Comparative Analyses of Stomatal Size and Density among Ecotypes of Aster hispidus (Asteraceae)

To determine the size and the density of stomata among different environments, we conducted anatomical analyses using Aster hispidus var. hispidus (open field), As. hispidus var. leptocladus (serpentine soil), and As. hispidus var. insularis (coastal). The stomatal size was not significantly different among these ecotypes but the density of stomata in the serpentine and coastal ecotypes was significantly lower than that of As. hispidus var. hispidus, which suggests that these ecotypes have experienced selection that reduced the density of stomata for adaptation to the dry conditions of serpentine and coastal areas.


Introduction
Stomata control the movement of gases in and out of a leaf, make carbon dioxide available for photosynthesis, and control the loss of water from the leaf through transpiration.Gas exchange is regulated by controlling the aperture of the stomatal pore and the number of stomata that form on the epidermis.The stomatal pores that perforate leaf surfaces are among the best-characterized examples of the fundamental biological relationship between form and function [1].The density of the stomata determines the stomatal conductance to CO 2 and H 2 O [2,3] because gaseous diffusion is regulated through turgor-mediated variation in the aperture of stomatal pores [4,5].The closure of stomata under dry atmospheric or soil conditions limits CO 2 diffusion from the atmosphere to chloroplasts; therefore, stomatal physiology is inextricably linked with stomatal physiology is inextricably linked to the physiology of photosynthesis [6].As a result, the patterning of stomata on leaf surfaces is correlated strongly with hydrological conditions [7,8].
Aster hispidus Thunb.(Asteraceae) is widely distributed across China, Korea, and Japan, and is found in lowland open fields and grasslands.This species has intraspecific taxa in Japan, namely As. hispidus var.hispidus, As. hispidus var.leptocladus (Makino) Okuyama, As. hispidus var.koidzumianus (Kitam.)Okuyama, As. hispidus var.tubulosus K. Asano ex T. Shimizu, and As.hispidus var.insularis (Makino) Mot.Ito [9,10].Of these varieties, As. hispidus var.leptocladus is found in serpentine areas of central Honshu, Kyushu, and Shikoku, and As.hispidus var.insularis is restricted to coastal areas along the Pacific Ocean of western Shikoku and eastern Kyushu [9,11].It is possible that the ecological transitions from open habitats (As.hispidus var.hispidus) to serpentine (As.hispidus var.leptocladus) or coastal conditions (As.hispidus var.insularis) has driven the evolution of stomatal patterning.The stomatal number may provide insights into evolution in terms of leaf anatomy and the response to different ecological changes.However, the extent to which the diversity of stomatal traits is linked to habitat remains unknown.The aim of this study was to characterize the anatomy of As.Hispidus var.hispidus, As. hispidus var.leptocladus, and As.hispidus var.insulari with regard to the occurrence of stomata on the abaxial leaf surface.The stomatal number changed (i.e., stomata density was reduced on the surface) as the ecotypes of As. hispidus moved into regions with distinct environments.

Materials and Methods
The samples used in this study were obtained from open (Aster hispidus var.hispidus), serpentine (As.hispidus var.leptocladus), and seashore fields (As.hispidus var.insularis) (Figure 1, Table 1).We analysed 58, 22, and 15 leaves from individuals of As. hispidus var.hispidus, As. hispidus var.leptocladus, and As.hispidus var.insularis, respectively.The middle part of a leaf blade along the midrib was examined.To identify the morphology of the guard cells and the density of the stomata, the abaxial leaf surface was replicated using Suzuki's Universal Micro-Printing (SUMP) method.Replicas of each leaf (1 mm 2 ) were prepared to determine the stomatal density and to measure the guard cell size and epidermal cell size (10 cells per leaf) using cellSens standard Imaging software (Olympus Co., Tokyo) (Figure 2. Moreover we treated as an individual value the average value of the measured data.The collected data were analysed using Tukey's honestly significant difference (HSD) test to compare the characters.The numerical data were subjected to statistical analysis based on [12].
Stomatal control of water loss allows plants to occupy habitats with fluctuating environmental conditions.A reduction in stomatal density may have occurred in the As.hispidus complex as an adaptation to reduce the evaporation and transpiration of water.In fact, plants growing in high-salinity habitats exposed to more or less constant water stress.Aster hispidus var.insularis occurs in coastal areas on the Pacific Ocean side.To survive coastal conditions, plants have developed growth strategies such as increased water-use efficiency via C 4 -or CAM metabolism [13], succulent growth, and extensive root systems [14].Therefore, As. hispidus var.insularis has adapted to coastal areas by developing succulent growth and by reducing stomatal density.
Geologically unique areas are often centres of biodiversity, and thus plant speciation.Serpentine soils present a generally stressful growing environment for plants because of a number of edaphic factors such as a low Ca/Mg ratio, low levels of essential plant nutrients, poor water retention, and high concentrations of several heavy metals [15].In particular, high levels of nickel in the soil block a plant's ability to take in soil nutrients.In fact, plants growing on serpentine soils were found to have a low Ca/Mg ratio and high concentrations of heavy metals [16].Moreover, because serpentine soils are shallow and low in organic matter and clay, they also cannot hold water well [15], suggesting that this environment is similar to the dry conditions.Our results indicate that the serpentine-adapted variety As. hispidus var.leptocladus had a lower density of stomata than As.hispidus var.hispidus (Table 2).This finding suggests that this stomatal type developed for adaptation to dry environments.Similarly, [17] reported that the stomatal size and density of the serpentine ecotype of Adenophora

Figure 1 .
Figure 1.Sampling localities used in this study.For other abbreviations, seeTable 1.

Table 1 . Sampling localities used in this study.
1.

Table 2 . Anatomical measurement (average ± standard deviation) of the Aster hispidus ecotypes.
Columns marked by different letters differ significantly according to the Tukey's HSD test (p < 0.05).