Field measurements were conducted in two study sites on Hainan Island in the south of China, which was considered a hotspot for mangrove research and conservation due to the highest mangrove diversity and the highest carbon stock of all mangrove forests in China (Li and Lee, 1997; Lin, 1999). Haikou (HK) and Sanya (SY), located in the Northern and Southern of Hainan Island, were selected as two main sites for this study. Haikou Dongzhaigang Mangrove Nature Reserve (113°57’E, 22°35’N) was established as the first National Mangrove Nature Reserve of China in 1986 and was then listed as one of the International Important Wetlands in 1992 (Qiu et al., 2011). Dongzhaigang Mangrove Nature Reserve is the first place where two exotic mangrove species (Sonneratia apetala, Laguncularia racemosa) were introduced and planted. The Laguncularia racemosa was introduced from Mexico in 1999 (Liao et al., 2006). Sanya Qingmeigang Mangrove Nature Reserve (109°36’E, 18°13’N) has a mangrove area of 64 ha^-1, which is the largest and best-preserved mangrove region in Sanya City. With a tropical monsoon climate, the mean annual temperature, mean annual precipitation and salinity were 23.8°C, 1685 mm, and 27‰ for the Haikou site, and 25.5°C, 1280 mm, 33‰ for the Sanya site, respectively.

Gas exchange parameters of leaves were measured using an LI-6400 portable photosynthesis system (Li-COR Inc., Lincoln, NE, USA). The exotic mangrove Laguncularia racemosa and the adjacent native mangroves (Avicennia marina, Aegiceras corniculatum, Bruguiera gymnorhiza, Kandelia obovata) with the same tree age (≈ two years) were chosen to measure the gas exchange parameters. All measurements were conducted between 09:00 and 15:00, with the ambient temperature ranging from 26 to 35°C in Summer. Conditions in the cuvette were maintained around 400 ppm CO₂ concentration, approximate local ambient CO₂ concentration, and photosynthetic active radiation was set at 1000 μmol photons m^-2 s^-1, with an airflow rate of 500 μmol s^-1. The assimilation rate (A), stomatal conductance (g_s ), transpiration rate (E), and CO₂ concentration (Ci) were recorded after stabilizing control conditions (≈ 30 mins) with a leaf inside the leaf cuvette. Instantaneous water use efficiency (WUEi) was calculated according to the ratio A/E. For each leaf, five consecutive measurements were averaged as one replicate value, and three to five replicates of individual plants were gathered for each mangrove species.

The mature and intact leaves of different species under the same environments were chosen to measure stomatal traits. For each leaf, a razor blade was used to scrape the leaf vein and mesophyll, leaving the leaf epidermis, which was then rinsed clean with deionized water. Small sections of the epidermis were cut, and drip stained on glass microscope slides. Under a fluorescence microscope (Nikon Eclipse Ci-L, Tokyo, Japan) at ×400 magnification, guard cell lengths were measured, and stomatal density was calculated as the number of stomata per field of view. Three fields were sampled for each leaf, and three to five leaves were measured for each species. Both sides of the leaf epidermis were imaged, and the stomatal density of both sides was calculated.

After measuring leaf gas exchange, we collected the plant samples from the exotic and the adjacent native mangroves. The leaf, stem, and root of each plant were separated and dried by oven at 70°C for 48 h until constant weight, then ground to powder using a grinding mill (Jingxin, JXFSTPRP-32, China), and passed through a 60-mesh sieve. The δ¹³C and δ¹⁵N were measured with an isotope ratio mass spectrometer (IRMS) (Delta V Advantage, Thermo Fisher Scientific, Inc., USA), the C and N content were measured by the elemental analyzer (Flash 2000 EA-HT, Thermo Finnigan, Bremen, Germany). The isotope composition of plant tissue (‰, in parts per thousand) was calculated as:

Where R_sample and R_standard are the isotope ratios of the plant tissue relative to PDB standard for δ¹³C and N₂-atm standard for δ¹⁵N, respectively. The δ¹³C and WUE were demonstrated as (Farquhar and Richards, 1984; Farquhar et al., 1989).

Where A is the assimilation rate of the plant, E is the transpiration rate. c_a and c_i are the partial pressures of CO₂ in the atmosphere and intercellular, respectively. v is the vapor pressure difference between the intercellular and the atmosphere.

where a is the fractionation caused by CO₂ diffusion in the air (4.4‰), b is the fractionation due to carboxylation (27‰), and δ¹³C_plant and δ¹³C_air are the carbon isotope composition of the plant and CO₂ in the atmosphere, respectively (Farquhar et al., 1982).

