xiong Guangxi Institute of Botany Longzhou, Guangxi D. cariniferum Zhongchen Xiong Guangxi Institute of Botany Tian’e, Guangxi D. gibsonii Zhongchen Xiong Guangxi Institute of Botany Baoshan, Yunnan D. hancockii Zhongchen Xiong Guangxi Institute of Botany Baise, Guangxi D. linawianum Zhongchen Xiong Guangxi Institute of Botany Jinxiu, Guangxi D. moschatum Zhongchen Xiong Guangxi Institute of Botany Jinghong, Yunnan D. crystallinum Zhongchen Xiong Guangxi Institute of Botany Tian’e, Guangxi Measurement of diurnal patterns of net CO 2 exchange Diurnal net CO 2 exchange was measured from 08:00 on October 7 to 06:00 on October 8, 2023. Healthy, fully expanded leaves located at the 3rd to 5th position from the shoot apex were selected for measurement. A portable photosynthesis system (LI-6400XT; LI-COR Biosciences, USA) was utilized under ambient CO 2 concentrations and natural light conditions. Measurements were conducted at 2-h intervals over a 24-h cycle, with each leaf measured three times to obtain a mean value. For each species, three individual plants were assessed. According to Beijing local time, sunrise and sunset on October 7 occurred at 06:33:15 and 18:20:17, respectively; on October 8, they were recorded at 06:33:41 and 18:19:17. Photosynthetically active radiation (PAR), air temperature ( T a ), and relative humidity (RH) were recorded simultaneously (Fig. 1 ). Measurement of carbon isotope ratio Carbon isotope ratios (δ 13 C) were determined according to the methodology established by Winter and Holtum ( 2002 ). Leaf samples were collected under mild drought stress, then ground in liquid nitrogen, freeze-dried, and weighed to a precision of 3 mg. The ratios of 13 C to 12 C were measured using a stable isotope ratio mass spectrometer (Delta V Advantage IRMS; Thermo Fisher Scientific, USA), with a precision of ± 0.05‰. The δ 13 C values were calculated based on the PDB standard: $$\:{{\delta\:}}^{13}\text{C}={\left(\frac{{({}_{\:}{}^{13}\text{c}/{}_{\:}{}^{12}\text{c})}_{\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}}}{{({}_{\:}{}^{13}\text{c}/{}_{\:}{}^{12}\text{c})}_{\text{s}\text{t}\text{a}\text{n}\text{d}\text{a}\text{r}\text{d}}}-1\right)}^{\:}\times\:\text{1,000}$$ Each sample was measured in triplicate, and the mean value was utilized for subsequent analysis. Measurement of titratable acidity Titratable acidity was measured following the methodology outlined by Eastmond and Ross ( 1997 ). Leaf samples were collected at dawn (06:00) and dusk (18:00) under mild drought stress conditions. Approximately 1 g of leaf tissue was frozen in liquid nitrogen and subsequently stored at − 80°C. The leaf tissues were ground in pre-chilled mortars, extracted with 5 mL of deionized water, boiled for 20 min, cooled to room temperature, and then centrifuged at 5,000 × g for 15 min. Two milliliters of the supernatant were diluted to a final volume of 25 mL. Using 0.2% bromothymol blue as an indicator, the samples were titrated with 0.01 M KOH until a color change from yellow to blue was observed (pH 7.2). Five replicates were measured for each sample, and the mean value was calculated. Measurement leaf anatomical structure For each species, three plants were selected, and three mature leaves per plant were sampled from the same orientation as those used for CO 2 exchange measurements. Paraffin sections were prepared according to the method described by Atkinson and Wells ( 2017 ). Anatomical observations and imaging were conducted using a light microscope. Leaf structural traits, including upper epidermal thickness (UET), lower epidermal thickness (LET), mesophyll thickness (MT), and total leaf thickness (LT), were measured using CaseViewer software. For each leaf, three sections were examined, with five randomly selected fields analyzed per section. Measurement of mesophyll succulent index The succulence index (Sm) was determined following Males ( 2017 ), based on the formula established by Kluge and Ting ( 1978 ): $$\:\text{s}\text{m}=\frac{\text{L}\text{e}\text{a}\text{f}\:\text{w}\text{a}\text{t}\text{e}\text{r}\:\text{c}\text{o}\text{n}\text{t}\text{e}\text{n}\text{t}\:\left(\text{g}\right)}{\text{T}\text{o}\text{t}\text{a}\text{l}\:\text{c}\text{h}\text{l}\text{o}\text{r}\text{o}\text{p}\text{h}\text{y}\text{l}\text{l}\:\text{c}\text{o}\text{n}\text{t}\text{e}\text{n}\text{t}\:\left(\text{m}\text{g}\right)}$$ Six to ten mature leaves were weighed for fresh weight (FW) and subsequently dried at 75°C until a constant weight was achieved (DW). Leaf water content (LWC) was calculated as follows: $$\:\text{L}\text{W}\text{C}=\frac{\text{F}\text{W}-\text{D}\text{W}}{\text{F}\text{W}}$$ A 0.2 g leaf sample was cut into pieces and extracted in 95% ethanol for 24 h in darkness. Absorbance at 665 nm and 649 nm was measured using a UV-Vis spectrophotometer (PerkinElmer, USA). Chlorophyll a and b concentrations were calculated using the following equations: Chl a = 13.95 × A 665 − 6.88 × A 649 ; Chl b = 24.96 × A 649 − 7.32 × A 665 , where A 665 and A 649 denote absorbance readings at their respective wavelengths. Total chlorophyll content (mg·dm -2 ) was calculated using the formula: Total Chl = (C × V × N) / LA, where C represents the pigment concentration, V is the extraction volume, N is the dilution factor, and LA denotes total leaf area.Three replicates were performed for each species. Data analyses Data on net CO 2 exchange, δ 13 C, ΔH + , leaf anatomical traits, and mesophyll succulent index were organized using Microsoft Excel 2019. Pearson correlation analyses (two-tailed) were conducted using SPSS Statistics 27.0 to evaluate linear relationships among variables. Graphing and curve fitting were performed using Origin 2021. Results Diurnal patterns of net CO 2 exchange The nine Dendrobium species exhibited distinct diurnal patterns of net CO 2 exchange (Fig. 2 ). D. cariniferum , D. gibsonii and D. hancockii maintained a negative net CO 2 exchange throughout the entire night, which aligns with a typical C 3 photosynthetic pattern. In contrast, the remaining six species, including D. parishii , D. aphyllum , D. anosmum , D. linawianum , D. moschatum and D. crystallinum , displayed positive net CO 2 exchange during part or all of the nighttime, indicating varying degrees of CAM-like characteristics. Among these species, D. parishii , D. aphyllum and D. anosmum exhibited high nighttime CO 2 exchange, each reaching its peak during the late-night or early-morning hours. D. linawianum and D. crystallinum demonstrated weak but detectable nighttime exchange primarily between 18:00 and 22:00. D. moschatum maintained a positive net CO 2 exchange throughout the entire 24-hour cycle, although the magnitude of exchange was low during the night. These results highlight substantial variation in diel carbon acquisition strategies among the studied species. Carbon isotope ratio and titratable acidity contents Carbon isotope ratios (δ 13 C) exhibited variability among the species studied (Fig. 3 ). D. parishii , D. aphyllum and D. anosmum presented δ 13 C values exceeding − 20‰, aligning with the characteristic range of CAM species. Conversely, the remaining species displayed δ 13 C values below − 20‰, a range typically associated with C 3 plants. While δ 13 C alone cannot conclusively differentiate C 3 species from potential C 3 /CAM intermediates, the observed patterns provides significant long-term evidence of variations in carbon assimilation strategies. The titratable acidity difference (ΔH + ) was positive across all species, indicating an increase in titratable acidity during the night (Fig. 3 ). D. parishii , D. aphyllum , D. anosmum and D. crystallinum recorded the highest ΔH + values, each above 45 µmol(H + )·g -1 FW, with D. aphyllum reaching a maximum value of 143.544 µmol(H + )·g -1 FW. In contrast, the other species exhibited lower ΔH + values, all below 19 µmol(H + )·g -1 FW, with D. hancockii showing the lowest ΔH + at 1.088 µmol(H + )·g -1 FW. Leaf anatomical structure All Dendrobium species exhibited fundamental anatomical characteristics, including a single-layered upper and lower epidermis, a compact mesophyll region and well-developed vascular tissues (Fig. 4 ). Notably, mesophyll cells lacked distinct palisade and spongy differentiation (Fig. 5 ). Variations in leaf thickness were observed among species, with D. parishii exhibiting the greatest total leaf thickness at 766.90 µm, including a mesophyll thickness of 714.07 µm. D. anosmum and D. aphyllum also displayed relatively thick leaves, both exceeding 500 µm. In contrast, D. cariniferum demonstrated moderate leaf thickness, while D. linawianum had the thinnest leaves, measuring 221.33 µm. These findings indicate that the species exhibited significant differences in their leaf anatomical parameters. Mesophyll succulent index The mesophyll succulent index (Sm) varied among the nine species (Fig. 6 ). D. parishii , D. anosmum , D. gibsonii and D. moschatum exhibited Sm values greater than 1.0. D. aphyllum , D. linawianum and D. crystallinum showed intermediate values of 0.98, 0.95 and 0.78 respectively. D. cariniferum and D. hancockii had lower values of 0.13 and 0.28. These results indicate that the species did not share the same degree of mesophyll succulence, with Sm values distributed across a broad range. Correlation analysis Nighttime net CO 2 exchange (NNEE) exhibited a strong positive correlation with δ 13 C (r = 0.894). Additionally, δ 13 C was positively correlated with ΔH + (r = 0.805), while NNEE showed a moderate positive relationship with ΔH + (r = 0.676). These three physiological indicators demonstrated coordinated changes across the species. Leaf thickness (LT) was positively related to these physiological parameters, with strong correlations observed NNEE (r = 0.877) and δ 13 C (r = 0.864), and a moderate correlation with ΔH + (r = 0.613). In contrast, the mesophyll succulent index (Sm) displayed weaker relationships with physiological traits, evidenced by correlation coefficients of 0.649 with δ 13 C and 0.521 with NNEE. These findings indicate that physiological traits are more closely interrelated than structural traits (Fig. 7 ). Discussion Judgment of photosynthetic carbon assimilation pathways Continuous monitoring of diurnal net CO 2 exchange provides a reliable means of determining whether plants exhibit nocturnal CO 2 exchange, a the key criterion for identifying CAM activity (van Tongerlo et al. 2021 ). In this study, D. parishii , D. aphyllum and D. anosmum maintained positive net CO 2 exchange throughout the night, demonstrating the characteristic sequence of CAM phases II, III and IV during the night-to-morning transition. This pattern reflects coordinated nocturnal CO 2 fixation and daytime decarboxylation, corroborating observations in typical CAM epiphytic orchid reported previously (Ceusters et al. 2008 ; Hogewoning et al. 2021 ). These species appear capable of fixing CO 2 at night through phosphoenolpyruvate carboxylase (PEPC), which supplies carbon for daytime metabolism while minimizing water loss, a strategy particularly advantageous in seasonally dry and warm environments (Zhang et al. 2014 ). In contrast, D. cariniferum , D. gibsonii and D. hancockii exhibited negative net CO 2 exchange from night to early morning, consistent with the C 3 pathway. Their diel rhythms align with those of typical C 3 plants, where CO 2 is assimilated through Rubisco during the day and released via respiration at night. These species are commonly found in shaded and humid habitats, where sufficient water availability supports continuous daytime CO 2 exchange and growth (Winter et al. 1983 ). D. linawianum , D. moschatum and D. crystallinum exhibited diel patterns intermediate between C 3 and CAM species. They relied primarily on C 3 photosynthesis during the day, although weak nocturnal CO 2 exchange was observed during certain nighttime periods. This suggests a limited capacity for nocturnal carboxylation that may be activated under specific environmental conditions to enhance carbon gain and water-use efficiency. Similar patterns have been documented in other facultative or intermediate C 3 /CAM species, such as Clusia minor and Mesembryanthemum crystallinum , which modify their carbon assimilation pathways in response to environmental fluctuations (Borland et al. 1993 ; Winter and Holtum 2007 ). This flexibility is regarded as a crucial adaptive trait in environments characterized by intermittent drought or periodic variations in water availability. Carbon isotope ratios provide additional long-term physiological evidence for distinguishing photosynthetic pathways. Typical C 3 plants generally exhibit δ 13 C values ranging from − 33 to − 22.1‰, while strong CAM species typically fall between − 22 and − 12‰ (Elheringer and Osmond 1989; Pearcy et al. 2012 ). Silvera et al. ( 2009 ) demonstrated a bimodal distribution of δ13C values in orchid, with C 3 species centered around − 28‰ and CAM species near − 16‰. Intermediate values reflect C 3 /CAM species that respond to environmental stress. Studies on Dendrobium and related orchid have also shown a clear bimodal pattern with an inflection point near − 20‰, and many recent works use δ 13 C values greater than − 20‰ as indicators of CAM activity (Messerschmid et al. 2021 ). In the present study, D. parishii , D. aphyllum and D. anosmum exhibited δ 13 C values of − 14.38‰, − 15.48‰ and − 15.95‰, respectively, which are higher than those typical of C 3 plants and suggest the presence of nocturnal CO 2 fixation. In contrast, D. cariniferum , D. gibsonii and D. hancockii displayed δ 13 C values below − 26‰, consistent with the C 3 pathway and aligned with their negative nocturnal CO 2 exchange. D. linawianum , D. moschatum and D. crystallinum had δ 13 C values ranging from − 27 to − 29‰, which fall within the C 3 range according to isotope-based classification. Although these values indicate a predominantly C 3 carbon source, δ 13 C alone may not fully capture the potential for inducible CAM activity, as intermediate C 3 /CAM species often exhibit C 3 -like δ 13 C signatures under non-stress conditions (Borland et al. 2009 ). Research on facultative CAM species has shown that δ13C values frequently resemble those of C 3 plants when CAM expression is weak and may only shift under conditions of severe drought or light stress (Ricalde et al. 2010 ). When considered alongside diel CO 2 exchange patterns, our results suggest that D. linawianum , D. moschatum and D. crystallinum primarily rely on C 3 photosynthesis under normal conditions but may