Fungal loop transfer of N depends on biocrust constituents and N form 2

Department of Plant and Wildlife Sciences, Brigham Young University, Provo, Utah 6 84602, USA Xinjiang Institute of Ecology and Geography, Key Laboratory of Biogeography and 8 Bioresource in Arid Land, Chinese Academy of Sciences, Urumqi 830011, China US Geological Survey, Southwest Biological Science Center, 2290 S. Resource 10 Blvd., Moab, UT 84532, USA 12 Running head: dark septate fungi translocate ammonium in biocrusts 14


Introduction 42
Fungi may act as conduits for biological networks connecting belowground ecosystem processes to plants.Soil fungi contribute to all aspects of litter constituents participating in fungal loops, including plants, are moisture-dependent and regulated by the magnitude and seasonality of episodic rainfall events.A 76 pulse-reserve paradigm (Collins et al. 2008) may explain biological activities where minor rainfall pulses stimulate microorganisms, generating reserves of resources to be 78 exploited during subsequent rainfall events (Huxman et al. 2004, Welter et al. 2005).
In such loops, minor rainfall events may stimulate N 2 fixation by free or 80 lichen-associated cyanobacteria (Belnap et al. 2003), N mineralization by bacteria and fungi (Cable andHuxman 2004, Yahdjian andSala 2010) and nitrification and possibly 82 denitrification (Wang et al. 2014) all increasing the levels of NH 4 + or NO 3 -.Fungal species, including fungal endophytes, may compete with mosses, lichen, 84 cyanobacteria, and other bacteria for newly released N. Once sequestered, the N may be Biogeosciences Discuss., https://doi.org/10.5194/bg-2017-433Manuscript under review for journal Biogeosciences Discussion started: 20 November 2017 c Author(s) 2017.CC BY 4.0 License.transformed into amino acids and transported within mycelium (Jin et al. 2012, Behie 86 and Bidochka 2014).Larger rainfall events may then activate plants, allowing the host to receive N from the fungi and transfer photosynthate to the fungal endophyte.If 88 fungal endophytes are poor competitors for newly released N, preferentially sequester one inorganic N form over another, or more efficiently transform and transport NH 4 + or 90 NO 3 -, biocrust constituents and N form may influence the translocation of N in fungal loops.92 The fungal endophytes most likely involved in the loop hypothesis are dark septate 94 fungi.Few arbuscular mycorrhizal fungi are found in biocrusts (Porras-Alfaro et al. 2011) or as endophytes in desert plants (Titus et al. 2002), due to mycorrhizae being 96 relatively sensitive to dry soil conditions (Aguilera et al. 2016).In contrast, the majority of biocrust fungi are Ascomycota, with the Pleosporales being widespread 98 and dominant (Bates et al. 2012, Porras-Alfaro et al. 2011).Pleosporales, along with other Ascomycota fungal orders, contain dark septate endophytes (Jumpponen and 100 Trappe 1998).Dark septate are thermal-and drought-tolerant fungi due to melanin-rich cell walls conferring protection from UV and drought stress (Gostincar 102 et al. 2010).Taken together, the prevalence of dark septate fungi in desert systems, along with their ability to maintain metabolic activity under low water potentials 104 (Barrow 2003), makes these endophytes excellent candidates to translocate resources in loops (Green et al. 2008).106  We simulated rainfall events containing two isotopically-labeled, inorganic N forms and tracked the movement of the label through our moss-dominated (Castle Valley) 140 and cyanobacteria-dominated biocrusts (USGS Station), and A. hymenoides.First, we randomly selected six circular plots per site with a radius of 1.0 m and at least 10 m 142 apart from each other.Three plots were assigned to be labeled with K 15 NO 3 (99 at.%) and the other three plots to be labeled with ( 15 NH 4 ) 2 SO 4 (99 at.%).Second, we 144 randomly selected five biocrust and five A. hymenoides along eight axes (e.g., N, NE, E, SE, S, SW, W, and NW) radiating from the center of each circular plot and 146 measured the radial distance to biocrusts or grasses.Third, we simulated a 2.5 mm rainfall event by spraying 3 mL of deionized water solution and either isotopic label 148 + and double NO 3 -concentrations to surface soils (Sperry et al. 2006).All additions were completed midday in April as A. hymenoides were 154 starting to set seed.156

