Biocrust Restoration in Drylands

OPERATIONAL MANUAL FOR BIOCRUST RESTORATION IN DRYLANDS

Akasha Faist, Colin Tucker, Sasha C. Reed, Anita Antoninka, Matt Bowker, Nichole Barger, Kara Dohrenwend, Natalie Day, Sue Bellagamba, Jayne Belnap, Michael Duniway, Stephen Fick, Ana Giraldo-Silva, Corey Nelson, Julie Bethany, Sergio Velasco-Ayuso, Ferran Garcia-Pichel Abstract The purpose of this manual is to synthesize current information about biological soil crust restoration for resource managers making decisions on the ground. Biological soil crusts (biocrusts) are communities of photosynthetic organisms (mosses, lichens, cyanobacteria, and/or microalgae) with accompanying populations of bacteria, archaea and fungi, that occupy the upper few millimeters of dryland or other sparsely vegetated soils across the globe. These communities play several fundamental roles in the functioning of ecosystems, being critical for soil stabilization and erosion prevention. Biocrusts may also affect vascular plant success, help dictate soil hydrology, and can be the dominant force for sustained fertility in some dryland soils. This manual is based on the experience gathered through work conducted as part of a Wildlife Conservation Society (WCS) project focused on elucidating the role of climate adaptation in biocrust restoration practice on the Colorado Plateau, as well as a Strategic Environmental Research and Development Program (SERDP) project assessing best practices for biocrust restoration carried out in the Chihuahuan and Great Basin deserts. The manual starts by introducing biocrusts, why biocrusts matter for ecological function of dryland ecosystems, and discussing the need for restoration of this important component of drylands in the face of anthropogenic pressures. The manual also explicitly addresses new options for biocrust restoration, including information about climate-adapted biocrust restoration, farm-grown biocrust inoculum sources, opportunities to acquire biocrust sources through salvage, and mechanizing the harvesting of biocrust. Methods for monitoring biocrust growth, development, and organismal composition are also provided. Finally, the operational manual ends with practical findings and potential areas that could benefit from further investigation as we progress in our understanding of best biocrust restoration practices for a changing world. Our understanding of biocrust restoration is rapidly improving, and this manual is expected to be a “living document” with improvements and updates made as our knowledge advances.

Acknowledgements We would like to thank the Wildlife Conservation Society Climate Adaptation Fund, which was established with funds provided by the Doris Duke Charitable Foundation, for their support of the restoration work that informed this manual. We are also grateful to the Strategic Environmental Research and Development Program (SERDP) for their support of research included in the manual, and to the U.S. Geological Survey Ecosystems Mission Area. Finally, we thank the resource managers who continue to help facilitate the research and biocrust collections from the Canyonlands Research Center (Kristen Redd and Matt Redd), on the Jornada Experimental Range (John Anderson), Fort Bliss (John Kipp), and the Utah Test and Training Range (Russ Lawrence, Mike Shane, and Jace Taylor). We are also indebted to the large number of biological science technicians from the U.S. Geological Survey, Northern Arizona University, Arizona State University, and the University of Colorado, Boulder who were critically important in implementing the field and laboratory work that was the foundation for this manual.

Cover photos: (upper left) Kristina Young, (all others) Anita Antoninka. OPERATIONAL MANUAL FOR BIOCRUST RESTORATION IN DRYLANDS

Akasha Faist1, Colin Tucker2,3, Sasha C. Reed3, Anita Antoninka4, Matt Bowker4, Nichole Barger5, Kara Dohrenwend6, Natalie Day3,7, Sue Bellagamba8, Jayne Belnap3, Michael Duniway3, Stephen Fick3,5, Ana Giraldo-Silva9, Corey Nelson9, Julie Bethany9, Ferran Garcia-Pichel9, Sergio Velasco-Ayuso9

1 Department of Animal and Range Sciences, New Mexico State University, Las Cruces, NM 2 U.S. Forest Service, Northern Research Station, Houghton, MI 3 U.S. Geological Survey, Southwest Biological Science Center, Moab, UT 4 School of Forestry, Northern Arizona University, Flagstaff, AZ 5 Department of Ecology and Evolutionary Biology, University of Colorado, Boulder, CO 6 Mayberry Native Plant Propagation Center, Rim to Rim Restoration, Moab, UT 7 U.S. Geological Survey, Colorado Water Science Center, Grand Junction, CO 8 The Nature Conservancy-Utah, Moab, UT 9 School of Life Sciences and Center for Fundamental and Applied Microbiomics, Arizona State University, Tempe, AZ

TABLE OF CONTENTS

SECTION 1: BIOCRUSTS AND RESTORATION 1 Introduction: What is a biocrust and why should we restore it?...... 1

SECTION 2: INOCULUM SOURCES, HARVESTING, PREPARATION, AND STORAGE 2.1 Introduction to inoculum sources, harvesting, preparation, and storage ...... 3 2.2 Propagule collection and growth ...... 4 2.2.1 Field collection ...... 4 2.2.2 Salvaged biocrusts ...... 4 2.2.3 Greenhouse-grown biocrusts ...... 5 2.2.4 Lab-grown biocrusts...... 8 2.2.5 Outdoor biocrust production...... 11 2.3 Climate adapted biocrusts ...... 12 2.4 Preparation after growth/collection...... 13 2.4.1 Hardening ...... 13 2.4.2 Sieving size considerations and instructions...... 14 2.4.3 Storage ...... 14 CLIMATE-ADAPTED BIOCRUST RESTORATION INFOGRAPHIC...... 15 SECTION 3: INOCULUM ADDITION 3.1 Introduction to inoculum additions...... 17 3.2 Ways of adding inoculum ...... 17 3.3 Amounts of inoculum to add...... 17 3.4 Season of inoculation...... 18

SECTION 4: HABITAT PREPARATION, MODIFICATION, AND SOIL STABILITY 4.1 Introduction to habitat preparation, modification, and soil stability...... 19 4.2 Habitat modifications...... 19 4.2.1 Surface roughening...... 19 4.2.2 Shading ...... 19 4.2.3 Irrigation and Water addition...... 19 4.2.4 Other habitat considerations...... 20 4.3 Soil stability considerations...... 20 4.3.1 Polyacrylamides ...... 20 4.3.2 Straw borders and checkerboards ...... 20 4.3.3 Psyllium and other plant-based stabilizers ...... 21

SECTION 5: MONITORING METHODS AND PROTOCOLS 5.1 Introduction to monitoring methods and protocols...... 22 5.2 Types of monitoring methods ...... 22 5.2.1 Biocrust cover ...... 22 5.2.2 Chlorophyll a...... 22 5.2.3 16S rRNA gene abundance ...... 23 5.2.4 Microbial community composition: 16S rRNA community diversity. . . 23 5.2.5 Soil aggregate stability ...... 23 5.2.6 Soil tensile strength and compaction ...... 24 5.2.7 Repeat photography...... 25 5.2.8 Level of development ...... 25 5.2.9 Soil nutrients ...... 25

SECTION 6: MAJOR FINDINGS 6.1 Major findings, challenges, and opportunities ...... 27 References...... 28 SECTION 1: BIOCRUSTS AND RESTORATION

1. Introduction: What is biocrust and why (Belnap 2006) by increasing their access to water, or should we restore it? decrease infiltration by increasing runoff. In addition to the potentially beneficial role of biocrusts in native Biological soil crusts (biocrusts) are a community vascular plant growth and establishment, there is of lichens, mosses, cyanobacteria, and other evidence that the presence of lichen biocrusts inhibits nonvascular photosynthetic organisms and associated the establishment of grasses including the exotic, decomposers living on soil surfaces worldwide invasive annual grass, Bromus tectorum (Leines et al. (see Garcia-Pichel 2003 for a primer; Belnap et al. 2007). Inhibition of annual grasses by well-developed 2001, Weber et al. 2016 for monographs). Wherever biocrusts has also been shown in the Great Basin soils have direct access to the sun, biocrusts have and Australia (Crisp 1975, Larsen 1995, Howell 1998). the potential to exist. Indeed, biocrusts occur on A recent metaanalysis of biocrust effects on plants all continents and across contrasting biomes (for shows that relationships are context-dependent, but example, polar, temperate, arid), and comprise a support the assertion that intact biocrust promotes majority of surface cover in many arid and polar native over exotic plant growth (Havrilla et al. 2019). systems (Belnap et al. 2016; Torres-Cruz et al. 2018). A recent global estimate suggested that biocrusts cover Biocrusts play a central role in stabilizing dryland soils. about 12% of the Earth’s terrestrial surface (Rodriguez- A matrix of filamentous, motile cyanobacteria (Garcia- Caballero et al. 2018), and these photosynthetic soil Pichel and Wojciechowski 2009) forms throughout communities play foundational roles in the ecosystems the uppermost soil layers, providing most of the where they occur (Belnap et al. 2016). cohesion (Belnap and Gardner 1993). Lichens and mosses physically shield soils with their aboveground Biocrusts are especially influential in numerous thalli and with anchoring structures that penetrate the aspects of dryland ecosystems, such as those in soil matrix (Sanders 1994). The resultant dense aerial the western United States. They help set a system’s and subterranean network reduces wind (Belnap soil stability (Belnap and Gillete 1997, Gaskin and and Gillette 1997) and water erosion (Gasking and Gardner 2001) and fertility (Johnson et al. 2007, Gardner 2001) to nearly undetectable levels (Belnap Thomazo et al. 2018), carbon uptake (Housman et al. 2003; Belnap 2006). This is a critical biocrust ecosystem 2006, Tucker et al. 2019) and storage capacity, and service in drylands, as soils are slow to form (<1 determine how water moves into, within, and out cm/1,000 years) but are quickly lost (Dregne 1983). of the landscape (Verrecchia et al. 1995, Rodriguez- Caballero et al. 2012, Faist et al. 2017). Biocrusts fix Unfortunately, while resistant to wind and water atmospheric carbon dioxide into living biomass, can erosion, biocrusts are highly susceptible to be the dominant source of nitrogen for plants and compressional forces, such as those generated by soils (Evans and Belnap 1999; Evans and Ehleringer vehicles, bikes, hooves from domestic livestock and 1993), and can increase the availability of many soil wildlife, and human foot traffic (Belnap and Eldridge nutrients (reviewed in Belnap et al. 2003, Beraldi- 2003). The microbial filaments that have such high Campesi et al. 2009), thus potentially benefitting both tensile strength (resisting even the strongest winds) plants and belowground decomposer communities are brittle and easily destroyed when compressed. (Darby et al. 2006, Ferrenberg et al. 2017, Albright Because light does not penetrate deeply into the et al. 2019). In some U.S. deserts, biocrust presence soil (Garcia-Pichel and Belnap 1996), burial by even a roughens the soil surface, thus increasing the capture small amount of loose soil will leave the phototrophic and retention of nutrient-rich dust (Reynolds et al. cyanobacteria, lichens, and mosses literally in the 2001), seeds, and organic matter (Rao and Burns dark. This, in turn, leads to a loss of the ecosystem 1990; Rogers and Burns 1994). Plant productivity, services the biocrusts provide, including carbon and survivorship, and the concentrations of most nitrogen inputs to the soils, water retention (Barger plant-essential nutrients are often higher in plants et al. 2006), and soil stability (Belnap and Eldridge growing in crusted soils than in those of adjacent, 2003). After mortality, full recovery rates for biocrusts uncrusted soils (Harper and Belnap 2001). Biocrusts under natural conditions can be slow, with published also influence many aspects of local and regional estimates ranging from decades to centuries (Belnap hydrologic cycling, which may benefit vascular plants and Eldridge 2003).

