Name: experimental protocol for manipulating plant-induced soil heterogeneity
Uploaded: 2014-03-13T15:00:00
Description: Watch this Scientific Journal Video about Experimental Protocol for Manipulating Plant-induced Soil Heterogeneity at JoVE.com

We thank Case Western Reserve University&#8217;s Squire Valleevue and Valley Ridge Farms, including A. Locci, C. Bond, and A. Alldridge, for help establishing the common garden. J. Hooks, L. Huffman, L. Gonzales, S. C. Leahy, B. Ochocki, A. Ubiles, C. Yu, X. Zhao, and N. M. Zimmerman provided field assistance. A.J.B. and J.H.B. were funded by startup funds from CWRU to J.H.B. G.A.D. was supported by a Summer Programs in Undergraduate Research grant from CWRU funded by the Howard Hughes Medical Institute. This work was also supported by National Science Foundation funding to J.H.B. (DEB 1250170).

1. Collect Field Soils to Produce Heterogeneous and Homogeneous Soil Treatments
<ol>
	<li>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).
	<ol>
		<li>For Rum...

<ol>
	<li>Agrawal, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filling+key+gaps+in+population+and+community+ecology.">Filling key gaps in population and community ecology.</a> Front. Ecol. Environ. 5, 145-152 (2007).</li><li>Suding, K. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+an+era+of+restoration+in+ecology:+successes,+failures,+and+opportunities+ahead.">Toward an era of restoration in ecology: successes, failures, and opportunities ahead.</a> Ann. Rev. Ecol. Evol. System. 42, 465-487 (2011).</li><li>Melbourne, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+in+a+heterogeneous+world:+resistance,+coexistence+or+hostile+takeover.">Invasion in a heterogeneous world: resistance, coexistence or hostile takeover.</a> Ecol. Lett. 10, 77-94 (2007).</li><li>Lundholm, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+species+diversity+and+environmental+heterogeneity:+spatial+scale+and+competing+hypotheses.">Plant species diversity and environmental heterogeneity: spatial scale and competing hypotheses.</a> J. Veg. Sci. 20, 377-391 (2009).</li><li>Pacala, S. W., Tilman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limiting+similarity+in+mechanistic+and+spatial+models+of+plant+competition+in+heterogeneous+environments.">Limiting similarity in mechanistic and spatial models of plant competition in heterogeneous environments.</a> Am. Natural. 143, 222-257 (1994).</li><li>Chesson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+theory+of+competitive+coexistence+in+spatially-varying+environments.">General theory of competitive coexistence in spatially-varying environments.</a> Theor. Pop. Biol. 58, 211-237 (2000).</li><li>Bever, J. D., Westover, K. M., Antonovics, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporating+the+soil+community+into+plant+population+dynamics:+the+utility+of+the+feedback+approach.">Incorporating the soil community into plant population dynamics: the utility of the feedback approach.</a> J. Ecol. 85, 561-573 (1997).</li><li>Kulmatiski, A., Beard, K. H., Stevens, J. R., Cobbold, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant-soil+feedbacks:+a+meta-analytical+review.">Plant-soil feedbacks: a meta-analytical review.</a> Ecol. Lett. 11, 980-992 (2008).</li><li>Bever, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rooting+theories+of+plant+community+ecology+in+microbial+interactions.">Rooting theories of plant community ecology in microbial interactions.</a> Trends. Ecol. Evol. 25, 468-478 (2010).</li><li>Brandt, A. J., de Kroon, H., Reynolds, H. L., Burns, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soil+heterogeneity+generated+by+plant-soil+feedbacks+has+implications+for+species+recruitment+and+coexistence.">Soil heterogeneity generated by plant-soil feedbacks has implications for species recruitment and coexistence.</a> J. Ecol. 101, 277-286 (2013).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> R: A language and environment for statistical computing. , (2012).</li><li>Crawley, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The R Book. , (2007).</li><li>Petermann, J. S., Fergus, A. J. F., Turnbull, L. A., Schmid, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Janzen-Connell+effects+are+widespread+and+strong+enough+to+maintain+diversity+in+grasslands.">Janzen-Connell effects are widespread and strong enough to maintain diversity in grasslands.</a> Ecology. 89, 2399-2406 (2008).</li><li>Reynolds, H. L., Haubensak, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soil+fertility,+heterogeneity,+and+microbes:+towards+an+integrated+understanding+of+grassland+structure+and+dynamics.">Soil fertility, heterogeneity, and microbes: towards an integrated understanding of grassland structure and dynamics.</a> Appl. Veg. Sci. 12, 33-44 (2009).</li><li>Sokal, R. R., Rohlf, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biometry, 3rd edn. , (1995).</li><li>Klironomos, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feedback+with+soil+biota+contributes+to+plant+rarity+and+invasiveness+in+communities.">Feedback with soil biota contributes to plant rarity and invasiveness in communities.</a> Nature. 417, 67-70 (2002).</li><li>Mommer, L., van Ruijven, J., Jansen, C., van de Steeg, H. M., de Kroon, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactive+effects+of+nutrient+heterogeneity+and+competition:+implications+for+root+foraging+theory?.">Interactive effects of nutrient heterogeneity and competition: implications for root foraging theory?.</a> Funct. Ecol. 26, 66-73 (2012).</li><li>Felsenstein, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenies+and+the+comparative+method.">Phylogenies and the comparative method.</a> Am. Natural. 125, 1-15 (1985).</li><li>Burns, J. H., Strauss, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=More+closely+related+species+are+more+ecologically+similar+in+an+experimental+test.">More closely related species are more ecologically similar in an experimental test.</a> Proc. Natl. Acad. Sci. U.S.A. 108, 5302-5307 (2011).</li><li>Fukami, T., Nakajima, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Community+assembly:+alternative+stable+states+or+alternative+transient+states.">Community assembly: alternative stable states or alternative transient states.</a> Ecol. Lett. 14, 973-984 (2011).</li></ol>