activate CAM activity when exposed to environmental stress. Similar responses have been documented in Agave deserti , which functions as a C 3 species in moist environments but exhibits weak CAM under drought or high irradiance (Hartsock and Nobel 1976 ). Therefore, while δ 13 C values provide valuable insights into long-term carbon assimilation patterns, additional physiological indicators are necessary for accurately determining photosynthetic pathways. As a central feature of the CAM pathway, nocturnal titratable acidity accumulation reflects the carboxylation activity of phosphoenolpyruvate carboxylase (PEPC) (Borland and Taybi 2004 ). In the present study, the analysis of ΔH + among nine Dendrobium species revealed differences among species, indicating diversity in their nocturnal organic acid metabolism. D. parishii , D. aphyllum and D. anosmum had ΔH + values of 143.54, 112.33 and 46.37 µmol(H + )·g -1 FW respectively, indicating day–night variations in organic acid content and suggesting that these species synthesize organic acids during the night. In contrast, D. cariniferum , D. gibsonii and D. hancockii variations low ΔH + values, with D. hancockii recording a mere 1.088 µmol(H + )·g -1 FW. These low values suggest that these species do not accumulate organic acids to a meaningful extent during the night, which aligns with the characteristics typically observed in C 3 plants. Although C 3 species generally do not build up large pools of organic acids overnight, small changes in acidity can occur due to of dark respiration and normal metabolic rhythms, potentially explaining their slightly positive ΔH + values rather than indicating CAM-related CO 2 fixation (Bräutigam et al. 2017 ). D. linawianum , D. moschatum and D. crystallinum had ΔH + values situated between those of the CAM-like and C 3 groups, indicating modest nocturnal organic acid accumulation and suggesting that these species may activate PEPC-mediated carboxylation specific particular environmental conditions. Silvera et al. ( 2005 ) examined 173 orchid species from Panama under drought conditions and reported broad and overlapping ranges of ΔH + , with obligate CAM species displaying values from 10.7 to 275.7 µmol(H + )·g -1 FW and facultative CAM species showing values from 1.7 to 36.1 µmol(H + )·g -1 FW. This pattern is broadly consistent with the trends observed in the present study. Although the ΔH + values of the intermediate Dendrobium species in this study are lower than those of strong CAM plants, their ability to accumulate organic acids during the night indicates that they may activate components of the CAM pathway in response to water or light stress. The integrated analysis of diurnal net CO 2 exchange, δ 13 C values and titratable acid accumulation provides a systematic basis for elucidating the carbon assimilation pathways of the nine Dendrobium species. These indicators represent complementary aspects of carbon metabolism, reflecting short-term gas exchange, long-term carbon source patterns and aspects biochemical processes. Their combined application facilitates the identification of C 3 , CAM and intermediate C 3 /CAM types (Table 2 ). D. parishii , D. aphyllum and D. anosmum exhibited consistent CAM characteristics across all indicators. Conversely, D. cariniferum , D. gibsonii and D. hancockii demonstrated full alignment with the C 3 pathway. Meanwhile, D. linawianum , D. moschatum and D. crystallinum displayed intermediate characteristics across all indicators and may activate components of the CAM pathway under specific environmental conditions. Table 2 Determination of photosynthetic carbon assimilation pathway types in nine Dendrobium species Species Net CO 2 exchange phenotype δ 13 C phenotype ΔH + phenotype Overall assessment D. parishii CAM CAM CAM CAM D. aphyllum CAM CAM CAM CAM D. anosmum CAM CAM CAM CAM D. cariniferum C 3 C 3 C 3 C 3 D. gibsonii C 3 C 3 C 3 C 3 D. hancockii C 3 C 3 C 3 C 3 D. linawianum CAM C 3 CAM C 3 /CAM D. moschatum CAM C 3 CAM C 3 /CAM D. crystallinum CAM C 3 CAM C 3 /CAM Discussion on leaf anatomical structure and the mesophyll succulent indexs Leaf thickness is an important anatomical characteristic associated with CAM activity, as CAM species are often linked to succulent tissues. Succulence has been documented in numerous CAM lineages across various plant families, including Geraniaceae , Orchidaceae and Clusiaceae (Jones et al. 2003 ; Barrera Zambrano et al. 2014 ; Zhang et al. 2018 ). Increased tissue succulence in many drought-adapted CAM plants offers advantages by enhancing water-storage capacity compared to C 3 and C 4 species. A comparative analysis of tropical orchid species indicated that obligate CAM plants typically exhibit the greatest leaf thickness (Silvera et al. 2005 ). In M. crystallinum , leaf succulence also increases during the transition from C 3 to CAM under salt treatment (Guan et al. 2020 ). Recent studies further suggest that the evolution of C 3 /CAM intermediate species may not necessitate major anatomical reconfiguration (Yang et al. 2019 ; Heyduk et al. 2021 ). Winter proposed that obligate CAM plants require substantial anatomical modification, whereas C 3 /CAM intermediate species may operate CAM without such extensive structural changes, indicating that the transition from C 3 to CAM may proceed with minimal alteration to C 3 type anatomy (Winter and Holtum 2014 ). In this study, the nine Dendrobium species exhibited considerable variation in leaf thickness, which may correlate with their differing carbon assimilation pathways. D. parishii , D. aphyllum and D. anosmum had the highest leaf thickness values (764.14, 584.20 and 551.83 µm, respectively), consistent with their classification as CAM species based on δ 13 C and net CO 2 exchange. Their well-developed leaf tissues likely provide the vacuolar space necessary for nocturnal malate storage and contribute to water conservation under fluctuating moisture conditions. However, certain observations deviate from traditional expectations. For example, D. cariniferum exhibited a relatively high leaf thickness of 445.29 µm, yet both its δ 13 C value (− 29.762‰) and net CO 2 exchange pattern clearly indicate a C 3 photosynthetic mode. This demonstrates that leaf thickness alone is not sufficient to determine the photosynthetic pathway in Dendrobium . Some C 3 species may develop thicker leaves as an adaptation to epiphytic microhabitats that experience high irradiance or nutrient limitation, without necessarily exhibiting CAM function. In contrast, species inferred to possess C 3 /CAM intermediate characteristics, such as D. crystallinum and D. linawianum , showed relatively low leaf thickness values (296.94 and 221.33 µm, respectively). These findings align with Winter’s hypothesis that intermediate species may operate inducible CAM within an essentially C 3 anatomical framework, relying on physiological and molecular regulation rather than major structural remodeling. Overall, leaf thickness in Dendrobium shows a general but not absolute association with CAM photosynthesis. Although obligate CAM species often possess thicker leaves, this trait does not reliably distinguish between C 3 , C 3 /CAM intermediate and CAM species on its own. The diversity of carbon assimilation pathways in the genus reflects long-term adaptation to heterogeneous microhabitats, shaped through coordinated evolution across anatomical, physiological and molecular levels. The mesophyll succulent index (Sm), a structural indicator reflecting the balance between water storage and photosynthetic investment (Ripley et al. 2013 ), provides additional anatomical evidence supporting the inferred photosynthetic types in this study. Sm values exhibited patterned variation across species groups, complementing interpretations based on physiological parameters. Species inferred to be typical CAM types ( D. anosmum , D. aphyllum and D. parishii ) presented high Sm values (1.27, 0.98 and 1.06, respectively), consistent with their CAM physiology. A high Sm indicates substantial water-storage capacity relative to photosynthetic tissue, aligning with the requirements for nocturnal CO 2 fixation and malate accumulation, thereby supporting survival under periodic drought conditions. In contrast, C 3 species such as D. gibsonii , D. hancockii and D. cariniferum exhibited varied Sm levels. The elevated Sm of D. gibsonii (1.02) suggests that enhanced water storage is not exclusive to CAM species and may represent a drought-tolerance strategy in certain C 3 taxa. Meanwhile, the low Sm values of D. hancockii (0.28) and D. cariniferum (0.13) correspond with their C 3 CO 2 exchange and minimal nocturnal acid accumulation. C 3 /CAM intermediate species ( D. linawianum , D. moschatum and D. crystallinum ) displayed intermediate Sm values (0.78, 1.10 and 0.95), spanning the ranges observed in both CAM and C 3 species. For D. crystallinum and D. linawianum , Sm values near 1 suggest sufficient but not extreme succulence to support inducible CAM under specific environmental conditions, aligning with their moderate nocturnal acid accumulation and δ 13 C values remaining within the C 3 range. Notably, D. moschatum had an Sm value similar to that of CAM species (1.10), yet its δ 13 C (− 28.453‰) and low ΔH + (6.435 µmol(H + )·g -1 FW) indicate only weak or inducible CAM expression. This suggests that certain Dendrobium species may develop anatomical characteristics suited to CAM, while the physiological engagement of CAM remains conditional. Correlation analysis between physiological and structural parameters The correlation analysis revealed that the physiological parameters employed to assess photosynthetic carbon assimilation types exhibited a high degree of consistency with one another. Nighttime net CO 2 exchange (NNEE), δ 13 C and the titratable acidity difference (ΔH + ) were positively correlated, indicating that nocturnal CO 2 exchange, long-term carbon isotope composition and overnight organic acid accumulation reflect similar trends in carbon assimilation across species. This coherence suggests that physiological indicators effectively capture the distinctions among C 3 , CAM and C 3 /CAM intermediate types, provideing a reliable basis for pathway determination. In contrast, the correlations between structural traits and physiological indicators were more variable. Leaf thickness (LT) demonstrated moderate correlations with certain physiological traits, while the mesophyll succulent index (Sm) exhibited weaker overall correlations, and consistency among structural traits was limited. These patterns indicate that structural characteristics can reflect aspects of CAM activity but are not determinative, as species may adopt different structural configurations to achieve similar photosynthetic outcomes. Consequently, structural parameters alone are insufficient for accurate discrimination among C 3 , C 3 /CAM intermediate and CAM pathways. Overall, the correlation results underscore the central role of physiological parameters in determining carbon assimilation type, while structural traits serve more appropriately as supplementary evidence. This distinction elucidate why some C 3 /CAM intermediate species display transitional or inconspicuous anatomical features and supports the perspective that photosynthetic pathway differentiation involves multidimensional regulation and diverse adaptive strategies (Borland et al. 2009 ). Ecological adaptability in photosynthetic carbon assimilation pathway variation This study reveals that the photosynthetic carbon assimilation pathways of nine Dendrobium species exhibit a high degree of correlation with the characteristics of their native habitats. Based on descriptions from Plants of the World Online and the Flora of China , these species are predominantly epiphytic or lithophytic orchids found in tropical and subtropical montane forests, open woodlands or rocky valleys (Table 3 ). Such habitats typically experience fluctuating water availability, nutrient-poor substrates and variable light conditions, all of which are known to promote CAM evolution in orchids (Nelson and Sage 2008 ; Silvera et al. 2010 ). In this study, species identified as typical CAM types ( D. parishii , D. aphyllum and D. anosmum ) are commonly found in environments characterized by pronounced dry–wet seasonality, high irradiance and epiphytic or lithophytic growth forms, aligning with ecological conditions that favor CAM metabolism. Conversely, species characterized as C 3 /CAM intermediates ( D. crystallinum , D. linawianum and D. moschatum ) often inhabit forest margins or areas with seasonally variable moisture, suggesting that their flexible photosynthetic strategies may reflect adaptive responses to these environments. In contrast, species classified as C 3 types ( D. cariniferum , D. gibsonii and D. hancockii ) are found in shaded, humid montane forests, ravines or high-elevation moist environments where water availability is relatively stable, consistent with C 3 photosynthesis. Collectively, these findings indicate that the diversity of carbon assimilation pathways among the nine Dendrobium species corresponds to distinct habitat preferences, reflecting ecological differentiation. Although this study did not quantitatively analyze climatic variables, comparisons with habitat descriptions suggest that environmental factors such as water availability, growth form (epiphytic or lithophytic) and light conditions may serve as important selective pressures shaping carbon assimilation strategies in Dendrobium . Future research that integrates distribution data with environmental parameters may elucidate the mechanisms linking habitat characteristics to the evolution of photosynthetic pathways. Table 3 Natural habitat distribution of nine Dendrobium species Species Native distribution Habitat type (as documented in the literature) References D. parishii Northeast India, Myanmar, Thailand, Laos, Vietnam, China (Yunnan) Epiphytic on trees in montane evergreen forests or on rocks; elevation 250–1200 m Flora of China;POWO D. aphyllum Nepal, Bhutan, India, Myanmar, Thailand, Laos, Vietnam, China (Yunnan) Epiphytic or lithophytic in tropical moist forests; elevation 200–1,500 m Flora of China;POWO D. anosmum Philippines, Malaysia, Laos, Vietnam, Myanmar, China (Yunnan) Epiphytic in lowland to montane forests, often near riverbanks or humid valleys New Guinea Orchids;POWO D. cariniferum China (Yunnan), Myanmar, Northeast India Epiphytic in humid montane forests; elevation 1,200–2,000 m Flora of China;POWO D. gibsonii