An Abstract of the Thesis Of

AN ABSTRACT OF THE THESIS OF

Matthew Hovland for the degree of Master of Science in Rangeland Ecology and Management presented on September 13, 2017

Title: Mycological Maintenance of Rangelands and the Impacts of a Thatch Forming Invasive Annual Grass (Taeniatherum caput-medusae)

Abstract approved: ______Ricardo Mata-Gonzalez

Arbuscular mycorrhizal fungi (AMF) may exert profound influences on ecosystem resilience and invasion resistance in western North American sagebrush steppe and other arid rangeland plant communities. Maintenance of plant community structure through ecological feedbacks such as facilitation of nutrient cycling and uptake by host plants, physical and chemical contributions to soil structural stability, and mediation of plant competition suggest AMF may act as keystone facilitators in stressful arid environments. A review of the existing scientific literature highlights the relevance to AMF in sagebrush steppe rangelands, with specific focus on impacts of land management, disturbance, and invasion on AMF communities. A mycologically- based approach to invasive plant management is proposed, based on an example root

AMF analysis of the native perennial bunchgrass Pseudoroegneria spicata and the exotic invasive annual grass Taeniatherum caput-medusae along an invasion gradient in eastern Oregon. This example demonstrates AMF colonization of the foundation bunchgrass species and limited colonization of the annual grass, at least in the site

studied. Taeniatherum caput-medusae may effect colonization rates of dark septate endophytes in the roots of neighboring P. spicata, although direction of influence varied between years. Hypothesis testing of possible effects of T. caput-medusae on soil structure and function was explored, finding correlations between increasing invasive plant cover, and increased soil moisture levels during periods of high soil moisture (i.e. April). During drought periods a relationship was only visible on one of three research hills, with increased invasive cover correlating with decreases in soil moisture percentage. Influences of soil texture on site invasibility is discussed, along with possible long term effects of a thatch-forming invasive annual grass with limited mycorrhizal colonization, and recommendations are made for future research.

Mycological Maintenance of Rangelands and the Impacts of a Thatch-Forming Invasive Annual Grass (Taeniatherum caput-medusae)

by Matthew Hovland

A THESIS

submitted to

Oregon State University

in partial fulfillment of the requirements for the degree of

Master of Science

Presented September 13, 2017 Commencement June, 2018

Master of Science thesis of Matthew Hovland presented on September 13, 2017

APPROVED:

Major professor, representing Rangeland Ecology and Management

Head of the Department of Animal and Rangeland Science

Dean of the Graduate School

I understand that my thesis will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my thesis to any reader upon request.

Matthew Hovland, Author

ACKNOWLEDGEMENTS

The author expresses sincere appreciation for the support of his family and friends, especially his wife Amy and son Leif who are the strongest and most beautiful. Leif’s grandparents are west coast rock hounds holding hands on the beach in vest coats. Doctor Toby Westberry is a role model surfing in a sea of phytoplankton. The great naturalist Howard Bruner paints the path with eloquence and provokes a thoughtful conversation. Advising plant physiologist Dr. Ricardo Mata-Gonzalez is a mid-range jump shot specialist. Committee members Dr. Jennifer Parke and Dr. David Myrold are like ice skating instructors who guide glidingly and inspire experimentation. Doctor Jane Smith knows the location of every hidden matsutake. If this work is of any merit it has been made possible by a community of scientific researchers willing to communicate their trials and observations, specifically those researchers interested in plant-fungi and plant-soil relationships. Sincere appreciation is extended to Dr. R. Paul Schreiner of the USDA Agricultural Research Service who provided the space and equipment necessary for observations of mycorrhizal colonization and treated the author with unrivaled kindness, generosity, truthfulness and respect. This project would not have been possible without the encouragement of Dr. Thomas J. Rodhouse and contributions from the National Park Service Upper Columbia Basin Network Cooperative Task Agreement P13AC00136. Those individuals who helped with sampling on the hills of Clarno at any time of year are in the author’s debt (i.e. the botanist Bob Hladky), as are those who were at any time unwillingly subjected to the author’s mycocentric soliloquys.

CONTRIBUTION OF AUTHORS

Ricardo Mata-Gonzalez served as adviser, discussing research weekly, and providing comments on all drafts of the two manuscripts composing this thesis. Thomas J. Rodhouse was involved in the initial conception of the study including site selection, design, and preliminary data collection, as well as providing feedback on multiple drafts. R. Paul Schreiner provided guidance, feedback, comments, intellectual discussions, laboratory space, equipment, and assistance in analysis of mycorrhizal colonization. David Eduardo Prado-Tarango assisted in field sampling and data collection, soil lab analysis, and provided comments and intellectual discussions concerning soil characteristics.

TABLE OF CONTENTS

Page

Thesis Introduction ...... ….... 1

Chapter 1: Mycorrhizal Maintenance of Rangelands: Conceptualizing the Relationship between Mycorrhizal Fungi and Ecosystem Resilience and Resistance to Invasion in Sagebrush Steppe ……………..…………...... ………………………………………… 3

Abstract ………………………………………………………..…………...... 4

Introduction ….…………………………...……………….………………..…6

AMF Mediated Ecosystem Resilience …….………….………….……….....11

Stress amelioration…...... …………….…………………...……..12

Soil functional and structural stability ……………..……….……….13

AMF mediated plant competition ...……...……………………….…14

AMF Disturbance Ecology ……...... …………………………..…16

Plant-AMF Associations in Sagebrush Steppe Plant Species …………….....18

AMF obligate keystone shrub – Artemisia tridentata …...…...... 18

Facultative keystone bunchgrass – Pseudoroegneria spicata ……....20

Facultative invasive annual grass – Bromus tectorum ……………....22

Invasive annual grass with limited colonization – Taeniatherum caput- medusae ……………………………………...... 23

AMF obligate invasive forb – Centaurea maculosa ……...... …25

Non-mycorrhizal invasive forbs – Sisymbrium species ……...... 26

Non-mycorrhizal invasive forb – Salsola kali ….…………………...27

Case Study: Mycorrhizal Colonization along a Medusahead Invasion Gradient ……………………………………………………………………...28

TABLE OF CONTENTS (Continued)

Page Implications for Research and Management: Advancing the Understanding and Utilization of AMF Ecology in Sagebrush Steppe Restoration and Management ……………………………………………………..……….….30

Suggested Methods ………………………………………..……...…31

Mycorrhizal Status and Growth Benefits ……………………31

AMF Community Structure ………………....………………33

Restoration ………………………………………………..…35

Conclusion……………………………………………………………..…….36

References …………………………………………………………………...38

Figures …………………………………………………………………….....61

Chapter 2: Investigating Soil Conditioning Effects of a Thatch-Forming Invasive Annual Grass along an Invasion Gradient in a Protected Area Oregon, USA …………………………………………………………………………..…..…68

Summary …………………………………………………………………….69

Introduction ……………………………………………………………….....71

Materials and Methods ………………………………………………………75

Site Description …………………………………………………...…75

Soil Moisture ……………………………………………………...…77

Soil Analysis ………………………………………………………...77

Root Analysis ………………………………………………………..78

Statistical Analysis …………………………………………………..79

Results ……………………………………………………………………….79

Soil Moisture ………………………………………………………...79

Texture …...... ….…………………………………………………….80

TABLE OF CONTENTS (Continued) Page Respiration……………... …………………………………………...81

pH ...………………………………………………………………….81

Percent root length colonization ……………………………….……81

Discussion ……..…………………………………………………………….82

Wet season soil moisture and vegetation cover ………………..……83

Drought season effects and clay percentage thresholds …………..…84

Mycorrhizal colonization …………………………………………....86

Further research ……………………………………………………..87

Conclusion …………………………………………………………..88

Acknowledgements ………………………………………………………….89

References ……………….…………………………………………………..90

Figures ……………………………………………………………………….96

Thesis Conclusion ………………………………………………………………….102

Comprehensive Bibliography …………………………………………………...…104

LIST OF FIGURES

Figure Page

1. Mycorrhizal feedbacks contributing to resilience/resistance……………………...61

2. Impacts of disturbance and mycorrhizal status of plant invaders...………………62

3. Mycologically based invasive plant management framework...………………….63

4. Dimorphism expressed in bluebunch wheatgrass roots……………………….….64

5. Paris-type colonization of bluebunch wheatgrass roots.....……………………….65

6. Simultaneous colonization of bluebunch wheatgrass roots by arbuscular mycorrhizal and dark septate fungi ….………………………………………………66

8. Medusahead root morphology…..………………………………………………..66

9. Medusahead colonization by AMF and DSE in June and July …….………….…67

10. Map of John Day Fossil Beds……. ………………………………...…………..96

11. Study site photos…..……………...…………………………………...……....97

12. Summary of soil characteristics …...……………………….…………………..98

13. Bluebunch wheatgrass colonization by AMF and DSE…….…………………..98

14. Hill 2 regression plots……………………...………………………………..…100

Introduction

The introduction to this thesis will remain brief, as the pages following are comprised of two manuscripts prepared for submission to two separate journals, both with abstracts and introductions of their own. The first manuscript is a synthesis paper which, one hopes, will serve to illuminate the importance of arbuscular mycorrhizal fungi in rangelands, at least to the point where land managers and researchers will consider the possible impacts of both land use and altered plant community structure on intact mycorrhizal networks, and subsequently on ecosystem structure, function, and resilience. If this manuscript accomplishes all it sets out to, it will provide a research framework to follow when attempting to understand plant invasion ecology from a mycological perspective, including considering the restoration and maintenance of mycorrhizal networks. The second manuscript attempts to follow the first few directives of the research framework, using the ongoing invasion of the annual grass medusahead (Taeniatherum caput-medusae) in central Oregon as an illustrative example. In this research paper we consider the impacts of medusahead on the mycorrhizal colonization of a neighboring perennial bunchgrass, as well as on soil characteristics which may influence mycorrhizal fungi community structure, or soil structural/functional integrity.

At this point in time, rangeland managers and arid land researchers in the western United States have been concerned with annual grass invasion for about 100 years. These grasses, namely cheatgrass (Bromus tectorum) and medusahead, are known to create vast monocultures, contribute to a thick thatch layer, and increase 2

fine fuel load and continuity. Historic fire return intervals have notably decreased

(meaning more frequent fires) due to this altered vegetative composition, and as the annual grasses are post-disturbance colonizers, a positive feedback loop results. As we search to understand the invasion ecology of plants in arid systems the narrative has shifted from landscape scale removal/restoration projects to identifying high quality native habitats and maintaining their integrity by managing for the characteristics of that site which contribute to resilience against disturbance and resistance to invasion. Enter arbuscular mycorrhizal fungi (AMF), obligate symbionts which colonize the roots of roughly 85% of vascular plant species (Wang and Qui,

2006). While it has become increasingly clear that the relationships between plants and AMF are complex in nature, the common ways in which AMF benefit their plant hosts will be explored at depth in the first half of the synthesis manuscript. In general,

AMF increase uptake of nutrients for the plant in exchange for photosynthates. In arid systems especially, their ability to ameliorate nutrient and drought stress in their plant hosts may be a defining factor for the maintenance of current plant communities. The effects of these organisms are so great that some plants will not survive environmental conditions of their current range, or will be outcompeted, without their fungal symbionts. We can only hope that a future reader of this thesis and ones like it will find even the most detailed descriptions of AMF form and function elementary and superfluous.

Mycorrhizal Maintenance of Rangelands: conceptualizing the relationship between arbuscular mycorrhizal fungi and ecosystem resilience and resistance to invasion in sagebrush steppe

A Synthesis Paper Prepared for Rangeland Ecology and Management

Matthew Hovland a,1, *, Ricardo Mata-Gonzalez b, R. Paul Schreiner c, Thomas J.

Rodhouse d a Graduate Student, Department of Animal and Rangeland Science, Oregon State

University, Corvallis, OR 97331 USA b Associate Professor, Department of Animal and Rangeland Science, Oregon State

University, Corvallis, OR 97331 USA c Plant Physiologist, USDA-ARS Horticultural Crops Research Laboratory, Corvallis,

OR 97330 USA d Ecologist, National Park Service, Oregon State University-Cascades, Bend, OR

97702 USA

* Correspondence: Matthew Hovland, Department of Animal and Rangeland Science,

Oregon State University, Corvallis, OR 97331 USA. Email address: [email protected]

1 Current address: Matthew Hovland, Department of Animal and Rangeland Science,

120 Withycombe Hall, Corvallis, OR 97331 USA.

Research was funded by the National Park Service Upper Columbia Basin Network

Cooperative Task Agreement P13AC00136.

