Quantification Of Stomatal Morphology And The Relationship Between Stomatal Size And Stomatal Density In 12 Magnoliaceae Species

Stomatal geometries are complex.A stoma consists of a pair of guard cells and the pore formed by those cells,which have the function of regulating the exchange of gases between the interior of the individual and the external environment.This function can affect water and carbon cycles in nature.The size and density of stomata on the plant epidermis reflects a key investment in the functional economics of plants,so an accurate description of stomatal geometry and the calculation of stomatal area are important for estimating leaf surface area allocation and investment in leaf morphogenesis.The Magnoliaceae family are the more primitive group of dicotyledons,which are widely distributed in Americas and Asia.In China,Magnoliaceae plants are an important component of temperate evergreen（deciduous）broad-leaved forests.Magnoliaceae plants have a significantly lower rate of evolution than other plants,and stomatal morphology,size and density vary across species,making the study of Magnoliaceae valuable in the phylogeny and evolution of flowering plants.In this study,12 Magnoliaceae species from Nanjing Forestry University Campus and Zhongshan Botanical Garden were selected and the leaves were treated by chromic acid-nitric acid dissociation method to obtain stomatal images of different positions of leaves.The images were processed by using Procreate,Photoshop,Image J and MATLAB software to obtain data including leaf marginal coordinates,stomatal marginal coordinates,stomatal length,stomatal width and stomatal pore length.The stomatal size（reflected by stomatal area）,stomatal density,maximum stomatal conductance and stomatal roundness index were analysed using the statistical software R.The stomatal geometry was fitted using the superellipse equation,and the goodness of fit of the Montgomery equation（ME）,a formulation assuming a proportional relationship between stomatal area and the product of stomatal length and width,and three other area calculation models was compared.The relationships between stomatal size and stomatal density and between maximum stomatal conductance(g_wmax)and leaf fresh mass per area（LFMA）for 12 Magnoliaceae species were fitted using ordinary least squares.The conclusions are as follows:（1）The estimated value of parameter n in the superellipse equation for Magnolia denudata,Magnolia stellata,Michelia chapensis,and Michelia martini were all greater than 2,indicating that the stomatal geometries of all four plants follow the superellipse equation but do not follow the ellipse equation.The adjusted root mean square error(RMSE_adj)of each of the 960 stomata was less than 0.04,i.e.,RMSE between the actual and predicted values of the stomatal radii did not exceed4%of the radii of the hypothetical circle whose area equal to the stomatal area.（2）The ME assumes that there is a simple proportionality coefficient between the stomatal area and the product of the stomatal length and width,which is known as the Montgomery parameter（MP）.ME can effectively calculate the stomatal area.The MPs for the four species of M.denudata,M.stellata,M.chapensis,and M.martini are 0.8111,0.8116,0.8016,and 0.8039,respectively,and the MP for the pooled data is 0.8071.This result suggests that the ME provides a more accurate estimate of stomatal area than the ellipse hypothesis.（3）The pooled data for 12 Magnoliaceae species showed a significant negative correlation between stomatal size and stomatal density（r=?0.8814,P<0.05）,with stomatal area decreasing as stomatal density increased.The data for individual species of 10 Magnoliaceae,except for M.cavaleriei var.platypetala and M.martini,also showed a significant negative correlation between stomatal size and stomatal density（P<0.05）.Stomatal density significantly affects stomatal size,resulting in interspecific differences in the stomatal size vs.density relationship.（4）There was no correlation between g_wmaxand LFMA（P=0.11）.g_wmax is influenced by a combination of multiple factors including stomatal density,aperture size,stomatal depth,and so on.Therefore,it cannot be used as a long-term characteristic indicator for plant photosynthetic potential.In other words,its interpretability is limited.（5）There are significant differences in stomatal size and stomatal density between different positions on the same leaf,generally with smaller stomatal area and higher density for the lamina sections near the midrib,and with larger stomatal area and lower density for the lamina sections at the leaf margin.It is important to understand the characteristics of stomata at different positions to reveal the adaptation strategies of plants to their environment.In summary,this study revealed interspecific differences in stomatal geometry,size and density,as well as a negative correlation between stomatal size and density,through the analysis of stomatal characteristics of 12 Magnoliaceae species.In addition,we demonstrated the validity of the ME in estimating stomatal area.We also explored the relationship between maximum stomatal conductance and LFMA,and the differences in stomatal characteristics at different positions on the same leaf.This study provides valuable information for the study of the evolution of Magnoliaceae and helps to further understand the adaptation strategies of plants to their environment.It is worthwhile to explore the relationship between stomatal characteristics and plant physiological and ecological traits further,based on a wider range of species and habitat types,in order to provide a more comprehensive basis for understanding plant responses to climate change.