Field collections and sampling strategies
All collections occurred at the Teller Wildlife Refuge in western
Montana, USA (46.3219 N 114.1292 W). The Teller Wildlife Refuge is a
525-ha property that comprises riparian, floodplain, wetland, and upland
habitat. Showy milkweed grows in numerous patches across the property,
concentrated near irrigation ditches, on the floodplain, and in old
fields where sub-irrigation provides enough water. We collected 6
individual leaves each from 20 randomly selected showy milkweed plants
once on May 18th, 2016 before flowering, and again on
September 6th, 2016 when seed pods were almost ripe.
Plants were selected from a 500 m2 area of a field
with a patchy distribution. All plants sampled were at least 3 m. apart.
During the September collection we also collected as much of the root
system as possible within the upper 20 cm of soil by destructively
sampling each plant. Tissue was kept on ice upon collection, and then
stored at -20 °C until further processing. Roots were first washed with
tap water to remove all visible soil, and fine roots (<1 mm in
diameter) were retained for further processing. Bulk root collections
weighed between 0.3 and 0.8 g dry weight, per plant. Bulk leaf
collections weighed between 1.2 and 2.5 g dry weight per plant. To
remove microbial DNA from root and leaf exterior and characterize only
colonizing microbes, all root and leaf tissue was surface sterilized in
70% ethanol for 1 min., 0.5% NaOCl for 1 min. and then rinsed 2X in
sterile water. Imprints of sterilized tissue were made on growth medium
to confirm the sterilization procedure. The absence of growth observed
indicated successful surface sterilization. After sterilization, leaves
and roots from the September collection were separated into two groups
based on the two sampling strategies we chose to compare.
For Strategy 1, homogenizing after subsampling , we removed one
leaf disc from 6 leaves per plant (at random locations) using an 11.5 mm
in diameter corkborer and placed the six discs per plant into single 1.2
ml Eppendorf tubes. This resulted in a total surface area of
approximately 62 mm2 per plant, with a dry weight of
approximately 30.0 mg of leaf tissue. For roots, approximately 30.0 mg
of dry root segments per plant was placed into individual 1.2 ml
Eppendorf tubes. As many DNA extraction kits recommend a maximum volume
of approximately 20-30.0 mg dry weight or 50.0 mg fresh weight, we have
found this to be a commonly used standard (e.g., Busby et al.,
2016; Gdanetz and Trail 2017; Haas et al ., 2018; Barge et
al., 2019; Bunn et al., 2019), however, many studies lack
sufficient detail on final extraction volumes. Subsampled root and leaf
tissues were then freeze-dried using a Labconco Freezone benchtop
freeze-dry system (Labconco, Kansas City, MO, USA). Subsampled tissue
was homogenized using a 1600 MiniG tissue
homogenizer and cell lyser (Spex SamplePrep, Metuchen, NJ, USA).
For Strategy 2, homogenizing before subsampling, all remaining
root and leaf tissue from the September collection was subsequently
freeze-dried and homogenized for each plant using the freeze-dry system
and MiniG as described above. Approximately 30.0 mg
dry weight per plant was then subsampled from the homogenized pool of
root or leaf tissue and placed in 1.2 ml Eppendorf tubes for DNA
extraction. All remaining foliar tissue from five plants collected in
September (> 40X more tissue per plant, equaling 1241-2467
mg per plant) was then divided into approximately 250 mg replicates for
additional DNA extractions to assess the extent of undersampling.