Abstract

Arbuscular mycorrhizal fungi (AMF) may exert profound influences on ecosystem resilience and invasion resistance in western North American sagebrush steppe and other arid rangeland plant communities. While scientific understanding of resilience and resistance dynamics in the sagebrush biome has developed rapidly in recent years it has done so by focusing primarily on abiotic soil structural and climatic linkages to above-ground plant community composition. Maintenance of plant community structure through ecological feedbacks such as facilitation of nutrient cycling and uptake by host plants, physical and chemical contributions to soil structural stability, and mediation of plant competition suggest AMF may act as keystone facilitators in stressful arid environments. This could translate into effects on state-transition dynamics in steppe communities by increasing native plant community resilience to drought, grazing, and fire, resistance to exotic plant invasion, and by reinforcing positive feedback loops in invaded sites. Invasive plant species have been shown to alter AMF community composition in ways which may decrease the effectiveness of restoration by decreasing seedling establishment, soil moisture potential, and nutrient availability. Here we review the existing scientific literature relevant to AMF in sagebrush steppe rangelands, with specific focus on impacts of land management, disturbance, and invasion on AMF communities. We present a case study as a motivating example of striking differences in mycorrhizal colonization between the native perennial bunchgrass foundational species

Pseudoroegneeria spicata and the exotic invasive annual grass Taeniatherum caput- medusae along an invasion gradient in eastern Oregon. Our review found compelling

evidence that exotic plant invasion impacts on AMF community structure vary with plant mycorrhizal host status and functional guild. We conclude by outlining the research needs to advance ecological knowledge of AMF in rangelands and enable better integration of this knowledge into the resilience to disturbance and invasion resistance paradigm of sagebrush steppe conservation and management. We encourage practitioners to explicitly integrate knowledge of plant-fungi relationships into invasive plant management strategies. Based on our review, it appears likely that current emphases on soil abiotic attributes and above-ground plant community attributes will be insufficient to effectively address lack of resiliency and invasion resistance in many rangeland ecosystems.

Keywords: biological invasion, resilience based management, stress gradient hypothesis, ecological feedbacks, plant-fungal interactions, sagebrush steppe

Introduction

Semi-arid regions in the United States such as the Intermountain West are experiencing landscape-scale shifts in plant community composition due to historic and prevailing land use practices and increased frequency and intensity of natural disturbances which allow for the invasion and persistence of non-native annual grasses and forbs. Invasive annual grasses such as cheatgrass (Bromus tectorum L.) and medusahead (Taeniatherum caput-medusae [L.] Nevski) are replacing historically fire-resilient sagebrush and bunchgrass steppe communities with monocultures of spatially contiguous fine fuels. The resulting positive feedback loop promotes the persistence and spread of disturbance associated species through a decreased fire return interval, while decreasing the quality of habitat for wildlife such as sage grouse (Pyke, 2011). Development and persistence of these kinds of annual grass dominated novel ecosystems has driven researchers to identify the causative agents that foster maintenance of native plant communities in order to define management strategies to promote resiliency and resistance to invasion (Bestelmeyer and Briske, 2012; Chambers et al., 2017). The extent to which a native plant community is able to maintain ecological function and/or return to its previously established composition following a disturbance is a measurement of that community’s resilience (Holling, 1973). While research has traditionally focused on the effects of feedback loops in terms of outcomes (alterations in quality of above ground habitat and forage, associated shifts in wildlife populations and stocking rates, and increases in the frequency and scale of wildfires), we are only beginning to understand the importance of plant-soil feedbacks (PSF), and the role of mycorrhizal

fungi in ecological feedback loops. Here we discuss both of these concepts, emphasizing the importance of mycorrhizal fungi as organisms which mediate ecosystem resilience by participating in both positive (community structure altering) and negative (community structure maintaining) feedback loops which ameliorate individual plant stress, contribute to soil structural stability, and mediate interspecific plant competition.

Arbuscular mycorrhizal fungi (AMF) are symbiotic root endophytes which influence host plant establishment and survival via increased uptake of mineral nutrients such as phosphorus and nitrogen, and micro-nutrients such as magnesium, zinc, and copper. There are two physiologically distinct components of the fungal partner in mycorrhizas. The external (extra-radical) hyphae, or soil hyphae phase, absorbs phosphorus and other nutrients from soil, essentially increasing absorptive root surface area of host plants into soil micropores (Smith and Read, 2008). Internal root colonizing hyphae form exchange sites (arbuscules and arbuscular coils) within cortical cells wherein nutrients are exchanged for host plant photosynthates. AMF, the primary organisms within the subphylum Glomeromycota (Spatafora et al., 2016), evolved at least 400 million years ago, possibly earlier (Hedges and Kumar, 2009), and appear to have helped early land plants colonize the terrestrial environment

(Smith and Read, 2008). Present day AMF are obligate biotrophs, and rely on obtaining reduced carbon from host plants in order to complete their life cycles.

Although plant-AMF interactions are often mutualistic, the contribution of AMF to host plant fitness may depend on several factors including plant functional guild

(Bunn et al., 2015), mycorrhizal status of neighboring plant competitors (Carey et al.,

2004), local plant-AMF adaptations (Rúa et al., 2016) soil nutrient levels (Johnson et al., 1997) and environmental stress levels (Wahl and Spiegelberger, 2016).

Furthermore, plants that are facultative hosts, or considered to be non-mycorrhizal are often negatively affected by AMF associations (Reynolds et al., 2006). While the topics of resource exchange and host selection are currently not well understood,

AMF root colonization and associated impacts on plant physiology and ecology depend on both plant and fungal species identity and fungal community composition

(van der Heijden et al., 1998).

Arbuscular mycorrhizal fungi may play a greater role in maintaining desired plant communities and mediating resiliency to disturbances in rangeland ecosystems than in other biomes due to the greater stress inherent in arid grassland ecosystems

(Allen, 2007). Arbuscular mycorrhizal fungi provide alleviation of plant stress through increased nutrient uptake and water use efficiency (Bolandnazar et al., 2007;

Pena-Becerril et al., 2016), and may promote the persistence of resilient plant communities by mediating competitive interactions between native and exotic plant species (Callaway et al., 2004). AMF also maintain soil structural and functional stability through increased soil aggregate formation which affects water availability, erodibility, carbon cycling and microbial microhabitats within aggregates (Rillig and

Mummey, 2006). While AMF play a role in resilience and resistance-promoting feedback loops, they are also an intermediary of plant-soil feedbacks, wherein a given plant species either promotes or reduces conspecific (same species) or heterospecific

(different species) colonization and performance through alteration in the soil biotic community, nutrient availability, and soil functional stability (Van der Putten et al.,

2013). While early seral plant species, including exotic annuals, may utilize plant-soil feedbacks to a greater degree than late seral species, these effects are often community structure altering, competitive effects, which may have evolved due to the lack of soil microbial mutualists in early seral, post-disturbance soils (Bauer et al.,

2015). Late seral feedbacks appear to be less extreme, since they are often community structure maintaining or facultative effects, but plants in late successional stages are particularly sensitive to shifts in AMF community structure (Koziol and Bever,

2016a), and often these are the communities for which we manage.

The relationships between AMF and their host plants have been of scientific interest in rangelands since at least the late 1970s (e.g., Fitter, 1977; Reeves et al.,

1979), with the majority of research focusing on invasion ecology, particularly AMF influenced competition between native and exotic plants (see Goodwin, 1992).

Although early work laid the foundation for considering AMF as mediators of resiliency, over the last decade technological advancements and decreased costs of genetic sequencing (Kohout et al., 2014; Lindahl et al., 2013) have revealed the complexity of mycorrhizal fungi communities, and allowed researchers to record subtle shifts in AMF community composition (Busby et al., 2013; Lekberg et al.,

2013). Broadly, AMF communities can be impacted in two general ways depending on the invading plant’s mycorrhizal status. Exotic mycotrophic (AMF host) invasive plant species may alter the AMF community by acting as a preferential hosts to a specific composition of AMF species, possibly increasing competitive effects over native plant species (Mummey and Rillig, 2006). Non-mycorrhizal exotic plant species are expected to reduce local AMF abundance, and therefore soil inoculum

potential (Bainard et al., 2009), since, inherently, they are reducing the abundance of symbiotic partners for the obligate biotrophic fungi. A monoculture of non- mycorrhizal invasive plants could potentially displace the AMF common mycelial network (CMN), a network of fungal hyphae within the soil that connects mycotrophic plants within the community (Smith and Read, 2008). Importantly, it is difficult to predict a directional shift in AMF community composition based solely on the invasion status of a plant community, as invasive plants are not a monophyletic group, and exhibit a broad range of competitive strategies and mycorrhizal dependencies (Bunn et al., 2015).

Given the considerable potential for below-ground biotic interactions to impact resilience and resistance and subsequent constraints on rangeland management and restoration actions, a need has emerged to consolidate what is currently known about AMF ecology, disseminate that information to the rangeland science and management communities, and outline next steps required to advance our understanding of and ability to harness AMF ecology within the context of sagebrush steppe conservation and management. We do so by reviewing AMF ecology in rangelands, which includes revealing landscape-scale shifts in belowground fungal diversity and abundance, and considering the consequences of such shifts on above- ground plant competition. We will begin by examining the role of AMF in mediating ecosystem resilience (Fig. 1), establishing whether or not there is a need to maintain native AMF communities to promote resilience, and emphasize the relationship between AMF and the sagebrush steppe keystone plant species including big sagebrush (Artemisia tridentata spp. Nutt.) and bluebunch wheatgrass

(Pseudoroegneria spicata [Pursh.] Á. Löve). Next, we will review AMF disturbance ecology including the effects of disturbance on AMF community structure and function (Fig. 2). We further explore potential shifts in AMF community composition by examining the relationships between AMF and common invasive plant species of the Intermountain West. We provide a motivating example from a case study in central Oregon where we sampled root AMF on the foundation bunchgrass, bluebunch wheatgrass, and invasive Eurasian annual grass, medusahead, along transects spanning invasion gradients, and contrast presence and composition of mycorrhizal colonies on these two species. We conclude with a conceptual framework of recommended steps to advance scientific understanding of AMF ecology in sagebrush steppe and the conservation and management community’s ability to utilize this growing understanding in restoration and management decision- making (Fig. 3).

AMF Mediated Ecosystem Resilience

In arid systems (<600 mm of annual precipitation) where primary productivity, and hence post-disturbance plant re-establishment, is limited by water

(Austin et al., 2004), local resilience may be mediated by microclimates that provide more moisture and less heat stress, such as sites located on steep north facing slopes in the northern hemisphere or beneath shrubs (Chambers et al., 2014; Germino et al.,

2016). This is described by the stress gradient hypothesis which proposes that the more a system is stressed, the greater its expression of, and dependence on biologically driven stress amelioration (facilitation) rather than competition (Brooker

and Callaghan, 1998). This hypothesis is supported in arid systems such as sagebrush steppe communities, where for example, perennial nurse plants (e.g. Artemisia tridentata, Pseudoroegneria spicata) mediate seedling establishment (Jackson and

Caldwell, 1993; Reisner et al., 2015). The formation of nurse plant mediated “islands of fertility” inherently coincides with a lack of vegetation between such areas, creating a patchy mosaic. This patchiness is maintained at least in part by biological soil crusts, which discourage seedling establishment of exotic annuals (Belnap, 2003;

Deines et al., 2007). Un-vegetated interstices reduce fuel continuity and severity of eventual wildfire events, contributing to system resiliency, however, increasingly arid regions of sagebrush steppe (i.e. Wyoming big sage communities) are more susceptible to invasion by exotic ruderal species exhibiting competitive strategies at high environmental stress levels (Chambers et al., 2014; Miller et al., 2013).

Considering that the optimal resource allocation model predicts that plant-soil feedbacks, which include AMF and rhizobacteria as intermediaries, will facilitate growth of neighboring plants in soils which are resource limited (Revillini et al.,

2016), we suggest AMF may function as belowground keystone facilitators in arid regions of the Intermountain West, contributing to plant community structural maintenance through stress amelioration, soil structural maintenance, and mediation of plant competitive effects.

Stress Amelioration

Rangelands in western North America are often characterized by low annual precipitation and nutrient poor soils with seasonally available nutrient pulses (Austin et al., 2004). Intuitively, environmental stress amelioration by AMF in arid

ecosystems is driven largely by increased nutrient uptake and associated increases in water uptake in the host plant. Drought stress can be ameliorated through multiple indirect mechanisms, such as AMF altered plant root morphology and regulation of stomatal conductance (Augé, 2001), possibly via altered expression of aquaporins which regulate cross membrane transfers (Ouziad et al., 2006; Uehlein et al., 2007).