1 BIOCRUST RESTORATION IN DRYLANDS Healthy biocrust growing at Canyonlands National Park on the Colorado Plateau. Photo Credit: W. Bowman

The need to restore biocrusts for ecosystem health succession of biocrust microbes (Garcia-Pichel and has long been acknowledged. Efforts to develop Wojciechowski 2009); 2) using organisms that had inoculants to speed this recovery have been not been “hardened off” before being seeded; and attempted repeatedly in the past (reviewed in Belnap 3) inoculating the soils with low-diversity or improper 2003, Bowker 2007). It was quickly recognized that species mixtures. harvesting of intact biocrusts to aid in the recovery Another issue of potential relevance is the quantity or of damaged crusts could result in over-harvesting concentration of inoculum. All restoration attempts of healthy biocrusts, given the high demands for require access to sufficient biological material to inoculum to facilitate quick recovery of biocrusts support recovery at the landscape scale (Duryea and in degraded soils elsewhere (biocrust inoculum is Dougherty 1990). In reforestation of wetter landscapes, biocrust vegetative or reproductive propagules one can rely on centuries of horticultural traditions introduced to stimulate recovery; Belnap 1993). to cultivate a large number of plant seedlings in a Early efforts focused on culturing common biocrust relatively short time. For arid landscapes, microbial cyanobacteria (for example, Microcoleus vaginatus, equivalent of tree nurseries can improve restoration Nostoc spp.) via growth of isolates in the lab (for potential: efficient biocrust nurseries that supply example, Lewin 1977, Tiedemann et al. 1980, Buttars inoculum of high fitness and reproductive capacity et al. 1998, Howard and Warren 1998, Wei 2005, grown under field conditions and in sufficient Maestre et al. 2006). These approaches succeeded quantities to inoculate large landscapes in need of in producing the inoculum at a small scale in the restoration. Unfortunately, we cannot rely on a long lab, but establishing cultures in the field continued history of “microbial horticulture”. With this in mind, to be problematic. Fortunately, we have learned this manual was created to synthesize our current much about the genetics, distribution, physiology, understanding of biocrust restoration, as well as to and ecology of biocrust organisms in the interim, provide a critical insight into the ease, relative cost, and we can see in hindsight where previous efforts and robustness of each of the available biocrust may have gone awry: 1) using microbial components restoration alternatives. that were not natural pioneers in the normal

BIOCRUST RESTORATION IN DRYLANDS 2 SECTION 2. INOCULUM SOURCES, HARVESTING, PREPARATION, AND STORAGE

Another consideration of collecting biocrust is a potential mismatch between the current and future climate that the collection and restoration sites experience. Climate change may pose a challenge for biocrust restoration if the rates of change of temperature and precipitation exceed the rates of biocrust development or population turnover. Healthy biocrust exposed to likely future climate conditions can suffer a severe decrease in biomass and species composition (Ferrenberg et al. 2015, Fernandes et al. 2018). Collecting biocrust from areas that climatically match climate predictions has the potential to be more successful in the long term and could provide climate adapted biocrust communities. Furthermore, this approach comes with the additional

Fig. 1 Greenhouse grown biocrust. Photo: A. Antoninka concern of releasing species outside of their native habitats, including invasive plant species. As with 2.1 Introduction to inoculum sources, vascular plants, using biocrust organisms that may be harvesting, preparation, and storage better adapted to future, more arid conditions could be of great value as climate continues to change, Biocrusts harvested directly from the field and added but more comprehensive assessment is needed to degraded sites in crumbled forms have proven before the climate-adapted transfer approach can be an effective inoculum source in multiple settings recommended or discouraged at larger scales. (Belnap 1993). Inoculating degraded areas with biocrust from the immediate vicinity makes sense, Two alternatives to field collection which are in that this approach uses organismal assemblages potentially more sustainable and scalable include: that may be optimally adapted to the local climate (1) greenhouse cultivation of whole community and soil. These assemblages grow together in biocrust from small amounts of locally field collected combinations that tend to be stable over time, and biomass (hereafter called “greenhouse-grown”) and form erosion-proof communities. Local sources also (2) large scale cultivation of relevant cyanobacteria in avoid potential problems of introducing non-native reared mixed isolates (hereafter called “lab-grown”). species or unintendedly spreading soil or plant Trade-offs in costs and analytical expertise exist diseases (Bethany et al, 2019). This type of inoculum with developing different approaches to inoculum is called “field collected”. A major drawback of development. They are discussed at length in Giraldo- this approach is the common lack of sufficient local Silva et al. (2019). The greenhouse-grown approach biocrusts to attain repopulation within realistic to inoculum development requires harvesting small time frames. Also, healthy intact crusts must be amounts of existing biocrust from field sites and then harvested in order to inoculate an area in need increasing the biomass under controlled greenhouse of restoration, thus, restoring larger scale areas conditions (Fig. 1), optimized to maximize growth requires the likely degradation of collection sites. without promoting severe community composition An alternative approach to finding a source of field shifts. This method may be easier for land managers collected inoculum that would otherwise remain and restoration professionals that have no prior intact is to salvage existing biocrusts from sites that microbiological training. A small trade-off in the are already slated for destructive land uses, such as greenhouse-grown method is that biocrusts must road construction, infrastructure building, or energy be harvested from the field resulting in impacts to a development. This allows collection of biocrust donor site, but this can be inconsequential since the inoculum without disturbing intact systems that would amounts needed are small. The need for extensive otherwise not be disturbed. greenhouse facilities, and the necessity to determine

3 BIOCRUST RESTORATION IN DRYLANDS optimal growth conditions, including the monitoring of final inoculum composition, are significant. The BOX 1: A simple protocol for salvage benefit of the lab-grown approach is that extremely harvest. small amounts of biocrust are used to isolate different 1. Document biocrust and vascular plant com- strains of biocrust organisms. Because the different munity at the salvage site prior to harvest. cultures are grown in isolation, there is virtually no 2. Identify and focus on large contiguous risk of deviation in community composition, as long patches of biocrust to increase efficiency. as the initial cultures have been pedigreed to be Scrape biocrust using either a cement trowel representative of field populations. These strains are 3. or flat-blade shovel, harvesting only the then scaled up to obtain larger quantities of inoculum. surface layer, avoiding large contaminants The lab-grown approach is best suited for those with such as rocks and plant litter. significant expertise in microbiology. This approach, 4. Manually crumble material and deposit however, holds much promise in future commercial biocrust in 5-gallon bucket after separating production of biocrust inoculum. The methods excess sub-crust soil. described in the following sections all focus on reducing 5. Transfer biocrusts from buckets to larger time and effort in producing biocrust inoculum. containers (e.g., 27-gallon totes) kept in a trailer for transport to the storage location. 2.2 Propagule collection and growth 6. Storage of biocrusts should optimally be in 2.2.1 Field collection a cool, dark, and dry space. Biocrust from the top 0.5 cm of the soil surface (Fig. 2) can be collected and crumbled into pea-sized 2) conducting the salvage, and 3) storing salvaged fragments (see section 2.3.1 for greater detail on material for later application. Salvage sites can be inoculum sieving) and homogenized for inoculum of identified opportunistically (finding nearby areas where a desired restoration area. Field collected inoculum biocrust is present, and permitting and logistics are should follow the same storage protocols as the straightforward) or strategically (requiring greater greenhouse-grown and lab-grown efforts (section 2.3.3). consideration of restoration goals and the ecology of source communities), but in both cases, building relationships with land owners and land managers prior to the restoration project will be critical to ensure access to potential salvage sites. Care should be taken not to inadvertently move problematic species (for example, exotic annual grasses or biocrust pathogens) from a donor site to a location where they did not previously occur. A simple protocol for conducting a salvage harvest is presented in Box 1. At present, salvage harvests of biocrusts have been Fig. 2 Biocrust scalping for field-collected inoculum. Photo: A. Antoninka conducted mostly using manual methods (in other words, shovels and buckets), but the method is 2.2.2 Salvaged biocrusts potentially highly amenable to upscaling using heavy Field collection of biocrust from one intact ecosystem equipment (Tucker et al. in review). Of the propagule to serve as inoculum for restoration of a degraded collection and cultivation methods presented in this site may not be sustainable for large restoration areas, manual, salvaging provides the greatest potential as an intact site needs to be sacrificed to restore the volume of source material, and the transporting degraded site. However, biocrusts salvaged from and storing the material may require special locations that are already slated for destructive land consideration. Storage of ~12 tons of salvaged use changes (for example, future solar development biocrust required use of a 10 x 20’ storage unit and and road construction sites) may provide a plentiful ~80 twenty-seven gallon totes (Tucker et al. in review). source of mature biocrust inoculum for restoration Because salvaging biocrusts may involve transfer of projects (Chiquoine et al. 2016, Tucker et al. in multiple species, along with soil and the associated review). The process of salvaging biocrusts has three seedbank, care may need to be taken to avoid phases: 1) identifying appropriate sites for salvage, causing unwanted species introductions.

