Methods copied directly from:
Graham, C.D.K., Warneke, C.R., Weber, M. et al. The impact of habitat fragmentation on domatia-dwelling mites and a mite-plant-fungus tritrophic interaction. Landsc Ecol 37, 3029–3041 (2022). https://doi.org/10.1007/s10980-022-01529-2
STUDY SITE
We worked within a long-running fragmentation experiment at the Savannah River Site (SRS), a
National Environmental Research Park located near New Ellenton, South Carolina. The experiment consists of seven replicated experimental landscapes (blocks), each of which is 50 ha in size and includes five ~ 1.4 ha longleaf pine savanna patches surrounded by longleaf (Pinus palustris Mill.) and/or loblolly (Pinus taeda L.) pine plantation with limited herbaceous ground cover (Fig. 1A). Savanna patches were originally created by clearing mature pine plantation forests and managing the clearings with prescribed fire every 2–3 years to maintain open patch structure, as is typical for longleaf pine savanna system (Jose et al. 2006). Six of the
seven blocks had been burned in the winter of 2017–2018 (the winter prior to our work), with the remaining block burned during the previous winter of 2016–2017. This design results in experimental patches of open savanna habitat surrounded by a forested matrix.
The design of the experiment isolates the effects of patch shape and connectivity while holding constant the area of individual patches and the total habitat area within each block. Each block includes a 100×100 m center patch with four peripheral patches each 150 m away from the center patch (Fig. 1A). A corridor (25 m wide and 150 m long) connects the center patch and one of the peripheral patches, and is included in the area of this “connected patch”. The unconnected patches are “rectangular” or “winged,” with equal areas (~ 1.4 ha, with the extra 0.4 ha area being the wings or back of the rectangle patch). Each experimental block has at least one rectangular and one winged patch and the remaining patch in each block was randomly assigned to be either a rectangular patch or a winged patch. Effects of connectivity can be distinguished through comparisons of connected and winged patches (which differ only in their connectivity), while patch shape is distinguished through comparing winged and rectangle patches (which differ only in their edge-to-area ratio).
FOCAL PLANT SPECIES
Quercus nigra (Fagaceae) is a deciduous oak tree species native to North America. This species is widespread within the experimental landscapes and has conspicuous mite domatia which take the form of
clusters of trichomes located at the junctions of major veins on the lower leaf surface. While this is the first
study to examine the mite communities associated with the domatia of Q. nigra, studies in other species (including oaks) have demonstrated that domatia are occupied by a group of largely fungivorous, but sometimes also predacious, mites from families such as Tydeidae and Phytoseiidae, which are considered plant mutualists (O’Dowd and Willson 1997). Due to recent prescribed fire, our sampled oaks were resprouting and relatively short (40–220 cm in height).
Q1 EXPERIMENTAL DESIGN
To test for fragmentation effects on mite communities, we sampled mites on leaves of Q. nigra plants
from across all treatments in the field experiment. We sampled four Q. nigra individuals in the peripheral patches of each of the seven replicate blocks (Fig. 1A) in late June through early August of 2018 (n=110
oaks in 28 patches, due to one patch that lacked the requisite number of oaks). We sampled two oaks
in each of two zones within each patch (Fig. 1B): the edge (within 12.5 m of the patch edge) and the
center (the patch area greater than 37.5 m from the patch edge). Within the two zones, we located oaks
that were healthy and similar in size. After selecting trees, we haphazardly sampled 10 fully-expanded,
undamaged leaves from each oak, and immediately placed these leaves in plastic bags containing moistened paper-towels for transport on ice from the field to the laboratory. Within the plastic bags, leaves did not come in contact with each other. Within 48 h of collection, we examined the undersides of all leaves with a dissection microscope, counting the number of domatia per leaf and the total number of mites per leaf. Additionally, we sorted mites into morphospecies; representatives of which were stored in 95% ethanol and stored at −20 °C for later DNA extraction and identification. The methods for mite DNA extraction and barcoding can be found in the supplement (Appendix S1).
Q2 EXPERIMENTAL DESIGN
To investigate whether fragmentation effects altered plant-mite-fungus interactions, we applied a mite exclusion treatment to two pairs of Q. nigra individuals, resulting in one control and one treatment oak
at both the edge and center of the patches (n=109 oak individuals among 28 patches, due to one mortality event and one patch that lacked the requisite number of oaks). We selected focal Q. nigra individuals for this experiment using the same edge and center zones and strategy as the mite diversity survey (Fig. 1B), although different individual plants were used. We excluded mites by blocking domatia with pruning tar, a standard manipulative technique for nearly eliminating domatia-dwelling mites on plant leaves (Norton et al. 2000, 2001; Monks et al. 2007). We applied pruning tar (TreeKote Wound Dressing, Walter E. Clark & Son, Inc., Orange, CT) to all of the domatia on the underside of every leaf of two treated trees in each patch, randomly assigned to the mite-exclusion treatment. For the remaining two Q. nigra trees in each patch, we conducted a control treatment to account for the presence of pruning tar (and any potential effects of the tar itself), by applying approximately the same number of tar spots that would have been applied to the domatia, but elsewhere on the lower leaf surface, such that the domatia were still available for mite colonization and use. Before applying the tar treatment, we pruned all focal Q. nigra individuals to a consistent size. We maintained the treatment on all leaves for six weeks, following initial tar application. Any new leaves that were produced during this time were removed, in order to maintain the treatment across the whole plant. After the six-week treatment period, we haphazardly sampled three fully-expanded, undamaged leaves from each oak. We immediately placed these leaves in plastic bags containing moistened paper towels for transport on ice from the field to the laboratory. All leaves were processed within 24 h of collection. For each leaf, we examined the leaf undersides under a dissection microscope, counting all domatia and the number of mites. To quantify fungal abundance on the leaf surface, we used established fungal peel methods (Harris 2000; Monks et al. 2007). In brief, following the counting of mites and domatia, we pressed the sticky side of a 19 mm wide piece of Matte Finish tape to the lower leaf surface (Skilcraft, Alexandria, Virginia, USA). We then placed the tape
on a slide and dyed the fungal hyphae on the slide mount using a solution of 0.5% (w/v) trypan blue in lactoglycerol (1:1:1 lactic acid, glycerol and water flitered at 0.45 μm). We counted the number of times hyphal threads crossed a transect from a standardized area of tape (that being the whole width of the tape,
from one side of the slide mount to the other).