China (Yunnan, Guangxi), India, Myanmar Epiphytic or lithophytic on cliffs, rocks or forest trunks; elevation 800–1,500 m Flora of China;POWO D. hancockii China (Yunnan, Guizhou, Guangxi), Vietnam, Northern Thailand Epiphytic or lithophytic in montane forests; elevation 700–1,500 m Flora of China;Smithsonian D. linawianum Taiwan, China (Guangdong) Epiphytic in montane evergreen forests; elevation 700–1,400 m Flora of China;POWO D. moschatum Northeast India, Nepal, Bhutan, Myanmar, China (Yunnan) Epiphytic in cliffs or montane moist forests; elevation 1,000–1,800 m Useful Tropical Plants;POWO D. crystallinum Thailand, Myanmar, Laos, Vietnam, China (Yunnan, Guizhou, Hainan) Epiphytic or lithophytic in montane forests with seasonal moisture variation; elevation 700–1,700 m Flora of China;POWO Conclusions In summary, D. parishii , D. aphyllum and D. anosmum exhibited characteristics consistent with typical CAM plants, including nocturnal net CO 2 exchange, δ 13 C values exceeding − 16‰ and substantial nocturnal titratable acidity accumulation (ΔH + > 46 µmol(H + )·g -1 FW), and their anatomical traits, such as increased leaf thickness and elevated mesophyll succulent indices, further substantiated their classification within the CAM category. Conversely, D. cariniferum , D. gibsonii and D. hancockii displayed characteristics indicative of C 3 plants, including the absence of nocturnal CO 2 exchange, δ 13 C values below − 26‰, and minimal overnight acid accumulation. D. linawianum , D. moschatum and D. crystallinum demonstrated intermediate C 3 /CAM characteristics; moreover, leaf thickness and mesophyll succulent inices were generally higher in most CAM species, although some overlap occurred with values from C 3 /CAM intermediates and C 3 species. Correlation analysis revealed that physiological indicators NNEE, δ 13 C and ΔH + were highly consistent with one another, reflecting a continuum of carbon assimilation strategies within the genus Dendrobium . In contrast, the associations between structural traits and physiological parameters were generally weaker. Leaf thickness exhibited moderate correspondence with specific physiological variables, while the mesophyll succulent index showed weak associations with physiological parameters and lacked a clear correlation with photosynthetic types. These findings indicate that structural traits alone are insufficient for reliably distinguishing between C 3 , C 3 /CAM intermediate and CAM species, and they should analyzed used in conjunction with physiological parameters for more accurate classification. The various photosynthetic types correspond to distinct habitat preferences: typical CAM species are commonly found in seasonally dry, high-light epiphytic or lithophytic environments; C 3 species are typically associated with humid and shaded conditions; while C 3 /CAM intermediate species thrive in habitats that experience alternating wet and dry conditions. This pattern reflects the relationship links between photosynthetic strategy and the availability of environmental moisture and light. Overall, the diversity of carbon assimilation pathways in Dendrobium illustrates both physiological plasticity and long-term ecological adaptation. This study offers a valuable foundation for ecological assessment, germplasm conservation and cultivation management of this orchid genus. Declarations Authors contributions Conceptualization, Z.Y., S.C. and H.P.; data curation, Z.Y. and N.C.; formal analysis, L.P.; investigation, N.C.; methodology, L.P. and Z.Y.; software, N.C. and Z.Y.; supervision, L.P., J.W., Q.J. and N.C.; writing—original draft, D.T.; writing—review and editing, Z.Y. and S.C. All authors have read and agreed to the published version of the manuscript. Funding This study was supported by the National Key Research and Development Program of China (2022YFF1300700), Guangxi Forestry Bureau Project (2024LYKJ01), and Guangxi Key Laboratory of Plant Functional Substances and Sustainable Utilization (ZRJJ2024-12). Data availability All data generated or analyzed during this study are included in this published article. The authors have no relevant financial or non-financial interests to disclose. Ethics, Consent to to participate Not applicable. Consent for publication All authors have approved the manuscript for submission to BMC Plant Biology. Clinical trial number Not applicable. Conflicts of Interest The authors declare no conflicts of interest. References Atkinson JA, Wells DM (2017) An updated protocol for high throughput plant tissue sectioning. Front. Plant Sci 8:1721. Barrera Zambrano VA, Lawson T, Olmos E, Fernández-García N, Borland AM (2014) Leaf anatomical traits which accommodate the facultative engagement of crassulacean acid metabolism in tropical trees of the genus Clusia. J Exp Bot 65:3513-3523. Blankenship RE (2021) Molecular mechanisms of photosynthesis. 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16:05:39","extension":"jpeg","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2345194,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8304845/v1/60eccbfbaf28bf5d2dcd2304.jpeg"},{"id":98810120,"identity":"77d7467d-a9ab-490d-90ca-cb2b28bfaf88","added_by":"auto","created_at":"2025-12-22 15:14:29","extension":"jpeg","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":247888,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8304845/v1/c9e41c9044a91bee9510b2c2.jpeg"},{"id":98810125,"identity":"5442a8d8-bf01-460c-83b1-a2b799b87ddb","added_by":"auto","created_at":"2025-12-22 15:14:30","extension":"jpeg","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3140124,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8304845/v1/fd6b3b6cb1b1e4bdfb590957.jpeg"},{"id":98810122,"identity":"5d7f4af6-2fd2-4d0b-a79d-410c5012a20a","added_by":"auto","created_at":"2025-12-22 15:14:29","extension":"jpeg","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1338764,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8304845/v1/1679cbeb21c71822ad53ab75.jpeg"},{"id":99307019,"identity":"eeced0cd-099b-4341-b7ad-922a11e531c3","added_by":"auto","created_at":"2025-12-31 16:05:21","extension":"jpeg","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":284699,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8304845/v1/6115aeb97fd2e69df40b6509.jpeg"},{"id":98810123,"identity":"4cfcb8c3-53b1-4c51-b9de-b73f6b75af94","added_by":"auto","created_at":"2025-12-22 15:14:29","extension":"xml","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":147224,"visible":true,"origin":"","legend":"","description":"","filename":"19c57c25ccf6416591be5a98756106781structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8304845/v1/2a94f4d2877967d478568447.xml"},{"id":98810124,"identity":"4c22da40-0d41-4deb-9f5b-3bca967f48c6","added_by":"auto","created_at":"2025-12-22 15:14:29","extension":"html","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":155690,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8304845/v1/8e1410217f1277969acb9e14.html"},{"id":98810109,"identity":"b948f703-300d-4305-b2b9-a0ac31cd8354","added_by":"auto","created_at":"2025-12-22 15:14:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":73048,"visible":true,"origin":"","legend":"\u003cp\u003eDiurnal variation curves of environmental factors\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8304845/v1/f74df75e1dc50badbbfe5d50.png"},{"id":98810114,"identity":"e2c91bc5-0ed0-4634-81f3-c497ea8d4fbf","added_by":"auto","created_at":"2025-12-22 15:14:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":122297,"visible":true,"origin":"","legend":"\u003cp\u003eThe diurnal variation in net CO2 exchange among nine \u003cem\u003eDendrobium\u003c/em\u003e species. Note: The white background denotes the daytime period, while the gray shaded area indicates the nighttime period. Each subfigure illustrates the diurnal and nocturnal dynamics of net CO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8304845/v1/2b8c63044960eae7df19b879.png"},{"id":98810107,"identity":"416523fc-7867-4b88-93d2-e1c93583cdde","added_by":"auto","created_at":"2025-12-22 15:14:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":44495,"visible":true,"origin":"","legend":"\u003cp\u003eCarbon isotope ratios and titratable acidity contents of the nine \u003cem\u003eDendrobium\u003c/em\u003e species. Note: that panel a) illustrates the carbon stable isotope ratios δ\u003csup\u003e13\u003c/sup\u003eC (‰). Different bar colors indicating interspecific variation in δ\u003csup\u003e13\u003c/sup\u003eC values. Green bars represent values associated with predominantly CAM-type carbon assimilation, while yellow bars indicate values linked to predominantly C\u003csub\u003e3\u003c/sub\u003e-type carbon assimilation. Panel b) displays leaf titratable acidity measured at dawn and dusk for each species, with blue bars representing acidity at dawn and red bars representing acidity at dusk. The yellow curve illustrates the difference in acidity between dawn and dusk.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8304845/v1/74f4a455f6b39d98d9823195.png"},{"id":99307151,"identity":"b2583592-4c47-49fe-a2f2-8e22559dc1e4","added_by":"auto","created_at":"2025-12-31 16:05:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":81787,"visible":true,"origin":"","legend":"\u003cp\u003eLeaf thickness and tissue composition in nine \u003cem\u003eDendrobium\u003c/em\u003e species. Note: The stacked bars represent the thickness (µm) of the upper epidermis (blue), mesophyll (yellow), and lower epidermis (red). The numerical values above each bar indicate total leaf thickness, and the percentages within each color segment denote the relative contribution of each tissue type.\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8304845/v1/81207f34e37bfe1cc987b8e2.png"},{"id":99307069,"identity":"b5e20651-62f9-4481-abb6-23795041723e","added_by":"auto","created_at":"2025-12-31 16:05:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":182708,"visible":true,"origin":"","legend":"\u003cp\u003eLeaf anatomical structure of nine \u003cem\u003eDendrobium\u003c/em\u003e species. Note: Images are presented in the correct anatomical orientation, with the upper (adaxial) epidermis positioned at the top and the lower (abaxial) epidermis at the bottom. Labels (a-i) correspond to the nine \u003cem\u003eDendrobium\u003c/em\u003e species examined in this study.\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8304845/v1/93c3ca7cd06e72a2b0dcb0f8.png"},{"id":98810116,"identity":"f07a4d27-9112-4e59-957d-d4bf8650b8bc","added_by":"auto","created_at":"2025-12-22 15:14:29","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":548426,"visible":true,"origin":"","legend":"\u003cp\u003eThe mesophyll succulence of nine \u003cem\u003eDendrobium\u003c/em\u003e species. Note:Each species is represented by a colored arc, with arc length proportional to its Sm value; larger Sm values correspond to longer.\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8304845/v1/4889673d8b3d257ad0eb3a87.png"},{"id":99307277,"identity":"b9423e0e-853b-49f0-b278-060aae75cc68","added_by":"auto","created_at":"2025-12-31 16:05:54","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":89558,"visible":true,"origin":"","legend":"\u003cp\u003eThe heatmap illustrates the correlations between physiological parameters and structural traits of nine \u003cem\u003eDendrobium\u003c/em\u003e species. Note: This heatmap illustrates the correlations among key physiological indicators (NNEE, Nighttime Net CO\u003csub\u003e2\u003c/sub\u003e Exchange; δ\u003csup\u003e13\u003c/sup\u003eC, Carbon Isotope Ratio; ΔH\u003csup\u003e+\u003c/sup\u003e, Titratable acidity difference) and leaf structural traits (LT, leaf thickness; Sm, mesophyll succulent index) . The color intensity reflects the strength of the correlations (−1 to +1), with red indicating positive correlations and blue indicating negative correlations. Greater color saturation corresponds to stronger correlation coefficients. Statistical significance is denoted as *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8304845/v1/389ec3a704210de0c4285f13.png"},{"id":99787998,"identity":"e91c6d19-7b78-410d-a232-abb4131ca4c7","added_by":"auto","created_at":"2026-01-08 12:43:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1880825,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8304845/v1/d8276a51-f5c2-494d-9479-130d82744c55.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Integrated Multi-Trait Analysis of Photosynthetic Carbon Assimilation Pathways and Adaptive Patterns in Dendrobium Species","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePhotosynthesis is the fundamental metabolic process through which plants convert light energy into chemical energy (Blankenship \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Nelson and Yocum \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Throughout evolution, plants have developed several distinct pathways for photosynthetic carbon assimilation, including the C\u003csub\u003e3\u003c/sub\u003e, C\u003csub\u003e4\u003c/sub\u003e and crassulacean acid metabolism (CAM) pathways, to adapt to diverse ecological environments (Ehleringer and Monson \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Sage \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). These pathways differ significantly in their in carbon-fixation efficiency, water-use characteristics and ecological adaptability. The C\u003csub\u003e3\u003c/sub\u003e pathway, being the most ancient and widespread form and predominates in temperate and humid regions; however, C\u003csub\u003e3\u003c/sub\u003e plants experience pronounced photorespiration under high temperatures, drought, and intense light. In contrast, C\u003csub\u003e4\u003c/sub\u003e pathway evolved as an adaptation to such stress conditions. This pathway relies on Kranz anatomy, a specialized leaf structure and a spatial CO\u003csub\u003e2\u003c/sub\u003e-concentrating mechanism that suppresses photorespiration, allowing plants to maintain efficient carbon fixation in hot and high-light habitats (Sage and Zhu \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). CAM represents another specialized adaptation that shifts CO\u003csub\u003e2\u003c/sub\u003e exchange to the night, storing it as organic acids. By closing their stomata during the day and relying on nocturnally accumulated malate for CO\u003csub\u003e2\u003c/sub\u003e release, CAM plants minimize water loss and can survive in extremely arid environments (Winter and Smith \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Borland et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Importantly, the C\u003csub\u003e3\u003c/sub\u003e, C\u003csub\u003e4\u003c/sub\u003e and CAM pathways are not entirely isolated from one another. C\u003csub\u003e3\u003c/sub\u003e/C\u003csub\u003e4\u003c/sub\u003e intermediate species are present in several plant groups. These intermediate forms reflect the intrinsic plasticity of photosynthetic carbon assimilation and provide valuable model systems for investigating how plants adjust their carbon-fixation strategies in response to environmental variations. The long-term evolution and distribution of these pathways have been influenced by changes in atmospheric CO\u003csub\u003e2\u003c/sub\u003e concentration, temperature and water availability. Since the Cenozoic era, global aridification, coupled with declining atmospheric CO\u003csub\u003e2\u003c/sub\u003e levels is believed to have promoted multiple independent origins of both C\u003csub\u003e4\u003c/sub\u003e and CAM photosynthesis (Sage et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Winter and Holtum \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The interplay of these biochemical mechanisms, along with their varying modes of spatio-temporal CO\u003csub\u003e2\u003c/sub\u003e concentration, has resulted in a diverse array of adaptive carbon assimilation strategies. This diversity forms the theoretical foundation for understanding the ecological drivers of photosynthetic evolution.