Soil moisture dynamics under two rainfall frequency treatments drive early spring CO2 gas exchange of lichen-dominated biocrusts in central Spain

Background Biocrusts, communities dominated by mosses, lichens, cyanobacteria, and other microorganisms, largely affect the carbon cycle of drylands. As poikilohydric organisms, their activity time is often limited to short hydration events. The photosynthetic and respiratory response of biocrusts to hydration events is not only determined by the overall amount of available water, but also by the frequency and size of individual rainfall pulses. Methods We experimentally assessed the carbon exchange of a biocrust community dominated by the lichen Diploschistes diacapsis in central Spain. We compared the effect of two simulated precipitation patterns providing the same overall amount of water, but with different pulse sizes and frequency (high frequency: five mm/day vs. low frequency: 15 mm/3 days), on net/gross photosynthesis and dark respiration. Results Radiation and soil temperature, together with the watering treatment, affected the rates of net and gross photosynthesis, as well as dark respiration. On average, the low frequency treatment showed a 46% ± 3% (mean ± 1 SE) lower rate of net photosynthesis, a 13% ± 7% lower rate of dark respiration, and a 24% ± 8% lower rate of gross photosynthesis. However, on the days when samples of both treatments were watered, no differences between their carbon fluxes were observed. The carbon flux response of D. diacapsis was modulated by the environmental conditions and was particularly dependent on the antecedent soil moisture. Discussion In line with other studies, we found a synergetic effect of individual pulse size, frequency, environmental conditions, and antecedent moisture on the carbon exchange fluxes of biocrusts. However, most studies on this subject were conducted in summer and they obtained results different from ours, so we conclude that there is a need for long-term experiments of manipulated precipitation impacts on the carbon exchange of biocrusts. This will enable a more complete assessment of the impacts of climate change-induced alterations in precipitation patterns on biocrust communities.

Results. Radiation and soil temperature, together with the watering treatment, affected the rates of net and gross photosynthesis, as well as dark respiration. On average, the low frequency treatment showed a 46 ± 3% (mean ± 1 SE) lower rate of net photosynthesis, a 13 ± 7% lower rate of dark respiration and a 24 ± 8% lower rate of gross photosynthesis. However, on the days when samples of both treatments were watered, no differences between their carbon fluxes were observed. The carbon flux response of D. diacapsis was modulated by the environmental conditions and was particularly dependent on the antecedent soil moisture. Discussion. In line with other studies, we found a synergetic effect of individual pulse size, frequency, environmental conditions and antecedent moisture on the carbon exchange fluxes of biocrusts. However, most studies on this subject were conducted in summer and they obtained results different from ours, so we conclude that there is a need for long-term experiments of manipulated precipitation impacts on the carbon exchange of biocrusts. This will enable a more complete assessment of the impacts of climate change-induced alterations in precipitation patterns on biocrust communities.

INTRODUCTION
to fix and store carbon (Belnap et al., 2004). However, we still know little about the impact of 84 changes in precipitation on the carbon uptake across biocrust communities in different dryland 85 regions and across seasons. In order to contribute to filling this knowledge gap, we have conducted 86 a short-term manipulative experiment in early spring to assess the effect of rainfall patterns on 87 photosynthesis and respiration of Diploschistes diacapsis, a lichen that generally is dominant in the 88 biocrust communities in central Spain (Maestre et al., 2011). We compared the carbon exchange 89 fluxes in response to two rainfall patterns that provided the same overall water amount; in one 90 of the treatments, single rainfall events were smaller, but more frequent; while in the other one, 91 single rainfall events were larger, but less frequent. By doing so, we seek to understand which 92 rainfall pattern could be more beneficial for carbon fixation by biocrusts, and to set the direction for Alcala del Olmo, 1990). Perennial plant coverage is lower than 40% and the site hosts a well-107 developed biocrust community dominated by lichens, such as D. diacapsis, Squamarina lentigera, 108 Fulgensia subbracteata and Buellia zoharyi (see Fig. S1).

