Amphistomy increases leaf photosynthesis more in coastal than montane plants of Hawaiian ʻilima

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 amphistomy informs its potential as a target for crop improvement and paleoenvironment reconstruction.

Methods

We developed a 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 surface (pseudohypostomy). Humidity modulated stomatal conductance and thus enabled comparing photosynthesis at the same total stomatal conductance. We estimated AA and leaf traits in six coastal (open, sunny) and six montane (closed, shaded) populations of the indigenous Hawaiian species ʻilima (Sida fallax).

Key results

Coastal ʻilima benefits 4.04 times more from amphistomy than montane leaves. Evidence was equivocal with respect to two hypotheses – that coastal leaves benefit more because 1) they are thicker and have lower conductance through the internal airspace, and 2) they benefit more because they have similar conductance on each surface, as opposed to most conductance being through the lower surface.

Conclusions

This is the first direct experimental evidence that amphistomy increases photosynthesis, consistent with the hypothesis that parallel pathways through upper and lower mesophyll increase CO2 supply 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 remains uncertain.

 

We identified 7 suitable natural populations of ʻilima on Oʻahu and 5 on Hawaiʻi Island by consulting Yorkston and Daehler (2006) and citizen scientist records on iNaturalist (Anon, 2022). We avoided sites that appeared to be cultivated. We visited sites between August and November 2022. For logistical reasons, the sites on Hawaiʻi were sampled during one three-day trip. We haphazardly sampled eight plants distributed evenly between the highest and lowest elevation plants along a transect at each site. For safety and conservation reasons, transects were along a trail or road. We did not sample small individuals if there was risk removing leaves would cause mortality. From each plant, we collected two fully expanded leaves for trait measurements. We sampled stomatal traits on all leaves; leaf thickness on one leaf from three randomly selected plants per site; and, due to limited time, a single leaf from a single plant at the middle of each transect for gas exchange measurements. 

 

 

 

We estimated the stomatal density and size on ab- and adaxial leaf surfaces from all leaves. For pubescent leaves (usually coastal), we dried and pressed leaves for 1 week (Hill et al., 2014), carefully scraped trichomes off with a razor blade, and rehydrated the leaf. Rehydration restores leaf area to its fresh value (Blonder et al., 2012). For glabrous leaves, we used fresh leaves. We applied clear nail polish to both leaf surfaces of fresh or rehydrated leaves in the middle of the lamina away from major veins. After nail polish dried, we mounted impressions on a microscope slide using transparent tape (Mott and Michaelson, 1991). We digitized a portion of each leaf surface impression using a brightfield microscope (Leica DM2000, Wetzlar, Germany). We counted all stomata and divided by the visible leaf area (0.890 mm2) to estimate density and measured guard cell length from five randomly chosen stomata per field using ImageJ (Schneider et al., 2012).

 

 

We cut thin sections using two razor blades taped together. We sectioned the leaf in a petri dish of water, wet-mounted sections onto a slide, and took digital micrographs using a brightfield microscope, as described above. Leaf thickness is measured as the length from upper cuticle to lower cuticle.

 

 

At each site, we selected one representative leaf from one plant near the middle of the transect for gas exchange measurements using a portable infrared gas analyzer (LI-6800PF, LI-COR Biosciences, Lincoln, Nebraska, USA). We estimated the photosynthetic rate (A) and stomatal conductance to water vapor (g_sw) at saturating light (photosynthetic photon flux density (PPFD) = 2000 μmol m^-2 s^-1}, ambient CO₂ (415 ppm), and T_leaf = 25.0-29.3 °C. The midday irradiance in coastal ʻilima typically meets or even exceeds a PPFD of 2000 μmol m-² s^-1 and previous experiments with sun leaves revealed that is always at or near saturating irradiance. Even though lower irradiance may be saturating for montane leaves, we used this higher value for all leaves to standardize conditions.