BIOCRUST RESTORATION IN DRYLANDS 4 Developing Biocrust

Upper Basin Containing Sand

Wicking Liner Poly Tubing Mainline Poly Tubing Lower Basin 1/2” Pump Drainage 1/4” Drip Line Reservoir

Fig. 3 Biocrust greenhouse growing diagram as described in Doherty et al. 2015.

2.2.3 Greenhouse-grown biocrusts key step in the recovery of biocrusts and associated ecosystem services. In particular, cultivation should There are many ways to cultivate biocrust in the focus on pioneer filamentous species of cyanobacteria, greenhouse. The key to success is to alleviate the such as M. vaginatus and M. steenstrupii, which are limitations to growth encountered in the field while the most abundant organisms in most early biocrust simultaneously minimizing community changes (e.g. communities, and are foundational for biocrust growth of ‘weedy’ species). In general, this means formation (Garcia-Pichel and Wojciechowski 2009, adding limiting nutrients, watering frequently while Belnap and Gardner 1993). Targeted growth of these subjecting biocrust organisms to dry-wet cycles components is a logical priority in creating biocrust (cyanobacterial biocrusts) (Velasco Ayuso et al. inoculum to seed in disturbed areas. 2017), or reducing or eliminating desiccation events. Through testing a variety of watering systems, watering intervals, nutrient additions, shading, and soil types, a few key components have shown themselves to be the most successful approaches. A basic design for an irrigation and wicking system is described in Doherty et al. 2015 (Fig. 3). Modifications that have been made since this publication suggest that deionized water is not a requirement, eliminating the need for water storage. Instead, filtered tap water can be used with good results (Bowker and Antoninka 2016). Cyanobacterial biocrust growth Cyanobacterial biocrusts are often the first step in biocrust successional maturity from bare soils, providing essential ecosystem services such as soil stabilization. Once stabilized by filamentous cyanobacteria, these cyanobacterial biocrusts can support the establishment of other biocrust organisms, such as mosses and lichens, that further mature the biocrust and enhance provided ecosystem services. Therefore, focusing on supporting rapid development of cyanobacterial biocrust in disturbed areas is a Fig. 4 Local biomass greenhouse-grown biocrust in Flagstaff, AZ. Photo: A. Antoninka

5 BIOCRUST RESTORATION IN DRYLANDS A two-step process to obtain cyanobacterial biocrust optimize biomass yields, while minimizing changes inoculum grown in the greenhouse is described in in the cyanobacterial community structure of the Velasco-Ayuso et al. (2017). This biocrust inoculum greenhouse grown biomass. Through doubling production approach is based on the enhancement of frequency of natural wetting events, a 60% reduction remnant biocrust populations to produce abundant in sunlight, inoculation by slurry, and nutrient biomass that is of high fitness and low ecological risk, additions (site specific), biocrust cyanobacterial in that grown microbes are of local origin. Here, high communities grown in the greenhouse can closely amounts of cyanobacterial biocrusts are produced mimic field cyanobacteria and serve as inoculum for from low levels of natural inoculum (Fig. 5) in relatively large scale field restoration projects. short time (4 to 5 months), by using factors that

Fort Bliss (FB) Jornada (J)

Nosecone (N) Burr Buttercup (BB)

Fig. 5 “Boxplots for the final phototrophic biomass (as aerial chl a content) obtained after greenhouse incubation of native soils from 4 sites (each panel shows a site) inoculated with natural biocrusts from their respective site under 18 different treatments. Boxes denote lower and upper quartiles (with median values depicted as black solid lines), and whiskers denote lower and upper extremes (n=3). Blue lines indicate the chl a content of field biocrust samples used as inoculum (INOC), and red lines indicate initial chl a content in the inoculated soils (INIT) (color solid lines indicate mean, and color dashed lines indicate standard deviations of n =3).” Directly from Velasco-Ayuso et al. (2017).

BIOCRUST RESTORATION IN DRYLANDS 6 Reuse of greenhouse-grown cyanobacterial desert sites consistently attained significant increases biocrust for continued inoculum production in biomass, with increased inoculum levels, there were progressively more marked shifts away from To reduce the need for additional field biocrust field cyanobacterial composition. In total, recycling collections, the reuse of greenhouse-grown biocrusts biocrusts for additional greenhouse or laboratory as a seed for recurrent biomass production has also growth is not advisable. If recycled inoculum is used, been tested (Bethany et al. 2019). Biocrusts from hot biocrusts should be closely monitored to ensure that desert sites seem especially unsuited to growth with greenhouse-grown cyanobacteria communities recycled inoculum, with biomass levels below initial match communities found in the field and sufficient inoculum levels (Fig. 6). While biocrusts from cold biomass is produced.

Fig. 6 Recycling biocrust inoculum from hot and cold deserts (Bethany et al. 2019).

Moss, lichen, and mixed biocrust growth soil stability (Chaudhary et al. 2009, Li et al. 2004), and nitrogen inputs by harboring nitrogen-fixing Huge strides in developing a cultivation technology cyanobacteria (Rousk et al. 2013). Mosses in particular that works for mosses and lichens have been made may be highly suitable for biocrust restoration due through an experimental cultivation system as to the fact that any vegetative tissue of a moss is described in detail in Doherty et al. (2015). Mosses a propagule that may grow into new moss plants are early successional in some systems, and late (totipotency) (Memon and Lal 1981), propagules can successional in others; lichens are most commonly be stored and retain viability for decades in the right mid-late successional. Thus, growing mosses and conditions (Stark et al. 2004), and mosses are highly lichens offers opportunities to restore biocrusts tolerant to desiccation. across multiple successional states, which would likely increase the ecosystem services the communities Cold desert mosses from the genus Syntrichia are providing (Housman et al. 2006). Mosses from respond to manipulation with water and nutrients. the genus Syntrichia can be targeted because they Most mosses and a few lichens are amenable to are common and abundant in biocrusts around the cultivation (Bowker and Antoninka 2016; Bowker et al. western U.S. and provide critical ecosystem services 2017). To process for cultivation, the moss and lichen such as water absorption (Chamizo et al. 2012), biocrusts are gently broken up, soil removed, and

7 BIOCRUST RESTORATION IN DRYLANDS cleaned with water over a 2 mm mesh sieve to establishment rates under field conditions(see remove the majority of mineral soil particles. Washing section 2.3.2 for details on hardening protocols). is followed by gentle shaking for 10 minutes in Although this method requires a laboratory with the water, repeating washing and shaking five times. ability to correctly identify and target the isolation Then mosses and lichens are carefully and slowly of cyanobacterial species that best represent local dried by gently patting them with a paper towel and field communities, developing the inoculum under spreading them on slotted trays over paper towels. laboratory controlled conditions ensures that it will The trays can then be placed in closed fume hoods match what is found in the field and could increase or any lighted, well-ventilated areas for maximal air inoculum success, as grown organisms are likely to flow. After drying, lichens and mosses can be broken be genetically preadapted to local site conditions. up by “grating” them over a 2 mm mesh sieve (see Moreover, it ensures that adventitious microbes are section 2.2.3 for additional sieving options). Using not part of the inoculum formulation. Additionally, an automated greenhouse cultivation system (for the cultivation of different community members example, Doherty et al. 2015) can help grow larger separately allows for the formulation of mixed amounts of inoculum. The moss/lichen greenhouse inoculants into an inoculum that matches field inoculum can then be added directly to the surface cyanobacterial community structure for each of the of the restoration site. targeted restoration sites. 2.2.4 Lab-grown biocrusts Protocols to isolate targeted cyanobacteria, and to obtain large quantities of biomass from the isolated Laboratory cultivation of cyanobacterial strains as cyanobacterial strains can be found below. inoculum supply for restoration efforts has also been pursued. In a multistep process, protocols for the Biocrust cyanobacteria isolation methods establishment of microbial nurseries to produce The protocols provided here describe two specific photosynthetic cyanobacterial inoculum (lab-grown pipelines for the isolation of the most common inoculum) for biocrust seeding at scale have been cyanobacteria inhabiting biocrusts communities in designed (for details see Giraldo-Silva et al. 2019a). the southwestern U.S. (for further details see Giraldo- These protocols comprise: a) specific methods for Silva et al. 2019a). The first isolation protocol targets the isolation and pedigree of cultures of the major the bundle-forming, non-nitrogen-fixing, biocrust biocrust forming cyanobacteria (Fig. 7a-e, examples pioneer cyanobacteria Microcoleus vaginatus and of cyanobacteria isolated from southwestern U.S. Microcoleus steenstrupii. For the isolation of these biocrust communities), b) methods for scaling up cyanobacteria, wet the biocrust and wait ~ 30 cyanobacterial isolates to produce large quantities minutes to allow for Microcoleus spp. to move to of biomass (see below), and c) hardening protocols the soil surface. Under a dissection microscope, pick to increase cyanobacterial biomass survival and cyanobacteria bundles directly from the biocrust

