DOI: 10.5061/dryad.2jm63xsv5
title: Moss establishment in restoration: the role of moss production method and short-term benefits of abscisic acid

description: Mosses may be useful in ecological restoration activities but are excluded
 from most native plant materials programs. Recent efforts have attempted
 to propagate mosses in controlled environments for deployment to boost the
 recovery of degraded field sites. Field re-entry and establishment have
 proven challenging, possibly because the moss materials are not
 field-ready. We compared the field establishment rates of mosses of the
 same species propagated using three methodologies carried out either in
 greenhouses or outdoors. In an attempt to chemically boost
 field-readiness, we amended each with either sucrose, an osmoprotectant,
 or abscisic acid, a stress response phytohormone, or neither. Mosses grown
 outdoors with only one initial fall irrigation event lost at least 30%
 less cover than outdoor-grown moss that was irrigated in spring and moss
 tissue grown in a fog chamber inside of a greenhouse. The addition of
 abscisic acid also induced a subtle difference, leading to about 10% less
 cover loss compared to controls. Ultimately, all treatments declined to
 only trace level moss cover at most after three years. From these results,
 we put forward the working hypothesis that growing methodologies more
 similar to field conditions and exposing mosses to environmental
 fluctuations are more likely to produce field-ready moss materials.
 Abscisic acid addition is promising as a way to delay the mortality of
 mosses introduced into a desiccating environment. To translate short-term
 relative differences to long-term success, these practices may need to be
 combined with techniques that reduce the stress experienced in the field.
description: Study
 Site  Our
 research was conducted on MPG Ranch in the Bitterroot Valley of Montana,
 in an experimental enclosure fenced to exclude herbivores (46.667°N,
 -114.007°W, 998 m). The mean annual temperature across the ranch is 7°C,
 and annual precipitation is 356 mm (modeled 30-year normals; Daly
 & Bryant 2013) mostly falling as rain and snow during cold
 winters. During our experimental period nearby the enclosure, the average
 annual temperature was 7.9° and annual precipitation was 233 mm. Vascular
 plant communities across the property range from shrub steppes,
 grasslands, and coniferous forests, in addition to former agricultural
 land. Biocrusts are ubiquitous across the ranch, averaging 19% cover
 across all sites (Durham et al., 2018). Biocrusts are variable and may be
 diverse, but in most locations are dominated by the moss
 <em>Syntrichia</em> <em>ruralis</em> and several
 lichens of the genus <em>Cladonia</em> (Durham et al.,
 2018). Moss materials
 We grew biomass of the
 moss <em>Syntrichia</em> <em>ruralis</em>, using
 three distinct production methods. All types were initially started using
 field-collected moss material, obtained in September 2017 from the MPG
 Ranch property (46.668°N, -114.017°W, 1047 m). To collect moss material,
 we located patches of <em>S. ruralis </em>and lifted the
 tissue away from the soil surface using flat-bottomed trowels. All
 collected moss material was disaggregated by passage through a 5 mm sieve
 while dry, resulting in a mixture of mostly individual shoots, small
 clumps of multiple shoots, and shoot fragments. Thus, mosses in the
 growing systems below grew mostly from individual shoots into colonies.
 “Greenhouse-fog” moss material was grown in the NAU research
 greenhouse over burlap cloth surfaces inside of a fog chamber (Figure 1),
 following the methods of Doherty et al. (2020). Briefly, growing surfaces
 housed within a PVC framed chamber were enclosed within a double layer of
 row cover cloth. Within the chamber, a fog emitter was submerged in a
 basin of water and the resultant fog was distributed by a fan throughout
 the chamber, nucleating droplets on moss tissues and hydrating them. We
 grew mosses on Vigoro brand natural burlap fabric (2 mm grid) for 10 weeks
 and then stored them for six months prior to this
 experiment. We grew moss tissue outdoors in the experimental
 enclosure at MPG Ranch; these also were initially seeded with moss tissues
 field-collected from the same site described above. We placed a thin layer
 of moss tissue in between two buffering substrates (Figure 1). The bottom
 materials were selected to discourage colonization by exotic invasive
 vascular plants. The upper fabric was either burlap or 50% shadecloth,
 intended to cool the growing mosses and extend hydration periods. Two
 variations of this technique were installed in October 2017.
 “Outdoor-passive” moss material was hand watered once at the initiation of
 the experiment using watering cans until full hydration was achieved. All
 additional water was obtained from natural rain and snowfall events. We
 harvested this material in May 2018 and stored it until use in our
 experiment. Because differences among different variations of top and
 bottom materials were not strong, we pooled moss biomass across treatments
 to provide inoculum for the present experiment.