\u003c/p\u003e \u003cp\u003eIn the crassulacean acid metabolism (CAM) pathway, plants exhibit distinctive adaptive advantages. Studies exhibit that the water-use efficiency of typical CAM species can be up to six times greater than that of C\u003csub\u003e3\u003c/sub\u003e plants and three times greater than that of C\u003csub\u003e4\u003c/sub\u003e plants (Nobel \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). This highly efficient water-saving strategy enables CAM plants to maintain a competitive edge in arid and semi-arid environments. Additionally, CAM species demonstrate significant tolerance to intense light and high temperatures, which collectively define their ecological roles in dry and semi-dry ecosystems (Silvera et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). CAM is characterized by a unique CO\u003csub\u003e2\u003c/sub\u003e fixation mechanism and operates through four distinct phases. During Phase I, which occurs at night, stomata open and CO\u003csub\u003e2\u003c/sub\u003e is fixed by phosphoenolpyruvate carboxylase to form malate, which is stored in vacuoles. Phase II occurs around dawn, when stomata are partially open, allowing both C\u003csub\u003e3\u003c/sub\u003e and C\u003csub\u003e4\u003c/sub\u003e carbon fixation to occur simultaneously. Phase III takes place during the day, when stomata remain closed and malate is decarboxylated, releasing CO\u003csub\u003e2\u003c/sub\u003e for the Calvin cycle through the action of Rubisco. Finally, Phase IV occurs in the late afternoon, when stomata reopen briefly and CO\u003csub\u003e2\u003c/sub\u003e is fixed directly via the C\u003csub\u003e3\u003c/sub\u003e pathway (Osmond \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1978\u003c/span\u003e; Ting \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1985\u003c/span\u003e). CAM expression can be further categorized into obligate CAM, which is constitutively expressed throughout the plant’s life cycle, facultative CAM, which is induced under drought or other stress conditions, CAM cycling, which involves nocturnal acid accumulation without net CO\u003csub\u003e2\u003c/sub\u003e exchange, and CAM idling, which occurs under extreme drought when stomata remain nearly completely closed and carbon recycling is maintained internally (Winter and Holtum \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). This diversity reflects the high degree of plasticity with which plants adapt to their environment. Research on CAM and the broader spectrum of carbon assimilation pathways is therefore essential for understanding plant stress physiology and offers new strategies for crop genetic improvement. Insights into the water-saving and heat-tolerant traits of CAM provide a foundation for breeding drought-resistant and water-efficient crops. Moreover, the carbon-fixation characteristics of CAM plants are enhancing for refining global carbon-cycle models and improving predictions of ecosystem function in the context of climate change.\u003c/p\u003e \u003cp\u003e \u003cem\u003eDendrobium\u003c/em\u003e is one of the largest genera within the Orchidaceae family, encompassing over 1,500 species that are widely distributed across tropical and subtropical regions of Asia (Hou et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Species within this genus hold significant ecological and economic value. Many \u003cem\u003eDendrobium\u003c/em\u003e species are integral components of forest ecosystems and serve as important medicinal and horticultural resources. Their survival status and physiological adaptability are directly related to biodiversity conservation and sustainable utilization (Cheng et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Recently, the diversity of photosynthetic carbon assimilation pathways and their environmental regulation have emerged as a major focus in plant physiological ecology (Sage et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Prior studies have demonstrated that the Orchidaceae family exhibits a wide range of photosynthetic types, spanning from typical C\u003csub\u003e3\u003c/sub\u003e species to strong CAM species (Silvera et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Evidence of plastic carbon assimilation in \u003cem\u003eDendrobium\u003c/em\u003e suggests that some species may modify their carbon-acquisition strategies in response to abiotic stress (Silvera et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This physiological plasticity may represent one of the key mechanisms that enable \u003cem\u003eDendrobium\u003c/em\u003e species to thrive in diverse habitats. Given the extensive distribution and varied ecological contexts of the genus, it is probable that a rich variety of photosynthetic types exists, particularly within the transitional range between CAM and C\u003csub\u003e3\u003c/sub\u003e pathways. Nonetheless, systematic research on photosynthetic carbon assimilation pathways in \u003cem\u003eDendrobium\u003c/em\u003e remains limited. Most existing studies concentrate on a few economically significant species, and comprehensive comparisons that integrate multiple indicators are still lacking.\u003c/p\u003e \u003cp\u003eTo address these issues, this study investigates nine native Chinese \u003cem\u003eDendrobium\u003c/em\u003e species, including \u003cem\u003eD. parishii\u003c/em\u003e, \u003cem\u003eD. aphyllum\u003c/em\u003e, \u003cem\u003eD. ellipsophyllum\u003c/em\u003e, \u003cem\u003eD. cariniferum\u003c/em\u003e, \u003cem\u003eD. gibsonii\u003c/em\u003e, \u003cem\u003eD. hancockii\u003c/em\u003e, \u003cem\u003eD. linawianum\u003c/em\u003e, \u003cem\u003eD. moschatum\u003c/em\u003e and \u003cem\u003eD. crystallinum.\u003c/em\u003e The photosynthetic carbon assimilation pathways of these species have not been previously examined. The innovation of this study lies in the application of a comprehensive analytical framework that integrates five categories of indicators: net CO\u003csub\u003e2\u003c/sub\u003e exchange, δ\u003csup\u003e13\u003c/sup\u003eC values, titratable acid accumulation (ΔH\u003csup\u003e+\u003c/sup\u003e), leaf anatomical traits and the mesophyll succulent index. This framework facilitates a systematic assessment of C\u003csub\u003e3\u003c/sub\u003e, CAM and intermediate C\u003csub\u003e3/\u003c/sub\u003eCAM photosynthetic types, revealing potential photosynthetic plasticity. Measurements of net CO\u003csub\u003e2\u003c/sub\u003e exchange enable the characterization of diel gas-exchange patterns (van Tongerlo et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Changes in titratable acidity serve as an important indicator of carbon metabolism and reflect the magnitude of nocturnal CO\u003csub\u003e2\u003c/sub\u003e fixation and daytime decarboxylation (Cushman and Borland \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Carbon isotope ratios distinguish among C\u003csub\u003e3\u003c/sub\u003e, C\u003csub\u003e4\u003c/sub\u003e, and CAM pathways, providing long-term information about water-use efficiency (Farquhar et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1982\u003c/span\u003e). Leaf anatomical traits are closely linked to photosynthetic pathways; for instance, C\u003csub\u003e4\u003c/sub\u003e plants typically possess Kranz anatomy, while CAM plants often exhibit thick leaves with well-developed water-storage tissue (Orsenigo et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). The mesophyll succulent index offers a quantitative measure of xeromorphic structure, reflecting the degree of tissue water storage. Higher succulence is commonly associated with typical CAM species and other plants adapted to arid habitats (Herrera \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). By integrating physiological, biochemical and anatomical indicators, this study aims to accurately classify the photosynthetic pathways of the nine species and identify potential intermediate types between C\u003csub\u003e3\u003c/sub\u003e and CAM. The findings provide insights into the environmental adaptation mechanisms of \u003cem\u003eDendrobium\u003c/em\u003e, offering a scientific basis for taxonomy, conservation and cultivation management. This is particularly pertinent in the context of climate change and the sustainable utilization of germplasm resources.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eStudy site and plant materials\u003c/p\u003e\u003cp\u003eThe experiment was conducted in 2023 at the Guangxi Institute of Botany, located in the Yanshan District of Guilin City, Guangxi, China (25°01′ N, 110°17′ E). The study site is situated at an elevation of approximately 180 m and is characterized by a subtropical monsoon climate. The region experiences a mean annual temperature of 18.8°C, with January being the coldest month, averaging 8.3°C, and July being the hottest month, averaging 28.3°C. Annual precipitation ranges from 1,900 to 2,000 mm, with over 70% occurring between April and August. The mean annual relative humidity is 78%, and the annual sunshine duration is approximately 1,500 h.\u003c/p\u003e\u003cp\u003eNine \u003cem\u003eDendrobium\u003c/em\u003e species were introduced from Guangxi and Yunnan and cultivated in the germplasm nursery of the Guangxi Institute of Botany. The species included \u003cem\u003eD. parishii\u003c/em\u003e, \u003cem\u003eD. aphyllum\u003c/em\u003e, \u003cem\u003eD. anosmum\u003c/em\u003e, \u003cem\u003eD. cariniferum\u003c/em\u003e, \u003cem\u003eD. gibsonii\u003c/em\u003e, \u003cem\u003eD. hancockii\u003c/em\u003e, \u003cem\u003eD. linawianum\u003c/em\u003e, \u003cem\u003eD. moschatum\u003c/em\u003e, and \u003cem\u003eD. crystallinum\u003c/em\u003e, as identified by Zhongchen Xiong of the Guangxi Institute of Botany. Detailed information regarding the plant materials is provided in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The plants were grown in plastic pots (height: 18 cm; inner diameter: 16 cm) filled with a substrate composed of tree bark, coconut fiber, and perlite in a ratio of 1:1:1. All test plants were three-year-old mature individuals maintained under mild drought stress. Shade nets with a transmittance of 30% were installed on the greenhouse roof, allowing the plants to grow under natural light conditions.Net CO\u003csub\u003e2\u003c/sub\u003e exchange measurements were conducted in early October 2023, and leaf samples were subsequently collected for analyses of carbon isotope ratios (δ\u003csup\u003e13\u003c/sup\u003eC), titratable acidity (ΔH\u003csup\u003e+\u003c/sup\u003e), leaf anatomical structure, and mesophyll succulent index.\u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eExperimental materials\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDeterminer\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDetermining Institution\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSite of Introduction\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eD.\u0026nbsp;parishii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZhongchen Xiong\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGuangxi Institute of Botany\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBaoshan, Yunnan\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eD. aphyllum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZhongchen Xiong\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGuangxi Institute of Botany\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePu’er, Yunnan\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eD. anosmum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZhongchen Xiong\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGuangxi Institute of Botany\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLongzhou, Guangxi\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eD. cariniferum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZhongchen Xiong\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGuangxi Institute of Botany\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTian’e, Guangxi\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eD. gibsonii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZhongchen Xiong\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGuangxi Institute of Botany\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBaoshan, Yunnan\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eD. hancockii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZhongchen Xiong\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGuangxi Institute of Botany\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBaise, Guangxi\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eD. linawianum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZhongchen Xiong\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGuangxi Institute of Botany\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJinxiu, Guangxi\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eD. moschatum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZhongchen Xiong\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGuangxi Institute of Botany\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJinghong, Yunnan\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eD. crystallinum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZhongchen Xiong\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGuangxi Institute of Botany\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTian’e, Guangxi\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003cp\u003eMeasurement of diurnal patterns of net CO\u003csub\u003e2\u003c/sub\u003e exchange\u003c/p\u003e\u003cp\u003eDiurnal net CO\u003csub\u003e2\u003c/sub\u003e exchange was measured from 08:00 on October 7 to 06:00 on October 8, 2023. Healthy, fully expanded leaves located at the 3rd to 5th position from the shoot apex were selected for measurement. A portable photosynthesis system (LI-6400XT; LI-COR Biosciences, USA) was utilized under ambient CO\u003csub\u003e2\u003c/sub\u003e concentrations and natural light conditions. Measurements were conducted at 2-h intervals over a 24-h cycle, with each leaf measured three times to obtain a mean value. For each species, three individual plants were assessed.