DOI: 10.5061/dryad.9p8cz8wf9
title: Data for: Testing an invasion mechanism for Eucalyptus globulus: is there evidence of allelopathy?

description: Premise of study- Sparse understory communities, in association with
 non-native tree species, are often attributed to allelopathy, the chemical
 inhibition of one plant by another. However, allelopathy is a difficult
 ecological phenomenon to demonstrate with many studies showing conflicting
 results. Eucalyptus globulus, a native tree to Australia, is one
 of the most widely planted trees around the world. Sparse understories are
 common beneath E. globulusplantations and are often attributed to
 allelopathy, but the ecological impacts of E. globuluson native
 plant communities are poorly understood.  Methods -To assess
 allelopathy as a mechanism of understory inhibition, we tested volatile-
 and water-soluble leaf extracts on seed germination of California native
 plants. We also quantified germination rates and early seedling growth of
 native plants grown in soil from E. globulusplantations versus
 soil from an adjacent native plant community.  Key results-
 Volatile compounds from E. globulusdid not significantly reduce
 germination for any species. Inhibition from water-soluble E.
 globuluscompounds was comparable to that of a native
 tree, Quercus agrifolia (10%).Eucalyptus globulussoil
 supported germination and early seedling growth of native species equal to
 or better than coastal scrub soil, although species responses were
 variable.  Conclusions- In contrast to previous
 studies, our results fail to support the hypothesis
 that E. globuluschemically inhibits germination of native
 species. California native plants germinate and grow well in soils
 from E. globulusplantations, which may have significant
 implications for management and restoration of land historically occupied
 by E. globulusplantations. 
description: Germination in the presence of volatile compounds - We attempted
 to germinate seeds of native species (Acmispon glaber (Vogel) Brouillet,
 Eschscholzia californica Cham., Lupinus succulentus Douglas ex K. Koch,
 Festuca microstachys Nutt., and Stipa pulchra Hitchc.) under an air
 control versus under the presence of volatile compounds from fresh leaves
 of E. globulus (treatment of interest), S. apiana (positive control;
 Muller and Muller, 1964; Muller et al., 1964; Muller, 1966), or Q.
 agrifolia (native tree treatment). Fresh leaf material was cut into 1.25
 to 2.5-cm-wide pieces, and approximately 2 g of fresh, cut leaf material
 was placed in a small glass dish. The small dish was then placed in the
 center of a 90-mm-diameter glass petri dish (large dish) lined with filter
 paper. The small petri dish functions to isolate the torn leaf material
 from interacting with the germinating seeds except through the effect of
 volatile compounds. Twenty seeds of one species were placed on the filter
 paper, watered with 7 mL of deionized water, sealed with parafilm, and
 left for two weeks, at room temperature in the dark, before we recorded
 germination for each seed. Ten replicate dishes were tested for each of
 the four treatments (E. globulus, S. apiana, Q. agrifolia, and no leaves)
 for each test species. In all germination tests, seeds were considered
 germinated if the seed coat was broken and a hypocotyl or radicle was
 visible. Germination in the presence of water-soluble
 compounds - We quantified germination rates of native species (A. glaber,
 E. californica, L succulentus, F. microstachys, and S. pulchra) in a water
 control versus in the presence of volatile compounds from fresh leaves of
 E. globulus, S. apiana (positive control), or Q. agrifolia (native tree
 treatment). Water-soluble compounds were extracted by macerating 10 g of
 fresh leaf material with 250 mL of deionized water for, 20 seconds in a
 blender. The filtrate was strained through four layers of fine-mesh
 cheesecloth and diluted with enough deionized water to bring each solution
 up to 500 mL. Twenty seeds of one test species were placed on filter paper
 in 90-mm-diameter glass petri dishes and watered with 7 mL of the prepared
 filtrate in the trial or with deionized water. We used five replicate
 dishes of each treatment (E. globulus, S. apiana, Q. agrifolia, and water)
 for each of the five native test species. All dishes were left at room
 temperature in the dark for two weeks before we recorded germination.   