Also an indirect mechanism, increased phosphorus availability (Payne et al., 1992;

Singh and Sale, 1991) and nitrogen-phosphorus ratios increase water use efficiency in some plants (Yan et al., 2016). Direct effects of increased water uptake through hyphal cytoplasm and along the exterior of extraradical hyphae have also been proposed (Allen, 2007; Wu et al., 2013), and although these effects have not been shown to significantly impact the host’s overall water budget, research on the topic is limited, especially in arid ecosystems where AMF ameliorated drought stress is likely more vital to plant health. Generally speaking, AMF can improve plant growth more in drier soils than wetter soils when compared to non-mycorrhizal plants (Augé et al.,

2015), and at least conceptually, when soil water potential exceeds -1.5 Mpa, soil water is restricted to micropores accessible only to plants via extraradical hyphal tips

(Allen, 2007). AMF mediated hydraulic redistribution could also be a factor in maintaining live roots in periods of drought when soil water potential is below -1.0

Mpa (Querejeta et al., 2007).

Soil functional and structural stability

Amelioration of drought by AMF is not only related to colonization of plant roots but also to hyphal colonization of the soil (Augé, 2004). One way AMF may alter available soil moisture is through increased soil aggregation, physically, via

hyphal enmeshment (Rillig et al., 2010), or chemically, through deposition of exudates including the glycoprotein glomalin (Rillig and Mummey, 2006). Glomalin is found in the extraradical hyphae and spores of AMF, and its presence in the soil as glomalin related soil proteins has been positively correlated with increased carbon and nitrogen sequestration (Wilson et al., 2009). Carbon inputs via AMF hyphal decomposition (Rillig and Mummey, 2006) contribute to increased soil organic carbon (SOC) pools, increasing soil moisture holding capacity (Rillig, 2004), which may also be integral in maintaining fire resilient bunchgrass communities tending to favor moist soil (Miller et al., 2013). Hyphal enmeshment and the creation of water stable soil aggregates also reduces soil erosion potential (Mardhiah et al., 2016) and is expected to increase infiltration rates. Importantly, soil aggregation and altered soil water characteristics likely play roles in shaping plant community structure in arid systems (Miller et al., 2013).

AMF mediated plant competition

Resistance to exotic plant invasion may be higher in AMF associated plant communities because of their lower susceptibility to environmental stress. However, other contributing factors of resistance are likely at play, especially when the invading species are poor hosts. While non-mycorrhizal plants may represent a fraction of the global population of vascular plants (around 15%), facultative, and non-mycorrhizal plants are common in rangelands as disturbance-dependent invasive annuals, ruderal forbs, and halophytes such as cheatgrass, Sisymbrium mustard species, and members of the non-mycorrhizal Chenopodiaceae family. The growth of non-mycorrhizal plants is often negatively affected by neighboring plants colonized

by AMF (Allen et al., 1989; Lambers and Teste, 2013; Veiga et al., 2013), however, plant identity as well as plant competitive interactions will contribute to the outcome

(Rinaudo et al., 2010). The deleterious effects on non-mycorrhizal species in the presence of AMF are likely due to parasitic hyphal colonization of roots without the formation of arbuscules or arbuscular coils, and the cost of defense without the benefit of nutrient exchange (Daisog et al., 2012; Veiga et al., 2013). Large, established plants colonized by AMF may also suppress nearby seedling growth of both mycorrhizal and non-mycorrhizal species (Janoušková et al., 2011), possibly due to reduction in soil nutrients adjacent to extraradical hyphae. This may provide a mechanism for the creation of invasion resistant bare soil interstices surrounding large plants, which would assist in the maintenance of landscape heterogeneity (i.e. patchiness), and moderated fuel continuity.

AMF likely play a role in plant competition in all instances where at least one of the competitors is either an obligate or facultative host, however, the extent to which AMF are actually altering the outcome of plant competition is, in many cases, yet unknown. The instances in which AMF are more inclined to facilitate native plant community maintenance are, intuitively, when non-mycorrhizal plants are invading a community dominated by obligate or facultative AMF hosts, or when facultative

AMF host plants are invading a community dominated by obligate AMF hosts.

However, when the invading plant is an obligate AMF host, an intact AMF community/network is less likely to contribute to native plant community maintenance. In fact, obligate AMF host plant invaders may utilize intact CMNs in order to either gain carbon from (Carey et al., 2004), or distribute allelopathic

exudates throughout the system (Achatz and Rillig, 2014). Plant competition and

AMF mediated effects could also be subject to adaptation of plant species over time.

Waller et al. (2016) examined the influence of AMF on competition between the exotic forb Centaurea solstitialis L. and the native bunchgrass Nassella pulchra

(Hitchc.) Barkworth. AMF colonization had a negative effect on the competitiveness of C. solstitialis plant genotypes selected from its native range when grown with N. pulchra, but the effect was less pronounced in C. solstitialis genotypes selected from its invaded range (i.e. North America). Therefore, as exotic species become more adapted to their invaded communities, the ability for the native community to resist that invasion may decrease.

AMF Disturbance Ecology

While AMF may mediate plant competition between exotic and native species, invasion is often not a bachelor event, and is instead preceded by disturbances such as fire or grazing which may alter AMF functionality prior to invasion. Different species of AMF have varying tolerance to abiotic stressors such as drought, extreme temperatures and salinity (Lenoir et al., 2016), as well as varying affinities for specific plant hosts, therefore, community structure is susceptible to alteration mediated by anthropogenic stressors of intensified disturbance (e.g. livestock grazing, fire) and to exotic plant invasion. Land use changes which involve mechanically disturbing the soil such as tilling may at least temporarily decrease inoculum potential because of damage to mycelial networks and roots of host plants

(Kabir, 2005). Similar reduction in AMF hyphal networks occur in fires which reduce

vegetation to bare soil, eliminating host presence (O’Dea, 2007a). Fire decreases

AMF spore diversity through increased soil temperatures, although subsequent alteration of soil chemical and physical properties may influence AMF community re- assemblage (Allen et al., 2011). Some studies have shown fire may have less of an effect on overall spore abundance (Eom et al., 1999) or density and hence inoculum potential (Longo et al., 2014), although the hyphal network may be more critical in some environments. Xiang et al. (2015) recorded a decrease in spore density a year post-fire in a semi-arid coniferous forest in northern China, however, the AMF community structure and spore numbers returned to pre-fire levels after 11 years. In a measure of mycorrhizal inoculum potential (MIP) in Bouteloa dominated grasslands of Arizona, AMF colonization rates decreased significantly after soils were subjected to low intensity prescribed burns (O’Dea, 2007a). The same author (O’Dea, 2007b) stresses the importance of the reestablishment of the AMF community following fire in terms of stabilizing soil which might otherwise be easily eroded.

The response of mycorrhizal plants to grazing varies, and likely depends on a combination of species specific tolerance of grazing, and responsiveness to AMF colonization (Allsopp, 1998). Walling and Zabinski (2006) recorded smaller plant sizes after simulated grazing in AMF colonized bluebunch wheatgrass and Idaho fescue (Festuca idahoensis Elmer; lower colonization rates than bluebunch) than non- colonized plants of the same species grown in sterile soils. AMF species identity also affects plant responses to grazing, for example, some species, or combinations of

AMF species, increased root and shoot biomass in clipped Bromus inermis Leyss. more than others (Klironomos et al., 2004). Grazing can be viewed as multiple

disturbances (trampling, herbivory, nutrient deposition) with individual and combined effects on AMF community structure dependent upon severity as well as plant host and AMF identity (Allsopp, 1998). Eom et al. (2001) found grazing to stimulate AMF root colonization in C4 grasses located in the Flint Hills prairie in northeast Kansas, however, reductions in percent root colonization may occur in some plants due to the resulting low photosynthetic rates and translocation of carbon to roots (Ilmarinen et al., 2008). Interestingly, Medina-Roldan et al. (2008) found no long-term effect on AMF root colonization in the grazing-tolerant species, Bouteloa gracilis (Willd. ex Kunth.) Lag. ex Griffiths, and Allsop (1998) found grazing to have less of an effect on extra-radical hyphae lengths in more grazing tolerant species.

Trampling in the absence of defoliation may increase AMF abundance (Liu et al.,

2015), as trampling may encourage reallocation of plant resources to root growth.

Disturbance of the soil surface through trampling may also increase spore dispersal, as will consumption of AMF spores by grazers, as shown in a community ecology study at Yellowstone National Park, where bison dung was used to successfully inoculate soil with AMF (Lekberg et al., 2011). Yellowstone is also the site of a 40- year old ungulate exclosure which allowed Murray et al. (2010) to detect a correlation between grazing and an increase in AMF diversity, while simultaneously recording a decrease in spore abundance.

Plant-AMF Associations in Sagebrush Steppe plant species

AMF Obligate Keystone Shrub – Artemisia tridentata

Artemisia tridentata (big sagebrush), may be the most “iconic” component of the sage-steppe ecosystem (Davies et al., 2007). As a keystone evergreen shrub, it provides habitat for a variety of fauna such as sage-grouse, while below canopy microclimates support establishment and facilitate persistence of bunchgrasses and forbs, especially at sites experiencing greater heat and drought stress (Davies et al.,

2007; Reisner et al. 2015). Historic removal of big sagebrush to extend grasslands for grazing, combined with invasion of exotic annuals and the associated altered fire regimes have led to a reduction of big sagebrush in the intermountain west, and restoration and reestablishment can be difficult due to small seed size, competition with annual grasses, and drought associated mortality (Meyer, 1992). Recently,

Davidson et al. (2016) found that the risk of mortality from cold and drought stress decreased by an average of 42 percent when big sagebrush seedlings were inoculated with native AMF species prior to transplanting, versus seedlings which relied solely on native AMF colonization. This correlates with a higher overall colonization rate

(84 percent) in the inoculated plants. The findings of Davidson et al. corroborate a previous study by Stahl et al. (1998) which reported root colonization increasing from

65 to 86 percent when grown with inoculum. The Stahl et al. (1998) study similarly highlighted the dependence of big sagebrush on AMF to mitigate drought stress, as mycorrhizal sagebrush seedlings tolerated soils up to -3.22 MPa while non- mycorrhizal seedlings succumbed at -2.77 MPa. Given that big sagebrush seedlings have poorer survival when not inoculated with AMF leads us to consider whether current climatic conditions (i.e. drought conditions) are exceeding the ameliorating effects of AMF at low levels of colonization. It is also possible that colonization

levels, and seedling survival, might be greater if seedlings were allowed to germinate beneath a nurse plant, in the presence of an intact CMN. In a discussion on the influence of mycorrhizal networks, Smith and Read (2008) propose that seedlings may benefit most from a CMN if the seeds are small, such as in big sagebrush, and the seedling establishes beneath a canopy where sunlight may be limited, as seen in nurse plant mediated microclimates. An earlier study by Stahl et al. (1988) suggested a relationship between mycelial networks and colonization levels, as sagebrush seedlings in disturbed soils had low levels of colonization, and gained little benefit from AMF association. A natural method of increased root colonization could coincide with insect assisted propagule density increases, as seen in ant mounds located near big sagebrush (Friese and Allen, 1993), but seedling survival has yet to be correlated with proximity to ant mounds.

Facultative Keystone Bunchgrass – Pseudoroegneria spicata

Pseudoroegneria spicata (bluebunch wheatgrass), is considered a foundation bunchgrass, due to its tall stature, dominance in the bunchgrass steppe community, and forage quality (Rodhouse et al., 2014). This tussock type bunchgrass forms habitats for small birds and mammals, as well as facilitates the growth of heterospecifics (Jackson and Caldwell, 1993), and has been shown to confer invasion resistance under some conditions in sagebrush steppe (Davies, 2008). Bluebunch wheatgrass is attributed with conditioning soils in a way which positively affects the growth of heterospecific plants (positive heterospecific PSF) in clayey soils, and negatively effects conspecific plants (negative conspecific plant PSF) in silty loams

(Perkins and Nowak, 2013). Meiman et al. (2006) recorded a positive plant soil

feedback on the emergence of spotted knapweed (Centaurea maculosa auct. non

Lam.), but a negative effect on diffuse knapweed (Centaurea diffusa Lam.). In a similar study, diffuse knapweed cover decreased from 18 percent when grown in soil conditioned by weedy species including itself and cheatgrass, to 5 percent cover when grown in soils conditioned by bunchgrass steppe community members including bluebunch wheatgrass, needle and thread grass (Hesperostipa comata [Trin. & Rupr.]