Sample collection and 15 N analyses
Biocrust and foliage samples were collected twenty-four hours after the simulated 158 rainfall event containing our different inorganic 15 N forms.Biocrusts were removed as three subsamples from each biocrust location with a soil corer (2 cm diameter × 5 160 cm length) to a depth of 2 mm.Crust distances away from the tracer application ranged from 22-97 cm.The composited soil sample was kept cold (5°C) in the field, 162 split in the lab, and a portion of the soil was frozen (-20°C) until we performed fungal and bacterial DNA analyses.We randomly selected five leaves from Achnatherum, 164 which ranged in distance anywhere from 29-120 cm away from the tracer application and in volume from 0.002-0.048m 3 .The leaves and remaining soils (sieved 2 mm) 166 were air-dried, ground in a reciprocating tissue homogenizer, and analyzed for 15 N using a PDZ Europa ANCA-GSL elemental analyzer, interfaced with a PDZ Europa 168 20-20 isotope ration mass spectrometer (Sercon Ltd., Cheshire, UK) at the University of California Davis Stable Isotope Facility (http://stableisotopefacility.ucdavis.edu).170 We expressed the resulting isotope ratios in δ notation as parts per thousand (‰) where: 172 sample.To track the movement of inorganic N forms through our two biocrust types (moss-lichen-dominated and cyanobacteria dominated biocrust) and into grasses, we 176 analyzed the relationships between δ 15 N present in crust and leaf tissue to the distance of the crust and Achnatherum by site using linear regression in SigmaPlot Version 178 13.0 (Systat Software, San Jose, CA).180

Biomass estimations of major fungal components
To investigate the potential for fungi to translocate our 15 N forms, we estimated the 182 biomass of two major divisions of fungi (Ascomycota and Basidiomycota) and bacteria in biocrusts using quantitative PCR.From the frozen biocrust samples, we 184 extracted genomic DNA using a DNeasy PowerLyzer PowerSoil Kit (Qiagen, MD, USA) and quantified the gene copy numbers of Ascomycota and Basidiomycota on a 186 Mastercycler EP Realplex qPCR (Eppendorf, Hamburg, Germany) with SYBR Green.
We amplified division-specific regions of the internal transcribed spacer (ITS) with 188 primer pair ITS5 (forward) and ITS4A (reverse) for Ascomycota (Larena et al. 1999) and ITS4B (forward) and 5.8sr (reverse) for Basidiomycota (Fierer et al. 2005).We 190 selected the universal bacterial 16S rRNA primer set EUB 338, forward, and Eub518, reverse, to estimate the biomass of bacteria (Aanderud et al. 2013).In 12.5 µl 192 reactions, using KAPA2G Robust PCR Kits (KAPA Biosystems, Wilmington, MA, USA), we amplified targeted genes using the following thermocycler condition: an 194 initial denaturation step at 94ºC for 3 min followed by 35 cycles of denaturation at  2013).The coefficients of determination (R 2 ) for our assays ranged 200 from 0.90 to0.99, and amplification efficiencies fell between 0.99 and 1.92.We analyzed the relationships between biocrust δ 15 N and the gene copy number of 202 Ascomycota and Basidiomycota using linear regression to investigate the potential for fungi to act as conduits for inorganic N. We tested for differences in our biomass 204 estimates between the crust types using multiple t-tests and a Benjamini-Hochberg correction to control for the false discovery rate associated with multiple comparisons 206 (Benjamini and Hochberg 1995).208

Biocrust fungal communities
To identify the fungal taxa participating in N translocation, we characterized fungal 210 communities in biocrusts using bar-coded sequencing.We PCR amplified the V9 region of the 18S rRNA gene using a universal eukaryote primer set, 1391F and 212 EukBr, with a unique 12-bp Golay barcode fused to EukBr (Amaral-Zettler et al. 2009, Hamady et al. 2008).Thermocycler parameters were similar to qPCR analyses and 214 consisted of a denaturation step at 94°C for 3 min, followed by 35 cycles of denaturation at 94°C for 45 s, an annealing step at 57°C for 60 s, elongation at 72°C for 216 90 s, and a final extension at 72°C for 10 min.We then purified and pooled PCR open-source software pipeline suitable for microbial community analysis (Caporaso et al. 2010).We removed barcodes and primers with a custom, in-house script previous to 226 joining paired-end reads by using fastq-join under default parameters (Aronesty 2011).
Joined reads were then de-multiplexed and checked for chimeras (Haas et al. 2011).We 228 then clustered the de-multiplexed reads into OTUs, applying a similarity threshold of 97%, using QIIME's default OTU clustering tool-uclust (Edgar et al. 2011).230 Taxonomies of representative OTUs were assigned using uclust and the 18S rRNA gene SILVA 128 database which was clustered into OTUs at 97% similarity (Quast et 232 al. 2013).To evaluate if biocrust type supported similar fungal composition, we calculated the relative recovery of 27 fungal orders, including dark septate lineages.We 234 tested for differences between biocrust types using t-tests and a Benjamini-Hochberg P=0.002, n=14, figure 1A).Surrounding the tracer application, δ 15 N was enriched upwards of 40‰ more than 20 cm away and continued to be enriched to 244 approximately 10‰ almost 100 cm away.To a lesser extent, 15 NO 3 -followed a similar pattern.δ 15 N decreased as the radial distance from the central application 246 point of 15 NO 3 -in cyanobacteria crusts increased, but the δ 15 N was never more enriched than 8‰ (R 2 =0.17,F=2.6, P<0.0001, n=15, figure 1B).248 In moss-dominated biocrusts, there was no relationship between δ 15 N and the radial distance from either the 15 NH 4 + (R 2 =0.01,F=0.13, P=0.73, n=15, figure 1A) or 250 15 NO 3 -addition (R 2 =0.03,F=0.46, P=0.51, n=15, figure 1B).There was no relationship between δ 15 N found in A. hymenoides leaves and the radial distance from 252 the 15 NH 4 + or 15 NO 3 -application with δ 15 N in leaves ranging from 3-18‰.The R 2 and F values from the regressions between leaves δ 15 N and isotopic distance was 254 0.01-0.21and 0.14-2.6(n=14-23) respectively (data not shown).256