A B C D E

Fig. 7a-e Examples of cyanobacteria isolated from Southwestern US biocrust communities. Photos: A. Giraldo-Silva

BIOCRUST RESTORATION IN DRYLANDS 8 using forceps and then clean the picked bundles by The second isolation protocol targets the isolation dragging them on 2% agar plates. This cleaning step of the non-motile, nitrogen-fixing, and scytonemin is important because it removes other cells attached (sunscreen pigment) producing cyanobacteria Nostoc to the bundle, such as filamentous cyanobacteria spp., Tolypothrix spp. and Scytonema spp. For the Schizothrix spp./Trichocoleus spp., Lyngbya spp. isolation of these cyanobacteria, take small pieces and, Leptoyngbya spp., that will otherwise rapidly of biocrust and add them to liquid media where

outcompete Microcoleus spp. in enriched cultures. N2 is the only nitrogen source (BG110). Incubate Transfer the cleaned bundles into liquid media enrichment cultures at 4, 25 and 35 °C for one week (BG11 or JM) (a 96 wells plate is recommended) and or until cyanobacterial growth is observed. The cover the transferred bundles (for example, with expectation is that at 4 °C the majority of the growing a Kimwipe), incubating them under culture room biomass belongs to Tolypothrix spp., and at 35 °C conditions: 25 °C, at ~ 20 μmol m-2 s-1, under a the majority of the biomass belongs to Scytonema 14h:10h light:dark photoperiod regime. After one species. Isolation of Nostoc spp. can be achieved at week, uncover the bundles, and add a few drops of 25 °C. Using a dissection microscope and forceps, pick new media to each well, then put them back to grow. grown biomass and streak it on nitrogen-free agar One week later, transfer all grown bundles to a bigger plates (BG110). Place plates back into their respective volume of media (a 24 wells plate is recommended). temperature until the defined colonies have grown. After bundles have grown to sufficient biomass, Transfer colonies to a new agar plate and allow them confirm with microscopy the identity of the desired to grow. Once growth is observed, confirm under the morphotype. Transfer the candidate isolates into a microscope the identity of the desired morphotype. larger volume of liquid media. Follow their growth Transfer the candidate isolates into liquid media. and transfer them at least three more times to Follow their growth and transfer them for at least three establish a new culture. more times to establish a new culture.

BOX 2: Floating cellulose technique (Giraldo-Silva et al. 2019a) Scale up technique to produce large quantities of the biocrust pioneer cyanobacteria Microcoleus vaginatus and Microcoleus steenstrupii. Culture room conditions: 25 ± 2 °C, 14h:10h photoperiod (light:dark), 20 - 30 μmol m-2 s-1 Keep a stock of cyanobacterial culture as an inoculum supply during the scale-up process. Maintain stock cultures in 1L Erlenmeyer flasks containing the desired volume (e.g. 200 mL) of JM or BG11 medium, in agitation (100 rpm), under culture room conditions. Replace culture medium every 2-3 weeks. To scale-up, inoculate sterile cellulose tissue (Kimwipes) with stock biomass and incubate it floating on liquid medium inside Petri plate, as follows: • Petri plate inoculation must be performed in the laminar hood (axenic conditions). • Cut cellulose tissue (Kimwipes) according to petri plate size and autoclave prior to use. • Add approximately 60 mL of medium (JM or BG11) to the bottom of a 14 cm diameter Petri plate. • Place the plate lid upside-down and, using forceps, put an autoclaved cellulose tissue inside it. • With the help of a cell spreader, homogenously distribute 4 mL of stock biomass onto the tissue. • Use forceps to transfer the cellulose tissue from the plate lid to the plate bottom (containing the medium). Avoid submerging the filter in the medium. Close and label the plate. • Incubate plates in culture room conditions above, and shade plates with a white paper (Kimwipes can be used for this step as well) during the first 24 h. • After 24 h, remove paper shading and allow 8 to 10 days to grow. Some strains may take longer to reach peak biomass. Keep track of the growing time as biomass may turn yellow if this time is exceeded. • After the growing period, remove the plates from the culture room open them inside a laminar hood to dry. Drying period ranges from 24 to 48 h. • Store biomass at room temperature in dark conditions. Note: cyanobacterial biomass grown on cellulose tissue must be shredded before blending it with native soils.

9 BIOCRUST RESTORATION IN DRYLANDS A BB M. steenstrupii M. vaginatus Day 0 Day 8 Day 0 Day 8

C M. vaginatus HSN003 D M. steenstrupii HS024 ) ) 2 2 m m / /

9 9 a a l l h h c c

3 3 (m g (m g n n l l a ss a ss 1 1 o m o m i 2 4 6 8 i 2 4 6 8 B B

0.3 0.3 Time (days) Time (days)

Fig. 8 Growing M. vaginatus and M. steenstrupii with the floating cellulose tissue technique.

Scaling-up lab-grown methods once the inoculum is seeded in the field (Giraldo- Silva et al. 2019b) (see section 2.3.2 for details on Traditional approaches to scaling up the culture of hardening protocols). cyanobacteria and algae (such as those used in the biofuels industry; Sharma et al. 2014), can be used As an alternative to the “floating cellulose to grow biomass from biocrust cyanobacteria such technique”, a fog-based watering system (the as Nostoc spp., Tolypothrix spp. and Scytonema fog chamber method) was developed to improve spp. However, when growing Microcoleus vaginatus cyanobacterial biomass yields and reduce time and and Microcoleus steenstrupii (biocrust pioneer labor costs associated with biomass scale-up (Nelson organisms), biomass yields using traditional methods et al. in prep). This method involves use of ultrasonic for scaling-up are very low. Following this, an mist-makers that fill semi-enclosed transparent alternative approach (the floating cellulose technique; plastic chambers with fog (Fig. 9) in order to saturate see Giraldo-Silva et al. 2019a) was developed native soil substrates without overwatering, and can to quickly grow these cyanobacteria. A detailed be utilized in both indoor and outdoor settings. By protocol is presented in Box 2. By implementing this new approach, exponential and rapid growth of the biocrust pioneers Microcoleus vaginatus and A B Microcoleus spp. Microcoleus steenstrupii can be obtained (Fig. 8). liquid culture Once biomass production is achieved for the isolates, the delivery strategy consists of blending the grown Sterile native soil biomass at a relative abundance that matches field saturated with cyanobacterial communities, and mixing them with medium sterile native soil to prepare an inoculum that mimics biocrust field populations. This mixture of cultured Petri Plate cyanobacterial biocrust organisms in native soils can additionally be conditioned to dry-wet cycles and increasing light exposure to increase inoculum Fig. 9 Fog-based watering system for culturing cyanobacteria. survival rate and speed of cyanobacterial recovery Photo: C. Nelson.

BIOCRUST RESTORATION IN DRYLANDS 10 using native soils, integrating desiccation cycles, and exposing grown biomass to harsher environmental conditions (“hardening” see section 2.3.2 for detail on hardening protocols), the fog chamber method increases cyanobacterial biocrust inoculum yields approximately 3-7 fold over those produced by the floating cellulose technique. This method of isolate- based biomass scaling is accessible to land managers A B due to the wide commercial availability of the fog- based watering system and the easily automated open air (non-sterile) growth chamber. This method has also been used to successfully cultivate whole cyanobacterial biocrust communities from remnant biocrust collected from field locales. 2.2.5 Outdoor biocrust production C D To date, biocrust inoculum has been successfully cultivated under lab and greenhouse conditions, but scaling up restoration efforts to larger areas could benefit from large-scale cultivation in outdoor nurseries or “biocrust farms”. In addition to the potential to propagate larger volumes of material, outdoor cultivation may provide multiple additional benefits. First, there are methods to “harden off” inoculum outlined in section 2.4.1, but organisms E F grown under outdoor nursery conditions are

subjected to a wider and more realistic range of Fig. 10 a-e. Open-row biogrust cultivation at Mayberry Native Plant Nursery. environmental stressors and are hardened during the Photos: a,c,e K. Dohrenwend; b,d C. Tucker; f S. Ruckman growing phase, meaning they may be better able soil for the next steps, as well as protection against to survive transplant to the restoration site. Second, wind and sheet flow erosion during growout. If only a subset of the most common organisms have harvest is to be mechanized, rows need to be made been shown to be easily cultivated under greenhouse at a width that fits harvesting equipment. and laboratory conditions, but this does not necessarily include the accompanying heterotrophic Next, the rows are prepared for inoculation. In components. Outdoor nursery cultivation may create each row, we suggest placing a layer of either better conditions to produce a broader subset of the biodegradable paper (which must be buried full biocrust community, which may also be important sufficiently because it is prone to wind damage) for improved establishment at restoration sites. or jute (a vegetable fiber that is woven into mats) atop black poly weed cloth, which is then covered Open row biocrust production by a shallow (0.5˝) layer of soil. Paper alone will Biocrusts can be cultivated at the large scale in long degrade quickly and necessitate harvest of the rows open rows: “farm-grown” biocrust. Figure 10 shows as crumbles. Poly weed cloth allows the rows to be multiple steps of the outdoor cultivation process. The rolled up for harvest. Rows can then be inoculated at first step is to prepare cultivation rows for inoculation the desired density by manually spreading inoculum by scraping soil to create a flat surface (Fig. 10a). over the rows (Fig. 10b). To facilitate more rapid Removing all vascular plants and their root systems, growth of biocrust, rows should be inoculated at a and reducing the seedbank before this step is highly minimum of 10 % cover of biocrust inoculum. Various recommended if at all possible. Figure 10a shows methods of creating a growing structure and shade rows created using a farm tractor with a drag blade system are being tested. These include burlap added leaving a ~ 6˝ high x 12˝ wide berm of soil on the as an overlay on the weed cloth for shade, and shade windward and uphill side of each row. This provides cloth (white) on top of the inoculum.