Barkworth), and species of the genus Lupinus L. (Kulmatiski et al., 2004). Soil feedbacks aside, bluebunch wheatgrass has shown greater negative competitive effects against spotted knapweed than did other bunchgrasses in the community

(Idaho fescue and prairie June grass [Koelaria macrantha]) which tend to be colonized by lower levels of AMF, and these effects dissipated after soils were treated with fungicide (Callaway et al., 2004).

In an effort to better understand bluebunch wheatgrass root systems, we unearthed whole plants from an Oregon, USA research site (see Case Study section) where medusahead is invading into a bluebunch wheatgrass community. Bluebunch wheatgrass exhibits morphologically distinct root types: shallow, laterally extending roots; and deeper, vertical roots of larger diameter (Fig. 4). This dimorphism was less distinct in clayey soils where vertically extending, large diameter roots dominated

(personal observation), lending itself to previous characterizations of bluebunch wheatgrass’ “architectural root plasticity” (Arredondo and Johnson, 2011). In our

2016 preliminary study, only the deeper roots appeared to be colonized by AMF at an average 51 percent root length colonization, with 2017 root sampling averaging closer to 60 percent (Hovland, et al. in preperation). Notably, the majority of the AMF

structures identified within the roots were arbusculate coils colonizing neighboring cortical cells, resembling the Paris-type AMF colonization pattern (Smith and Read,

2008) (Fig. 5). The presence of arbusculate coils does not signify colonization by a particular AMF species, rather, this morphology is likely driven by a combination of plant and fungal species identity, and possibly environmental/edaphic factors (Smith and Read, 2008). However, Paris-type colonization is rare in some ecosystems (e.g.

Majewska et al., 2015; Velazquez et al., 2010; Zubek et al., 2011), but dominates in the roots of ferns and lycopods (Lara-Pérez et al., 2015; Zubek et al., 2010). Dark septate endophyte (DSE) hyphae and microsclerotia were also observed in some bluebunch wheatgrass roots in addition to AMF (Fig. 6).

Facultative invasive annual grass - Bromus tectorum

One of the most widespread invaders of the Intermountain West is cheatgrass

(Bromus tectorum), an exotic annual grass which creates vast stands and alters fire return intervals (Antonio and Vitousek, 1992). One of the proposed mechanisms for invasion involves increased available nitrogen, possibly by altering the soil nitrifying community (Blank and Morgan, 2016), although Perkins and Nowak (2013) found no evidence for positive conspecific feedbacks in their study. Considered a facultative

AMF host, cheatgrass biomass increases are not correlated with AMF root colonization (Allen, 1984), and in some cases AMF colonization may actually decrease cheatgrass performance (Owen et al., 2013). Mycorrhizal root colonization of cheatgrass is lower in salt shrub desert communities than in sagebrush grasslands

(Haubensak et al., 2014). However, it is common that AMF root colonization of rangeland plants decreases with increasing soil salinity (Aliasgharzadeh et al., 2001).

AMF community composition in the soil differs between sites invaded by cheatgrass and sites dominated by native plant species (Busby et al., 2013; Hawkes et al., 2006). Also, more saprobic and pathogenic fungal species have been observed in cheatgrass-dominated soil compared to soils associated with big sagebrush (Weber et al., 2015). Following the findings of Weber et al. (2015), Gehring et al. (2016) investigated the association between cheatgrass and dark septate endophytes (DSE).

These fungi in the phylum Ascomycota are identified by melanized septate hyphae, are facultative biotrophic symbionts, and may serve as mycorrhizal mutualists in certain instances (Barrow, 2003; Mandyam and Jumpponen, 2005). DSE can also translocate nutrients between soil crusts and vascular plants (Green et al., 2008).

However, DSE-plant interactions likely fall on a continuum ranging from mutualistic to parasitic (Mandyam and Jumpponen, 2005). Cheatgrass roots were highly colonized by DSE, with a root length colonization average of 60 percent (Gehring et al. 2016). Furthermore, colonization of DSE in roots of big sagebrush was greater when roots analyzed were sampled from areas invaded by cheatgrass versus areas lacking cheatgrass cover (Gehring et al., 2016). This raises the question as to what role DSE fungi may play in mediating plant competitive interactions, which may in turn influence plant community composition, and invasion by exotic species.

Invasive annual grass of questionable mycorrhizal status - Taeniatherum caput- medusae

Introduced to the Intermountain West in 1897, medusahead is viewed as one of the most problematic invasive plants by rangeland managers (Johnson et al., 2011).

Its long, barbed awns cause problems to livestock when becoming lodged in eyes or

noses, and its high silica content (Swenson et al., 1964) makes medusahead a poor forage. The silica content of dead plant matter resists decay, encouraging thatch creation which shades out heterospecifics and contributes to light-fuel loading and increased fuel continuity, altering fire regimes (Nafus and Davies, 2014). Although

Gornish et al. (2016) found no effects of medusahead invasion on soil microbe community structure, medusahead invasion alters soil organic matter content (Nafus and Davies, 2014), which is a known environmental filter affecting AMF community structure (Zhu et al., 2016). Early studies seemed to indicate correlations between soil microbe communities and medusahead invasion (Trent et al., 1992). A comparison of medusahead performance when grown in native and invaded soils, including autoclaved soils, revealed that soil microbes may play a role in invasibility (Blank and Sforza, 2007), however, follow up research found no correlation between the bacterial and fungal ratio and medusahead invasion (Blank et al., 2008). Furthermore, clayey soils conditioned by medusahead result in a negative heterospecific plant-soil feedback (Perkins and Nowak, 2013), yet, once again, there was no evidence of microbial community conditioning when comparing bacterial/fungal ratios. However, any initial shift in AMF community structure attributed to invasive annuals such as medusahead and cheatgrass may be of less significance than the impact of altered fire regimes on mycelial networks and subsequent aboveground recolonization by low quality plant hosts. Medusahead root samples obtained by the authors at the Oregon study site (see Case Study section) revealed a fine root structure with extensive root hairs (Fig. 7), characteristic of non-mycorrhizal species (Brundrett, 2009). No AMF structures were identified within cortical cells after clearing and staining roots

sampled late in the season (early July) in 2016, while roots sampled in June 2017 exhibited sporadic colonization, at an average of 8.5 percent of total root length (Fig.

8). Medusahead roots sampled in July 2016 and June 2017 were colonized with DSE at average rates of 20 percent and 44 percent total root length respectively (Fig. 8).

Further research is needed to validate these results, but we hypothesize that medusahead presence AMF and DSE colonization in neighboring bluebunch wheatgrass roots.

AMF Obligate invasive forb - Centaurea maculosa

Centaurea maculosa aka stoebe (spotted knapweed) is an allelopathic invasive forb with a close relationship with AMF. Spotted knapweed is considered AMF dependent, as colonization increases competitive effects against native bunchgrasses such as Idaho fescue and bluebunch wheatgrass (Callaway et al., 2004; Marler et al.,

1999). The pathway to competitive dominance includes utilizing interspecies hyphal networks to gain carbon from neighboring plants. Carey et al. (2004) traced stable isotopes and estimated that 15 percent of spotted knapweed’s carbon was supplied by

Idaho fescue, while Callaway et al. (2004) recorded decreased facilitation by neighboring bunchgrasses after applying fungicide. Allelopathic root exudates

(catechins) decrease growth rates of surrounding bunchgrasses and alter soil bacterial communities (Thorpe et al., 2009; Thorpe and Callaway, 2011), but the direct effects of catechins on AMF communities are unknown. However, spotted knapweed invasion does alter the soil AMF community (Mummey and Rillig, 2006), as well as the AMF colonization and community composition of neighboring plant roots (Klein et al., 2006; Mummey et al., 2005). This alteration is not always represented as a

negative shift in diversity, as AMF diversity may increase or decrease in soil conditioned by spotted knapweed depending on the initial composition (Bunn et al.,

2014; Lekberg et al., 2013). Interestingly, although spotted knapweed contains much higher phosphorus levels than bunchgrass steppe natives, it may elevate, rather than decreases phosphorus levels in invaded soils (Thorpe et al., 2006).

Non-mycorrhizal invasive forbs - Sisymbrium species

Sisymbrium species such as S. officinale (L.) Scop., S. loeselii L., and S. altissimum L. are annual or biennial exotic forbs that invade disturbed areas, loose soil, and roadsides throughout the Intermountain West. Sisymbrium altissimum stands create massive seed banks, and at one time were considered the second most invasive species in the Great Basin next to cheatgrass (Howard, 2003). Sisymbrium belong to what is considered a non-mycorrhizal plant family, the Brassicaceae, or mustard family (Brundrett, 2009). Interestingly, Lambers and Teste (2013) describe two functional guilds of non-mycorrhizal plants, the Brassicacea and the Proteaceae.

Brassicaceae type non-mycorrhizal plants lack specialized roots characteristic of the

Proteaceae type, but the high amount of available phosphorus and nitrogen in the soils they typically inhabit allows Brassicaceae type plants ample nutrient uptake without the cost of specialized root morphology or carbon tax of fungal endophytes. However, it is possible that some Brassicaceae in the genera Biscutella and Thlaspi may form mycorrhizal associations during certain life stages (Orlowska et al., 2002; Regvar et al., 2003). Many mustards, including species of Allaria, Sisymbrium, Thlaspi,

Brassica, and Cardaria produce glucosinates, a class of anti-fungal root exudates

(Schreiner and Koide, 1993). Sisymbrium officinale, or hedge mustard, and tall hedge

mustard (Sisymbrium loesili) are both confirmed allelopaths (Blažević et al., 2010;

Bainard et al., 2009). Glucosonate degradation products identified from S. loeselii suppressed spore germination of one species of AMF in a lab experiment, and a reduction in inoculum potential was apparent in soil conditioned by S. loeselii in the field (Bainard et al., 2009). Seed germination and growth of bunchgrass steppe perennials was also reduced by S. loeselii (Bainard et al., 2009). Although Fontela et al. (1999) included Sisymbrium officinale in their study on the negative impact of non-mycorrhizal plants on percent root colonization of the AMF species Glomus mosseae in Pisum sativum, research supporting S. officinale impacts on mycorrhizal communities seem inadequate due to its invasion status and potential for allelopathy.

Non-mycorrhizal invasive forb - Salsola kali

Plant species in the family Chenopodiaceae are often considered non- mycorrhizal (Brundrett 2009). Although there is evidence of colonization by AMF hyphae, and even vesicles in many Chenopodiaceae species, colonization by arbuscules or arbusculate coils is rare (Zhao et al. 2017). Atriplex and Sarcobatus are two genera of Chenopodiaceae native to the salt desert rangelands in North America, which grow in environments characterized by highly saline and alkaline soils (Mata-

González et al., 2017). The exotic forb Salsola kali is a common invader of heavily disturbed areas such as roadsides or abandoned cropland (Mata-González et al.,

2012). Unlike Sisymbrium, Salsola kali has not been found to exude fungicides, although it is allelopathic to some extent (Lodhi, 1979), containing a list of phenolic acids in the roots and foliage (Anna et al., 2009). Salsola kali may potentially increase AMF spore production in the rhizosphere of recently disturbed soils low in

inoculum potential (Schmidt and Reeves, 1984). As with many non-mycorrhizal species, AMF appear to have a direct negative effect on S. kali’s growth, with the plant exhibiting reduced stomatal conductance when competing with bunchgrasses in soil inoculated with AMF (Allen and Allen, 1984). Salsola kali grown in mining tailings with AMF inoculum exhibited decreased growth when the soils were amended with high levels of phosphorus (Johnson, 1998), and the plant seems to actively reject colonization as roots infected with hyphae quickly turn yellow and detach (Allen et al., 1989).

Case Study: mycorrhizal colonization along a medusahead invasion gradient

We examined root morphology and AMF colonization on medusahead plants and bluebunch wheatgrass plants collected during 2 years along survey transects placed on north-facing slopes of the Clarno Unit of the John Day Fossil Beds

National Monument, Oregon (Hovland et al. in preparation). Two transects measuring 30 m and 60 m on each of three hillslopes started in medusahead monoculture stands and were extended upslope through the invasion gradient into medusahead-free bluebunch wheatgrass-dominated stands. We collected root samples of medusahead (n=12) and bluebunch wheatgrass plants (n=18) in sampling events in

2016 and 2017. Once at the greenhouse we washed the roots carefully, and preserved them in an acetic acid and ethyl alcohol solution. Samples were then cleared with

10% (w/v) KOH for 20 minutes in a 90 degree Celsius bath and rinsed with water before sitting for 5 minutes in a 1% HCl solution, then stained with Trypan blue for 5 minutes, and finally de-stained at room temperature for 48 hours (Schreiner, 2003).

Further methodology and study site description can be found in (Hovland et al in preparation).