Dark septate fungi as major components of biocrusts
Four of the nine fungal orders contained known dark septate endophyte members and 278 were present in both biocrust types with the Pleosporales and Pezizales being dominant taxa.In biocrusts: fungi comprised much of eukaryotic community 280 (cyanobacteria=30% ±4.7 and moss=33% ±4.0), Ascomycota was the most common fungal division (cyanobacteria=82% ±2.8 and moss=87% ±2.9), and orders with 282 known dark septate members accounted for at least 67% of the Ascomycota Biogeosciences Discuss., https://doi.org/10.5194/bg-2017-433Manuscript under review for journal Biogeosciences Discussion started: 20 November 2017 c Author(s) 2017.CC BY 4.0 License.

4 Discussion
In biological networks, the magnitude and direction of resource transfer in fungi is 298 predominantly thought to be influenced by the physiological source-sink gradients created by individual plants (Fellbaum et al. 2014) or between plants (Weremijewicz 300 et al. 2016).However, fungi may be more than just passive conduits and exert control over resources due to their own sink-source resource needs (Simard and Durall 2004).302 Our finding suggest that a minor rainfall event stimulated fungi, likely dark septate endophytes, to rapidly translocate N at a rate of 40 mm h -1 in the absence of a plant 304 sink for N.In the absence of a large rainfall event to stimulate plant activity, none of as Ascomycota biomass increased.Eighty-three percent of the Ascomycota were from 310 four fungal orders containing known dark septate endophytes and 66% of these taxa were from one order, the Pleosporales.Taken together, our results suggest that fungal 312 loops are structured by fungal constituents, especially Pleosporales, translocating N from NH 4 + over NO 3 -.314

Fungal loops only in cyanobacteria-dominated crusts 316
Although the moss, S. caninervis, appeared to hinder N transfer between biocrusts and plants, our findings suggest that fungal loops do occur in cyanobacteria-dominated 318 biocrusts.Lichen-dominated biocrusts remain to be tested.Our results are consistent with Green et al. (2008) whose previous work identified loops in cyanobacteria 320 biocrusts across the Chihuahuan Desert grassland and showed comparable distances of N movement within biocrusts (Green et al. 2008=44 mm h -1 ).Biocrust components 322 are known to fix and secrete up to 50% of their newly fixed C and 88% newly fixed N to surrounding soils within minutes to days of fixation, depending on precipitation 324 characteristics (Belnap et al. 2003), and thus, would likely be available to other biocrust constituents, such as fungi, for translocation.Bacteria and fungi were found 326 in both crust types, albeit in different amounts and species compositions, but mosses Biogeosciences Discuss., https://doi.org/10.5194/bg-2017-433Manuscript under review for journal Biogeosciences Discussion started: 20 November 2017 c Author(s) 2017.CC BY 4.0 License.mosses and lichens only occurred in one crust type.Mosses, in particular, change the 328 N cycling characteristics of arid lands.When S. caninervis was lost from this system, a dramatic increase in NH 4 + , which ultimately nitrifies to NO 3 -, was observed (Reed et 330 al. 2012).The decomposition of dead mosses most likely contributed to the increase of N; however, after the mosses died, inorganic N pooled in the remaining 332 cyanobacteria-dominated biocrust.Thus, mosses may be effective scavengers for N and outcompete fungal endophytes for newly N. The ability of mosses to 334 scavenge N is well recognized in other systems (Liu et al. 2013, Fritz et al. 2014).
Further, rhizoids, stem cells, and thalli of bryophytes may contain fungal associations 336 (Pressel et al. 2010).If desert mosses have fungal associations, then fungi have the potential to move sequestered N to the mosses in a new kind of loop.Unlike plants 338 that may require a larger rainfall event to become active, fungi and mosses, including S. caninervis, are stimulated by minor rainfall events (Wu et al. 2014) and dark 340 septate endophytes do colonize mosses (Day and Currah 2011).Thus, the exchange of photosynthate and N may occur in a tighter, more localized loop.Another explanation 342 may lie in the microtopography of the two biocrusts.The moss-dominated crust was pinnacled, while the cyanobacteria-dominated crust was smooth.Therefore, transport 344 distance between our application point and target plant was significantly further in mosses than cyanobacteria crusts, potentially slowing the movement of N. We found that NH 4 + , but not NO 3 -, was rapidly translocated within crusts.The enrichment of δ 15 N, from 15 NH 4 + , in cyanobacteria biocrusts was related to the 350 Ascomycota and potentially dark septate fungi due to their dominance.We explain the negative relationship between Ascomycota gene copy number and δ 15 N signal as a 352 simple dilution-the higher the biomass of Ascomycota, the more spread in the 15   (Govindarajulu et al. 2005, Garcia et al. 2016).Quantum dots 362 (fluorescent nanoscale semiconductors) have tracked the flow of organically derived N into arbuscular mycorrhizae and into Poa annua in less than 24 hours (Whiteside et 364 al. 2009) and arbuscular colonization can also increase uptake of multiple other amino acids (e.g., phenylalanine, lysine, asparagine, arginine, histidine, methionine, 366 tryptophan, and cysteine) by their host plants (Whiteside et al. 2012).NO 3 -did move in our cyanobacteria crusts but not nearly to the extent reported by Green et al. 368 (2008).Besides fungal preferences, other factors may play a role in the uptake of N, such as the increase in mobility of NO 3 -in soils, differences in soil cation exchange 370