\u003c/p\u003e\u003cp\u003eAccording to Beijing local time, sunrise and sunset on October 7 occurred at 06:33:15 and 18:20:17, respectively; on October 8, they were recorded at 06:33:41 and 18:19:17. Photosynthetically active radiation (PAR), air temperature (\u003cem\u003eT\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e), and relative humidity (RH) were recorded simultaneously (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMeasurement of carbon isotope ratio\u003c/p\u003e\u003cp\u003eCarbon isotope ratios (δ\u003csup\u003e13\u003c/sup\u003eC) were determined according to the methodology established by Winter and Holtum (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Leaf samples were collected under mild drought stress, then ground in liquid nitrogen, freeze-dried, and weighed to a precision of 3 mg. The ratios of \u003csup\u003e13\u003c/sup\u003eC to \u003csup\u003e12\u003c/sup\u003eC were measured using a stable isotope ratio mass spectrometer (Delta V Advantage IRMS; Thermo Fisher Scientific, USA), with a precision of ± 0.05‰. The δ\u003csup\u003e13\u003c/sup\u003eC values were calculated based on the PDB standard:\u003c/p\u003e\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:{{\\delta\\:}}^{13}\\text{C}={\\left(\\frac{{({}_{\\:}{}^{13}\\text{c}/{}_{\\:}{}^{12}\\text{c})}_{\\text{s}\\text{a}\\text{m}\\text{p}\\text{l}\\text{e}}}{{({}_{\\:}{}^{13}\\text{c}/{}_{\\:}{}^{12}\\text{c})}_{\\text{s}\\text{t}\\text{a}\\text{n}\\text{d}\\text{a}\\text{r}\\text{d}}}-1\\right)}^{\\:}\\times\\:\\text{1,000}$$\u003c/div\u003e\u003c/div\u003e\u003cp\u003eEach sample was measured in triplicate, and the mean value was utilized for subsequent analysis.\u003c/p\u003e\u003cp\u003eMeasurement of titratable acidity\u003c/p\u003e\u003cp\u003eTitratable acidity was measured following the methodology outlined by Eastmond and Ross (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Leaf samples were collected at dawn (06:00) and dusk (18:00) under mild drought stress conditions. Approximately 1 g of leaf tissue was frozen in liquid nitrogen and subsequently stored at − 80°C. The leaf tissues were ground in pre-chilled mortars, extracted with 5 mL of deionized water, boiled for 20 min, cooled to room temperature, and then centrifuged at 5,000 × g for 15 min. Two milliliters of the supernatant were diluted to a final volume of 25 mL. Using 0.2% bromothymol blue as an indicator, the samples were titrated with 0.01 M KOH until a color change from yellow to blue was observed (pH 7.2). Five replicates were measured for each sample, and the mean value was calculated.\u003c/p\u003e\u003cp\u003eMeasurement leaf anatomical structure\u003c/p\u003e\u003cp\u003eFor each species, three plants were selected, and three mature leaves per plant were sampled from the same orientation as those used for CO\u003csub\u003e2\u003c/sub\u003e exchange measurements. Paraffin sections were prepared according to the method described by Atkinson and Wells (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Anatomical observations and imaging were conducted using a light microscope. Leaf structural traits, including upper epidermal thickness (UET), lower epidermal thickness (LET), mesophyll thickness (MT), and total leaf thickness (LT), were measured using CaseViewer software. For each leaf, three sections were examined, with five randomly selected fields analyzed per section.\u003c/p\u003e\u003cp\u003eMeasurement of mesophyll succulent index\u003c/p\u003e\u003cp\u003eThe succulence index (Sm) was determined following Males (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), based on the formula established by Kluge and Ting (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1978\u003c/span\u003e):\u003c/p\u003e\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:\\text{s}\\text{m}=\\frac{\\text{L}\\text{e}\\text{a}\\text{f}\\:\\text{w}\\text{a}\\text{t}\\text{e}\\text{r}\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{e}\\text{n}\\text{t}\\:\\left(\\text{g}\\right)}{\\text{T}\\text{o}\\text{t}\\text{a}\\text{l}\\:\\text{c}\\text{h}\\text{l}\\text{o}\\text{r}\\text{o}\\text{p}\\text{h}\\text{y}\\text{l}\\text{l}\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{e}\\text{n}\\text{t}\\:\\left(\\text{m}\\text{g}\\right)}$$\u003c/div\u003e\u003c/div\u003e\u003cp\u003eSix to ten mature leaves were weighed for fresh weight (FW) and subsequently dried at 75°C until a constant weight was achieved (DW). Leaf water content (LWC) was calculated as follows:\u003c/p\u003e\u003cdiv id=\"Equc\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equc\" name=\"EquationSource\"\u003e\n$$\\:\\text{L}\\text{W}\\text{C}=\\frac{\\text{F}\\text{W}-\\text{D}\\text{W}}{\\text{F}\\text{W}}$$\u003c/div\u003e\u003c/div\u003e\u003cp\u003eA 0.2 g leaf sample was cut into pieces and extracted in 95% ethanol for 24 h in darkness. Absorbance at 665 nm and 649 nm was measured using a UV-Vis spectrophotometer (PerkinElmer, USA). Chlorophyll a and b concentrations were calculated using the following equations: Chl a = 13.95 × A\u003csub\u003e665\u003c/sub\u003e − 6.88 × A\u003csub\u003e649\u003c/sub\u003e; Chl b = 24.96 × A\u003csub\u003e649\u003c/sub\u003e − 7.32 × A\u003csub\u003e665\u003c/sub\u003e, where A\u003csub\u003e665\u003c/sub\u003e and A\u003csub\u003e649\u003c/sub\u003e denote absorbance readings at their respective wavelengths. Total chlorophyll content (mg·dm\u003csup\u003e-2\u003c/sup\u003e) was calculated using the formula: Total Chl = (C × V × N) / LA, where C represents the pigment concentration, V is the extraction volume, N is the dilution factor, and LA denotes total leaf area.Three replicates were performed for each species.\u003c/p\u003e\u003cp\u003eData analyses\u003c/p\u003e\u003cp\u003eData on net CO\u003csub\u003e2\u003c/sub\u003e exchange, δ\u003csup\u003e13\u003c/sup\u003eC, ΔH\u003csup\u003e+\u003c/sup\u003e, leaf anatomical traits, and mesophyll succulent index were organized using Microsoft Excel 2019. Pearson correlation analyses (two-tailed) were conducted using SPSS Statistics 27.0 to evaluate linear relationships among variables. Graphing and curve fitting were performed using Origin 2021.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eDiurnal patterns of net CO\u003csub\u003e2\u003c/sub\u003e exchange\u003c/p\u003e \u003cp\u003eThe nine \u003cem\u003eDendrobium\u003c/em\u003e species exhibited distinct diurnal patterns of net CO\u003csub\u003e2\u003c/sub\u003e exchange (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). \u003cem\u003eD. cariniferum\u003c/em\u003e, \u003cem\u003eD. gibsonii\u003c/em\u003e and \u003cem\u003eD. hancockii\u003c/em\u003e maintained a negative net CO\u003csub\u003e2\u003c/sub\u003e exchange throughout the entire night, which aligns with a typical C\u003csub\u003e3\u003c/sub\u003e photosynthetic pattern. In contrast, the remaining six species, including \u003cem\u003eD. parishii\u003c/em\u003e, \u003cem\u003eD. aphyllum\u003c/em\u003e, \u003cem\u003eD. anosmum\u003c/em\u003e, \u003cem\u003eD. linawianum\u003c/em\u003e, \u003cem\u003eD. moschatum\u003c/em\u003e and \u003cem\u003eD. crystallinum\u003c/em\u003e, displayed positive net CO\u003csub\u003e2\u003c/sub\u003e exchange during part or all of the nighttime, indicating varying degrees of CAM-like characteristics. Among these species, \u003cem\u003eD. parishii\u003c/em\u003e, \u003cem\u003eD. aphyllum\u003c/em\u003e and \u003cem\u003eD. anosmum\u003c/em\u003e exhibited high nighttime CO\u003csub\u003e2\u003c/sub\u003e exchange, each reaching its peak during the late-night or early-morning hours. \u003cem\u003eD. linawianum\u003c/em\u003e and \u003cem\u003eD. crystallinum\u003c/em\u003e demonstrated weak but detectable nighttime exchange primarily between 18:00 and 22:00. \u003cem\u003eD. moschatum\u003c/em\u003e maintained a positive net CO\u003csub\u003e2\u003c/sub\u003e exchange throughout the entire 24-hour cycle, although the magnitude of exchange was low during the night. These results highlight substantial variation in diel carbon acquisition strategies among the studied species.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCarbon isotope ratio and titratable acidity contents\u003c/p\u003e \u003cp\u003eCarbon isotope ratios (δ\u003csup\u003e13\u003c/sup\u003eC) exhibited variability among the species studied (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). \u003cem\u003eD. parishii\u003c/em\u003e, \u003cem\u003eD. aphyllum\u003c/em\u003e and \u003cem\u003eD. anosmum\u003c/em\u003e presented δ\u003csup\u003e13\u003c/sup\u003eC values exceeding \u0026minus;\u0026thinsp;20\u0026permil;, aligning with the characteristic range of CAM species. Conversely, the remaining species displayed δ\u003csup\u003e13\u003c/sup\u003eC values below \u0026minus;\u0026thinsp;20\u0026permil;, a range typically associated with C\u003csub\u003e3\u003c/sub\u003e plants. While δ\u003csup\u003e13\u003c/sup\u003eC alone cannot conclusively differentiate C\u003csub\u003e3\u003c/sub\u003e species from potential C\u003csub\u003e3\u003c/sub\u003e/CAM intermediates, the observed patterns provides significant long-term evidence of variations in carbon assimilation strategies.\u003c/p\u003e \u003cp\u003eThe titratable acidity difference (ΔH\u003csup\u003e+\u003c/sup\u003e) was positive across all species, indicating an increase in titratable acidity during the night (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). \u003cem\u003eD. parishii\u003c/em\u003e, \u003cem\u003eD. aphyllum\u003c/em\u003e, \u003cem\u003eD. anosmum\u003c/em\u003e and \u003cem\u003eD. crystallinum\u003c/em\u003e recorded the highest ΔH\u003csup\u003e+\u003c/sup\u003e values, each above 45 \u0026micro;mol(H\u003csup\u003e+\u003c/sup\u003e)\u0026middot;g\u003csup\u003e-1\u003c/sup\u003e FW, with \u003cem\u003eD. aphyllum\u003c/em\u003e reaching a maximum value of 143.544 \u0026micro;mol(H\u003csup\u003e+\u003c/sup\u003e)\u0026middot;g\u003csup\u003e-1\u003c/sup\u003e FW. In contrast, the other species exhibited lower ΔH\u003csup\u003e+\u003c/sup\u003e values, all below 19 \u0026micro;mol(H\u003csup\u003e+\u003c/sup\u003e)\u0026middot;g\u003csup\u003e-1\u003c/sup\u003e FW, with \u003cem\u003eD. hancockii\u003c/em\u003e showing the lowest ΔH\u003csup\u003e+\u003c/sup\u003e at 1.088 \u0026micro;mol(H\u003csup\u003e+\u003c/sup\u003e)\u0026middot;g\u003csup\u003e-1\u003c/sup\u003e FW.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLeaf anatomical structure\u003c/p\u003e \u003cp\u003eAll \u003cem\u003eDendrobium\u003c/em\u003e species exhibited fundamental anatomical characteristics, including a single-layered upper and lower epidermis, a compact mesophyll region and well-developed vascular tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Notably, mesophyll cells lacked distinct palisade and spongy differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Variations in leaf thickness were observed among species, with \u003cem\u003eD. parishii\u003c/em\u003e exhibiting the greatest total leaf thickness at 766.90 \u0026micro;m, including a mesophyll thickness of 714.07 \u0026micro;m. \u003cem\u003eD. anosmum\u003c/em\u003e and \u003cem\u003eD. aphyllum\u003c/em\u003e also displayed relatively thick leaves, both exceeding 500 \u0026micro;m. In contrast, \u003cem\u003eD. cariniferum\u003c/em\u003e demonstrated moderate leaf thickness, while \u003cem\u003eD. linawianum\u003c/em\u003e had the thinnest leaves, measuring 221.33 \u0026micro;m. These findings indicate that the species exhibited significant differences in their leaf anatomical parameters.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMesophyll succulent index\u003c/p\u003e \u003cp\u003eThe mesophyll succulent index (Sm) varied among the nine species (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). \u003cem\u003eD. parishii\u003c/em\u003e, \u003cem\u003eD. anosmum\u003c/em\u003e, \u003cem\u003eD. gibsonii\u003c/em\u003e and \u003cem\u003eD. moschatum\u003c/em\u003e exhibited Sm values greater than 1.0. \u003cem\u003eD. aphyllum\u003c/em\u003e, \u003cem\u003eD. linawianum\u003c/em\u003e and \u003cem\u003eD. crystallinum\u003c/em\u003e showed intermediate values of 0.98, 0.95 and 0.78 respectively. \u003cem\u003eD. cariniferum\u003c/em\u003e and \u003cem\u003eD. hancockii\u003c/em\u003e had lower values of 0.13 and 0.28. These results indicate that the species did not share the same degree of mesophyll succulence, with Sm values distributed across a broad range.