We found bluebunch wheatgrass roots to exhibit dimorphism in some plants sampled, with deeper, thicker diameter roots colonized by Paris-type arbusculate coils, as well as dark septate endophyte hyphae and microsclerotia. Shallow roots of bluebunch wheatgrass were of smaller diameter than deep roots, had abundant root hairs, were mat forming, and only rarely colonized by AMF. The shallow, horizontally extending roots of bluebunch wheatgrass were, in some soils, visible on the surface, and appeared to be competing for resources at the same depths as medusahead. Medusahead roots were morphologically more similar to the shallow roots of bluebunch wheat grass, but of even smaller diameter, and with longer, denser root hairs. Both species were sampled during early July when soil moisture was still available to bluebunch wheatgrass roots, and summer medusahead recruits from seedlings that had taken advantage of early summer rains in order to germinate. Dark septate endophytes were observed colonizing these medusahead roots, but only at about 20% of root length surveyed, while AMF structures were rarely visible (9 percent of total root length on average) and only in one of the two study years (Fig.

9). In contrast, bluebunch wheatgrass was heavily colonized by AMF hyphae, by >50% in both years.

These findings reveal striking differences in AMF and DSE colonization rates between these two species. If this differential is typical, it further underscores the likely importance of soil fungi community structure and function on invasion

potential for medusahead, and encourages further studies of the effects of mycorrhizal colonization on both species.

Implications for Research and Management: Advancing the Understanding and

Utilization of AMF Ecology in Sagebrush Steppe Restoration and Management

The current understanding of AMF ecology in relation to annual grass infestations in the Intermountain West suggests that a deeper and more explicit integration of knowledge about soil fungi will greatly complement existing above- ground oriented approaches such as Ecologically Based Invasive Plant Management

(EBIPM) framework (James et al., 2010; Sheley et al., 2010). Conceptually, a mycologically-based approach to invasive plant management can be viewed as a subset of EBIPM and similar programs, whereby site specific attributes of competitive interactions and ecosystem function degradation include an awareness of and understanding of the possible influences of AMF in order to identify the drivers and alterations of native plant community maintaining ecological feedbacks (Fig. 3).

It is increasingly clear that, in general, plant community restoration and management strategies will benefit from synthetic understanding of below- and above-ground community biotic and abiotic conditions and interactions (Heneghan et al., 2008;

Koziol and Bever, 2016b) We suggest research attempting to utilize and contribute to the growth of this framework be accomplished in the field as much as possible, through high replication studies along invasion gradients (Bansal and Sheley, 2016;

Bunn et al., 2014) including reciprocal transplants of both plant and fungal species.

Hypothesis testing should focus on answering questions pertaining to the degree of

reliance on AMF for plant species’ fitness and community resilience, including maintenance of functional stability with a focus on soil structure and function. First, researchers and managers should question the mycorrhizal status of both desired natives and invading exotic plants. This should be followed by an assessment of the mycorrhizal growth benefits (MGB) incurred during competition. Plant competitive strategies such as resource requirements and acquisition, root morphology, allelopathy, and environmental stress tolerance should also be considered as aspects of plant interactions which may either be altered by AMF associations, or independent of MGB. Finally, invasive species soil conditioning effects should be examined in order to quantify impacts on native soil feedbacks, including alterations of AMF community structure.

Suggested Methods

Mycorrhizal Status and Growth Benefits

The process of clearing and staining plant roots is a method which has changed little since its inception and provides basic information concerning the level of plant root colonization by endophytes. However, quantifying different AMF structures within roots, particularly arbuscules (the site of nutrient transfer between plants and AMF) can provide a measure of active resource exchange (Schreiner and

Scagel, 2016). We stress the need to carefully identify roots by tracing back to crowns when collecting AMF colonization data from mixed plant communities. Inferences of plant or AMF benefits simply through observation of fungal structures in roots cannot be made without other quantifiable data, as hyphae, vesicles and even spores in roots remain long after they were formed. Klironomos et al. (2004) advises using the metric

of total root length colonized instead of percent of root length colonized, as they saw no effect in their grazing study without factoring in overall root length. However, this measure does not always lead to a better understanding, as both roots and AMF root colonization patterns can both grow and expand, at different rates. External hyphal length has been argued to be a better predictor AMF associated benefits that root length colonization (Miller et al., 1995), as external hyphae are directly linked to plant phosphorus uptake (Jakobsen et al., 1992) and soil aggregate stability (Kohler et al., 2017), however, it may be difficult to fully quantify the extent of external hyphae, or the benefits, if common mycelial networks are preferentially distributing nutrients and carbon.

Quantifying MGBs is accomplished by measuring total plant biomass of different individuals of the same plant species over time, under two main growth conditions: one in which the growth medium is either native soil, or has been inoculated with AMF, and one in which the growth medium has been sterilized of soil fungi. Johnson et al. (1997) outline an approach to assess MGBs which stresses the importance of utilizing contrasting phosphorus concentrations since this can affect the expression of parasitism. We suggest that in order to fully understand MGBs, subsets of growth conditions should include at least the environmental variables encountered at the site in question, which in rangelands may include, along with varying nutrient concentrations, assessing MGBs under conditions of increased drought, temperature, or salinity. This would be conducted in a greenhouse, and might include using soil collected from the field along an invasion gradient, or via the creation of an artificial inoculum potential gradient.

AMF Community Structure

The decrease in cost of genetic analysis is one of the major reasons for the advancement in understanding structure and function of the soil microbial community

(Lindahl et al., 2013). The creation of species specific primers (i.e. Krüger et al.,

2009; see Kohout et al., 2014 for a comparison of primer sets) for analyzing AMF community composition is ultimately what allows for the identification of community constituents at a scale fine enough to illuminate complex relationships including host preference and correlations between plant performance and subtle shifts in AMF community composition/diversity. High throughput next generation sequencing (i.e.

Illumina) allows for quick, inexpensive analysis utilizing short amplicon lengths, but high numbers of reads (Lindahl et al., 2013). Pooling multiple reads is highly advised when analyzing fungal diversity (Schmidt et al., 2013). Amplicon length of previously established series for 454 pyrosequencing may be inadequate for Illumina sequencing, and currently, shorter amplicon lengths based on NS31 and AML2 primers are emerging (J. Zhang et al., 2016). Alternatively, broader fungal primers may be necessary to capture the breadth of mycorrhizal endophytes which could include members of the phylum Ascomycota such as dark septate endophytes (DSE).

While less is known about how DSE affect plant performance and community composition, their ecosystem functions are hypothesized to mirror AMF functionality

(Barrow and Osuna, 2002; Green et al., 2008; Mandyam and Jumpponen, 2005) and

DSE likely compose a high percentage of soil fungi in rangelands due to a melanin content enabling survival in harsher (e.g. arid, alkaline) environments. In this

instance, the broad fungal primers used in the past have been ITS1 and ITS4, however, recent use of ITSoF7 and ITS4R has achieved genus level identification of both AMF and DSE (Gehring et al., 2016; Kohout et al., 2014). The more frequent use of rRNA over rDNA has provided information at a finer temporal scale than previously available. Remnant and extracellular DNA may identify members of the community which are non-active, or locally extinct. The quickly deteriorating characteristics of rRNA provide a snapshot of the present, active community.

Phospholipid fatty acid (PLFA) analysis can be an inexpensive way of determining the ratio of bacteria and fungi in the soil by identifying cell membrane constituents, and presents a broad view of the soil microbial community. The fatty acid 16:1 omega 5c is used as an indicator of AMF presence (Olsson, 1999), but overall the analysis lacks the ability to interpret functionality. Soil respiration readings from a CO2 flux chamber cannot be attributed to AMF species alone, and instead include respiration of other soil microbes as well as live roots. Another common measure of AMF is the identification of spores present in soils through extraction and identification by microscopy (Walker, 1992). The taxonomy of AMF is based on spore morphology and has been largely supported by molecular phylogenies (Redecker et al., 2013). Furthermore, spore microscopy may result in similar results when compared with some molecular methods of AMF community analysis (Gamper et al., 2008; Wetzel et al., 2014). However, identifying AMF spores, or fungal DNA in soil samples does not mean that the species identified will colonize the plant roots at the site in question (Hempel et al., 2007).

In order to best understand the effects of above ground community composition on AMF we must have an understanding of interactions between fungi and plant species, including colonization rates, nutrient requirements and autecology of the plant species, and the individual responses of AMF species to altered soil environments. Similarly, the soil environment itself must be quantified in terms of

AMF relevant conditions including nitrogen and phosphorus content, SOM, pH, soil moisture, and temperature. As seasonality affects AMF activity it would be wise to sample during biologically active periods, during the time of year when the function in question is most pertinent, or multiple times throughout a year to acquire average annual functionality. Seasonality will affect percent root length colonization and spore surveying, but likely isn’t an issue if analyzing glomalin as it appears to be a more stable pool (Lutgen et al., 2003).

Restoration

AMF inoculum is currently being used in restoration efforts across ecosystems in order to increase establishment and growth of planted natives (Koziol and Bever,

2016b; Torrez et al., 2016; White et al., 2017), but in the context of managing for resilient arid land plant communities, simply mixing in commercial inoculum may not be the most effective strategy. Managing AMF in arid steppe systems may include maintaining presence of inoculum or appropriate levels of colonization through soil transplants and greenhouse inoculated seedlings, but also maintaining appropriate plant hosts, and the associated soil characteristics (e.g. texture, chemistry) to which the native/desired AMF are adapted through revegetation and soil amendments.

Johnson (1998) suggests that for larger scale projects restoring the soil to a condition

which favors AMF presence through organic matter amendments may be more viable than forms of inoculation. If attempting to inoculate with AMF species, Maltz and

Tresseder (2015) stress the importance of local inoculum, while Richter et al. (2002) warns against assuming high plant recovery rates correlate directly to high inoculum potential. Similarly, a more is better approach to AMF inoculum potential may not net the desired plant community. If an undesired plant community is currently gaining competitive benefits from an AMF network, fungicide may be used as a management tool as McCain et al. (2011) demonstrated when promoting forb establishment in a C4 grass dominated tall-grass prairie. The restoration methods utilized prior to re-seeding should also be considered, as fire and tilling have the potential to damage spores as well as mycelial networks, and herbicides may decrease spore viability and root colonization for at least a month after application in a grassland system (Druille et al.,

2013). Ultimately, AMF restoration is likely most impactful on small scales at sites identified as high priority links, remnants, or restoration islands.

Conclusion

Although this review is rangeland-centric, all plant communities are susceptible to climatic shifts, abiotic and biotic stresses, and exotic plant invasion.

While soils in more mesic ecosystems may be less susceptible to above-ground shifts in alternative stable states (Bansal and Sheley, 2016), and associated altered soil microbe community composition, it is possible that shifts in global climate and land use patterns will create locally novel stress and disturbance scenarios analogous to those driving state-transition dynamics in arid ecosystems (Millar and Stephenson,

2015). Furthermore, all soil ecosystems experience some level of conditioning via plant uptake, exudates, and decomposition, which are often integral components to feedback driven community maintenance. In closing, plant-fungal relationships at the species and community levels are highly complex and without a better understanding of these relationships we can’t estimate the impact of AMF or disturbance associated alteration of AMF communities. The current challenges are to clearly define those relationships and their outcomes, and not only identify shifts in AMF community structure, but to identify any associated alterations in plant growth rate, nutrient uptake, competitive effects, plant community composition, and soil functional stability which might accompany these shifts.

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Figures

Figure 1 – Conceptual diagram illustrating the role of arbuscular mycorrhizal fungi in maintaining a disturbance resilient old growth sage steppe community. Community maintenance is mediated through alleviation of environmental stresses in plant hosts, enhancing seedling establishment through common mycelial networks, maintaining soil structural/functional stability, and maintaining bare soil interstices through enhanced competitive effects of plant hosts and decreased plant performance in non- mycorrhizal invaders. The potential for non-mycorrhizal plant communities to negatively affect AMF mycelial networks is also shown. Arrows pointing in two directions are feedback mechanisms affecting both AMF obligate and non- mycorrhizal plants. Plus (+) signs next to text indicate an increase while minus (-) signs indicate a decrease. Arrow and line thickness illustrates the amount of research available on the impact of the feedback mechanism, while dashed lines indicate more research is needed.

Figure 2 – Conceptual diagram illustrating possible impacts to arbuscular mycorrhizal fungi (AMF) and ecosystem function through disturbance mediated invasion of exotic plant species of varying mycorrhizal status. Plant communities on the left side of the diagram utilize subtle AMF mediated negative feedback loops, while plant communities on the right side of the diagram either utilize AMF mediated positive feedback loops, or create their own positive feedback loops through other methods of soil conditioning and alteration of fire regimes. Plus (+) signs next to text indicate an increase while minus (-) signs indicate a decrease.

