Background and Aims Ottelia alismoides (Hydrocharitaceae) is a freshwater macrophyte that, unusually, possesses three different CO2-concentrating mechanisms. Here we describe its leaf anatomy and chloroplast ultrastructure, how these are altered by CO2 concentration and how they may underlie C4 photosynthesis. Methods Light and transmission electron microscopy were used to study the anatomy of mature leaves of O. alismoides grown at high and low CO2 concentrations. Diel acid change and the activity of phosphoenolpyruvate carboxylase were measured to confirm that CAM activity and C4 photosynthesis were present. Key Results When O. alismoides was grown at low CO2, the leaves performed both C4 and CAM photosynthesis whereas at high CO2 leaves used C4 photosynthesis. The leaf comprised an upper and lower layer of epidermal cells separated by a large air space occupying about 22 % of the leaf transverse-section area, and by mesophyll cells connecting the two epidermal layers. Kranz anatomy was absent. At low CO2, chloroplasts in the mesophyll cells were filled with starch even at the start of the photoperiod, while epidermal chloroplasts contained small starch grains. The number of chloroplasts in the epidermis was greater than in the mesophyll cells. At high CO2, the structure was unchanged but the thicknesses of the two epidermal layers, the air space, mesophyll and the transverse-section area of cells and air space were greater. Conclusions Leaves of O. alismoides have epidermal and mesophyll cells that contain chloroplasts and large air spaces but lack Kranz anatomy. The high starch content of mesophyll cells suggests they may benefit from an internal source of CO2, for example via C4 metabolism, and are also sites of starch storage. The air spaces may help in the recycling of decarboxylated or respired CO2. The structural similarity of leaves at low and high CO2 is consistent with the constitutive nature of bicarbonate and C4 photosynthesis. There is sufficient structural diversity within the leaf of O. alismoides to support dual-cell C4 photosynthesis even though Kranz anatomy is absent.

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Acclimation to variable CO 2 was studied in floating leaves of the freshwater monocot Ottelia cordata grown in either low or high CO 2. The most striking anatomical variations responding to high CO 2 included the enlarged upper epidermal cells and the decreased area of epidermal chloroplasts. Stomata that distributed on the upper surface, and the stomatic chamber area, showed no significant response to high CO 2. pH-drift experiments indicated that floating leaves of O. cordata were able to use bicarbonate regardless of CO 2 concentrations. Photosynthetic enzyme activities and patterns of organic acids fluctuation confirmed that floating leaves of O. cordata can operate CAM only at low CO 2 , and perform C 4-like metabolism at both high and low CO 2. Overall, the present results imply that the floating leaves of O. cordata does not just rely on the atmospheric CO 2 for its inorganic carbon, but is also dependent on CO 2 and bicarbonate in the water. By showing these effects of CO 2 variation, we highlight the need for further experimental studies on the regulatory mechanisms in O. cordata floating leaves, that prevent futile cycling among the three CO 2 concentrating mechanisms (bicarbonate use, C 4 , and CAM metabolism) and the strategy for exploiting atmospheric CO 2 , as well as studies on the detailed biochemical pathway for C 4 and CAM metabolism in this species.