We applied linear and non-linear regressions to test the significance of correlations (p<0.05) between stomatal density and stomatal size. One-way analysis of variance (ANOVA) was used to evaluate the differences in gas exchange parameters among different mangrove species means. A Tukey’s HSD multiple comparison was used to test significance at α=0.05 level. Mean and standard error (S.E.) values of replicates were calculated for each variable. A multi-linear regression model and spearman correlation heat-matrix were created to explore the relationships among stomatal traits, photosynthesis characters by using the “ggplot2” and “ggcor” package in R 4.0.5. The conceptual diagram was made to summarize the physiological link of mangrove stomatal traits and other factors by using Adobe illustrator CC2018 (Adobe Systems Inc., San Jose, CA, USA). The statistical analyses and figures were carried out using Origin software 2019b. (OriginLab Corp., Northampton, MA, USA).

For the native mangrove K. obovata, the stomata were only found on the lower epidermis ( Figures 1A, C ). However, stomata are distributed on both the lower and upper epidermis of leaves in L. racemosa ( Figures 1B, D ). Furthermore, the stomatal density of the upper epidermis was higher than those of the lower epidermis in L. racemosa. Stomatal density tended to increase with the stomatal guard cell length decline, the negative relationship was best estimated (R^2 = 0.66, p<0.001) by a power function ( Figure 1E ). The stomatal density of exotic mangrove species is significantly higher than that of native mangrove species. However, the stomatal guard cell length of exotic mangroves is lower than that native mangrove species ( Figure 1E ).

Gas exchange parameters varied significantly between species. Specifically, the leaf CO₂ assimilation rate was significantly higher in exotic mangroves (Lr) compared with other native mangrove species ( Figure 2A ). Consistently, the stomatal conductance and transpiration of L. racemosa were significantly higher than those of native species ( Figures 2B, C ). Instantaneous water use efficiency in L. racemosa showed no significant difference when compared with B. gymnorhiza but was lower than other native mangrove species ( Figure 2D ). Remarkably, L. racemosa has the largest stomata density but the smallest stomata size ( Figures 2E, F ).

The δ ¹³C of leaf, stem, root in L. racemosa were all significantly lower than native mangrove both in the Haikou site and Sanya site ( Figures 3A, B ). In addition, the ⊿leaf of L. racemosa was significantly higher than those of native mangroves across two sites ( Figures 3C, D ). For the Haikou site, the value of δ ¹³C in L.racemosa increased in the order of root>stem>leaf, whereas the value of δ ¹³C in K. obovata was leaf >stem>root. the largest difference (≈3 ‰) in the carbon isotope discrimination between L. racemosa and K. obovata was observed in roots. The δ¹⁵N of L. racemosa was not significantly different from those of native mangroves ( Figures 3A, B ). For Sanya site, the δ ¹³C of L. racemosa were more negative than the natives species, but had a different pattern compared with the δ ¹³C of L. racemosa in the Haikou site, the value of δ ¹³C increased in the order of root >stem>leaf ( Figure 3B ). Furthermore, the range of δ ¹⁵N (root-stem-leaf) discrimination in L. racemosa (4.2‰-6.7‰) was wider than those in C. tagal (4.5‰-5.5‰) but lower than those of L. racemosa in Haikou site (6 ‰-10 ‰). And the leaf δ¹⁵N of L. racemosa was lower than those of all native mangrove species. Generally, the δ ¹³C of exotic mangrove (L. racemosa) was more negative than native mangrove. Meanwhile, the δ ¹³C discrimination of mangroves in Haikou was lower than mangroves in Sanya, whereas the δ ¹⁵N discrimination of mangroves in Haikou was higher than mangroves in Sanya.

The spearman correlation analyses show that stomata size negatively related to stomata density (R=-0.78). This pattern was similarly observed in WUE (R=-0.56) ( Figure 4A ). Generally, the stomata density was significantly positively correlated with A, E, Gs, whereas the negative coefficients were observed between stomata size and A, E, Gs ( Figure 4A ). However, the relationships among stomata size, stomata density and A, E, Gs were nonlinear ( Figure 4B , Figure S1 ). For instance, the WUE decreased slowly with low density but sharply declined with high density, which differed from the trend of stomata size ( Figure 4B ). With the increase of stomata density, conductance increased rapidly with low density and then stably rose with high density, thus leading to an increase in photosynthesis rate and transpiration ( Figure 4B ). However, the transpiration increased more in high-density samples compared to the trend of stomatal conductance and photosynthesis rate ( Figure 4B ).