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After collection, we covered the bottom of the soil cores with a fine-meshed fabric to avoid soil 110 loss and then took them to the lab, where they were watered to full saturation with low mineralized 111 water and drained for 24 hours to determine the weight at saturation water content. The undisturbed 112 cores were placed on a structure that allowed water to drain from the cores under a transparent roof whereas temperature and radiation remained similar to those at ambient conditions. Five days after 116 placing the cores under the roof, we started to apply a daily water pulse of 5 mm to six of the samples 117 (high frequency treatment), and a 15 mm pulse at a three-day interval to the other six samples (low 118 frequency treatment). In total, all samples received a water amount of 60 mm during twelve days 119 (from March 21 st 2017 until April 1 st 2017). Although D. diacapsis can activate the photosynthetic 120 system at smaller rainfall pulses (Lange et al., 1997;Pintado et al., 2005), the watering patterns were 121 chosen such that they were sufficient to provide enough water to both stimulate a pulse of lichen 122 activity and wet the soil beneath the sample. Also, we wanted to ensure that the pulses exceed the 123 threshold for ecologically effective precipitation, which has been reported to be 5 mm in a semiarid

Measurements
One of the six samples from each treatment was used to monitor soil temperature at 3 cm depth with 127 a temperature sensor (UP Umweltanalytische Produkte GmbH, Cottbus, Germany). In the other 128 five soil cores, we conducted gas exchange measurements using a Li-6400 portable photosynthesis 129 system (Li-Cor, Lincoln, NE, USA). Measurements were taken every day after the water application 130 and started earliest at 9:30 AM. The mean time between water application and the first measurement 131 of the samples was 77 min (SD = 13 min). In each sample, light and dark measurements were paired.

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First, the net CO 2 flux was measured by placing a transparent chamber on the sample (hereafter 133 referred to as net photosynthesis), waiting for equilibrium to be reached between the chamber (see  to stabilize before taking the measurements, a process which took ca. 3-5 min. We assume, that 141 after this time of dark-acclimation and stabilization, there is no longer a dynamic evolution of 142 gas exchange due to a decrease in residual photosynthesis (compare Smith and Griffiths (1996) 143 and Smith and Griffiths (1998)). Between each measurement, the infrared gas analyzers were 144 standardized using the "match" procedure of the measuring device. With these constraints, the 145 paired measurements were taken at the closest time possible to avoid a shift in the environmental 146 conditions between them. The difference between the paired light and dark measurements was 147 calculated, and will be referred to as gross photosynthesis hereafter. As the underlying soil was not 148 removed from the crusts, the measurements comprise the fluxes from both biocrusts and underlying 149 soil. With this, we follow an ecological approach, taking into account the contribution of the whole 150 soil profile and avoiding any mechanical disturbance to the lichen (e.g. by thallus clipping) that 151 could affect its functioning.

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Paired measurements were taken alternatingly between the samples receiving a high and low 153 frequency watering treatment to minimize the differences in environmental variables between the 154 measurements. In total, three paired measurements were taken daily for every sample with a mean 155 interval of 88 min between measurements. The last measurements were taken with a mean time 156 of 4.5 h after the watering of that day (for detailed information on the mean times since the last 157 watering see Table S1). On the 23 rd of March, only one paired measurement was made for each 158 sample due to snow and heavy wind being registered. Further measurements on that day were 159 cancelled to avoid damage to the measuring device. After daily gas exchange measurements, the 160 sample weights were determined to calculate the volumetric soil moisture. An average of on-site 161 bulk density measurements of 1.03 g cm -3 was used for all calculations.

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The occurrence of dew could potentially influence the photosynthetic activity in our microcosms. 163 Therefore, we estimated dewpoint temperature according to Lawrence (2005). For the calculation, 164 we used the relative air humidity and temperature recorded by the Li-6400 device and compared it 165 to the lichen surface temperature which was recorded by the device as well (see Fig. S3 for relative 166 humidity, dewpoint and lichen surface temperature during the measurements). Relative humidity did 167 not exceed 76% and the dewpoint never was reached. Hence, we assume that dew did not confound 168 the response of the microcosms to the applied watering treatments. Manuscript to be reviewed Data analysis 170 We applied linear mixed effect models (LMEs) to evaluate the effect of the high and low watering 171 frequency treatment and the environmental conditions on dark respiration, net and gross photosyn-172 thesis. We used the R statistical software (R Core Team, 2018) with the "nlme" package (Pinheiro 173 et al., 2018) to conduct these analyses. Due to a high correlation between PAR, air and soil tempera-174 ture (T soil ), we used PAR as a covariate in the models of net and gross photosynthesis, and T soil in 175 the model of respiration. Because of problems with the measuring device, soil temperature data on 176 the first day were only available for the low frequency treatment from 11 to 12 AM. We used the 177 average of these temperatures to fill the day's measurement gaps because soil temperature did not 178 differ substantially between treatments. The watering treatment, PAR or T soil and their interaction 179 were included as fixed factors in the models. We followed a protocol for model selection based on  In this way, we selected a final model that only contained terms significant at the 5% level. The 189 final model was refitted with restricted maximum likelihood estimators. 190 We performed Wilcoxon rank sum tests to compare the soil moisture of the two treatments 191 on day 0, 1, and 2 since the last watering of the low frequency treatment. Because data were not 192 normally distributed, paired Wilcoxon rank sum tests were used to compare CO 2 fluxes of dark 193 respiration, net and gross photosynthesis between the two treatments overall and on the different 194 days since the last watering. The same test was also used to compare soil temperatures between 195 treatments during the measurement times. This data analysis was also performed using R statistical 196 software (R Core Team, 2018).

