Experimental Protocol for Manipulating Plant-induced Soil Heterogeneity

Summary

Understanding the role of environmental heterogeneity in species coexistence has typically focused on types of heterogeneity that are extrinsic to the community’s species composition. We provide novel detailed methods for creating soil heterogeneity treatments using soils subject to plant-soil feedback conditioning, or heterogeneity intrinsic to the community composition.

Abstract

Coexistence theory has often treated environmental heterogeneity as being independent of the community composition; however biotic feedbacks such as plant-soil feedbacks (PSF) have large effects on plant performance, and create environmental heterogeneity that depends on the community composition. Understanding the importance of PSF for plant community assembly necessitates understanding of the role of heterogeneity in PSF, in addition to mean PSF effects. Here, we describe a protocol for manipulating plant-induced soil heterogeneity. Two example experiments are presented: (1) a field experiment with a 6-patch grid of soils to measure plant population responses and (2) a greenhouse experiment with 2-patch soils to measure individual plant responses. Soils can be collected from the zone of root influence (soils from the rhizosphere and directly adjacent to the rhizosphere) of plants in the field from conspecific and heterospecific plant species. Replicate collections are used to avoid pseudoreplicating soil samples. These soils are then placed into separate patches for heterogeneous treatments or mixed for a homogenized treatment. Care should be taken to ensure that heterogeneous and homogenized treatments experience the same degree of soil disturbance. Plants can then be placed in these soil treatments to determine the effect of plant-induced soil heterogeneity on plant performance. We demonstrate that plant-induced heterogeneity results in different outcomes than predicted by traditional coexistence models, perhaps because of the dynamic nature of these feedbacks. Theory that incorporates environmental heterogeneity influenced by the assembling community and additional empirical work is needed to determine when heterogeneity intrinsic to the assembling community will result in different assembly outcomes compared with heterogeneity extrinsic to the community composition.

Introduction

One of the primary goals of community ecology is to explain and predict the processes governing community assembly. However, plant communities are frequently more diverse than predicted by coexistence theory¹, and restoration ecologists need to understand coexistence mechanisms to successfully restore diverse native communities². Environmental heterogeneity is a theoretically important mechanism that can help explain high levels of community diversity, but experimental manipulations of heterogeneity are infrequent³ and focus on abiotic heterogeneity (e.g. reviewed in Lundholm⁴). Theory that incorporates heterogeneit....

Protocol

1. Collect Field Soils to Produce Heterogeneous and Homogeneous Soil Treatments

1. Identify two cooccurring plant species in similar habitats and soil types for study (e.g. Figure 1a). Determine amount of soil needed based on soil patch size to use in the experiment (e.g. one-third to one-half of the focal species' primary root zone, or volume encompassed by the root ball) and the number of patches needed for the experiment (see Protocol 2 and 3, below).
   1. For Rumex spp. (Figure 1a), use soil patches of 10 cm x 10 cm x 18 cm (length x width x depth) for approximate root zone of 10 cm radiu....

Results

Species responded to plant-induced soil heterogeneity in diverse ways at both the population and individual level (Figures 3 and 4), with implications for community assembly. To determine whether plant populations respond to plant-induced soil heterogeneity, a field experiment was established as in Protocol 2 using three congeneric pairs of species. Plant populations were censused weekly for three months and the total proportion of planted seeds that germinated and the total proportion o.......

Discussion

Plant-induced soil heterogeneity is highly likely in natural communities because plants have large and often species-specific effects on their soil environment and the subsequent plants that experience that soil (e.g. Petermann et al.¹³). However, our understanding of the role of this type of heterogeneity on plant communities is minimal^10,14. Here, we present a method for manipulating plant-induced soil heterogeneity, using soil from different origins (i.e. zones of root .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Shovel(s)	Any	NA	It is helpful to have at least two shovels, one for each species of soil origin.
Trowel(s)	Any	NA	It is necessary to have at least two trowels of identical size, one for each species of soil origin.
Gloves	Any	NA	Gardening gloves can be used.
Diluted bleach	Any	NA	We use an ~1:10 concentration of household bleach (containing 5-10% NaClO) to water to sterilize all equipment between soil collections.
Plastic grid(s)	Any	NA	CUSTOM. We used plastic sheeting from the construction of greenhouse walls to create the grid used in the field experiment. However any stiff plastic that can be manipulated can be used. It is helpful to have three grids to produce reciprocal heterogeneous treatments and a homogeneous treatment without needing to sterilize between each experimental unit.
Plastic dividers	Any	NA	CUSTOM. We used stiff sheets of plastic, cut to fit the pot minimum width, such that they can slide down to the bottom of the pot for the greenhouse experiment. It is helpful to have at least two dividers, one for heterogeneous and one for homogeneous treatments, if investigators want to randomize the order in which experimental units within a block are filled without needing to sterilize the divider in between each experimental unit.
Buckets or wheelbarrows	Any	NA	Any container for transporting soils.
Seeds	Any	NA	We collect seeds in the field by hand. Seeds can also be ordered from horticultural suppliers, if appropriate.
Plastic toothpicks	Soodhalter Plastics, Inc.	805KP	We plant individual seeds glued on toothpick in the field experiment to facilitate monitoring germination and survival of individuals.
Water soluble glue	Elmer's	Elmer's Glue-all	Any water soluble glue can be used to adhere seeds to plastic toothpicks.
Pots	Any	NA	Pot size will depend on experimental plants used and number of soil patches desired (e.g. 2 or 6).
Sand	Any	NA	Coarse sand may be mixed with field soils to improve drainage in pots.
Lab tape	Any	NA	Tape may be used to label equipment used in handling soils with the species of origin.
Pin flags	Any	NA	Flags can be used to identify individuals in the field prior to soil collection.
Landscape fabric	Any	NA	Landscape fabric can be used in the field to minimize the growth of plants outside experimental plots.