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.

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 Syntrichia ruralis and several lichens of the genus Cladonia (Durham et al., 2018).

Moss materials

We grew biomass of the moss Syntrichia ruralis, 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 S. ruralis 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 < 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 Syntrichia cover or none at all, in addition to a few trace occurrences of lichens (Cladonia spp.) and other mosses (Gemmabryum caespiticium). 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 Syntrichia ruralis from our two monitoring points within the first year using linear mixed-effects models, assuming a Gaussian distribution with the lme4 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 S. ruralis remained after three years, we converted these data to presence-absence, and conducted logistic regression to detect if treatment effects were still present (stats 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.