DOI: 10.5061/dryad.rxwdbrvfw
title: Amphistomy increases leaf photosynthesis more in coastal than montane plants of Hawaiian ʻilima

description: 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.
description: <strong>Plant sampling and
 climate</strong>   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. 
   <strong>Leaf
 traits</strong>  
 <strong>Stomata</strong>
   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).   <strong>Leaf
 thickness</strong>  
 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.  
 <strong>Gas exchange
 measurements</strong>  
 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<sub>sw</sub>) at saturating
 light (photosynthetic photon flux density (PPFD) = 2000 μmol
 m<sup>-2</sup> s<sup>-1</sup>}, ambient
 CO<sub>2</sub> (415 ppm), and T<sub>leaf </sub>=
 25.0-29.3 °C. The midday irradiance in coastal ʻilima typically meets or
 even exceeds a PPFD of 2000 μmol m-<sup>2</sup>
 s<sup>-1</sup> 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.
   <strong>Literature
 Cited</strong>   
 Anon. 2022. iNaturalist.  
 Blonder, B., V. Buzzard, I. Simova, L. Sloat, B. Boyle, R.
 Lipson, B. Aguilar-Beaucage, et al. 2012. The leaf-area shrinkage effect
 can bias paleoclimate and ecology research. American Journal of Botany 99:
 1756–1763.   Hill, K. E., G.
 R. Guerin, R. S. Hill, and J. R. Watling. 2014. Temperature influences
 stomatal density and maximum potential water loss through stomata
 of<em> Dodonaea viscosa</em> subsp. Angustissima along a
 latitude gradient in southern Australia. Australian Journal of Botany 62:
 657.   Mott, K. A., and O.
 Michaelson. 1991. Amphistomy as an adaptation to high light intensity in
 <em>Ambrosia cordifolia</em> (Compositae). American Journal of
 Botany 78: 76–79.  
 Schneider, C. A., W. S. Rasband, and K. W. Eliceiri. 2012. NIH
 Image to ImageJ: 25 years of image analysis. Nature Methods 9:
 671–675.   Yorkston, M., and
 C. C. Daehler. 2006. Interfertility between Hawaiian ecotypes of
 <em>Sida fallax</em> (Malvaceae) and evidence of a hybrid
 disadvantage. International Journal of Plant Sciences 167:
 221–230.
description: # Data from: Amphistomy increases leaf photosynthesis more in coastal than
 montane plants of Hawaiian ʻilima (*Sida fallax*)
 This dataset contains leaf
 trait data for each individual 'ilima individual and/or leaf in our
 sample. ## Description of the data and file structure The data are
 structured as a comma separated value (CSV) file with 185 rows and 27
 columns: * `genus`: genus name * `species`: specific epithet *
 `authority`: authority * `exposition`: sampled from 'natural
 environment' or 'experimental growth conditions' *
 `plant_maturity`: 'juvenile' or 'mature' plant *
 `leaf_age`: 'young', 'youthful', 'mature',
 'old', or 'senescent' leaf according to [Bowman and
 Box 1983](https://doi.org/10.1111/j.1442-9993.1983.tb01515.x) * `site`:
 freeform site name * `site_code`: four-letter site code * `site_type`:
 'coastal' or 'montane' site type * `island`: island
 where site is located (oahu = O'ahu; hawaii = Hawai'i, aka Big
 Island) * `latitude_degree`: approximate site latitude in decimal degrees
 * `longitude_degree`: approximate site longitude in decimal degrees *
 `elevation_m_site`: site elevation in meters above sea level *
 `elevation_m_plant`: plant elevation in meters above sea level *
 `date_sampled`: date of sampling in YYYY-MM-DD format * `plant_id`: id of
 individual plant within site (i1, i2, ...) * `leaf_id`: id of individual
 leaf within plant (l1, l2, ...) * `lower_number_of_stomata`: count of
 stomata on lower (abaxial) surface per 0.890 mm^2 field *
 `upper_number_of_stomata`: count of stomata on upper (adaxial) surface per
 0.890 mm^2 field * `lower_stomatal_density_mm2`: stomatal density per mm^2
 on lower (abaxial) surface, calculated as `lower_number_of_stomata` /
 0.890 mm^2 * `upper_stomatal_density_mm2`: stomatal density per mm^2 on
 upper (adaxial) surface, calculated as `upper_number_of_stomata` / 0.890
 mm^2 * `lower_gcl_um`: lower (abaxial) guard cell length (gcl) in um *
 `upper_gcl_um`: upper (adaxial) guard cell length (gcl) in um *
 `leaf_thickness_um`: leaf lamina thickness from upper cuticle to lower
 cuticle in um * `Tleaf`: leaf temperature for `A` and `gsw` in degree C *
 `A`: photosynthetic CO2 assimilation rate in umol / m^2 / s * `gsw`:
 stomatal conductance to water vapor in mol / m^2 / s Missing data are
 indicated by `NA`. ## Sharing/Access information Links to other publicly
 accessible locations of the data: * ##
 Code/Software All code associated with these data and related publications
 is available on GitHub and archived on Zenodo: *
 *