11 BIOCRUST RESTORATION IN DRYLANDS Irrigation is then installed. In Figure 10c, two strips Field cultivation to maximize relative humidity of irrigation tape were laid out along each 3’ x 100’ and minimize ultra-violet (UV) radiation damage row, which was sufficient to water the full width of In cold deserts, biocrusts tend to be active during each row. For cool and cold desert biocrusts, water the cooler wetter months, whereas outdoor should be applied in the cold months only — when cultivation activity often has to occur during frost free temperatures are below 26° C — because research summer months. This high UV/ low relative humidity shows negative effects of warm season watering on environment can be stressful for biocrusts, leading mosses and lichens (Reed et al. 2012). Preliminary to less than optimal growth. This can be countered evidence suggests that 3 days/week watering by adding shade cloth, either directly on top of the promotes higher growth than 1 day/week watering, irrigation lines, or by creating low hoop houses. Fick but optimal watering regimes likely vary by biocrust et al. (in press) demonstrated rapid growth under type, soil type, and the propagation site’s climate. these conditions, but trials to test viability in the field When snow and rain fall naturally, irrigation is not are still underway. necessary and those irrigation cycles can be skipped. Watering during the cold months favors winter- 2.3 Climate adapted biocrusts germinating annual weeds if they are present on Over the past century the Southwest U.S. dryland site or in the inoculum. These weedy species need region has grown 0.9 °C hotter, and climate model to be addressed before they go to seed. Weeds ensembles predict that by the middle of the 21st germinating in the rows outside the inoculum may century these regions may see a 2.6 - 2.8 °C increase be treated with herbicide. Preliminary results suggest in temperatures and a 3.6 – 5.4 ° C increase by the that herbicides that work as amino acid synthesis end of the century (Garfin et al. 2013). Even without inhibitors (such as glyphosate) cannot be used changes to precipitation (which are hard to predict), directly on biocrusts (Lydia Bailey, unpublished data), hotter conditions can result in drier soils. Dryland so these weeds must be pulled or clipped manually. ecosystems are defined by hot and dry conditions, Weed management can be a very time-consuming but climate manipulation experiment results suggest issue in farm cultivation of biocrust (as in any other that organisms in these systems are often living on the type of agriculture) (Fig. 10d). Preparing the rows to edge of their physiological tolerances for extremes reduce seeds in the parent material can reduce this of heat and aridity (Fig. 11). Accordingly, further shifts work, as can harvesting inoculum from areas with few in the climate regime to hotter and drier conditions weeds present. Dealing with exotic invasive seeds is might have large impacts on the abundance and particularly important to avoid spreading unwanted distribution of biocrust organisms, and thus of the seeds to new sites. This is an area that needs further ecosystem functions associated with these organisms research and experimentation to refine techniques. (Reed et al. 2016, Fernandes et al. 2018). The final step is harvesting the cultivated biocrust. In a study conducted by the U.S. Geological Survey Depending on the row set up and if there is structure in Utah, long-term experimental climate warming to allow for biocrust rolling, harvesting can be resulted in major changes to the ecosystem, including done either by scraping the biocrust layer as in dramatic loss of biocrust mosses, which provide section 2.2.2, or by harvesting long rolls of biocrust high levels of ecosystem services (Ferrenberg et al. sandwiched between layers of weed cloth (Fig. 10e). 2015). These patterns are in line with biocrust losses The rolling process results in a biocrust “sod” with a with warming climate that have been suggested at base layer of weed cloth serving to hold the rolled- the global scale (Rodriguez-Caballero et al. 2018). up section of row intact so that it can be unrolled The patterns captured in these results may pose an partially intact at the restoration site. A surface layer important challenge: how can we restore ecosystem of biodegradable shade cloth (such as jute) can be functions associated with biocrust in a way that will be included in this biocrust sod. The sod application successful both now and in a warmer, drier future? process allows the added biocrust to immediately The answer to this question is at present beyond the start stabilizing soils at the restoration site (Fig. 10f), scope of a manual, but several different approaches as well as to ameliorate habitat for additional biocrust can be posed. First, habitat modification may allow growth (see sections 4.2 and 4.3).

BIOCRUST RESTORATION IN DRYLANDS 12 more similar to projected future climates of the restoration site. This approach (assisted migration or “prestoration”) may present a viable climate- adaptive restoration approach (Butterfeld et al. 2017), although risk assessment should be conducted to balance problems associated with long distance transfer of organisms against the potential benefits for climate adaptation (Mueller and Hellman 2008). None of these approaches have been conclusively demonstrated to enhance resilience of restored biocrust communities to climate change, and more data are needed to address this important management question. There is reason to believe that, in particular for cyanobacterial crusts, the rates of crust development and community turnover (years) may be much faster than the rates of significant climatic changes (decades). In sum, climate-adapted communities offer important options for dryland restoration, and research will continue to assess the best organisms and practices for biocrust restoration in a changing world. 2.4 Preparation after growth/collection 2.4.1 Hardening Just prior to field deployment, a step-wise hardening treatment can be initiated for both greenhouse– and lab-grown inoculum, which may potentially increase biocrust organisms’ tolerance of field conditions. The fact that biocrust organisms are grown under much milder conditions from those expected in the field encouraged the idea of implementing hardening Fig. 11 Experimental climate manipulation effects on cover of biocrust moss, procedures with the goal of increasing inoculum lichen and cyanobacteria (Ferrenberg et al. 2015). field adaptation and survival rates. Hardening can be implemented by increasing exposure to natural biocrusts to establish and persist under sub-optimal environmental stressors such as intense sunlight climate conditions. Second, restoration projects can (including UV), desiccation, and low nutrients, focus effort on organisms from the target ecosystem before formulation both as a dried or a wet slurried that are less likely to face negative effects of climate inoculum for dispersal in the field. Hardening is an warming. For instance, experimental warming studies, active area of research, and as of yet, whether or not as well as the geographic distribution of organisms, the effort is necessary remains as an open question suggest that several biocrust cyanobacteria and for greenhouse-grown moss-dominated biocrust lichen species may be more tolerant of climate (Antoninka et al. 2018). However, experiments warming than are some mosses (Garcia-Pichel et designed to assess the potential beneficial effects al. 2013, Ferrenberg et al. 2015, Tucker et al. 2019). of hardening on lab-grown inoculum (cyanobacterial Similarly, the responses to modified precipitation isolates), support the notion that hardening will regimes and increased drought is different for speed cyanobacterial establishment and recovery different crust forming cyanobacteria (Fernandes et rates under field conditions (Giraldo-Silva et al. al. 2018). The third option is moving organisms or 2019b). The tested hardening protocol comprise the whole communities from hotter deserts with climates exposure of the cyanobacterial biomass to multiple

13 BIOCRUST RESTORATION IN DRYLANDS dry-wet cycles, a gradual increase in light intensity, instead of the sky, the organism cannot survive. If and a shift from visible-only artificial light to full solar larger pieces are preferred, some amount of flipping spectral radiation. biocrust pieces over to ensure they are face up might be worth the effort. A detailed hardening protocol for lab-grown cyanobacterial biomass is provided in Box 3 to allow 2.4.3 Storage for a comparison of options and choice of what is It is important to collect inoculum dry, and to available for application. maintain an air-dry state throughout storage. Prior 2.4.2 Sieving size considerations and to field implementation, store all inoculum in a cool, instructions dark, dry location. Also, when storing prepared biocrust inoculum, containers should ideally have a Different sieve sizes can be used (for example, 2 mm, lid to prevent contamination from other inoculum or 5.6 mm) for all inoculum placed out into the field. sources and outside non-biocrust impurities. There is However, larger intact pieces are less likely to blow evidence that biocrust moss viability is reduced over or wash away, and so may provide a faster recovery time in storage, so shorter storage duration should time and sieving may not be a necessary step. An be balanced against other project considerations alternative concern of larger inoculum is that pieces (Guo et al. 2018). landing upside-down may not survive. For example, if a lichen’s photosynthetic cells are facing the ground

BOX 3: Hardening protocol for cyanobacterial cultures – lab-grown biocrust (Giraldo-Silva et al. 2019b) The hardening protocol includes exposure of scaled-up cyanobacterial biomass to 12 dry-wet cycles, stepwise increases in light intensity, and a shift from visible-only artificial light to full solar spectral radiation. Hardening treatments are performed on individual lab-grown cyanobacterial isolates embedded in native soils. Protocols take place consecutively in three different locations: culture room, greenhouse, and outdoors, for a total duration of 12 days. Deionized water is used in dry-wet cycles (wetting cycle should be gentle enough to fully saturate the biomass-soil mixture, but should not create pools), and different pore size shade cloth is used during the light acclimation part of the protocols: • The produced cyanobacterial biomass (mixed with native soil) is divided into as many containers as needed. The blended mixture height should not be more than 0.5 cm and containers must be transparent. • Culture room (four days), greenhouse (four days), and outdoors (four days): place all of the containers under the same growing conditions used during the scale up process. Wet biomass in the morning and let it air dry over the course of the day. Repeat the same wet-dry cycle every day. Using the appropriate shade cloth, progressively increase light exposure from 20% (day 1), to 60% (day 2), to 100% (days 3 and 4). At the end of the hardening (day 12), allow the inoculum to air dry for about 48 hours. Then sieve the mixture (recommend: 0.5 cm). Cyanobacterial inoculants grown separately can then be mixed to match local cyanobacterial community structure, or used individually to fortify greenhouse grown inoculum with respect to important biocrust components.

BIOCRUST RESTORATION IN DRYLANDS 14 Climate-adapted Biocrust Restoration

Why restore biocrust?