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&#160;et al.13). However, our understanding of the role of this type of heterogeneity on plant communities is minimal10,14. Here, we present a method for manipulating plant-induced soil heterogeneity, using soil from different origins (i.e. zones of ...

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 heterogeneity typically assumes that heterogeneity is extrinsic to the assembling community. Extrinsic heterogeneity is governed by factors such as landscape typology, which are independent of the community composition. Extrinsic heterogeneity can result in coexistence through niche partitioning (reviewed in Melbourne&#160;et al.3, e.g. Pacala and Tilman5&#160;and&#160;Chesson6). However, much of the environmental heterogeneity relevant to plant communities may be intrinsic to the community, developing as the community assembles and depending on the identity of the species in the community. Intrinsic heterogeneity can result from biotic feedbacks, which can lead to coexistence through negative frequency-dependence (e.g. Bever&#160;et al.7). Here, we describe a novel method for manipulating plant-induced soil heterogeneity, a type of soil heterogeneity that is intrinsic to the community and arises from plant-soil feedbacks.
Plant-soil feedbacks (PSF) occur when plants influence the soil structure, chemistry, or biota in a manner that affects subsequent plant performance in that soil, and PSF have large mean effects on plant performance in native plant communities8. Studies of PSF have typically either collected soils from the field or conditioned soils experimentally, then asked how plants perform in conspecific soil relative to heterospecific or sterilized soil9. If plants perform better in conspecific soil relative to reference soils, then PSF are positive, while if plants perform better in reference soils, PSF are negative. Reciprocal negative PSF can lead to frequency-dependent coexistence between species7. While the mean effects of PSF are well-characterized8, the effects of spatial heterogeneity in PSF are poorly understood10.
Because PSF occur on the scale of individual plants7 and because plants are often nonrandomly distributed in space and time, PSF are likely to lead to soil heterogeneity, which we call plant-induced soil heterogeneity. Unlike many other forms of heterogeneity (e.g. landscape topology), this heterogeneity is intrinsic to the assembling community and may thus influence community assembly differently than more extrinsic forms of heterogeneity. In order to understand the influence of this form of heterogeneity on plant performance and coexistence, we need experimental methods that manipulate plant-induced soil heterogeneity. Here, we demonstrate such a method, which uses soils conditioned by two species to create a heterogeneous treatment with separate patches from two soil origins and a homogeneous treatment, which is a mixture of the two soil origins. This soil mixing could represent at least two plausible scenarios in the field: (1) disturbance (e.g. rodent, agriculture) which mixes soils of different origins or (2) plants of two species growing in close proximity, such that their zones of root influence intermingle and homogenize.
We present two example experiments that use plant-induced soil heterogeneity to answer key questions at different levels of ecological organization: (1) Do plant populations respond to plant-induced soil heterogeneity? and (2) Do individual plants respond to plant-induced soil heterogeneity? We describe a field experiment using 6 soil patches to address the first question and a greenhouse experiment using 2 soil patches to address the second question. Quantifying both population and individual plant responses to soil heterogeneity is essential to understanding how heterogeneity influences community assembly.