Figure 3 – Conceptual diagram outlining the steps, methods, and hypotheses of current importance in utilizing mycorrhizal ecology as the basis for an invasive plant management framework that explicitly considers this. A mycologically-based approach to invasive plant management seeks to understand mycorrhizal fungi mediated interactions between native plants, invasive plants and the environment in order to conserve, promote, and restore native plant community maintaining fungal feedbacks and will complement current above-ground oriented paradigms

Figure 4 - Morphological differences in bluebunch wheatgrass (Pseudoroegneria spicata) roots revealing fine lateral surface roots with extensive root hairs (above) and thicker, vertically extending roots (below). Only rare occurrences of arbuscular mycorrhizal colonization were detected in the laterally extending surface roots, while vertical roots were moderately (50 percent root length) colonized.

Figure 5 - Paris-type colonization of bluebunch wheatgrass (Pseudoroegneria spicata) cortical cells at two different magnifications. Arbuscular coils (dark blue) can be seen colonizing adjacent cortical cells.

Figure 6 – Cleared and stained bluebunch wheatgrass (Pseudoroegneria spicata) roots revealing simultaneous colonization of arbuscular mycorrhizal fungi (AMF) and dark septate endophytes (DSE). AMF structures (dark blue) include vesicles, hyphae, and arbuscular coils. DSE structures (dark brown) include hyphae and microsclerotia.

Figure 7 -Medusahead root morphology under a dissecting scope revealing extremely fine roots and long root hairs. This type of root morphology is consistent with plant species lacking associations with arbuscular mycorrhizal fungi (AMF).

Figure 8 – Data for each sample analyzed for percent root length colonization in medusahead (Taeniatherum caput-medusae) roots. Samples were collected either in July of 2016 or June of 2017. Colonization by arbuscular mycorrhizal fungi (AMF) was only observed in roots collected in June of 2017, and even then, at relatively low (including none) intensity. Colonization by dark septate endophytes (DSE) was more common throughout both sampling periods, but average colonization was higher (44.57 vs 20.09) in 2017.

Investigating soil conditioning effects of a thatch-forming invasive annual grass along an invasion gradient in a protected area, Oregon, USA.

Matthew Hovland a,1, *, Ricardo Mata-Gonzalez b, David Eduardo Prado-Tarango a, R.

Paul Schreiner c, Thomas J. Rodhouse d

An original research paper prepared for New Phytologist

a Graduate Student, Department of Animal and Rangeland Science, Oregon State

University, Corvallis, OR 97331 USA b Associate Professor, Department of Animal and Rangeland Science, Oregon State

University, Corvallis, OR 97331 USA c Plant Physiologist, USDA-ARS Horticultural Crops Research Laboratory, Corvallis,

OR 97330 USA d Ecologist, National Park Service, Oregon State University-Cascades, Bend, OR

97702 USA

* Correspondence: Matthew Hovland, Department of Animal and Rangeland Science,

Oregon State University, Corvallis, OR 97331 USA. Email address: [email protected]

1 Current address: Matthew Hovland, Department of Animal and Rangeland Science,

120 Withycombe Hall, Corvallis, OR 97331 USA.

Research was funded by the National Park Service Upper Columbia Basin Network

Cooperative Task Agreement P13AC00136.

Summary

 Soil conditioning effects of invasive plant species have been implicated in

some species’ abilities to outcompete natives. The invasive annual grass

medusahead (Taeniatherum caput-medusae) is an exotic characterized by long

barbed awns, a high silica content, and the ability to dominate disturbed sites

in the sage-steppe ecosystem of the Intermountain West of North America.

 We established transects on three adjacent hill slopes in the Clarno Unit of the

John Day Fossil Beds National Monument, Oregon, USA which spanned

medusahead invasion gradients from dense monocultures to intact bunchgrass-

steppe dominated by bluebunch wheatgrass (Pseudoroegneria spicata). We

measured soil moisture levels throughout these gradients and analyzed soil

physical and chemical characteristics. Statistical analyses is restricted to

simple linear regression, with outcomes of analysis serving as hypothesis

generating rather than hypothesis testing.

 We found evidence that percent root length colonization by dark septate

endophytes in bluebunch wheatgrass differed significantly between native and

invaded plots. Medusahead cover was positively correlated with soil moisture

percentage when observed during a period of high overall soil moisture (about

40%), and negatively correlated with soil moisture percentage in drought

months, but only in soils composed of over 80 percent clay.

 The soil conditioning effects of a thatch building invasive annual grass may

only be apparent in a field study observing multi-generational effect, and

mechanisms of competitive dominance may include alteration of neighboring

plant root fungal community composition, and the reduction of soil moisture

retention in certain soil types.

Keywords (8): AMF, DSE, medusahead, plant-soil feedback(s), sage steppe, soil conditioning, soil moisture, thatch

Introduction

Invasive plant species are a non-monophyletic group, and there is no one strategy contributing to invasion success, however, plants utilizing plant-soil feedbacks (PSFs)(Bever, 1994) which negatively affect heterospecific growth rates are likely to gain a competitive advantage if establishment is successful. The term plant-soil feedbacks, although generally applied to multigenerational plant-soil microbe interactions, may be used to describe any effect on individual plant vital rates or community composition attributed to conspecific or heterospecific soil conditioning, including alterations to soil structure and chemistry via mechanisms such as root exudates (Bainard et al., 2009; Thorpe and Callaway, 2011), resource acquisition (Ashton et al., 2008; Ehrenfeld, 2010), and decomposition of plant litter

(Aponte et al., 2013; Farrer and Goldberg, 2009; N. Zhang et al., 2016). Currently, conceptual frameworks for examining plant-soil feedbacks have become finer grained, utilizing molecular analysis of microbial communities (Gornish et al., 2016;

Weber et al., 2015), with a focus on analyzing the relationships between microscopic decomposers, pathogens, and mutualists (Putten et al., 2016). While immediate effects on plant vitality can be extrapolated to fit larger community scales (Suding et al., 2013), the compounding effects of soil conditioning on soil chemistry and structure may, over time, lead to the breakdown of soil structural and functional stability in ways which surmount traditional above ground restoration strategies (i.e. lasting soil legacies) (Cramer et al., 2008; Hawkes et al., 2005). Therefore, in order to understand the broad reaching effects of plant-soil relationships on ecosystem

functionality, studies of soil conditioning should include aspects of belowground ecology involved in maintaining soil functional/structural integrity and observed in the field, under natural laboratory conditions (e.g. invasion gradients), where soils have been conditioned for multiple generations (Hawkes et al., 2013; Kardol et al.,

2013; Van der Putten et al., 2013).

Invasive annual plants which create thatch layers composed of slowly decomposing litter may severely alter multiple aspects of the invaded ecosystem including plant community structure via altered germination rates (Loydi et al., 2013), fire return intervals due to increased fine fuel continuity (Mack and D’Antonio,

1998), soil surface microclimates in a newly created region between litter and soil

(i.e. sublectica), and the soil microbial community due to altered organic inputs

(Facelli and Pickett, 1991). Some thatch effects may not be visible in limited time span studies, especially in instances of altered organic matter input, and associated soil moisture holding and retention capacity. Estimating effects of thatch accumulation on soil organic matter (SOM) may be difficult since initially there may be reduced rates of decomposition due to a reduction in carbon inputs (Fontaine et al.,

2004). Decomposition rates will also be affected by any changes in soil moisture and temperature caused by thatch accumulation, as well as shifts in the soil microbe community (Hattenschwiler et al., 2005). Furthermore, shading effects of thatch, and the creation of the sublectica microclimate could buffer against decreased SOM by reducing heating and evaporation, and maintaining higher soil moisture levels than non-thatched areas, at least at shallow depths (Facelli and Pickett, 1991; Jensen and

Gutekunst, 2003). Further complicating soil moisture retention, mycorrhizal status of

plant invaders may also effect soil carbon inputs, and soil structural integrity.

Mycorrhizal fungi contribute soil carbon pools, and to soil aggregate formation via hyphal enmeshment as well as production of the glycoprotein glomalin (Augé 2001;

Rillig et al., 2001), and monocultures of non-mycorrhizal plant species may, overtime, reduce mechanisms of soil aggregation by displacing soil fungi which require suitable symbiotic plant partners.

The Great Basin of the western United States provides an illustrative example of how thatch forming annual grasses can alter ecosystem structure and function. The increase in wildfire frequency and reduction of native sagebrush-steppe plant communities, and associated wildlife habitat, has been largely attributed, in part, to the annual invasive thatch forming grass, Bromus tectorum L. (cheatgrass). While cheatgrass has been the focus of several soil conditioning studies (Belnap and

Phillips, 2001; Decrappeo et al., 2017; Hawkes et al., 2005; Weber et al., 2015), the soil conditioning effects of other, co-invading invasive annual grasses are less well known. The annual grass medusahead (Taeniatherum caput-medusae [L.] Nevski) is currently a co-dominant in annual grass invaded regions of the semi-arid western

United States, and has the potential to impact ecosystem functionality to an even greater degree than cheatgrass due to its comparable root phenology and germination traits which confer competitive advantages over some perennial bunchgrasses (Harris,

1977), coupled with a high silica content (Swenson et al., 1964) which contributes to its lack of palatability and tendency to form dense thatch layers. Native to the

Mediterranean region of France and Italy, medusahead is a winter germinating annual which was introduced to North America in the late 1800s and is characterized by long

barbed awns which can assist in seedling dispersal, but may be harmful to potential grazers. It tends to dominate disturbed sites in the sage-steppe ecosystem of the

Intermountain West of North America, and has been viewed as problematic for ranchers due to its lack of palatability for livestock (Johnson et al., 2011). Ecologists have focused on its tendency to create monocultures through extensive seed production and alter fire regimes through the creation of a dense thatch layer which acts as a contiguous fine fuel (Nafus and Davies, 2014). The competitive advantage of medusahead, especially in clayey soils, is of current interest, as it appears to alter the soil in a way which negatively affects native species establishment and growth

(Perkins & Nowak, 2013). This alteration, termed a negative heterospecific plant-soil- feedback, has been observed in relatively short timespans in a greenhouse experiment

(Perkins & Nowak, 2013), but in some sites in the Intermountain West medusahead has been persisting in monocultures for decades. Since plant soil feedbacks may increase over time (Diez et al. 2010), the observation made by Perkins and Nowak may be compounding generationally, or currently undocumented feedbacks may only be taking place at decadal time scales. Although a recent study in a California annual grassland found no correlation between medusahead cover and changes in soil microbial community (Gornish et al., 2016), medusahead is consider a poor quality host of mycorrhizal fungi, and in some sites and seasons completely lacks AMF colonization (Hovland et al. in preperation).

In this study we begin to examine the soil conditioning effects of medusahead on clay dominated soils in a central Oregon bunchgrass-steppe system. The goal of this research is to illuminate the mechanisms contributing to medusahead’s

persistence and dominance, including those that could potentially drive negative heterospecific plant-soil feedbacks, which in this study include effects on moisture, pH, respiration, and mycorrhizal colonization of a neighboring bunchgrass.

Furthermore, and more generally, we are investigating the effects of a thatch building invasive annual grass on soil functional stability (i.e. soil moisture retention). We consider a mycologically based invasive plant management strategy of systematically analyzing the influence of and the effects on mycorrhizal fungi by first observing the colonization rates of both native and invasive species, and investigating soil properties along a medusahead invasion gradient. We hypothesize that mycorrhizal

(AMF and DSE) colonization of a perennial bunchgrass, Pseudoroegneria spicata

(Pursh.) Á. Löve (bluebunch wheatgrass) will differ between plots with high and low percentages of medusahead cover. We further hypothesize that a negative correlation will be observed with soil moisture as medusahead increases in percent cover. With this research we hope to understand the invasion ecology of medusahead in such a way that mechanisms of resilience, or further methods of restoring native bunchgrass habitat (such as soil transplants, inoculation or amendment) may be revealed.

Materials and Methods

Site Description

The Clarno Unit of the John Day Fossil Beds National Monument in central

Oregon (JODA) is a protected area managed by the U.S. National Park Service.

