Greenhouse experiment and phenotype measurements
Seedling traits that may be related to drought tolerance include height
growth, root length, dry shoot weight, dry root weight, root-to-shoot
dry mass ratio, and specific root length. Allocation to roots versus
shoots can affect a seedling’s ability to take up water (Brunner &
Godbold 2007; Markesteijn & Poorter 2009; Moran et al. 2017a),
while the number and size of needles affect the area over which they may
lose water through the stomata (Parker 1949). Two other traits related
to this latter point are stomata density and the number of rows on the
adaxial versus abaxial sides of the needles. However, it should be noted
that lower stomatal conductance can be achieved with either fewer
stomata, smaller stomata, or by closing the stomata more often (Irvineet al. 1998; Ryan 2011).
Seeds were collected from a subset of 50 genotyped parent trees in the
summer of 2018. We placed 2-3 mature cones from each mother tree into
paper bags and put them in a warm, dry place until seeds were released.
We aimed to have ten seedlings from each of the fifty maternal families
in wet and dry treatments, 1000 seedlings in total. Forty-eight of the
50 families had enough seeds in their cones to be included in the
experiment (Fig. S1). During winter 2018, the seeds were stratified to
break dormancy by placing them in aerated water for 48 hours, then
surface-drying them and storing them in plastic bags in the refrigerator
(~1.7°C) for six weeks. Because pines are
wind-pollinated and outcrossing (Williams 2009), seeds from the same
tree are mostly half-siblings, occasionally full-sibs.
Because the maximum first-year seedling root length observed in a pilot
experiment was more than 110 cm, we used plastic tubes with an 8 cm
width and 120 cm depth for planting. The bottom of each tube was capped
with mesh to prevent the soil from falling out while allowing drainage.
The lightweight clear tubes were wrapped in black plastic to keep roots
in the dark. The planting soil was a mixture of 70% sand, 20%
vermiculite, and 10% organic-rich potting mix to mimic the coarse
texture of the soil of many Sierra Nevada conifer forests (Baleset al. 2011). To keep tubes upright, we used PVC pipes to build
10 frames that could each hold 100 tubes. Two seeds from each family
were planted in each tube in February 2019, and two tubes from each
family were randomly placed within each frame. In April 2019, we
replanted more stratified seeds of the correct family in tubes without
seedlings. All the tubes were watered every other day during the
germination and seedling establishment period (February through June).
At the end of June 2019, all but one seedling per tube was removed, and
alternating frames were assigned to the wet treatment and the dry
treatment (5 frames containing up to 500 seedlings per treatment) (Fig.
S1). The wet treatment group was watered twice weekly, and the drought
treatment group was watered once every three weeks until mid-October
(3.5 months). While wild ponderosa pine seedlings would receive little
to no precipitation during the summer, this occasional watering was
necessary for the greenhouse environment. Temperatures inside the
greenhouse in the low-elevation environment of Merced, CA, reached as
high as 37°C on the hottest days and the soil volume of the tubes was
limited, with no access to groundwater, both of which made evaporation
and drought stress more intense than the no-precipitation condition in
the wild.
Multiple phenotypic traits were measured during and after the greenhouse
experiment. Only 42 out of 48 mother trees had enough germination to
carry out these measurements across both treatments. We calculated shoot
growth as final height minus height at the initiation of the treatments.
The length of fresh roots was measured from the soil surface to the
taproot tip immediately after the harvesting to avoid shrinkage.
Following harvest, needles, fresh stems, and fresh roots of all the
seedlings were separately put into paper bags and dried at 75 °C for 48
hours. We measured root dry mass (RW) and shoot weight (SW, total of
stem and needles). We then calculated the root-shoot ratio (R2S) as
RW/SW. Specific Root length (SRL) was calculated as root length/root
weight.
Before harvest, we also collected 3-4 fresh needles from living
seedlings to calculate stomatal density. In pines, stomata are arranged
into longitudinal rows. We put each needle on a slide and photographed
it at 100x magnification using a Leica DME compound microscope equipped
with a Leica DFC290 digital camera. All counts were conducted near the
middle of the needle to avoid variation that might occur at the base and
the tip. Approximately 1.96 mm lengths of the needle were surveyed for
the number of stomata and stomatal rows on their adaxial (upper) and
abaxial (lower) surfaces. Needle width was measured in magnified images
using the line measure tool in the Leica software. Then we calculated
the stomata density on each side as the number of stomata divided by
1.96*width of needle. An average density and number of rows were
calculated for each individual across sampled needles.