<table border="0" cellpadding="0" cellspacing="0" width="1119">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Shovel(s)</td>
			<td style="width:136px;">Any</td>
			<td style="width:71px;">NA</td>
			<td style="width:696px;">It is helpful to have at least two shovels, one for each species of soil origin.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trowel(s)</td>
			<td style="width:136px;">Any</td>
			<td style="width:71px;">NA</td>
			<td style="width:696px;">It is necessary to have at least two trowels of identical size, one for each species of soil origin.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gloves</td>
			<td style="width:136px;">Any</td>
			<td style="width:71px;">NA</td>
			<td style="width:696px;">Gardening gloves can be used.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Diluted bleach</td>
			<td style="width:136px;">Any</td>
			<td style="width:71px;">NA</td>
			<td style="width:696px;">We use an ~1:10 concentration of household bleach (containing 5-10% NaClO) to water to sterilize all equipment between soil collections.</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:216px;">Plastic grid(s)</td>
			<td style="width:136px;">Any</td>
			<td style="width:71px;">NA</td>
			<td style="width:696px;">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.</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Plastic dividers</td>
			<td style="width:136px;">Any</td>
			<td style="width:71px;">NA</td>
			<td style="width:696px;">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.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Buckets or wheelbarrows</td>
			<td style="width:136px;">Any</td>
			<td style="width:71px;">NA</td>
			<td style="width:696px;">Any container for transporting soils.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Seeds</td>
			<td style="width:136px;">Any</td>
			<td style="width:71px;">NA</td>
			<td style="width:696px;">We collect seeds in the field by hand. Seeds can also be ordered from horticultural suppliers, if appropriate.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Plastic toothpicks</td>
			<td style="width:136px;">Soodhalter Plastics, Inc.</td>
			<td>805KP</td>
			<td style="width:696px;">We plant individual seeds glued on toothpick in the field experiment, to facilitate monitoring germination and survival of individuals.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Water soluble glue</td>
			<td style="width:136px;">Elmer&#39;s</td>
			<td style="width:71px;">Elmer&#39;s Glue-all</td>
			<td style="width:696px;">Any water soluble glue can be used to adhere seeds to plastic toothpicks.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pots</td>
			<td style="width:136px;">Any</td>
			<td style="width:71px;">NA</td>
			<td style="width:696px;">Pot size will depend on experimental plants used and number of soil patches desired (e.g. 2 or 6).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sand</td>
			<td style="width:136px;">Any</td>
			<td style="width:71px;">NA</td>
			<td style="width:696px;">Coarse sand may be mixed with field soils to improve drainage in pots.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lab tape&#160;</td>
			<td style="width:136px;">Any</td>
			<td style="width:71px;">NA</td>
			<td style="width:696px;">Tape may be used to label equipment used in handling soils with the species of origin.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pin flags</td>
			<td style="width:136px;">Any</td>
			<td style="width:71px;">NA</td>
			<td style="width:696px;">Flags can be used to identify individuals in the field, prior to soil collection.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Landscape fabric</td>
			<td style="width:136px;">Any</td>
			<td style="width:71px;">NA</td>
			<td style="width:696px;">Landscape fabric can be used in the field to minimize the growth of plants outside experimental plots.</td>
		</tr>
	</tbody>
</table>

experimental protocol for manipulating plant-induced soil heterogeneity

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.

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...

Watch this Scientific Journal Video about Experimental Protocol for Manipulating Plant-induced Soil Heterogeneity at JoVE.com

Experimental Protocol for Manipulating Plant-induced Soil Heterogeneity

Understanding the role of environmental heterogeneity in species coexistence has typically focused on types of heterogeneity that are extrinsic to the community&#8217;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.

Coexistence theory has often treated environmental heterogeneity as being independent of the community composition; however biotic feedbacks such as ...

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