Biological soil crusts (biocrusts) are a mix of mosses, lichens, and cyanobacteria that form a critical living skin between the soil and atmosphere in dryland environments around the world. Biocrust organisms may be small, but they play very large roles in the functioning of these ecosystems. For example, biocrusts provide soil stability, which reduces erosion; they increase soil fertility and Left: Healthy Healthy biocrust reduces erosion, maintains nutrient cycling, and provides provide habitat for other organisms; habitat for many other organisms. they maintain water; and they aid in Right: Loss of biocrust from disturbance carbon storage. Research suggests or climate change results in less healthy that increased temperatures and ecosystems with increased erosion and dust. drought conditions could result in the loss of some Biocrust restoration on the Colorado plateau Restoring the landscape biocrusts and their associated Canyonlands National Park This study will help evaluate the best ecosystem functions. Collecting and growing biocrust A. restoration practices. The biocrust Already, biocrusts have There are two biocrust restoration sites in this area, near inoculum collected from more arid source been significantly impacted Castle Valley, UT and here at the Canyonlands Research locations were propagated (grown) on 1.5 by damaging land-use Center. The source biocrust (inoculum) for these sites acres at the local Mayberry Native Plant practices, offering important were collected from other source locations further B. Propagation Center. Rapid growth was opportunities for biocrust south and west. Biocrusts from hotter deserts like the promoted with irrigation, shade, and weed restoration. Research, here and Mojoave and Sonoran deserts may fare better on the prevention. The field-grown biocrusts were across the Colorado Plateau, Colorado Plateau, because they are already adapted then harvested and applied at the two explores how to succesfully to more arid climates. The inoculum source locations in the Mojave and 211 restoration sites (approximately 20 acres). restore biocrusts and assist Southwest Aridity Index Sonoran deserts (left) were determined based on their Two different methods were used to install adaptation to climate change Biocrust collection sites Indian Creek the new biocrust – including broadcast climate and on model predictions of the Colorado A. Biocrust restoration site in a way that will be successful Restoration sites near dispersal of dried, crumbled biocrust and a Canyonlands Research Center Plateau’s future climate. B. Canyonlands Research Center now and in the future. “turf” roll technique.

Monitoring success Chemical Properties The future of biocrust restoration A. Exopolysaccharides are microscopic The goal for this biocrust restoration polymers that protect cyanobacteria Biocrusts perform critical functions in our dryland landscapes and thus, when they are lost is to successfully establish a climate- and stabilize soil particles, they are the due to disturbance or climatic change, restoration efforts are needed to help bring back biocrust “glue” that holds soils together, these important communities and the services they provide. Biocrusts that are adapted to adapted community that can enhance and they are measured using chemical key ecosystem functions. To evaluate its lab techniques. the climate we predict for the future offer new opportunities for successful restoration. Now success an initial survey of the degraded C. B. Soil samples are collected to measure the is an exciting time for biocrust restoration science and practice and the innovative work at site is compared to the physical and A. total carbon and nitrogen concentrations. the Canyonlands Research Center has significantly advanced our understanding of how to chemical properties of the soil and successfully restore biocrusts. biocrust after restoration occurs and Physical Properties over time (right). Soil stability; moisture C. Soil stability is measured with soil Funding and collaboration: control; habitat and species composition; aggregate stability tests (rapid wetting This project was funded by support from the soil carbon and nutrient fertility will be to test soil dissolution). Wildlife Conservation Society Climate Adaptation monitored under current and future D. Transects are used to characterize Fund, which was established with funds provided climate conditions. landscape-scale biocrust community by the Doris Duke Charitable Foundation. B. D. composition.

Illustration: Brooke Weiland Studios LLC SECTION 3: INOCULUM ADDITION

3.1 Introduction to inoculum additions moderated by targeting additions during periods with low forecasted wind speeds, as well as by Addition of inoculum to the restoration site is a spraying the inoculated surface with water to help the critical step in the restoration process. The goal biocrust bind to the soil surface. during inoculum addition is generally to get enough biocrust inoculum added in the right place at the Trials using a slurry addition approach have met with right time to facilitate establishment of a self- some success for the cultivation of cyanobacterial sustaining biocrust layer. Beyond the method of biocrusts (Lorite et al. 2019, Wang et al. 2009). In inoculum addition, the amount of inoculum to be slurry, cultured biocrust is mixed with a larger quantity added is a key consideration, as is the seasonal of water and sprayed or poured onto the soil surface, timing of inoculum addition. facilitating growth and adhesion of biocrust inoculum. To date, one project has attempted to add inoculum using a drill-seeder and imprinter. Drill seeding was less effective than hand broadcasting inoculum, but imprinting followed by broadcasting improved the establishment of biocrust mosses (Doherty et al. 2019). Addition of biocrust inoculum either embedded in fabric or on top of weed cloth rolled up as a sod (harvested from outdoor cultivation as described in section 2.2.5) shows significant promise as an approach to restoring high priority restoration areas (Fig. 10e). Fig. 12 Inoculum addition at different plot sizes. Photo: A. Antoninka The applied fabric or weed cloth serves multiple functions. First, it allows the cultivated biocrust to 3.2 Ways of adding inoculum be applied to the field without entirely removing it from the conditions under which it grew, as well as Many approaches to inoculum addition have been maintaining some of the spatial organization of the evaluated, including 1) broadcasting dry, crumbled cultivated biocrust community. Second, it serves to inoculum, 2) application of a wet slurry, 3) use of hold the soil below it in place, reducing erosion to a drill-seeder or imprinter, and 4) application of an extent while the biocrust establishes. Third, it can biocrust embedded in or atop rolls of weed cloth as prevent establishment and competition by weedy a sod. The first two of these approaches have some annuals. A downside to this approach is that it is demonstrated efficacy under the right conditions inherently more costly and spatially restrictive than (Antoninka et al. 2018), and one machine-assisted broadcast or slurry dispersal methods. approach was beneficial (Doherty et al. 2019), while the last shows significant promise for restoration of 3.3 Amounts of inoculum to add high priority areas (Doherty et al. 2019, Lorite et al. There are different ways to determine how much 2019). The most frequently used method of inoculum inoculum should be added to the area of restoration. addition is to manually broadcast dry, crumbled One way is by adding a percentage of chlorophyll a and sieved biocrust over the restoration area. This found in later successional biocrusts in the area (mg approach is simple and requires only readily available of chl a equivalents by area, Box 4). By adding mg tools such as containers for measuring a known of chl a to a known area, additions are not related volume of biocrust to spread over a known area to directly to the volume of inoculum, but rather to the achieve the target density as described below in photosynthetic potential. This method does however section 3.3. One critical consideration is that dry require the lab capabilities to assess chlorophyll a crumbles are highly vulnerable to being blown away concentrations. in windy conditions, which has resulted in complete failure of a restoration trial in at least one case If the ability to assess chlorophyll a is not present or (C. Tucker, personal observation). This has been feasible, an alternative method of determining the

17 BIOCRUST RESTORATION IN DRYLANDS and delivered as evenly as possible across the BOX 4: Example chlorophyll a calculation restoration area to achieve 10 % biocrust cover. This for inoculation. method is easily adapted to different assumptions • The amount of soil needed from each location to of baseline biocrust depth and density. If volume cover 14.4 m2 at 200% field chl a levels (14.4 m2 at of inoculum is chosen over chlorophyll a quantity 200% = 72 m2 inoculated at 20% field chl a levels). additions, the soil surface cover (for example, 10 From here we calculated the mass of inoculum %, 20 %, or 40 %) can be added (Fig. 13). Inoculum needed to cover 1 m2 at 20%. at a 10 % cover was identified as being optimal in • That 72 m2 is coming from the initial established balancing effort and cost to efficiency of restoration area to restore. (Fig. 13), although experience suggests that higher inoculum levels produce higher restoration success. • In order to easily measure the amount of inoculum needed for each location, enough soil from each 3.4 Season of inoculation location was measured to cover 14.4 m2. The appropriate biomass was mixed with each of the Considering the timing of inoculation is critically soils so that final inoculum chl a levels matched important to success. Biocrusts need water to 200% field chl a levels. The correct mass of the grow and establish, but ideally these would be concentrated inoculum was calculated so that lighter, consistent rains with lower wind so as field locations would be inoculated with 20% not to disturb the newly-added biocrust surface. field chl a levels. Inoculating in monsoon season can lead to loss of propagules through overland flow (Young et al. 2019). Instead, choosing periods with mild temperatures, amount of inoculum to be applied to the restoration high relative humidity, and light but consistent area uses a volume to area conversion assuming a precipitation is preferred. These patterns can be baseline 1 cm depth of biocrust. This method can different across different deserts. be calculated by converting the volume necessary to cover a known area with biocrust to 1 cm depth and In North American deserts, these conditions are then reducing it to the desired percent which relates common in the fall and winter, making late-fall an to that volume (see section 4.3 below for efficacy of appropriate time to inoculate in most cases (Fick different percent cover). For instance, to cover an et al. 2019). High levels of interannual variation in area with 10 % biocrust cover one would first calculate the timing and duration of favorable conditions the volume required to add biocrust to 0.1 cm depth for biocrust growth may necessitate planning for over that area. Intact biocrust could thus be collected repeated inoculum applications across years, from an area 1/10th the size of the restoration area if possible.

Fig. 13 Biocrust cover resulting from different levels of initial inoculation.

BIOCRUST RESTORATION IN DRYLANDS 18 SECTION 4. HABITAT PREPARATION, MODIFICATION, AND SOIL STABILITY

4.1 Introduction to habitat preparation, 4.2.2 Shading modification, and soil stability Because biocrusts are often limited by water, shading Preparing the restoration site prior to addition can be used as a method to enhance the length of of biocrust inoculum may by a critical step in the time that water is present by suppressing evaporative restoration process. There are many opportunities losses, and can also reduce stress caused by radiation associated with preparing the restoration habitat prior to (Figs. 15 and 16; Antoninka et al. 2019; Sorochkina et adding biocrust communities that may facilitate biocrust al. 2018). This can be achieved through a variety of recovery. However, this is a topic that needs more work, means, including the use of local materials, such as and our understanding of what modifications are slash from nearby vegetation. Depending on the size most likely to yield benefits remains incomplete. of the project, if a small-scale shading effort is needed, ½ inch PVC frames (10 cm wider than the area to be 4.2. Habitat modifications covered) can be used with cut shade cloth or screens that reduces light by 50-60 % (Fig. 16). Shade cloth is attached using a fabric stapler. At larger scales, shade cloth can be placed directly on top of the biocrust, or placed over wire row cover frames, although this approach may be conducive to loss of shade cloth during high winds (Fick et al. 2019).

