DOI: 10.5061/dryad.4f4qrfjb4
title: Leaf hair tufts function as Domatia for mites in Quercues agrifolia (Fagaceae)

description: An identifying feature of Quercus agrifolia Ne ́e (Fagaceae) is the
 presence of hair tufts on lower leaf surfaces. In other plant species,
 hair tufts act as domatia for arthropods such as mites, which in turn feed
 on leaf fungi or small herbivores and possibly benefit plant health.
 However, this mutualistic relationship remains untested in Q. agrifolia.
 In this study two primary questions were addressed within a natural stand
 of Q. agrifolia in San Luis Obispo, CA: 1) Do hair tufts act as domatia
 for mites? and 2) Does the removal of hair tufts impact mite abundance,
 herbivory or fungal pathogens on leaves? In an observational study of 377
 leaves from 20 trees, we found a significant association between the
 presence of hair tufts and the presence of mites. When we experimentally
 removed hair tufts, we found a significant reduction in mites, yet there
 was no impact on leaf herbivory or necrosis. We conclude leaf
 hair tufts on Q. agrifolia serve as domatia for mites, but we found no
 evidence that mites reduce herbivory or fungal pathogens. Thus, while
 mites likely benefit from housing provided by hair tufts on Q. agrifolia,
 it is unclear that the tree benefits from the mites, i.e., whether this is
 a mutually beneficial relationship.
description: This study took place in a natural stand of <i>Quercus
 agrifolia </i>in “Poly Canyon” at California Polytechnic University,
 San Luis Obispo, California (latitude 35.3057, longitude -120.6581). To
 address our first study question, we conducted an observational study on
 22 February 2019. Twenty teams, each of 2-4 students, were assigned a
 random GPS coordinate within the study population and sampled leaves from
 the nearest tree. Random GPS coordinates were obtained by creating a
 roughly 9-acre polygon around oak woodland habitat in Poly Canyon using
 ArcGIS software (ESRI 2018) and randomly sampling coordinates from within
 the polygon. Thirty leaves were haphazardly sampled, each from a unique
 branch within the sub-canopy.  For each leaf, the petiole was cut using a
 razor blade to avoid flinging any mites off the leaf and leaves were
 placed individually in a plastic bag. Bags were carefully transported back
 to the lab in plastic trays so as to cause minimal disturbance to any
 mites. In the lab, each team of students recorded data
 on 2-3 leaves from each of the twenty sample trees. This was done in order
 to avoid confounding the effects of student group and sample tree. For
 each leaf, the number of leaf vein axils with hair tufts was recorded. 
 Hair tufts were defined as clusters of three or more hairs.  For example,
 in Fig. 2, leaf vein axils 3 and 6 contain hair tufts.  To determine the
 association between hair tufts and mites, a single axil on each leaf was
 observed for the presence or absence of mites.  All observations were made
 with a dissecting microscope at 10-40X magnification. Mite presence
 included larvae, nymph, or adult mites (eggs were excluded due to
 uncertainty in their identification). In order to sample approximately the
 same number of ‘no hair tuft” as “hair tuft” leaf axils for each tree,
 each leaf was pre-assigned to either the “no hair tuft” or “hair tuft”
 category.  If “no hair tuft” category was assigned, an axil without a hair
 tuft was selected for observation, always beginning with the second leaf
 vein axil from the bottom and to the right of the mid vein (Fig. 2, axil
 2), and moving counterclockwise until an axil without hair tufts was
 located (in Fig. 2 this would be axil 2). Upon locating a suitable axil,
 the absence or presence of mites was recorded.  The same protocol was used
 for sampling leaves pre-assigned to the “hair tuft” category (in Fig. 2
 this would be axil 3). Next, we performed a
 manipulative experiment to determine whether removal of hair tufts
 impacted the abundance of mites and leaf health. This experiment took
 place between 25 February through 10 May 2019, using a paired study design
 on 20 haphazardly sampled trees. Within each tree, 10 branches were
 selected that met the following criteria: branches contained at least 5
 fully expanded leaves, were close enough to the ground to allow easy
 access by researchers, and were not in contact with any neighboring
 branches (to reduce the opportunity for mites to move from untreated to
 treated branches). Branches were then randomly assigned as removal or
 control treatment. On the ‘removal treatment’ branches, all hair tufts
 located in leaf vein axils on the abaxial surface were removed by gently
 scraping the leaf surface with a sharp tool (e.g. a scalpel). This was
 performed on every leaf within the branch. On ‘control treatment’
 branches, abaxial leaf surfaces were scraped immediately adjacent to but
 just outside all leaf vein axils with hair tufts, thus leaving the hair
 tufts intact. The control treatment accounts for any effect of leaf
 scraping, for example, potential damage to the epidermis of the
 leaf. Treatments were left untouched for 4-6 weeks, at
 which point we returned to assess mite abundance and leaf health. On each
 branch, we removed three leaves, always sampling the 3rd, 4th, and 5th
 fully expanded leaf from the apical meristem. The three leaves were placed
 in a plastic bag, transported back to the laboratory, and assessed within
 24 hours of collection. Using a dissecting scope, on each leaf we recorded
 the number of each leaf vein axils with: hair tufts, mites, herbivory,
 necrosis due to fungal or bacterial pathogens, and fly larvae/larvae scars
 (Fig. 1). Herbivory was defined as leaf areas with visible physical
 damage, while necrosis was defined as leaf areas with visible
 discoloration or decay.  Fly larvae/larvae scars were not included in
 downstream analyses as only 27 of 450 leaves had any presence of
 larvae/larvae scars.