   Germination & growth in field-collected soil
 - We conducted two greenhouse experiments using field-collected soil to
 document the capacity for native species to germinate and establish in the
 presence of naturally-occurring concentrations of potentially allelopathic
 chemicals from E. globulus. We sampled soil from two plantations of E.
 globulus as well as from two adjacent coastal scrub communities (scrub
 soil). We collected soil in two different seasons, in April, 2015 (spring
 collection/experiment) and December, 2015 (winter collection/experiment)
 to capture any potential seasonal differences in accumulation of chemical
 extracts (del Moral and Muller, 1970). During each soil collection, duff
 and leaf litter were brushed off the soil surface, and soil was collected
 from the surface to, 20 to 25 cm depth. In the spring soil experiment, we
 germinated seeds in both soil types from each site and measured seed
 germination and total biomass at the end of the experiment. Ten seeds of
 each of three native species were sown together in 12 large (38cm2 x 25 cm
 deep) plastic flats, with three replicate flats for each of the four soil
 collection locations. We tested seed from species common to the native
 coastal scrub and grassland plant communities that are most likely
 impacted by the establishment of E. globulus plantations throughout
 California. We used seed from Lupinus succulentus Douglas ex K. Koch,
 Stipa pulchra Hitchc., and Festuca microstachys Nutt.. Two other species,
 Baccharis pilularis DC and Salvia mellifera Greene seed were also sown but
 failed to germinate across all treatments, so were excluded from the
 study. Seeds were obtained from S&S Seeds (Carpinteria,
 California) and came from locally-sourced stock from the Central Coast of
 California. The seeds of each native species were planted in a randomly
 selected grid location within each 38-cm flat. Each flat received enough
 water to maintain saturated conditions, between, 200 and 600 mL once or
 twice daily for the duration of the experiment. We recorded germination
 weekly and measured above-ground biomass two months after planting. The
 above-ground biomass of germinated plants was harvested, placed in
 individual paper bags, dried for 72 hours at 49 C°, and weighed.     In
 the winter soil experiment, our design and species selection were slightly
 different to increase our statistical power.  We used seeds of A. glaber,
 E. californica, Lupinus succulentus, and Stipa pulchra. Festuca
 microstachys was not used in the winter experiment due to insufficient
 seed availability. Ten seeds of a single native species were planted in
 10-cm pots in a replicated block design, with each block containing each
 native species and each soil type (n=10). Each pot received the same
 amount of water at each watering, approximately 18 to 25 mL every two to
 three days. We scored germination on all seeds over a 60-day germination
 period and calculated total germination over the study period.     Data
 analyses - To determine whether water soluble or volatile compounds of E.
 globulus inhibited germination, we used factorial generalized linear
 models (GLMs, logit link function, binomial distribution) in the R base
 stats package (R Core Team, 2018).  Two separate models were performed,
 one for the volatile experiment and one for the water-soluble experiment.
  For each model, the proportion of seeds germinated out of total seeds per
 dish was the response variable, while treatment, species, and the
 interaction of treatment and species were fixed-effect predictors. Tukey
 tests were carried out to compare germination across all pairwise
 treatment comparisons (glht function in the R package multcomp, Hothorn et
 al., 2008).  Given a significant interaction of treatment and species,
 pre-planned independent contrasts were used to determine all pairwise
 treatment comparisons for each species separately for a total of 30
 independent contrasts (glht function in the R package multcomp, Hothorn et
 al., 2008). These contrasts compare the effect of each treatment/control
 to all other treatments/controls within a species, allowing us to compare
 any observed inhibition in E. globulus to that of the control (air/water),
 Q. agrifolia (native tree), and S. apiana (positive control). Due to the
 possibility of false discovery from multiple comparisons, we employed a
 Benjamini–Hochberg correction when determining significance of pre-planned
 contrasts (Benjamini & Hochberg, 1995).     To determine whether
 soil collected from under E. globulus inhibits germination, we again used
 a factorial generalized linear model with a logit link function, and
 binomial distribution.  The proportion of germinated out of total seed per
 pot was the response variable, while soil type (coastal scrub,
 Eucalyptus), season (winter, spring), species, site, and all two and three
 way interactions between the soil, season, and species were included as
 fixed-effect predictors. Given a significant interaction of soil type and
 species, pre-planned independent contrasts were again used to compare
 germination by soil type for each species separately for a total of five
 independent contrasts.     Finally, to determine whether soil collected
 from under E. globulus reduces above ground biomass following germination,
 we used a factorial generalized linear model with a natural log link
 function, gamma distribution. A gamma distribution is suitable for
 left-skewed response variables such as plant biomass. The total biomass
 for each pot was the response variable, while soil (coastal scrub,
 Eucalyptus), species, site, and the interaction between soil and species
 were included as fixed-effect predictors.
description: See R Script for annotations.