Located 18 miles west of the town of Fossil in Wheeler County, Oregon (Fig. 1), its exposed volcanic geology harbors paleontological specimens dating back at least 40

million years, to a time when the local climate was subtropical. Today, the climate at

Clarno is semi-arid, with annual precipitation averaging 250 mm, and occurring mostly in the late winter and spring. Sagebrush steppe is the dominant plant community in the area, however, recent fire activity, including a 2011 wildfire which impacted 98% of the vegetated surface of the park, has reduced the sagebrush cover at Clarno significantly, and the native vegetation, where it can be found, is now dominated by perennial bunchgrasses such as Pseudoroegneria spicata and Poa secunda J. Presl, with Achnatherum thurberianum (Piper) Barkworth and Festuca idahoensis Elmer occurring less frequently (Rodhouse, 2013). Due in part to the recent wildfire activity, this unit of JODA is significantly impacted by both cheatgrass and medusahead, yet some north-facing slopes harbor intact stands of perennial bunchgrasses (Rodhouse et al., 2014).

Study sites are located on three consecutive north-facing hillslopes of differing soil type and texture (Fig. 2, Fig. 3). Further evidence of soil heterogeneity at Clarno can be seen in a survey conducted in the late 1980s and early 1990s outlining lithostratigraphic units within the park, where Hill 1 is labeled as

Conglomerate of Hancock Canyon and Conglomerate of Palisades, Hill 2 is mostly

Claystone of Red Hill, with Upper Andesite of Horse Mountain, while Hill 3 is predominantly Andesite of Hancock Canyon (Bestland et al., 1999). Sites consist of two permanent transects per hill, spanning a medusahead invasion gradient from heavily invaded, commonly at the lower slope position, to non-invaded, intact old growth bunchgrass steppe characterized in part by soil crusts and other non-vegetated interstices. Transects are 30 and 60 meters in length with fifteen, evenly spaced, 1 x 1

meter monitoring plots located throughout the length of the transects. Transects of 60 meters are meant to capture the breadth of the transitional zone, and, substituting space for time, capture temporal, additive effects of medusahead soil conditioning and thatch creation. Transects of 30 meters in length, with shorter distances between plots (every 2 meters) are established to capture fine scale differences in soil characteristics.

Soil Moisture

Soil moisture was measured using a hand-held reflectometer (Hydrosense II,

Campbell Scientific, Inc., Logan, UT) utilizing 12 cm rods. Moisture was measured at a depth of 12 cm at three different locations within each 1x1m plot and averaged for that plot. Data were collected through multiple seasons, once preliminarily in August of 2015 (data not shown), then again in October of 2015, when soils were still extremely dry (5% soil moisture on average). An attempt to collect soil moisture was made in April of 2016, however, recent rains made sampling impractical due to both highly saturated clay soils, and the potential for erosion caused by walking on the hillslopes. Sampling resumed in June of 2016, and then in April of 2017 wet season data was successfully collected, followed by another round in June 2017. Plant cover was recorded in June 2016 within all 90 plots of the study area, identifying each plant inside a plot to species, and estimating percent cover using Daubenmire cover classes

1-6 (Daubenmire, 1959), with the addition of a 0.5 cover class to represent single plants covering less than 1% of the plot. For the purpose of this analysis only cover classes for medusahead and bluebunch wheatgrass will be used.

Soils analysis

Soil samples were collected in June 2016. A 2 cm diameter soil corer was used to limit disturbance within plots, and sample depth was limited to 12 cm. Five subsamples were collected in each plot (one at each corner and one in the center) and combined in the field. Samples were labeled and kept in a cooler for a day before being stored at -80° C. Samples were first sieved while frozen to remove 20 g for potential future molecular analysis, then dried and sieved again to achieve at least 100 grams per sample. All dried and sieved samples were analyzed in the Central

Analysis Laboratory at Oregon State University in Corvallis, Oregon. Texture was determined through the hydrometer method, utilizing 20 grams of soil. Soil respiration was determined through a 24 h carbon dioxide burst test with the use of a

Picarro isotopic analyzer, wherein CO2 concentration is measured 5 min and 24 h after wetting 40 g of soil with water. Soil pH was measured with a Hanna Instruments

HI5522 probe.

Root analysis

Percent root length colonization required the excavation of whole root balls of both native (bluebunch wheatgrass) and invasive (medusahead) grasses. While there is little difficulty in collecting entire root samples of medusahead (average root length of approximately 10 cm) bluebunch wheat grass roots were typically collected at depths of 15 to 20 cm, and exhumed with a maximum diameter of 20 cm. Two bluebunch wheatgrass plants were sampled per transect, and were selected based on size, neighboring plant type (native or invasive) and proximity to plots. Plants were not sampled directly from plots, but rather just adjacent, in order to maintain integrity

of vegetative cover in plots for further studies. A total of 18 bluebunch wheatgrass plants were sampled over the entre two-year study.

For both species, unearthed roots were rinsed and preserved in an ethyl alcohol / acetic acid solution, then cleared with KOH for 20 minutes in a 90 degree

Celsius bath, 5 minutes in a 1% HCl solution, stained with Trypan blue for 5 minutes, then de-stained at room temperature for 48 hours (Schreiner, 2003). Percent root length colonization was observed on a random selection of fine roots squash mounted under coverslips. Percent root length was measured using line intercept method with greater than 100 root crosses per slide. Fungal structures observed were hyphae, coils, arbusculate coils/arbuscules, and vesicles of arbuscular mycorrhizal fungi (AMF) and hyphae and microsclerotia of dark septate fungi.

Statistical Analysis

We used simple univariate linear regression available in the Data Analysis toolpak in

Microsoft Excel (2013) to calculate R2 and p-values. Correlations with p-values less than 0.05 were considered statistically significant.

Results

Soil moisture

Average soil moisture percentage across all sites for October 2015 is 5.0,

11.309 for June 2016, 10.466 for June 2017, and 40.838 for April 2017 (Fig. 3).

Average soil moisture percentage across all sampling periods is 37.909 for Hill 1,

44.016 for Hill 2, and 40.59 for Hill 3 (Fig. 3). Counter to our hypothesis,

medusahead cover was not a significant indicator of soil moisture percentage as averaged across all three hills during dry months, however, there was a slight positive correlation with medusahead cover and soil moisture (R2 = 0.174; p < 0.001) during

April of 2017 (Table 1), when soil moisture levels were high (on average 40%). April

2017 was also the only sampling period in which bluebunch wheatgrass cover was negatively correlated with soil moisture (R2 = 0.234; p < 0.001) (Table 1). If each slope is analyzed individually, Hill 2 stands out as the only site where medusahead cover is negatively correlated with soil moisture in the drought months of June and

October. Combining June 2016 and June 2017 data, Hill 2 soil moisture percentages were negatively correlated with medusahead cover with an R2 = 0.338 (p < 0.001)

(Fig. 5). Soil moisture percentage and slope position were only slightly correlated according to June 2017 data (R2 = 0.0815; p < 0.001), and even less of a relationship was observed in April 2017 (R2 = 0.0459; p < 0.001) (Table 1).

Texture

Soil texture (as represented by percent clay), predictably, has a positive correlation with June soil moisture percentage across all sites (R2 = 0.175; p = 0.016) (Table 1).

While medusahead cover was greater in plots located at lower slope positions (R2 =

0.435; p < 0.001), clay percentage showed no relationship with medusahead or slope position (Table 1). However, while analysis of Hill 2 initially revealed only a small correlation (R2 = 0.10; p < 0.001) between clay percentage and medusahead cover, the removal of a single outlier in the Hill 2 regression plot revealed a linear relationship with an R2 of 0.29 (Fig. 5). This outlier was a soil sample composed of

54% clay, much lower than the Hill 2 average of 86%. Soil samples from Hills 1 and

3 contained much lower average percentages of clay than Hill 2, with 56.2% and

49.7% respectively (Fig. 3). Interestingly, there was no correlation between clay percentage and soil moisture on Hill 2 in June 2016 and 2017 (p = 0.004) (Fig. 5).

Respiration

Soil respiration, a measurement related to soil microbial biomass and organic matter, and expressed as µg CO2-C g-1 soil d-1, averaged 11.03 across all sites (Table

1). Hills 1 and 3 were similar in average respiration, at 12.006 and 12.448 respectively, while Hill 2 was significantly lower at 8.678 (Table 1). There was no significant relationship found between medusahead cover and respiration across all sites, however, there was a slight positive correlation with bluebunch wheatgrass cover and respiration (R2 = 0.108; p = 0.0024) (Table 1). pH

Average soil pH levels ranged from close to neutral for Hill 1 (7.05) to slightly alkaline for Hill 2 (7.95) and Hill 3 (8.02) (Table 1). What initially appeared to be correlations between pH and soil moisture percentage in April and June are insignificant due to high p-values (0.688 and 0.148 respectively) (Table 1). A negative correlation is observed with bluebunch wheatgrass cover with an R2 = 0.142

(p < 0.001), which might imply bluebunch wheatgrass either competes well in, or creates soil conditions where pH is closer to neutral.

Percent root length colonization

Preliminary root sampling in July of 2016 of two plants per hill slope revealed bluebunch wheatgrass roots were colonized by AMF at an average of 50%, and no evidence of AMF colonization was found in the roots of medusahead (Fig. 4). Dark

septate endophytes colonized the roots of both species, however, bluebunch wheatgrass roots sampled from areas dominated by medusahead were colonized by a higher percentage of DSE (Fig. 4). While not as drastic a correlation, AMF hyphal, coil, and arbusculate coil colonization was also lower in roots from medusahead invaded areas. The results from 2017 sampling corroborated almost all of the findings from the previous year. Again, although not convincingly, the roots from medusahead invaded areas were slightly less colonized by AMF hyphae (56.2 vs 61.2%), coils, and arbusculate coils, however the differences in colonization were not statistically significant (Fig. 4). Contrary to 2016 sampling, roots from medusahead invaded areas exhibited lower levels of DSE colonization (38%) than roots from uninvaded areas

(56%) in 2017 (Fig. 4). While the difference between invaded and native plant community AMF colonization wasn’t statistically significant, 50 percent of the variation in colonization data was explained by soil moisture percentage (p = 0.0004), while 21.5% of the DSE variation is explained by soil moisture (p = 0.0009) (Table

1). Respiration and mycorrhizal colonization levels were correlated with an R2 of about 0.11 (p = 0.0012), however, unexpectedly, there was no correlation between pH and DSE, which are melanized endophytic fungi that appear to thrive in harsh environments, including alkaline soils (Table 1). Furthermore, a slight negative correlation is evident (R2 = .110; p = 0.0058) between AMF colonization and pH

(Table 1).

Discussion

The exploratory nature of this study must be emphasized, as analysis relied solely on simple linear regression, when multivariate analysis of covariance should be utilized to fully account for all variables with possible influences on soil moisture

(e.g., slope, texture, plant cover). The outcomes of simple linear regression presented in this paper should be viewed more as hypothesis generating outcomes rather than hypothesis testing, confirmatory correlations. With that being said, this study has revealed the variability of both soil moisture percentages and mycorrhizal colonization over space and time, suggesting that future studies place a premium on large sample sizes.

Wet season soil moisture and vegetation cover

While we found no correlation between vegetative cover and soil moisture levels across all sites during June and October, counter to our hypothesis, analysis of

April 2017 data revealed a positive correlation between soil moisture percentage and medusahead cover, and a negative correlation between soil moisture and bluebunch wheatgrass cover. This is possibly explained by the period of active precipitation in

April coinciding with a period of active growth (Daubenmire, 1972). The extensive root systems of perennial bunchgrasses such as bluebunch wheatgrass allow these plants to uptake greater amounts of moisture from surrounding soils than annual grasses. Soils dominated by medusahead could be less affected by plant uptake of water, since their root systems were rarely observed to extend greater than 10 cm at the study sites. It is also possible that medusahead thatch cover was actively reducing evaporation of soil moisture, especially at the depths our probe was sampling (12 cm). In our study area medusahead is more likely to be found in monocultures at

lower slope positions (Table 1), therefore one might expect to observe a correlation with medusahead/lower slopes and soil moisture. However, since the already scant correlation between soil moisture and slope position seen in drought months (R2 =

0.081) was reduced in April 2017 (R2 = 0.045) other explanations may be more appealing (Table 1).

Drought season effects and clay percentage thresholds

The textural heterogeneity observed across our three hills allowed us to largely exclude texture as a possible confounding variable for medusahead cover effects on soil conditions. Percent clay differed throughout transects and between hills, but only had correlations with slope position or medusahead cover on Hill 2.