 “Outdoor-irrigated” moss material
 was composed of only materials grown on a cardboard substrate with a
 burlap covering. These were allowed to continue growing in spring with
 irrigation provided by a sprinkler on an automated timer providing daily
 10-minute watering at sunrise. We initiated irrigation in March 2018 after
 the danger of frost had passed and continued until May 2018. We terminated
 irrigation due to a worse-than-expected proliferation of exotic vascular
 plants; nevertheless, mosses did continue to grow and we were able to
 harvest tissue immediately prior to the initiation of the present
 experiment.  Experimental design 
 In our experiment, we crossed two
 factors. To implement the first factor, we added 2.5 g of moss-grown using
 one of the three production methods to each experimental unit by
 sprinkling them from envelopes evenly over the surface area. This created
 an average initial cover of about 74% in our plots. Prior to the addition,
 mosses from each production method were disaggregated by passing the
 tissue through a 5 mm sieve, tissues (inclusive of aboveground
 gametophytes, belowground rhizoids, and fragments of both) collected on a
 2 mm sieve, and then were thoroughly mixed. Since our experiment was
 focused on the relative performance of moss tissues, we did not establish
 uninoculated plots. Uninoculated areas of the study site (e.g. in between
 experimental units) were free of mosses and remained so. An experimental
 unit was a 15 x 15 cm plot, marked with landscape staples on two diagonal
 corners. To implement the second factor, we added either
 sucrose, abscisic acid solutions, or neither to the moss material. We
 added 1 mL of 0.75M sucrose or 10μM abscisic acid with a hand pump sprayer
 that delivered atomized solutions evenly across the plots. These
 molarities were selected based on results from prior work demonstrating
 improvement in desiccation tolerance among mosses when abscisic acid
 (Werner et al., 1991) and sucrose (Pence, 1998) were added. Controls
 received an equivalent amount of water. 
 The entire experimental area was
 overlain by a jute net, including all plots and the spaces in between.
 Jute net is known to improve moss and biocrust establishment (Bowker et
 al., 2020; Condon & Pyke, 2016; Slate et al., 2020) and is used as
 an erosion control and plant establishment aid in the study area. The
 study site was weeded over the first year of the
 experiment.  Monitoring
 We monitored moss cover using
 ocular estimation, with the aid of a quadrat that snapped into place
 between the corner markers. Cover estimates to the nearest 5%, or nearest
 1% if &lt; 5%, were obtained on September 19th, 2018 (immediately
 following installation), May 2nd, 2019, and September 15th, 2019.
 We
 revisited the plots 3 years after the start of the experiment to resurvey
 them using the same methods. Nearly all plots at this time had
 ＜1% of <em>Syntrichia</em> cover or none
 at all, in addition to a few trace occurrences of lichens
 (<em>Cladonia</em> spp.) and other mosses
 (<em>Gemmabryum</em> <em>caespiticium</em>).
 Thus, we added a trace
 occurrence category, quantitatively expressed as 0.5% cover. At this
 sampling, one corner staple was missing from 9 plots, particularly in one
 portion of the study site. In these cases, we placed the quadrat as well
 as we could, estimating the plot position based on the remaining corner
 marker and the position of nearby plots. In some cases, both markers were
 missing, or there was visible damage in the plot, and the quadrat simply
 could not be read. Data Analysis
 Because our treatments initially
 varied in cover, due to differences in moss density by cultivation method,
 we standardized subsequent cover measurements, expressing them as a
 proportion of the initial value. We analyzed the standardized cover values
 of <em>Syntrichia</em> <em>ruralis</em> from our
 two monitoring points within the first year using linear mixed-effects
 models, assuming a Gaussian distribution with the
 <em>lme4</em> R package. We included three-way and lower order
 interactions of osmoprotectant-phytohormone treatment, growth method, and
 observation period in our model, as well as the main effects. We assigned
 a random effect to observational unit identities to mitigate temporal
 autocorrelation from repeat observations. We assessed the global effects
 of osmoprotectant treatment, production method, and observation period by
 conducting 3-way ANOVA (type II from the car R package). We conducted
 Tukey-Kramer post-hoc tests to assess pairwise differences among groupings
 of factor levels to further aid interpretation. 
 Because only trace amounts of
 <em>S. ruralis</em> remained after three years, we converted
 these data to presence-absence, and conducted logistic regression to
 detect if treatment effects were still present (<em>stats</em>
 R package). We conducted 2-way ANOVA (type II) to assess the global
 importance of factors and performed Tukey-adjusted post-hoc tests to
 reveal differences among factor level groupings.