Fig. 14 Surface roughening (diagonal lines) and polymer application. Photo: A. Antoninka

4.2.1 Surface roughening Bowker et al. (2002) demonstrated the importance of microaspect in shaping the biocrust community and function. In an attempt to provide a mix of biocrust microhabitat types, a cement trowel can be used to make ~1˝ troughs diagonally across plots every 2˝ at a NNE-SSW direction (Fig. 14). This is easily achieved Fig. 16 Shading using PVC frame and 50 % shade cloth. Photo: A. Antoninka in a fine textured soil, but troughs will not hold up in coarse soil without additional support. 4.2.3 Irrigation and water addition Water addition could be beneficial at the time of inoculation to encourage propagules to bind with and grow into the soil. Again, there are many ways to achieve this, through a fine scale spray misting the surface or through mimicking rainfall droplets. It would be important to add enough water that the biocrust can utilize the moisture for growth, but not so much that runoff and erosion are created weakening the surface soil stability. However, some biocrust restoration irrigation work has not found a longer-term benefit of watering (Condon and Pyke

Fig. 15 Chlorophyll a levels (a proxy for photosynthetic potential) over three 2016), and thus this step may not be necessary if years for different habitat modifications as compared to recently scraped and watering is unfeasible or logistically challenging. completely intact (From Chock et al. 2019).

19 BIOCRUST RESTORATION IN DRYLANDS 4.2.4. Other habitat considerations been shown to stabilize entire sand dunes in inner Mongolia and China, and were found to be one of the It is important to evaluate a site to determine the best habitat modification methods for the effort (for barriers to success that may be present at that example, shading requires a high level of effort) for location (Bowker 2007). It is also important to biocrust recovery in the different methods described consider the order of activities. For example, it is wise in this operational manual. However, recent work to do all soil manipulations prior to inoculation, so as suggests that adding straw checkerboards where not to disturb the biocrust once it is added. Although soil is stable can actually lead to suppression of not studied yet in great detail, growing biocrust on biocrust establishment (Antoninka et al. 2019) and so jute has been shown promising in providing benefits considering soil stability is important in deciding if to biocrust restoration (Condon & Pyke 2016, Slate straw borders are needed. et al. in press, and Bowker et al. in press). In the context of habitat considerations, growing biocrusts on jute and adding them to the restoration site intact can help modify the environment the biocrusts experience and may increase success.

Fig. 18 Straw border. Photo: A. Antoninka

BOX 5: Example Polymer delivery protocol Dilution: 1:9 polymer to water ratio Fill sprayer designated for polymer with 1 part polymer to 9 parts water to assure that we are getting 1 liter of water on the soil surface and the Fig. 17 Addition of polymer to experimental plot. Photo: A. Faist polymer is not too dilute. **Make sure the water used is filtered through the “pool filter” as this removes chemicals and minerals not wanted 4.3. Soil stability considerations in the water** 4.3.1 Polyacrylamides Application rate: Polyacrylamides are commonly used as a method to Add 1 liter of polymer and solution per 1m2 of soil reduce dust on roads, as well as to stabilize road banks surface. or other soils subject to erosion. Polyacrylamides Method of application: can be selected based on the following criteria: Apply polymer solution in a grid like pattern for 1) documented use and effectiveness at soil even distribution and coverage. stabilization in the peer reviewed literature, 2) UV Time of application: and biodegradability, and 3) variety in chemical Polymer is ideally applied directly before adding composition. Through these criteria, three polymers the inoculum to also serve as the water treatment. can be used: 1) Dirt Glue (aqueous acrylate polymer Make sure when inoculating that the polymer has emulsion), 2) SoilTac (vinyl copolymer emulsion), and 3) not dried and is still “sticky.” TerraLoc (polyvinyl alcohol), although other companies Equipment needed: offer comparable products. Polymer 4.3.2 Straw borders and checkerboards Pool hose filter (to filter out pollutants and chlorine that may be harmful for biocrust species) Straw borders assembled from straw and inserted Containers to hold filtered water vertically into sandy soils (Fig. 18, Box 6) can slow down Polymer sprayer (use only polymer sprayer as particle movement over the soil surface, providing a spray can get clogged) more stable area for biocrust recovery. Large arrays Measuring cup to make polymer mixture. of straw borders, forming straw checkerboards, have Polymer addition checklist

BIOCRUST RESTORATION IN DRYLANDS 20 4.3.3 Psyllium and other plant-based BOX 6: Straw border implementation stabilizers using the shovel method. Psyllium is a plant-based stabilizer made from Implementation: the protective coating of the plantago (Plantago Set a thin layer of straw along the soil surface (~10- insularis) plant’s seed. It is used in hydroseeding 12 inches long). Use the edger (or flat shovel) to applications, to stabilize soils, and for dust control. push the straw into the slit so it folds upon itself Ecology Controls M-Binder has been used in biocrust and forms a vertical fence like barrier held in place restoration projects to provide temporary soil by the soil. stabilization with no negative and sometimes positive Equipment needed: effects on biocrust survival in one study (Fick et al. Straw in press). Psyllium powder can be applied by hand String and 4 nails to set up straight lines and gently raked into the soil to increase the depth Bucket to put straw in of stability. Other plant-based stabilizers, including guar gum and xantham gum, either interfered Clippers to cut straw to correct size with biocrust growth or created undesirable soil Edger (or flat shovel) conditions. A protocol for application of psyllium is Straw check sheet provided in Box 7. Providing biocrust inoculum grown on jute, a plant- based woven cloth, also adds additional soil stability. Because the biocrust community on jute is a cohesive mat (Fig. 10e), it can stabilize the degraded soils beneath while the biocrust community establishes. Being plant-based, the jute can break down and disappear over time.

BOX 7: Psyllium delivery protocol Application rate: 60 g/m2. May need to adjust based on soil type and slope (i.e., less for fine soils and more for greater slopes). Method of application: Sprinkle psyllium powder evenly across the area and gently rake into soil by hand. Apply biocrust inoculum. Spray soil with 1L of water per m2 with a pump sprayer. Equipment needed: Psyllium powder Pool hose filter (to filter out pollutants and chlorine that may be harmful for biocrust species) Containers to hold filtered water Pump sprayer Measuring cup for psyllium Container to use for psyllium application (e.g., Tupperware or yogurt container)

21 BIOCRUST RESTORATION IN DRYLANDS SECTION 5: MONITORING METHODS AND PROTOCOLS

5.1 Introduction to monitoring methods and protocols A variety of monitoring methods exist and can be tailored to fit desired restoration objectives. For instance, if the restoration goal is to return biocrust diversity and multiple levels of successional growth (for example, cyanobacteria, lichens, and mosses), assessing the cover of surface area by biocrust at the species level may be the best option. Biocrust grouping categories could also be used, such as proportion of cover that is moss, lichen, or cyanobacteria. Alternatively, if biocrust function is the highest priority, testing soil aggregate stability, shear strength, and/or soil organic matter or nutrient concentrations may be a preferred option. Fig. 19 Cover frame on lichen-rich biocrust in the Mojave desert. Optimally, however, one should monitor both species Photo: C. Tucker composition and function, if at all possible. This operational manual provides overviews of primary 5.2.2 Chlorophyll a monitoring methods used in biocrust restoration. Chlorophyll a concentrations can serve as a proxy A more comprehensive treatment of monitoring for the photosynthetic biomass found in the soil. The biocrusts in a restoration context is provided in higher the chlorophyll a, the higher the photosynthetic Mallen-Cooper et al. (2019). potential and hence a higher level of biocrust recovery. 5.2 Types of monitoring methods This metric has been calculated in a variety of ways (for example, Ritchie et al. 2006, Castle et al. 2011) 5.2.1 Biocrust cover with the primary goal being to break up the individual Cover estimates can be a good representation of cells to extract chlorophyll a from the phototrophic different stages of recovery. Many different cover components and to avoid interference by other estimates are available. A common way to reduce pigments present in crusts. Box 8 provides an example bias and establish cover estimates is through the protocol of how to accomplish this, and Figure 20 point intercept method (Fig. 19). This method collects shows the extracted chlorophyll a in a solution. data from a grid pattern where the pin intercepts the biocrust species at the substrate level. Another method is ocular cover estimation where an observer stands directly over the plot (sizes usually vary from 1 m2 to 0.25 m2) and records estimates at the functional group level (in other words, mosses, lichens and well-developed surface cyanobacteria). This approach does not detect subsurface populations of cyanobacteria or algae, and will miss microbial populations that do not coalesce into macroscopic assemblages that are visible to the naked eye.

