Triplett, G.; Buckley, T. N.; Muir, C. D.

Premise of the study The adaptive significance of stomata on both upper and lower leaf surfaces, called amphistomy, is unresolved. A widespread association between amphistomy and open, sunny habitats suggests the adaptive benefit of amphistomy may be greatest in these contexts, but this hypothesis has not been tested experimentally. Understanding why amphistomy evolves can inform its potential as a target for crop improvement and paleoenvironment reconstruction. Methods We developed a new method to quantify \"amphistomy advantage\", AA, as the log-ratio of photosynthesis in an amphistomatous leaf to that of the same leaf but with gas exchange blocked through the upper (adaxial) surface, which we term \"pseudohypostomy\". We used humidity to modulate stomatal conductance and thus compare photosynthetic rates at the same total stomatal conductance. We estimated AA and related physiological and anatomical traits in 12 populations, six coastal (open, sunny) and six montane (closed, shaded), of the indigenous Hawaiian species ilima (Sida fallax). Key results Coastal ilima leaves benefit 4.04 times more from amphistomy compared to their montane counterparts. Our evidence was equivocal with respect to two hypotheses - that coastal leaves benefit more because 1) they are thicker and therefore have lower CO2 conductance through the internal airspace, and 2) that they benefit more because they have similar conductance on each surface, as opposed to most of the conductance being on the lower (abaxial) surface.Conclusions This is the first direct experimental evidence that amphistomy per se increases photosynthesis, consistent with the hypothesis that parallel pathways through upper and lower mesophyll increase the supply of CO2 to chloroplasts. The prevalence of amphistomatous leaves in open, sunny habitats can partially be explained the increased benefit of amphistomy in \'sun\' leaves, but the mechanistic basis of this observation is an area for future research.