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Environmental conditions during the experiment 199 The temporal dynamics of the measured environmental variables (PAR, soil and air temperature and 200 soil moisture) were highly variable during the experiment (see Fig. 1 and Fig. 2). PAR values ranged temperature and therefore evaporation were low and thus moisture accumulated in the samples.

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When air temperature exceeded 20 • C, evaporation was high enough for soil moisture to decrease.

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On the days when the samples of both treatments were watered, soil moisture was 13% higher for  Table S2 for details).  During these days, temperature and radiation were higher and the largest flux differences were 229 associated with the largest differences in soil moisture between treatments. 24 ± 8% lower gross photosynthesis (W=17602, p<0.001) (see Fig. 3B).

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The relative difference between treatments changed with the days since the last watering of the 237 low frequency treatment (see Table 1 and Fig. S4). On days when both treatments were watered, 238 no significant flux differences between them were found (see Fig. 4 (A-C), net photosynthesis: 239 W=1032, p=0.391; dark respiration: W=980, p=0.635; gross photosynthesis: W=931, p=0.909).

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In the low frequency treatment, the CO 2 fluxes for dark respiration, net and gross photosynthesis 241 decreased on the days following water application. For dark respiration the difference between 242 the two treatments was significant on the first and second day after watering (W=587, p=0.016).

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Net and gross photosynthesis were more variable among samples, therefore means only differed 244 significantly on the second day following water application (net photosynthesis: W=1034, p<0.001, 245 gross photosynthesis: W=1118, p<0.001).

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The linear mixed model analysis (see Table S3 for results of final model) showed a significant p=0.005 for dark respiration). Therefore, we could not exclude the single effects from the model.

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In contrast, the interaction between these variables was not found to be significant for gross The carbon balance of biocrust communities depends on the patterns of moisture availability, which 259 are likely to be altered by climate change. Therefore, it is important to assess how different biocrust 260 communities respond to an alteration in the rainfall event size and frequency and how regional 261 and seasonal differences can influence this response. In this study, we assessed how two different 262 precipitation frequency patterns providing the same overall amount of water (i.e. 5 mm/day vs.  The asterisks indicate significant differences between the two treatments, calculated with a paired Wilcoxon rank sum test (p<0.05 *, p<0.001 ***, ns = not significant, n=60 for 0 and 1 days since the last watering and n=50 for 2 days since the last watering). Note the significant difference in dark respiration on day 1 since the last watering despite largely overlapping notches, which arises due to the paired nature of the test. An unpaired test did not show significant differences in this case. we conclude that the main driver of the differences is water availability but that soil temperature 282 differences also contributed to the observed patterns. Manuscript to be reviewed

Individual pulse sizes
We did not find differences in the mean photosynthetic and respiratory response between the two

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Applying the larger pulse on previously wetted soil was not beneficial in comparison to a smaller 310 pulse that was already large enough to sufficiently wet the sample to trigger photosynthetic activity.

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Inter-pulse frequency 312 When looking at the simulated rainfall patterns, we found that smaller, more frequent watering 313 pulses were beneficial in terms of net photosynthesis. The observed differences between treatments 314 increased with differences in soil moisture, and were higher during the last days of the experiment The interacting effect of season and temperature with alterations in rainfall patterns on the 328 performance of biocrust constituents is a common observation in many studies. The correlation 329 between rainfall frequency and biocrust growth was found to be positive for winter rain and 330 negative for summer rain areas in a study across southern African sites (Büdel et al., 2009). In the 331 southwestern United States, the carbon balance of S. caninervis in response to rainfall frequency and of different rainfall patterns should also be studied during these phases of high biocrust activity.

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In accordance with other studies, we showed that precipitation frequency plays an important role lichens and mosses, our results indicate that at moderate temperatures, a higher rainfall frequency 364 is beneficial given the same overall water amount over a short period. This clearly shows that the 365 gas exchange response to different rainfall frequencies is modulated by radiation and temperature 366 conditions leading to seasonal differences. We therefore call for detailed cross-site and cross-season Manuscript to be reviewed ACKNOWLEDGMENTS