Fig. 20 Chlorophyll a suspended in solution before absorbances are measured. Photo: N. Barger

BIOCRUST RESTORATION IN DRYLANDS 22 5.2.4 Microbial community composition: BOX 8: Chlorophyll a (and scytonemin) 16S rRNA community diversity. extraction from biological soil crust Analyses of community composition by bioinformatic samples (solvent: 90% acetone in water) analyses of ribosomal genes are now standard in 1. Take biocrust cores in replicates (1 cm diameter microbial ecology, and they have been successfully and 5 mm deep is recommended). Record core used to monitor community composition of inoculum weight and area. produced in microbial nurseries, to compare to the 2. Grind each core in solvent with a mortar and original field-collected communities, and also to pestle for 3 minutes (making sure the core analyze which isolates to use in laboratory cultivation becomes a powder). Add as much solvent as so that they genetically match the target field needed when grinding the sample (3 mL are populations (see for example Velasco-Ayuso et al. recommended). 2017; Bethany et al. 2019). No other methods offer 3. Transfer the powder slurry to a clean Falcon the level of resolution provided by this approach. tube. Use more solvent when transferring if Again here, while the level of specialization of this needed (2 mL are recommended). technique is significant, commercial sample and data 4. Adjust final volume of the solvent in Falcon processing are becoming more common tube as needed. and affordable. 5. Vortex the biocrust-solvent mixture for 30 sec. 6. Incubate for 12-18 h, in the dark at 4° C, 5.2.5 Soil aggregate stability ensuring consistent incubation times across Soil aggregate stability can be used as a key indicator samples, treatments, replicates, etc. of resistance to erosion and of soil health (Herrick et 7. After incubation, centrifuge the biocrust-solvent al. 2001, 2005) and is taken to be also an indicator mixture (recommended: 7000 rpm, 5 min, 15 °C). of biocrust development. Soil aggregate stability is Note: Ensure that the supernatant looks clear (depending measured using the method as presented in Box 9. on the edaphic origin of the soil, small particles can remain in the column). If particle-free supernatant is problematic to obtain, filter the supernatant using a 0.2 µm pore diameter DMSO-safe nylon filter. BOX 9: Soil aggregate stability field 8. Proceed to read the extract absorbance spectra in a spectrophotometer. Absorbance must be methods: recorded at 384, 490 and 663 nm (and 1010 nm- Because the primary area of interest is the to reduce noise in the turbidity signal). biological soil crust stability take the top most Note: Trichromatic equation according to Garcia- soil layer in the samples. While the number of sub Pichel & Castenholz (1991) should be used to discount samples per area can be determined on site, repeat interference from scytonemin and carotenoids. samples in a single area are beneficial for better *Acetone must be used as the solvent if scytonemin understanding recovery variability. extraction is desired. Equipment needed: DI water 5.2.3 rRNA gene abundance Soil stability test kits (Slake kits) An alternative method to assess crust development Stop watch is by the use of counts of total rRNA genes in the Datasheets soil by quantitative polyemerase chain reaction Pencils (PCR; see Fernandes et al. 2018, for example). While Stopwatch this requires principally sophisticated methods, the Slake checklist analyses are becoming available commercially in specialized microbiome facilities, and are swiftly becoming cheaper. They can specifically target bacteria (including cyanobacteria) and algal plastids, archaea, and eukaryotes, including fungi.

23 BIOCRUST RESTORATION IN DRYLANDS 2 cm

3 cm 2.5 cm diameter pvc 3 cm B 0.5 cm

5 cm 1.65 mm mesh Stability class table 1 cm (see table 1) C

3.5 cm

A 10.5 cm 21 cm drawn by Tye Lightfoot

Fig. 21 Soil aggregate stability (also know as slaking) test kit from Herrick Fig. 22 Food pressure tester, or also called “pocket penetrometer” to obtain et al. (2001). downward soil strength. Photo: Certified material testingproducts.com

5.2.6 Soil tensile strength and compaction recorded in kg. When a 0 is recorded in the down column, this means the strength of the soil was The fruit pressure tester (Fig. 22) can be used to too low to be read by the fruit pressure tester. determine the downward pressure needed to Alternatively, the soil shear strength can be calculated penetrate the surface of the soil, which is a proxy using a Torvane (for example, see Durham Geo- for soil tensile strength. The smaller of the two tips enterprises Inc. instructions, Fig. 23). should be used, and all measurements are to be

Fig. 23 Torvane to obtain surface sheer strength. Photo: http://www.durhamgeo.com/pdf/m_test-pdf/manuals/IC-S-160%20Torvane%20Instructions.pdf

BIOCRUST RESTORATION IN DRYLANDS 24 5.2.8 Level of Development The level of development (LOD) (Belnap et al. 2008) can be an inexpensive means of tracking the stages of biocrust recovery through visual comparisons (Fig. 25). LOD allows rapid evaluation of biocrust recovery over larger areas by restoration practitioners with limited familiarity with biocrust species. 5.2.9 Soil nutrients If the project is focused on returning soil fertility through the restoration of biocrust, monitoring soil nutrients along the recovery gradient could provide useful information. Equipment needed for field collection is basic and can be modified according to what is available. Basically, soils are simply collected in a way that allows samples to be taken to a given depth. If lab equipment is not provided/available by the restoration team, a variety of national or local labs can receive soil samples for processing. Depending on labs and nutrient analyses, desired costs can vary greatly. Assessing concentrations of soil nitrogen and organic matter are common analyses for determining soil fertility. Here, an example field collection and lab Fig. 24 Plot overview to show general biocrust recovery and overhead plot analysis protocol is provided to show general level of photos showing fine scale recovery. Photo: A. Faist effort needed to obtain soil nutrients (Box 10).

5.2.4 Repeat Photography Repeat photography provides a good visual representation of how the biocrust community may be changing over time, and allows for both qualitative and quantitative assessment of the state of biocrust. Different camera angles can capture different aspects of the biocrust recovery. With a broader picture overview (for example, plot overview in Fig. 24 - Top Panel), larger changes can be cataloged; with a direct overhead photo, smaller scale changes can be captured (Fig. 24 - Bottom panel). When looking from directly overhead, if the camera has a high enough resolution and is accompanied by a color chart, visual biocrust cover estimates can be calculated after returning from the field. If this method is to be used for cover estimates, the area of interest must be shaded, either using an umbrella or a veil, a color chart must be present, and a direct overhead photo must be taken. Photography sequences after wetting the soil improve the detectability of cyanobacterial populations that otherwise inhabit refugia in the microscopic subsurface of the soil (Sorochkina et al. 2018). Fig. 25 Level of development as shown in Belnap et al. (2008).

25 BIOCRUST RESTORATION IN DRYLANDS BOX 10: Soil fertility field collection and lab processing protocols Field Methods: Use soil auger (Metal soil core that looks like a “T”) to drill down to 10 cm. When pulling up the Auger make sure that no soil comes out the bottom of the core. Then separate the soil core into 3 segments (0-2 cm, 2-5 cm, and 5-10 cm). Then place the separated core segments into their own labeled whirlpack. **Note: if samples are wet then keep bags open to dry** Labeling example (follow own labeling procedures for specific restoration project): For example, on whirlpack bag write: What is being sampled, Date, Location (site, soil type and block), Plot (e.g., 147-LB-PM) + what sub sample from the plot (A or B) and what depth the sample is from (0-2, 2-5, 5-10). Lab Methods: After returning from the field, dry samples at 60° before grinding to a fine powder either in a mortar and pestle or with a ball mill grinder. Weigh samples into tin capsules and run on the Elemental Analyzer (EA) for C and N content. Inorganic C was determined by measuring CO2 produced on addition of 6M HCl (Pressure Calcimetry method). Organic C was defined as the difference between the EA Carbon and the IC value. Soil fertility field equipment: Meter tape (for transect line) Metal soil auger (1” diameter preferred) Ruler Whirlpacks Sharpie Soil fertility sampling checklist Soil fertility lab equipment: Drying oven capable of 60° Soil grinder Scale capable of μg precision Tin capsules (5mm X 9mm) Tweezers and small spatula/scoopula Elemental Analyzer capable of C and N determination (e.g., Vario Micro Cube)

BIOCRUST RESTORATION IN DRYLANDS 26 SECTION 6: MAJOR FINDINGS

Major Findings • Relatively simple, fast, and effective methods now exist to grow biocrust inoculum in greenhouses from field- collected samples (Velasco–Ayuso et al. 2017, Doherty et al. 2015). These approaches still require monitoring for inoculum quality with respect to final community composition.

• Exciting opportunities for lab-grown biocrusts exist, using laboratory grown cultures isolated from native communities. Protocols include methods to isolate and select cyanobacterial strains that resemble the most abundant cyanobacterial population at each field location; methods for scaling-up biomass production from cultured isolates to larger volumes for restoration; and methods for inoculum preconditioning treatments that increase exposure to solar radiation and temperature, and recurrent wet-dry cycles to pre-acclimate grown cyanobacterial isolates to the extreme conditions expected in the field.

• The fog chamber method, an alternative to the floating cellulose technique, represents an improvement on current lab-grown cyanobacterial biocrust inoculum production methods, through both increases in cyanobacterial biomass yields and significantly reduced associated time and cost commitments.

• Although inoculation of soils with lab and greenhouse grown biocrusts can enhance biocrust recovery (Antoninka et al. 2017), significant barriers and challenges still exist. More time is needed to evaluate these patterns and the inoculum’s ability to return to fully natural conditions, and new advances are made regularly.

• Biocrust inoculum grown outside may offer valuable options for growing mixed biocrust communities that are already hardened for survival in the restoration site. More work is needed to assess the utility of this approach and to compare the strengths and weaknesses to those of lab and greenhouse grown biocrust inoculum.

• Salvage harvests provide powerful opportunities to collect biocrust inoculum material that would otherwise be destroyed, but, as with all inoculation methods, risks with material transfer must be evaluated.

• Growth of climate-adapted biocrust for restoration has the potential for significant success in the face of climate change, although the longer-term outcomes of climate-adapted vs. local biocrusts will take time to emerge. Potential risks associated with assisting in the migration of biocrusts (for example, unintentionally bringing foreign species or disease) must be carefully weighed against the risks associated with climate-induced loss of existing biocrust communities and their function. The benefits of this approach will depend on an eventual assessment of rates of population turnover and natural spread in relation to rates of climate change.

• Biocrust restoration is a worthwhile goal and represents a component of restoration science that is rapidly growing. New advances will likely continue to emerge and new options made available for resource managers and restoration practitioners. Now is an exciting time for biocrust restoration.

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