Hill 2 is characterized by an extremely high percentage clay soil (average 86%), the highest average soil moisture percentages, and the lowest average soil respiration level compared with the other two hills (Fig. 3). While not quantified in the data, the lower slope of Hill 2 is dominated by the largest contiguous medusahead monoculture out of all 3 hills (Fig. 2). The medusahead stand is so strikingly contrasted with an intact perennial bunchgrass stand that the site was chosen for the original August

2015 preliminary sampling efforts. Out of all three hills, Hill 2 is the only site where there is a significant correlation between medusahead cover and clay percentage. Hill

2 is also the only site where there is a correlation between slope position and clay percentage. While it is clear that what makes Hill 2 different is the soil texture, and that clay percentage is a major driver of medusahead dominance (Dahl and Tisdale,

1975; Young and Evans, 1970) the mechanism responsible for the correlation between low soil moisture and high medusahead cover on Hill 2 in drought months is

unclear. It is likely that the high clay percentage vertisols described by Young and

Evans (1970) which favor medusahead dominance may have lower soil moisture readings in June compared with other soil types as they are shrink-swell soils, prone to cracking in the summer. The only problem with that theory for our site is (contrary to the rest of the data set) on Hill 2 in June, there is no correlation between clay percentage and soil moisture (Fig. 4). In April on Hill 2 the level of correlation is similar to the relationship observed between clay and moisture across the entire study area (R2 = 0.175), but not in June.

Previous literature describing the affinity of medusahead for clay, coupled with the lack of correlation between medusahead and soil texture in Hills 1 and 3 leads to a hypothesis concerning the importance of a clay percentage threshold, in which medusahead has a greater competitive advantage in soils with high percentages of clay. Dahl and Tisdale (1985) described the sites where medusahead dominated as grumusols or other similar soils with high clay content, but the highest recorded percentage was 64% clay. Perkins and Nowak (2013) conditioned sandy loams and clay soils by growing medusahead and other rangeland grasses in a PSF study, and only observed negative heterospecific feedbacks from medusahead when grown in the clay soils. In our study, a significant correlation between medusahead and clay content wasn’t evident until Hill 1 and 3 were removed from the equation, and our current estimate for a soil clay percentage threshold leading to medusahead dominance and possible alteration of soil structural stability would be around 75%.

The question is then, at this threshold, would medusahead be more likely to contribute to a loss in SOM, or negatively affect soil moisture levels in June? A

recent manipulative study in litter accumulation effects of medusahead revealed a decrease in soil dissolved organic carbon (Mariotte et al., 2017) with increased litter depth. Decreased decompositional rates of medusahead thatch, over time, could hypothetically alter soil organic carbon levels enough to decrease soil moisture retention. While whatever mechanism is driving the Hill 2 June correlation between soil moisture and medusahead cover, it is strong enough to negate the positive correlation between clay percentage and soil moisture during that time period. We require further evidence to claim that either a loss of AMF mediated aggregation, or reduced organic matter input is altering soil moisture retention.

Mycorrhizal colonization

The potential effect of medusahead on neighboring plant root fungal community structure would not be unprecedented. Plants which exude allelopathic chemicals from their roots may alter neighboring colonization, perhaps to the benefit of the allelopathic plant (Mummey et al., 2005). The invasive annual grass, B. tectorum was recently the target of a study revealing that it may somehow increase the level of dark septate endophyte colonization in neighboring plant roots (Gehring et al., 2016). While preliminary sampling in 2016 revealed an increase in DSE colonization in bluebunch wheatgrass plants surrounded by medusahead, we observed the opposite in the follow up study in 2017. While both studies suffered from small sample sizes, the AMF colonization data in both years was almost identical, with average hyphal colonization increasing from 50% to 60% in 2017 (Fig. 4). Assuming the 2016 data is not representative, the reasons for the 2017 shift in DSE colonization percentage is still of interest, but perhaps not as cryptic. Dark septate endophytes are

mycorrhizal fungi which may serve similar ecological functions as AMF, but, due in part to their melanized hyphae, persist in more extreme environments including arid rangelands (Mandyam and Jumpponen, 2005). While AMF are known to increase phosphorus uptake in host plants, DSE may play a larger role in translocation of nitrogen, specifically, in rangeland ecosystems, from biological soil crusts to perennial bunchgrasses (Green et al., 2008). In the case of bluebunch wheatgrass colonization by DSE, the increase may be more related to the variable of slope position (R2 = 0.428; p < 0.001) (Table 1), since biological soil crusts at these sites are generally located on the upper slopes. If the 2016 colonization data is included in the discussion, the slope position and proximity to soil crust argument is no longer as attractive, and more complex hypotheses are required. While it is a perplexing problem, and a third year of sampling should occur, hypotheses which consider precipitation timing and fluctuation in nitrogen fixation as well as plant host phenology might be a relevant starting point. Dark septate endophytes are known to preferentially colonize plants more intensely at the end of a plant’s lifespan or seasonal growth period, and shifts in plant available nitrogen may also influence colonization rates (Mandyam and Jumpponen, 2008).

Further research

According to the framework for studying invasive species from a mycological perspective, the next steps would include determining the mycorrhizal growth benefits of both native and invasive species. For this study, that would include observing effects of varying levels of DSE colonization on bluebunch wheatgrass, with and without competition from medusahead. A comparison of mycorrhizal

colonization of medusahead in its native and invaded range would assist hypothesis generation concerning benefits conferred from either enemy escape, or utilization of novel microbial associations. A study of inoculum potential throughout the invasion gradients which utilized bluebunch wheatgrass as the host would also assist us in understanding both medusahead impacts on mycorrhizal networks, and the reliance of bluebunch wheatgrass on such networks. Extraradical hyphal length could be quantified for further data on mycorrhizal network alteration. We would like to further address the issue of possible medusahead soil conditioning effects in high clay percentage soils in order to both understand the mechanisms at work more clearly, and to further define a possible medusahead dominance threshold for soil texture.

This may include a PSF study with medusahead and bluebunch wheatgrass grown in an artificial clay percentage gradient.

Conclusion

In this study we observed possible soil conditioning effects of an invasive annual grass along an invasion gradient. This research highlights the merit of a practical soil conditioning field study designed to account for multiple interacting variables of interest which contribute to soil functional stability. The invasion gradient transect concept allows for large quantities of data to be collected relatively quickly, and to be analyzed using simple linear regression. We would promote this, as well as the consecutive hillslope structure, especially in regions characterized by heterogeneous soils, in order to reduce confounding environmental variables such as precipitation, temperature, and solar insolation, but retain variability of soil chemical and physical characteristics. While this and other studies have not completely

revealed the mechanisms driving the dominance of medusahead in its invaded range, we feel by following a framework which encourages incremental steps toward a complete comprehension of plant-soil and plant-microbe interactions we are illuminating the intricacies surrounding these relationships, and building the knowledge base necessary to manage uninvaded areas for resistance against invasion, and increase positive post-restoration outcomes.

Acknowledgements

The authors would like to acknowledge the assistance of the National Park Service, and the USDA Agricultural Research Service. Research was funded by the National

Park Service Upper Columbia Basin Network Cooperative Task Agreement

P13AC00136. Sincere thanks to the John Day Fossil Beds National Monument superintendent, Shelley Hall, and the managing staff at the Hancock Field Station.

We would like to thank Dr. Jennifer Parke and Dr. David Myrold of Oregon State

University for their comments and suggestions regarding this manuscript.

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Figures

Fig. 1 – A map of the three units of the John Day Fossil Beds (JODA) National

Monument in Central Oregon, USA. The study area for this research is the Clarno

Unit of JODA, 18 miles west of the town of Fossil.

Fig. 2 – Clockwise from top left: overview of the three study locations at the Clarno

Unit of the John Day Fossil Beds National Monument; Hill 1; Hill 2; Hill 3. All photographs were taken during drought period sampling efforts, from the bottom of the hills with medusahead (Taeniatherum caput-medusae) in the foreground. Two transects were established on each hill, measuring 30 and 60 m in length, and oriented with the hillslope.

Fig. 3 – Average soil characteristics for each study site (Hill 1, 2, and 3), including soil moisture percentage, soil nutrients (potassium, calcium, and magnesium), pH, respiration, and clay percentage.

Fig. 4 – Average percentages of root length colonization by arbuscular mycorrhizal fungi (AMF) and dark septate fungi (DSE). The upper chart shows the outcome for preliminary sampling in 2016 for bluebunch wheatgrass (Psuedoroegneria spicata) roots from native (n=3) and invaded (n=3) communities, as well as shallow, mat forming roots of bluebunch wheatgrass (n=3) and TACA (Taeniatherum caput- medusae), or medusahead (n=6). The lower chart compares percent root length colonization of bluebunch wheatgrass from native (n=6) and invaded (n = 6) communities by AMF and DSE from July 2017 sampling efforts.

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Fig. 5 – Hill 2 regression plots illustrate the relationship between soil moisture percentage and medusahead (Taeniatherum caput-medusae) cover (top left; R2 =

0.3384). Although it was originally hypothesized to be a function of shrink-swell clays, since this was the only site where medusahead was positively correlated with clay percentage (bottom left), Hill 2 during June is the only site and time when a correlation between clay percentage and soil moisture is not evident (top right; R2 =

0.0039). When the clay percentage outlier (54 percent) is removed from the bottom two plots, R2 exceed 0.20.

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Soil Soil Medusahead P. spicata AMF DSE moisture moisture Cover Cover colonization colonization June April R2 x 100 p-value R2 x 100 p-value R2 x 100 p-value R2 x 100 p-value R2 x 100 p-value R2 x 100 p-value Soil Moisture June (-) 4.1 4.9667E-10 0 2.4599E-08 (+) 50.28 0.00048795 (+) 21.5 0.00091812 NA NA NA NA Soil Moisture April (+) 17.48 4.1269E-05 (-) 23.46 1.199E-11 NA NA NA NA NA NA NA NA Clay% 0 1.2147E-32 (-) 3.49 4.1175E-08 0 0.00541884 0 0.00357701 (+) 17.54 0.01612459 (+) 12.54 4.9227E-25 Respiration 0 1.1851E-23 (+) 10.81 0.00247845 (+) 11.0 0.0012648 (+) 22.2 0.00208866 0 5.2717E-12 (-) 3.76 1.1353E-41 pH (+) 5.88 3.8111E-82 (-) 14.28 3.1066E-06 (-) 11.07 0.0058374 0 0.36875721 (+) 9.67 0.14885622 (+) 24.34 0.6882442 Slope position (-) 43.55 1.8005E-35 (+) 15.69 0.00022663 0 3.6046E-05 (+) 42.87 3.8672E-05 (+) 8.15 3.3523E-11 (-) 4.59 3.1523E-47

Table 1 – A table of correlation values across all study sites, expressing positive or negative correlations (+ or -), and the percent of variation in the data explained by the relationship.

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Conclusion

As we await the return of the rain, soil moisture levels for our central Oregon clays are still low, and we are perhaps left kicking the dusty hills with more questions than at the onset of our preliminary sampling two years ago. We welcome the questions though, because the framework is there. We have only scratched the surface of medusahead invasion ecology. Future research has already been discussed, but it may be relevant to reiterate. The goal of the framework is to understand, and within that understanding might be a hint, a clue, to the reason behind medusahead’s ability to invade, or the native community’s ability to resist. In terms of utilizing mycorrhizal fungi, we know that the native perennial bluebunch wheatgrass is colonized, but we do not know to what extent this colonization is benefiting the plant, and if an alteration in colonization percentage, or colonizing fungal community structure, might affect plant vitality in competition with medusahead. We know that medusahead experiences colonization limitations, but do AMF colonization benefit medusahead, or do the fungi act more parasitic? Medusahead doesn’t appear to require assistance from AMF since it can outcompete practically all other plants if the soil has enough clay. Medusahead’s ability to dominate in clayey soils is likely part root morphology, part phenology, and part soil conditioning. The invasive little plant has such fine roots, with such fine long hairs, that it is able to extract the resources it needs from clay soils with microscopic pore spaces too small for most other roots. It is able to grow those thin roots and fine hairs in dense clay, because when it is growing roots, the soil is saturated with moisture, in fact it is the most saturated soil 103

on the hill. And then something mysterious happens. After medusahead has been established there is likely a change in the soil. How long does it take? One generation? Two? Perhaps there are changes in nutrients, changes in carbon inputs, lost mycorrhizal network connectivity, reduced inoculum potential. The soil changes, we know that, because, all plants condition the soil in which they grow. I will leave you with my top three questions: Does mycorrhizal status differ between medusahead in the United States versus medusahead in its native range, the Mediterranean region of southern Europe? This includes response to AMF colonization in and out of competition with other plants. What are the mechanisms behind medusaheads soil conditioning, and do they change throughout a clay percentage gradient? And, how does multi-generational soil conditioning affect seedling establishment and growth rate of bluebunch wheatgrass? Test this last one by growing the bunchgrass in a greenhouse in soil collected from the field along the medusahead invasion gradient, and make sure to check for percent colonization in the bluebunch roots for inoculum potential.

There is much work to be done. Perhaps the work in these pages will assist someone else with similar interests. If that is you, good luck.

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