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCorrelation analysis\u003c/p\u003e \u003cp\u003eNighttime net CO\u003csub\u003e2\u003c/sub\u003e exchange (NNEE) exhibited a strong positive correlation with δ\u003csup\u003e13\u003c/sup\u003eC (r\u0026thinsp;=\u0026thinsp;0.894). Additionally, δ\u003csup\u003e13\u003c/sup\u003eC was positively correlated with ΔH\u003csup\u003e+\u003c/sup\u003e (r\u0026thinsp;=\u0026thinsp;0.805), while NNEE showed a moderate positive relationship with ΔH\u003csup\u003e+\u003c/sup\u003e (r\u0026thinsp;=\u0026thinsp;0.676). These three physiological indicators demonstrated coordinated changes across the species. Leaf thickness (LT) was positively related to these physiological parameters, with strong correlations observed NNEE (r\u0026thinsp;=\u0026thinsp;0.877) and δ\u003csup\u003e13\u003c/sup\u003eC (r\u0026thinsp;=\u0026thinsp;0.864), and a moderate correlation with ΔH\u003csup\u003e+\u003c/sup\u003e (r\u0026thinsp;=\u0026thinsp;0.613). In contrast, the mesophyll succulent index (Sm) displayed weaker relationships with physiological traits, evidenced by correlation coefficients of 0.649 with δ\u003csup\u003e13\u003c/sup\u003eC and 0.521 with NNEE. These findings indicate that physiological traits are more closely interrelated than structural traits (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eJudgment of photosynthetic carbon assimilation pathways\u003c/p\u003e \u003cp\u003eContinuous monitoring of diurnal net CO\u003csub\u003e2\u003c/sub\u003e exchange provides a reliable means of determining whether plants exhibit nocturnal CO\u003csub\u003e2\u003c/sub\u003e exchange, a the key criterion for identifying CAM activity (van Tongerlo et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In this study, \u003cem\u003eD. parishii\u003c/em\u003e, \u003cem\u003eD. aphyllum\u003c/em\u003e and \u003cem\u003eD. anosmum\u003c/em\u003e maintained positive net CO\u003csub\u003e2\u003c/sub\u003e exchange throughout the night, demonstrating the characteristic sequence of CAM phases II, III and IV during the night-to-morning transition. This pattern reflects coordinated nocturnal CO\u003csub\u003e2\u003c/sub\u003e fixation and daytime decarboxylation, corroborating observations in typical CAM epiphytic orchid reported previously (Ceusters et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Hogewoning et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These species appear capable of fixing CO\u003csub\u003e2\u003c/sub\u003e at night through phosphoenolpyruvate carboxylase (PEPC), which supplies carbon for daytime metabolism while minimizing water loss, a strategy particularly advantageous in seasonally dry and warm environments (Zhang et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In contrast, \u003cem\u003eD. cariniferum\u003c/em\u003e, \u003cem\u003eD. gibsonii\u003c/em\u003e and \u003cem\u003eD. hancockii\u003c/em\u003e exhibited negative net CO\u003csub\u003e2\u003c/sub\u003e exchange from night to early morning, consistent with the C\u003csub\u003e3\u003c/sub\u003e pathway. Their diel rhythms align with those of typical C\u003csub\u003e3\u003c/sub\u003e plants, where CO\u003csub\u003e2\u003c/sub\u003e is assimilated through Rubisco during the day and released via respiration at night. These species are commonly found in shaded and humid habitats, where sufficient water availability supports continuous daytime CO\u003csub\u003e2\u003c/sub\u003e exchange and growth (Winter et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e1983\u003c/span\u003e). \u003cem\u003eD. linawianum\u003c/em\u003e, \u003cem\u003eD. moschatum\u003c/em\u003e and \u003cem\u003eD. crystallinum\u003c/em\u003e exhibited diel patterns intermediate between C\u003csub\u003e3\u003c/sub\u003e and CAM species. They relied primarily on C\u003csub\u003e3\u003c/sub\u003e photosynthesis during the day, although weak nocturnal CO\u003csub\u003e2\u003c/sub\u003e exchange was observed during certain nighttime periods. This suggests a limited capacity for nocturnal carboxylation that may be activated under specific environmental conditions to enhance carbon gain and water-use efficiency. Similar patterns have been documented in other facultative or intermediate C\u003csub\u003e3\u003c/sub\u003e/CAM species, such as \u003cem\u003eClusia minor\u003c/em\u003e and \u003cem\u003eMesembryanthemum crystallinum\u003c/em\u003e, which modify their carbon assimilation pathways in response to environmental fluctuations (Borland et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Winter and Holtum \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). This flexibility is regarded as a crucial adaptive trait in environments characterized by intermittent drought or periodic variations in water availability.\u003c/p\u003e \u003cp\u003eCarbon isotope ratios provide additional long-term physiological evidence for distinguishing photosynthetic pathways. Typical C\u003csub\u003e3\u003c/sub\u003e plants generally exhibit δ\u003csup\u003e13\u003c/sup\u003eC values ranging from \u0026minus;\u0026thinsp;33 to \u0026minus;\u0026thinsp;22.1\u0026permil;, while strong CAM species typically fall between \u0026minus;\u0026thinsp;22 and \u0026minus;\u0026thinsp;12\u0026permil; (Elheringer and Osmond 1989; Pearcy et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Silvera et al. (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) demonstrated a bimodal distribution of δ13C values in orchid, with C\u003csub\u003e3\u003c/sub\u003e species centered around \u0026minus;\u0026thinsp;28\u0026permil; and CAM species near \u0026minus;\u0026thinsp;16\u0026permil;. Intermediate values reflect C\u003csub\u003e3\u003c/sub\u003e/CAM species that respond to environmental stress. Studies on \u003cem\u003eDendrobium\u003c/em\u003e and related \u003cem\u003eorchid\u003c/em\u003e have also shown a clear bimodal pattern with an inflection point near \u0026minus;\u0026thinsp;20\u0026permil;, and many recent works use δ\u003csup\u003e13\u003c/sup\u003eC values greater than \u0026minus;\u0026thinsp;20\u0026permil; as indicators of CAM activity (Messerschmid et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In the present study, \u003cem\u003eD. parishii\u003c/em\u003e, \u003cem\u003eD. aphyllum\u003c/em\u003e and \u003cem\u003eD. anosmum\u003c/em\u003e exhibited δ\u003csup\u003e13\u003c/sup\u003eC values of \u0026minus;\u0026thinsp;14.38\u0026permil;, \u0026minus;\u0026thinsp;15.48\u0026permil; and \u0026minus;\u0026thinsp;15.95\u0026permil;, respectively, which are higher than those typical of C\u003csub\u003e3\u003c/sub\u003e plants and suggest the presence of nocturnal CO\u003csub\u003e2\u003c/sub\u003e fixation. In contrast, \u003cem\u003eD. cariniferum\u003c/em\u003e, \u003cem\u003eD. gibsonii\u003c/em\u003e and \u003cem\u003eD. hancockii\u003c/em\u003e displayed δ\u003csup\u003e13\u003c/sup\u003eC values below \u0026minus;\u0026thinsp;26\u0026permil;, consistent with the C\u003csub\u003e3\u003c/sub\u003e pathway and aligned with their negative nocturnal CO\u003csub\u003e2\u003c/sub\u003e exchange. \u003cem\u003eD. linawianum\u003c/em\u003e, \u003cem\u003eD. moschatum\u003c/em\u003e and \u003cem\u003eD. crystallinum\u003c/em\u003e had δ\u003csup\u003e13\u003c/sup\u003eC values ranging from \u0026minus;\u0026thinsp;27 to \u0026minus;\u0026thinsp;29\u0026permil;, which fall within the C\u003csub\u003e3\u003c/sub\u003e range according to isotope-based classification. Although these values indicate a predominantly C\u003csub\u003e3\u003c/sub\u003e carbon source, δ\u003csup\u003e13\u003c/sup\u003eC alone may not fully capture the potential for inducible CAM activity, as intermediate C\u003csub\u003e3\u003c/sub\u003e/CAM species often exhibit C\u003csub\u003e3\u003c/sub\u003e-like δ\u003csup\u003e13\u003c/sup\u003eC signatures under non-stress conditions (Borland et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Research on facultative CAM species has shown that δ13C values frequently resemble those of C\u003csub\u003e3\u003c/sub\u003e plants when CAM expression is weak and may only shift under conditions of severe drought or light stress (Ricalde et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). When considered alongside diel CO\u003csub\u003e2\u003c/sub\u003e exchange patterns, our results suggest that \u003cem\u003eD. linawianum\u003c/em\u003e, \u003cem\u003eD. moschatum\u003c/em\u003e and \u003cem\u003eD. crystallinum\u003c/em\u003e primarily rely on C\u003csub\u003e3\u003c/sub\u003e photosynthesis under normal conditions but may activate CAM activity when exposed to environmental stress. Similar responses have been documented in \u003cem\u003eAgave deserti\u003c/em\u003e, which functions as a C\u003csub\u003e3\u003c/sub\u003e species in moist environments but exhibits weak CAM under drought or high irradiance (Hartsock and Nobel \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1976\u003c/span\u003e). Therefore, while δ\u003csup\u003e13\u003c/sup\u003eC values provide valuable insights into long-term carbon assimilation patterns, additional physiological indicators are necessary for accurately determining photosynthetic pathways.\u003c/p\u003e \u003cp\u003eAs a central feature of the CAM pathway, nocturnal titratable acidity accumulation reflects the carboxylation activity of phosphoenolpyruvate carboxylase (PEPC) (Borland and Taybi \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). In the present study, the analysis of ΔH\u003csup\u003e+\u003c/sup\u003e among nine \u003cem\u003eDendrobium\u003c/em\u003e species revealed differences among species, indicating diversity in their nocturnal organic acid metabolism. \u003cem\u003eD. parishii\u003c/em\u003e, \u003cem\u003eD. aphyllum\u003c/em\u003e and \u003cem\u003eD. anosmum\u003c/em\u003e had ΔH\u003csup\u003e+\u003c/sup\u003e values of 143.54, 112.33 and 46.37 \u0026micro;mol(H\u003csup\u003e+\u003c/sup\u003e)\u0026middot;g\u003csup\u003e-1\u003c/sup\u003e FW respectively, indicating day\u0026ndash;night variations in organic acid content and suggesting that these species synthesize organic acids during the night. In contrast, \u003cem\u003eD. cariniferum\u003c/em\u003e, \u003cem\u003eD. gibsonii\u003c/em\u003e and \u003cem\u003eD. hancockii\u003c/em\u003e variations low ΔH\u003csup\u003e+\u003c/sup\u003e values, with \u003cem\u003eD. hancockii\u003c/em\u003e recording a mere 1.088 \u0026micro;mol(H\u003csup\u003e+\u003c/sup\u003e)\u0026middot;g\u003csup\u003e-1\u003c/sup\u003e FW. These low values suggest that these species do not accumulate organic acids to a meaningful extent during the night, which aligns with the characteristics typically observed in C\u003csub\u003e3\u003c/sub\u003e plants. Although C\u003csub\u003e3\u003c/sub\u003e species generally do not build up large pools of organic acids overnight, small changes in acidity can occur due to of dark respiration and normal metabolic rhythms, potentially explaining their slightly positive ΔH\u003csup\u003e+\u003c/sup\u003e values rather than indicating CAM-related CO\u003csub\u003e2\u003c/sub\u003e fixation (Br\u0026auml;utigam et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). \u003cem\u003eD. linawianum\u003c/em\u003e, \u003cem\u003eD. moschatum\u003c/em\u003e and \u003cem\u003eD. crystallinum\u003c/em\u003e had ΔH\u003csup\u003e+\u003c/sup\u003e values situated between those of the CAM-like and C\u003csub\u003e3\u003c/sub\u003e groups, indicating modest nocturnal organic acid accumulation and suggesting that these species may activate PEPC-mediated carboxylation specific particular environmental conditions. Silvera et al. (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) examined 173 \u003cem\u003eorchid\u003c/em\u003e species from Panama under drought conditions and reported broad and overlapping ranges of ΔH\u003csup\u003e+\u003c/sup\u003e, with obligate CAM species displaying values from 10.7 to 275.7 \u0026micro;mol(H\u003csup\u003e+\u003c/sup\u003e)\u0026middot;g\u003csup\u003e-1\u003c/sup\u003e FW and facultative CAM species showing values from 1.7 to 36.1 \u0026micro;mol(H\u003csup\u003e+\u003c/sup\u003e)\u0026middot;g\u003csup\u003e-1\u003c/sup\u003e FW. This pattern is broadly consistent with the trends observed in the present study. Although the ΔH\u003csup\u003e+\u003c/sup\u003e values of the intermediate \u003cem\u003eDendrobium\u003c/em\u003e species in this study are lower than those of strong CAM plants, their ability to accumulate organic acids during the night indicates that they may activate components of the CAM pathway in response to water or light stress.\u003c/p\u003e \u003cp\u003eThe integrated analysis of diurnal net CO\u003csub\u003e2\u003c/sub\u003e exchange, δ\u003csup\u003e13\u003c/sup\u003eC values and titratable acid accumulation provides a systematic basis for elucidating the carbon assimilation pathways of the nine \u003cem\u003eDendrobium\u003c/em\u003e species. These indicators represent complementary aspects of carbon metabolism, reflecting short-term gas exchange, long-term carbon source patterns and aspects biochemical processes. Their combined application facilitates the identification of C\u003csub\u003e3\u003c/sub\u003e, CAM and intermediate C\u003csub\u003e3\u003c/sub\u003e/CAM types (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). \u003cem\u003eD. parishii\u003c/em\u003e, \u003cem\u003eD. aphyllum\u003c/em\u003e and \u003cem\u003eD. anosmum\u003c/em\u003e exhibited consistent CAM characteristics across all indicators. Conversely, \u003cem\u003eD. cariniferum\u003c/em\u003e, \u003cem\u003eD. gibsonii\u003c/em\u003e and \u003cem\u003eD. hancockii\u003c/em\u003e demonstrated full alignment with the C\u003csub\u003e3\u003c/sub\u003e pathway. Meanwhile, \u003cem\u003eD. linawianum\u003c/em\u003e, \u003cem\u003eD. moschatum\u003c/em\u003e and \u003cem\u003eD. crystallinum\u003c/em\u003e displayed intermediate characteristics across all indicators and may activate components of the CAM pathway under specific environmental conditions.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDetermination of photosynthetic carbon assimilation pathway types in nine \u003cem\u003eDendrobium\u003c/em\u003e species\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNet CO\u003csub\u003e2\u003c/sub\u003e exchange\u003c/p\u003e \u003cp\u003ephenotype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eδ\u003csup\u003e13\u003c/sup\u003eC\u003c/p\u003e \u003cp\u003ephenotype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eΔH\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003cp\u003ephenotype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eOverall assessment\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eD.