The relationships between stomata size and stomata density exert a significant influence on plant growth and productivity through regulating stomatal conductance and transpiration, which control the carbon, water, nutrient cycling between plant and environment. We found that the relationship between stomata size and stomata density in mangrove was best described by a negative power function ( Figure 1E ), which was consistent with the previous studies (Franks and Beerling, 2009). Besides, we observed that stomata can be found on both the lower and upper epidermis of the leaves of L. racemosa, whereas stomata exist only on the lower epidermis in most native mangrove species ( Figure 1 ). Also, the exotic mangrove species tend to have higher stomata density and smaller stomata size than the native mangrove species. The negative correlation between stomata size and stomata density of mangrove may be explained by the physical constraints and energy strategy (Franks et al., 2009). On one hand, the limited space of leaves requires the allocation of stomata with the sufficient density and size to achieve the optimized conductance for photosynthesis. On the other hand, the plants balance photosynthetic productivity (energy gain) and respiration (energy loss) through the evolution of stomatal properties.

The higher stomata density corresponding to a higher photosynthetic rate, and stomatal conductance was observed in the L. racemosa ( Figures 2A, B, F ). The same trend can be found in other mangrove species, namely, that higher stomata density tends to increase stomatal conductance and photosynthetic rate, and that lower stomata density corresponds with a down-regulation of conductance ( Figure 4 ). This trend is consistent with the previous study by (Franks et al., 2009), which showed an enhancement of photosynthetic rate and conductance limited by the relationship between stomatal size and density. This can be explained by the negative relationship between stomata size and density, the allocation traits of stoma constrained by the space-optimized rule. In order to enhance stomatal conductance, plants tend to allocate the small size but high density stoma to optimize the space (Dow et al., 2014). With the higher conductance and photosynthesis of L. racemosa, the transpiration increase results in a decline in instantaneous water use efficiency (WUEi) compared with native mangroves. Although increasing conductance supplies additional CO₂ and water for carboxylation, more water is lost through the more numerous stomata. Several previous studies have verified that conductance plays an important role in regulating the WUE, leading to a negative relationship between conductance and WUE ( Figure 4 ) (Franks et al., 2015; Dunn et al., 2019).

However, it should be pointed out that mangroves are restricted by salinity due to the saline environment imposing a negative effect on photosynthesis and conductance through regulating the opening and closure of stoma (Ball and Farquhar, 1984; Parida et al., 2004). This suggests that stomata traits and gas exchange parameters of mangrove will shift with environmental changes (Sperry et al., 2016; Dong et al., 2020). Mangroves, therefore, have evolved salt-tolerant function such as salt exclusion in the root, salt secretion by salt glands, osmolyte accumulation to adapt to the high salinity of seawater (Parida and Jha, 2010; Jiang et al., 2017; Cheng et al., 2020). Even so, only a few mangrove species, such as Aegiceras corniculatum, Avicennia marina, have specialized salt glands. Interestingly, we found that L. racemosa has a good salt secretion function owing to abundant salt glands and aqueous tissues distributed in the leaves (Chen et al., 2018; Li and Lin, 2006). Thus, we concluded that despite the lower WUE, there is no strong limit on water supply for L. racemosa compared to native mangroves. These results may explain why L. racemosa possesses a high salt tolerance.

The evidence from the isotope also confirmed that L. racemosa has a lower WUE (more negative δ¹³C) but higher photosynthetic capacity compared with other native mangrove species ( Figure 3 ). The higher nitrogen (N) content and lower carbon-nitrogen ratio (C:N) of L. racemosa suggest a higher nitrogen uptake efficiency and photosynthetic nitrogen use efficiency (NUE) compared to native species ( Figure S2 ). Moreover, we found that a spatial pattern exists in the δ¹³C and δ¹⁵N values, namely, the δ¹³C discrimination of mangrove in Haikou site (low salinity) were lower than mangroves in Sanya site (high salinity), whereas the δ¹⁵N discrimination of mangroves in Haikou were higher than mangroves in Sanya ( Figure 3 ). The lower foliar δ¹³C of mangrove reflect a lower WUE in Haikou sites due to the lower salt stress. Foliar δ¹⁵N values serve as an indicator of the supply of N relative to plant N demand, and the lower δ¹⁵N values represents a greater N limitation (Craine et al., 2018). The results of δ¹⁵N shows that the mangroves in Haikou exhibited a lower N limitation compared to the mangroves in Sanya, which was caused by lower salt stress and higher N availability in Haikou mangrove habitats (Bai et al., 2021).