\u0026nbsp;parishii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCAM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCAM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCAM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCAM\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eD. aphyllum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCAM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCAM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCAM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCAM\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eD. anosmum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCAM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCAM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCAM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCAM\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eD. cariniferum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eC\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eD. gibsonii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eC\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eD. hancockii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eC\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eD. linawianum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCAM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCAM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eC\u003csub\u003e3\u003c/sub\u003e/CAM\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eD. moschatum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCAM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCAM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eC\u003csub\u003e3\u003c/sub\u003e/CAM\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eD. crystallinum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCAM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCAM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eC\u003csub\u003e3\u003c/sub\u003e/CAM\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eDiscussion on leaf anatomical structure and the mesophyll succulent indexs\u003c/p\u003e \u003cp\u003eLeaf thickness is an important anatomical characteristic associated with CAM activity, as CAM species are often linked to succulent tissues. Succulence has been documented in numerous CAM lineages across various plant families, including \u003cem\u003eGeraniaceae\u003c/em\u003e, \u003cem\u003eOrchidaceae\u003c/em\u003e and \u003cem\u003eClusiaceae\u003c/em\u003e (Jones et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Barrera Zambrano et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Increased tissue succulence in many drought-adapted CAM plants offers advantages by enhancing water-storage capacity compared to C\u003csub\u003e3\u003c/sub\u003e and C\u003csub\u003e4\u003c/sub\u003e species. A comparative analysis of tropical \u003cem\u003eorchid\u003c/em\u003e species indicated that obligate CAM plants typically exhibit the greatest leaf thickness (Silvera et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). In \u003cem\u003eM. crystallinum\u003c/em\u003e, leaf succulence also increases during the transition from C\u003csub\u003e3\u003c/sub\u003e to CAM under salt treatment (Guan et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Recent studies further suggest that the evolution of C\u003csub\u003e3\u003c/sub\u003e/CAM intermediate species may not necessitate major anatomical reconfiguration (Yang et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Heyduk et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Winter proposed that obligate CAM plants require substantial anatomical modification, whereas C\u003csub\u003e3\u003c/sub\u003e/CAM intermediate species may operate CAM without such extensive structural changes, indicating that the transition from C\u003csub\u003e3\u003c/sub\u003e to CAM may proceed with minimal alteration to C\u003csub\u003e3\u003c/sub\u003e type anatomy (Winter and Holtum \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In this study, the nine \u003cem\u003eDendrobium\u003c/em\u003e species exhibited considerable variation in leaf thickness, which may correlate with their differing carbon assimilation pathways. \u003cem\u003eD. parishii\u003c/em\u003e, \u003cem\u003eD. aphyllum\u003c/em\u003e and \u003cem\u003eD. anosmum\u003c/em\u003e had the highest leaf thickness values (764.14, 584.20 and 551.83 \u0026micro;m, respectively), consistent with their classification as CAM species based on δ\u003csup\u003e13\u003c/sup\u003eC and net CO\u003csub\u003e2\u003c/sub\u003e exchange. Their well-developed leaf tissues likely provide the vacuolar space necessary for nocturnal malate storage and contribute to water conservation under fluctuating moisture conditions. However, certain observations deviate from traditional expectations. For example, \u003cem\u003eD. cariniferum\u003c/em\u003e exhibited a relatively high leaf thickness of 445.29 \u0026micro;m, yet both its δ\u003csup\u003e13\u003c/sup\u003eC value (\u0026minus;\u0026thinsp;29.762\u0026permil;) and net CO\u003csub\u003e2\u003c/sub\u003e exchange pattern clearly indicate a C\u003csub\u003e3\u003c/sub\u003e photosynthetic mode. This demonstrates that leaf thickness alone is not sufficient to determine the photosynthetic pathway in \u003cem\u003eDendrobium\u003c/em\u003e. Some C\u003csub\u003e3\u003c/sub\u003e species may develop thicker leaves as an adaptation to epiphytic microhabitats that experience high irradiance or nutrient limitation, without necessarily exhibiting CAM function. In contrast, species inferred to possess C\u003csub\u003e3\u003c/sub\u003e/CAM intermediate characteristics, such as \u003cem\u003eD. crystallinum\u003c/em\u003e and \u003cem\u003eD. linawianum\u003c/em\u003e, showed relatively low leaf thickness values (296.94 and 221.33 \u0026micro;m, respectively). These findings align with Winter\u0026rsquo;s hypothesis that intermediate species may operate inducible CAM within an essentially C\u003csub\u003e3\u003c/sub\u003e anatomical framework, relying on physiological and molecular regulation rather than major structural remodeling. Overall, leaf thickness in \u003cem\u003eDendrobium\u003c/em\u003e shows a general but not absolute association with CAM photosynthesis. Although obligate CAM species often possess thicker leaves, this trait does not reliably distinguish between C\u003csub\u003e3\u003c/sub\u003e, C\u003csub\u003e3\u003c/sub\u003e/CAM intermediate and CAM species on its own. The diversity of carbon assimilation pathways in the genus reflects long-term adaptation to heterogeneous microhabitats, shaped through coordinated evolution across anatomical, physiological and molecular levels.\u003c/p\u003e \u003cp\u003eThe mesophyll succulent index (Sm), a structural indicator reflecting the balance between water storage and photosynthetic investment (Ripley et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), provides additional anatomical evidence supporting the inferred photosynthetic types in this study. Sm values exhibited patterned variation across species groups, complementing interpretations based on physiological parameters. Species inferred to be typical CAM types (\u003cem\u003eD. anosmum\u003c/em\u003e, \u003cem\u003eD. aphyllum\u003c/em\u003e and \u003cem\u003eD. parishii\u003c/em\u003e) presented high Sm values (1.27, 0.98 and 1.06, respectively), consistent with their CAM physiology. A high Sm indicates substantial water-storage capacity relative to photosynthetic tissue, aligning with the requirements for nocturnal CO\u003csub\u003e2\u003c/sub\u003e fixation and malate accumulation, thereby supporting survival under periodic drought conditions. In contrast, C\u003csub\u003e3\u003c/sub\u003e species such as \u003cem\u003eD. gibsonii\u003c/em\u003e, \u003cem\u003eD. hancockii\u003c/em\u003e and D. \u003cem\u003ecariniferum\u003c/em\u003e exhibited varied Sm levels. The elevated Sm of \u003cem\u003eD. gibsonii\u003c/em\u003e (1.02) suggests that enhanced water storage is not exclusive to CAM species and may represent a drought-tolerance strategy in certain C\u003csub\u003e3\u003c/sub\u003e taxa. Meanwhile, the low Sm values of \u003cem\u003eD. hancockii\u003c/em\u003e (0.28) and D. cariniferum (0.13) correspond with their C\u003csub\u003e3\u003c/sub\u003e CO\u003csub\u003e2\u003c/sub\u003e exchange and minimal nocturnal acid accumulation. C\u003csub\u003e3\u003c/sub\u003e/CAM intermediate species (\u003cem\u003eD. linawianum\u003c/em\u003e, \u003cem\u003eD. moschatum\u003c/em\u003e and \u003cem\u003eD. crystallinum\u003c/em\u003e) displayed intermediate Sm values (0.78, 1.10 and 0.95), spanning the ranges observed in both CAM and C\u003csub\u003e3\u003c/sub\u003e species. For \u003cem\u003eD. crystallinum\u003c/em\u003e and \u003cem\u003eD. linawianum\u003c/em\u003e, Sm values near 1 suggest sufficient but not extreme succulence to support inducible CAM under specific environmental conditions, aligning with their moderate nocturnal acid accumulation and δ\u003csup\u003e13\u003c/sup\u003eC values remaining within the C\u003csub\u003e3\u003c/sub\u003e range. Notably, \u003cem\u003eD. moschatum\u003c/em\u003e had an Sm value similar to that of CAM species (1.10), yet its δ\u003csup\u003e13\u003c/sup\u003eC (\u0026minus;\u0026thinsp;28.453\u0026permil;) and low ΔH\u003csup\u003e+\u003c/sup\u003e (6.435 \u0026micro;mol(H\u003csup\u003e+\u003c/sup\u003e)\u0026middot;g\u003csup\u003e-1\u003c/sup\u003e FW) indicate only weak or inducible CAM expression. This suggests that certain \u003cem\u003eDendrobium\u003c/em\u003e species may develop anatomical characteristics suited to CAM, while the physiological engagement of CAM remains conditional.\u003c/p\u003e \u003cp\u003eCorrelation analysis between physiological and structural parameters\u003c/p\u003e \u003cp\u003eThe correlation analysis revealed that the physiological parameters employed to assess photosynthetic carbon assimilation types exhibited a high degree of consistency with one another. Nighttime net CO\u003csub\u003e2\u003c/sub\u003e exchange (NNEE), δ\u003csup\u003e13\u003c/sup\u003eC and the titratable acidity difference (ΔH\u003csup\u003e+\u003c/sup\u003e) were positively correlated, indicating that nocturnal CO\u003csub\u003e2\u003c/sub\u003e exchange, long-term carbon isotope composition and overnight organic acid accumulation reflect similar trends in carbon assimilation across species. This coherence suggests that physiological indicators effectively capture the distinctions among C\u003csub\u003e3\u003c/sub\u003e, CAM and C\u003csub\u003e3\u003c/sub\u003e/CAM intermediate types, provideing a reliable basis for pathway determination. In contrast, the correlations between structural traits and physiological indicators were more variable. Leaf thickness (LT) demonstrated moderate correlations with certain physiological traits, while the mesophyll succulent index (Sm) exhibited weaker overall correlations, and consistency among structural traits was limited. These patterns indicate that structural characteristics can reflect aspects of CAM activity but are not determinative, as species may adopt different structural configurations to achieve similar photosynthetic outcomes. Consequently, structural parameters alone are insufficient for accurate discrimination among C\u003csub\u003e3\u003c/sub\u003e, C\u003csub\u003e3\u003c/sub\u003e/CAM intermediate and CAM pathways. Overall, the correlation results underscore the central role of physiological parameters in determining carbon assimilation type, while structural traits serve more appropriately as supplementary evidence. This distinction elucidate why some C\u003csub\u003e3\u003c/sub\u003e/CAM intermediate species display transitional or inconspicuous anatomical features and supports the perspective that photosynthetic pathway differentiation involves multidimensional regulation and diverse adaptive strategies (Borland et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEcological adaptability in photosynthetic carbon assimilation pathway variation\u003c/p\u003e \u003cp\u003eThis study reveals that the photosynthetic carbon assimilation pathways of nine \u003cem\u003eDendrobium\u003c/em\u003e species exhibit a high degree of correlation with the characteristics of their native habitats. Based on descriptions from \u003cem\u003ePlants of the World Online\u003c/em\u003e and \u003cem\u003ethe Flora of China\u003c/em\u003e, these species are predominantly epiphytic or lithophytic orchids found in tropical and subtropical montane forests, open woodlands or rocky valleys (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Such habitats typically experience fluctuating water availability, nutrient-poor substrates and variable light conditions, all of which are known to promote CAM evolution in orchids (Nelson and Sage \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Silvera et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In this study, species identified as typical CAM types (\u003cem\u003eD. parishii\u003c/em\u003e, \u003cem\u003eD. aphyllum\u003c/em\u003e and \u003cem\u003eD. anosmum\u003c/em\u003e) are commonly found in environments characterized by pronounced dry\u0026ndash;wet seasonality, high irradiance and epiphytic or lithophytic growth forms, aligning with ecological conditions that favor CAM metabolism. Conversely, species characterized as C\u003csub\u003e3\u003c/sub\u003e/CAM intermediates (\u003cem\u003eD. crystallinum\u003c/em\u003e, \u003cem\u003eD. linawianum\u003c/em\u003e and \u003cem\u003eD. moschatum\u003c/em\u003e) often inhabit forest margins or areas with seasonally variable moisture, suggesting that their flexible photosynthetic strategies may reflect adaptive responses to these environments. In contrast, species classified as C\u003csub\u003e3\u003c/sub\u003e types (\u003cem\u003eD. cariniferum\u003c/em\u003e, \u003cem\u003eD. gibsonii\u003c/em\u003e and \u003cem\u003eD. hancockii\u003c/em\u003e) are found in shaded, humid montane forests, ravines or high-elevation moist environments where water availability is relatively stable, consistent with C\u003csub\u003e3\u003c/sub\u003e photosynthesis. Collectively, these findings indicate that the diversity of carbon assimilation pathways among the nine \u003cem\u003eDendrobium\u003c/em\u003e species corresponds to distinct habitat preferences, reflecting ecological differentiation. Although this study did not quantitatively analyze climatic variables, comparisons with habitat descriptions suggest that environmental factors such as water availability, growth form (epiphytic or lithophytic) and light conditions may serve as important selective pressures shaping carbon assimilation strategies in \u003cem\u003eDendrobium\u003c/em\u003e. Future research that integrates distribution data with environmental parameters may elucidate the mechanisms linking habitat characteristics to the evolution of photosynthetic pathways.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eNatural habitat distribution of nine \u003cem\u003eDendrobium\u003c/em\u003e species\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNative distribution\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHabitat type (as documented in the literature)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eReferences\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eD.