Previous studies have reported that L. racemosa was more invasive than other mangrove species due to fast growth and high salinity tolerance (Gu et al., 2019). However, the potential mechanism of fast growth is still unclear. As Figure 5 shows, the foliar stomata density of L. racemosa is higher than that of native mangrove species, particularly on the upper epidermis, which yields two important physiological functions for its fast growth. On one hand, high stomata density enhances the conductance thus supplies more CO₂ and H₂O to support the carboxylation. On the other hand, having numerous stomata on the upper epidermis (chloroplasts distribution) shortens the diffusion path of CO₂ and H₂O. Consequently, more CO₂ diffuses into the chloroplasts thus enhancing the rate of photosynthesis. Meanwhile, the transpiration increases with the conductance increasing, which results in more water loss thus decline of WUE ( Figures 2D , 3 ). This is consistent with the study by Tian et al. (2016). The study shows that stomatal density was positively correlated with vein density thereby balancing the leaf-level water transpiration and supply. As mentioned above, however, the lower WUE has no strong restriction on water supply due to the efficient salt secretion function and aqueous tissues of L. racemosa. Furthermore, the increasing transpiration alters the water potential through the plant and accelerates the water transport and nutrient uptake in L. racemosa. It thereby maintains foliar nutrient supply and water balance (Cernusak et al., 2011; Ocheltree et al., 2016; Lu et al., 2018). As a result, the N content of the L. racemosa is higher than those of native mangroves but the C:N ratio is significantly lower than native species, especially for foliar N content ( Figure S2 ), which provides further evidence to verify the advantages of water and nutrient transport and allocation in L. racemosa.

The ongoing rising atmospheric CO₂ (C_a), is expected to affect the stomatal traits of plants, and thus regulate the gas-exchange and energy balance, such as carbon, water, and nutrient cycling of plants (Voelker et al., 2016). Studies from experiments have reported that the water use efficiency tends to increase with a rise in atmospheric CO₂ by decreasing the stomatal conductance, thus enhancing assimilation rates (Keenan et al., 2013; Franks et al., 2015). The trend was further corroborated by the increase of ¹³C/¹²C isotopic discrimination of plant photosynthesis (Keeling et al., 2017). Whether this trend exists for mangrove plants is not clear. Several studies indicated that elevated CO₂ imposes a positive effect on mangrove productivity (Farnsworth et al., 1996; Ball et al., 2010; Reef and Lovelock, 2015). Furthermore, the above- and belowground production of mangroves have different responses to elevated CO₂ in combination with other environmental factors (e.g. N availability, salinity, temperature) (McKee and Rooth, 2008; Reef et al., 2016). Normally, the optimized assimilation rate is constrained by the balance of maximum C gain and minimum water loss. However, L. racemosa shows higher photosynthesis but lower WUE due to higher conductance. To some degree, there is minimal water limitation of the L. racemosa. Hence, we hypothesize that the rising atmospheric CO₂ is expected to significantly enhance the assimilation rate of L. racemosa and thus facilitate the expansion of L. racemosa into native mangrove habitats. Certainly, more evidence is needed to further test this hypothesis.

Furthermore, more nutrient input could enhance the mangrove productivity in a short term. Aquaculture is a major threat to mangrove forest degradation (Rahman et al., 2013; Richards and Friess, 2016). Aquaculture not only directly reduces the mangrove cover but also increases nutrient input into the neighboring mangroves. Especially in China, the pollution and deforestation caused by aquaculture are the main threats leading to over 50% of mangroves loss (Dan et al., 2016). The increased nutrient loading might also strengthen the CO₂–nutrient fertilized effect of exotic mangroves because of the higher assimilation rate and nutrient use efficiency compared to the native mangroves. Those results suggest that climate change and human activity also impose widespread effects and synergistic interactions to accelerate the expansion of exotic mangroves.