\u0026nbsp;parishii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNortheast India, Myanmar, Thailand, Laos, Vietnam, China (Yunnan)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEpiphytic on trees in montane evergreen forests or on rocks; elevation 250\u0026ndash;1200 m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFlora of China;POWO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eD. aphyllum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNepal, Bhutan, India, Myanmar, Thailand, Laos, Vietnam, China (Yunnan)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEpiphytic or lithophytic in tropical moist forests; elevation 200\u0026ndash;1,500 m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFlora of China;POWO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eD. anosmum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhilippines, Malaysia, Laos, Vietnam, Myanmar, China (Yunnan)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEpiphytic in lowland to montane forests, often near riverbanks or humid valleys\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNew Guinea Orchids;POWO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eD. cariniferum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChina (Yunnan), Myanmar, Northeast India\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEpiphytic in humid montane forests; elevation 1,200\u0026ndash;2,000 m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFlora of China;POWO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eD. gibsonii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChina (Yunnan, Guangxi), India, Myanmar\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEpiphytic or lithophytic on cliffs, rocks or forest trunks; elevation 800\u0026ndash;1,500 m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFlora of China;POWO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eD. hancockii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChina (Yunnan, Guizhou, Guangxi), Vietnam, Northern Thailand\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEpiphytic or lithophytic in montane forests; elevation 700\u0026ndash;1,500 m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFlora of China;Smithsonian\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eD. linawianum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTaiwan, China (Guangdong)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEpiphytic in montane evergreen forests; elevation 700\u0026ndash;1,400 m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFlora of China;POWO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eD. moschatum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNortheast India, Nepal, Bhutan, Myanmar, China (Yunnan)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEpiphytic in cliffs or montane moist forests; elevation 1,000\u0026ndash;1,800 m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUseful Tropical Plants;POWO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eD. crystallinum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThailand, Myanmar, Laos, Vietnam, China (Yunnan, Guizhou, Hainan)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEpiphytic or lithophytic in montane forests with seasonal moisture variation; elevation 700\u0026ndash;1,700 m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFlora of China;POWO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, \u003cem\u003eD. parishii\u003c/em\u003e, \u003cem\u003eD. aphyllum\u003c/em\u003e and \u003cem\u003eD. anosmum\u003c/em\u003e exhibited characteristics consistent with typical CAM plants, including nocturnal net CO\u003csub\u003e2\u003c/sub\u003e exchange, δ\u003csup\u003e13\u003c/sup\u003eC values exceeding \u0026minus;\u0026thinsp;16\u0026permil; and substantial nocturnal titratable acidity accumulation (ΔH\u003csup\u003e+\u003c/sup\u003e \u0026gt; 46 \u0026micro;mol(H\u003csup\u003e+\u003c/sup\u003e)\u0026middot;g\u003csup\u003e-1\u003c/sup\u003e FW), and their anatomical traits, such as increased leaf thickness and elevated mesophyll succulent indices, further substantiated their classification within the CAM category. Conversely, \u003cem\u003eD. cariniferum\u003c/em\u003e, \u003cem\u003eD. gibsonii\u003c/em\u003e and \u003cem\u003eD. hancockii\u003c/em\u003e displayed characteristics indicative of C\u003csub\u003e3\u003c/sub\u003e plants, including the absence of nocturnal CO\u003csub\u003e2\u003c/sub\u003e exchange, δ\u003csup\u003e13\u003c/sup\u003eC values below \u0026minus;\u0026thinsp;26\u0026permil;, and minimal overnight acid accumulation. \u003cem\u003eD. linawianum\u003c/em\u003e, \u003cem\u003eD. moschatum\u003c/em\u003e and \u003cem\u003eD. crystallinum\u003c/em\u003e demonstrated intermediate C\u003csub\u003e3\u003c/sub\u003e/CAM characteristics; moreover, leaf thickness and mesophyll succulent inices were generally higher in most CAM species, although some overlap occurred with values from C\u003csub\u003e3\u003c/sub\u003e/CAM intermediates and C\u003csub\u003e3\u003c/sub\u003e species. Correlation analysis revealed that physiological indicators NNEE, δ\u003csup\u003e13\u003c/sup\u003eC and ΔH\u003csup\u003e+\u003c/sup\u003e were highly consistent with one another, reflecting a continuum of carbon assimilation strategies within the genus \u003cem\u003eDendrobium\u003c/em\u003e. In contrast, the associations between structural traits and physiological parameters were generally weaker. Leaf thickness exhibited moderate correspondence with specific physiological variables, while the mesophyll succulent index showed weak associations with physiological parameters and lacked a clear correlation with photosynthetic types. These findings indicate that structural traits alone are insufficient for reliably distinguishing between C\u003csub\u003e3\u003c/sub\u003e, C\u003csub\u003e3\u003c/sub\u003e/CAM intermediate and CAM species, and they should analyzed used in conjunction with physiological parameters for more accurate classification. The various photosynthetic types correspond to distinct habitat preferences: typical CAM species are commonly found in seasonally dry, high-light epiphytic or lithophytic environments; C\u003csub\u003e3\u003c/sub\u003e species are typically associated with humid and shaded conditions; while C\u003csub\u003e3\u003c/sub\u003e/CAM intermediate species thrive in habitats that experience alternating wet and dry conditions. This pattern reflects the relationship links between photosynthetic strategy and the availability of environmental moisture and light. Overall, the diversity of carbon assimilation pathways in \u003cem\u003eDendrobium\u003c/em\u003e illustrates both physiological plasticity and long-term ecological adaptation. This study offers a valuable foundation for ecological assessment, germplasm conservation and cultivation management of this orchid genus.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAuthors contributions\u003c/p\u003e\n\u003cp\u003eConceptualization, Z.Y., S.C. and H.P.; data curation, Z.Y. and N.C.; formal analysis, L.P.; investigation, N.C.; methodology, L.P. and Z.Y.; software, N.C. and Z.Y.; supervision, L.P., J.W., Q.J. and N.C.; writing\u0026mdash;original draft, D.T.; writing\u0026mdash;review and editing, Z.Y. and S.C. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Key Research and Development Program of China (2022YFF1300700), Guangxi Forestry Bureau Project (2024LYKJ01), and Guangxi Key Laboratory of Plant Functional Substances and Sustainable Utilization (ZRJJ2024-12).\u003c/p\u003e\n\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003eEthics, Consent to to participate\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eAll authors have approved the manuscript for submission to BMC Plant Biology.\u003c/p\u003e\n\u003cp\u003eClinical trial number\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eConflicts of Interest\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAtkinson JA, Wells DM (2017) An updated protocol for high throughput plant tissue sectioning. 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Plant Physiol 143:98-107. \u003c/li\u003e\n\u003cli\u003eWinter K, Holtum JA (2014) Facultative crassulacean acid metabolism (CAM) plants: powerful tools for unravelling the functional elements of CAM photosynthesis. J Exp Bot 65:3425-3441. \u003c/li\u003e\n\u003cli\u003eYang X, Liu D, Tschaplinski TJ, Tuskan GA (2019) Comparative genomics can provide new insights into the evolutionary mechanisms and gene function in CAM plants. J Exp Bot 70:6539-6547. \u003c/li\u003e\n\u003cli\u003eZhang S, Yang Y, Li J, Qin J, Zhang W, Huang W, Hu H (2018) Physiological diversity of orchids. Plant Diversity 40:196-208. \u003c/li\u003e\n\u003cli\u003eZhang S, Li J, Shen Y, Korkor LN, Pu Q, Lu J, Shakeela B, Kong D, Ou L, Zeng G (2020) Physiological responses of \u003cem\u003eDendrobium officinale\u003c/em\u003e under exposure to cold stress with two cultivars. Phyton 89:599. \u003c/li\u003e\n\u003cli\u003eZhang Z, He D, Niu G, Gao R (2014) Concomitant CAM and C\u003csub\u003e3\u003c/sub\u003e photosynthetic pathways in \u003cem\u003eDendrobium officinale\u003c/em\u003e plants. J Am Soc Hortic Sci 139: 290-298.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Dendrobium species, carbon assimilation pathways, crassulacean acid metabolism, ecological adaptability, leaf anatomy","lastPublishedDoi":"10.21203/rs.3.rs-8304845/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8304845/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTo analyze the diversity of photosynthetic carbon assimilation pathways and ecological adaptation characteristics in \u003cem\u003eDendrobium\u003c/em\u003e species, this study selected nine native Chinese \u003cem\u003eDendrobium\u003c/em\u003e species. A comprehensive approach was employed, integrating diurnal dynamics of net CO\u003csub\u003e2\u003c/sub\u003e exchange, carbon isotope ratios (δ\u003csup\u003e13\u003c/sup\u003eC), diurnal titratable acid accumulation (ΔH\u003csup\u003e+\u003c/sup\u003e), leaf anatomical structure, and mesophyll succulent index (Sm). The results indicate: that \u003cem\u003eDendrobium parishii\u003c/em\u003e, \u003cem\u003eDendrobium aphyllum\u003c/em\u003e, and \u003cem\u003eDendrobium anosmum\u003c/em\u003e maintain positive net CO\u003csub\u003e2\u003c/sub\u003e exchange at night, with δ\u003csup\u003e13\u003c/sup\u003eC values exceeding \u0026minus;\u0026thinsp;16\u0026permil; and high nocturnal acid accumulation. Combined with their substantial leaf thickness (LT) and Sm, these species are classified as typical CAM plants; \u003cem\u003eDendrobium cariniferum\u003c/em\u003e, \u003cem\u003eDendrobium gibsonii\u003c/em\u003e, and \u003cem\u003eDendrobium hancockii\u003c/em\u003e exhibited no positive CO\u003csub\u003e2\u003c/sub\u003e exchange throughout the night, with δ\u003csup\u003e13\u003c/sup\u003eC values below \u0026minus;\u0026thinsp;26\u0026permil; and low ΔH\u003csup\u003e+\u003c/sup\u003e levels, demonstrating physiological characteristics consistent with the C\u003csub\u003e3\u003c/sub\u003e pathway; Additionally, \u003cem\u003eDendrobium linawianum\u003c/em\u003e, \u003cem\u003eDendrobium moschatum\u003c/em\u003e, and \u003cem\u003eDendrobium crystallinum\u003c/em\u003e exhibited weak nocturnal CO\u003csub\u003e2\u003c/sub\u003e exchange and moderate to low acid accumulation. However, their δ\u003csup\u003e13\u003c/sup\u003eC values remained within the C\u003csub\u003e3\u003c/sub\u003e range, indicating they are not stable CAM types but may exhibit plastic expression under specific environmental conditions, displaying characteristics of intermediate C\u003csub\u003e3\u003c/sub\u003e/CAM photosynthetic plants. The LT and SM exhibited partial overlap between C\u003csub\u003e3\u003c/sub\u003e/CAM and strictly C\u003csub\u003e3\u003c/sub\u003e species, highlighting the limitations of using morphological traits as sole diagnostic criteria. Correlation analyses further revealed that although LT was significantly associated with Nighttime Net CO\u003csub\u003e2\u003c/sub\u003e Exchange (NNEE) and δ\u003csup\u003e13\u003c/sup\u003eC, the Sm displayed only weak correlations with key physiological indicators. In contrast, NNEE, δ\u003csup\u003e13\u003c/sup\u003eC, and ΔH\u003csup\u003e+\u003c/sup\u003e were strongly aligned with one another, underscoring that structural characteristics can serve only as supplementary references, while reliable classification must depend on the coordinated variation of physiological metrics. Furthermore, the photosynthetic types of the examined \u003cem\u003eDendrobium\u003c/em\u003e species closely corresponded to moisture availability and light conditions in their native habitats, reflecting the ecological plasticity of their carbon-assimilation strategies. In summary, \u003cem\u003eDendrobium\u003c/em\u003e species exhibit remarkable diversity and environmental plasticity in their photosynthetic carbon assimilation strategies. This study, through multi-indicator integrated assessment, not only clarifies the photosynthetic types of different species but also provides important references for identifying photosynthetic carbon assimilation pathways and cultivating \u003cem\u003eDendrobium\u003c/em\u003e species.\u003c/p\u003e","manuscriptTitle":"Integrated Multi-Trait Analysis of Photosynthetic Carbon Assimilation Pathways and Adaptive Patterns in Dendrobium Species","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-22 15:14:24","doi":"10.21203/rs.3.rs-8304845/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-01-11T11:50:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"96098291226201952697249505211545197550","date":"2026-01-04T00:38:34+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-31T03:09:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"163323168901188381816888764901730700568","date":"2025-12-30T08:10:59+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-19T09:19:47+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-10T17:13:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-09T12:27:37+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-09T12:26:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2025-12-08T07:30:31+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9523309b-6a5d-45e5-8202-1221346c4407","owner":[],"postedDate":"December 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-12-22T15:14:24+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-22 15:14:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8304845","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8304845","identity":"rs-8304845","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}