Dark septate and Pleosporales as conduits
Our results support the idea that Pleosporales are the most likely conduits for N. Four 376 of the nine fungal orders we identified contained known dark septate endophyte members but one order was the most abundant.The Pleosporales accounted for 66% of 378 the Ascomycota taxa in cyanobacteria crusts.Based on the relationship between δ 15 N and Ascomycota biomass, the overwhelming abundance of Pleosporales, and the 380 universal occurrence of Ascomycota in biocrusts, the Pleosporales assumedly a role in fungal loops.We are not the first to reach this conclusion.Green et al. (2008) 382 also identified Pleosporales as being the primary candidate involved in fungal loops.In their semi-arid grassland, Pleosporales were the most common taxa on Bouteloua roots, 384 in the rhizosphere, and in biocrusts.We found 799 operational taxonomic units, based on 97% similarity, with all of the identifiable sequences, belonging to three genera: 386 Leptosphaeria (1.6% of Pleosporales sequences), Morosphaeria (3.8% of Pleosporales sequences), and Ophiosphaerella (8.1% of Pleosporales sequences; data not shown).388 Leptosphaeria and Ophiosphaerella may be pathogenic endophytes on grass species (Martin et al. 2001, Yuan et al. 2017), but may also be beneficial by delaying and 390 reducing the symptoms of other fungal pathogens (Yuan et al. 2017).However, 86% of Biogeosciences Discuss., https://doi.org/10.5194/bg-2017-433Manuscript under review for journal Biogeosciences Discussion started: 20 November 2017 c Author(s) 2017.CC BY 4.0 License.investigate the potential for biocrust constituents and N form to influence the movement of N through the putative fungal loops, we created minor, localized rainfall 110 events and measured δ 15 N, from 15 N-NH 4 + and 15 N-NO 3 -, within surrounding cyanobacteria-and moss-dominated crusts, and grass, Achnatherum hymenoides 112 (Indian ricegrass).In tandem with 15 N analyses, we estimated the biomass of two major division of fungi (Ascomycota and Basidiomycota) and bacteria, and 114 characterized fungal communities by sequencing the 18S rRNA gene to identify potential link between fungal taxa and 15 N movement.We conducted our study in two cold desert ecosystems of the Colorado Plateau, UT. 120 One site was near Castle Valley (40°05'27.43"N-112°18'18.24"W)and the other was adjacent to the US Geological Survey (USGS), Southwest Biological Science Center 122 Research Station in Moab, UT (40°05'27.43"N-112°18'18.24"W).Rugose crusts consisting of moss Syntrichia caninervis and cyano-lichens Collema tenax and 124 Collema coccophorum cover the Castle Valley site (Darrouzet-Nardi et al. 2015), while smooth, light algal crusts of one cyanobacterium, Microcoleus vaginatus, cover 126 the USGS site.Specifically, biocrust cover across the Castle Valley was 50% cyanobacteria, 22% S. caninervis, and 5-7% Collema spp.(Zelikova et al. 2012), and 128 100% cyanobacteria for the USGS.Across both sites, vegetation is dominated by Biogeosciences Discuss., https://doi.org/10.5194/bg-2017-433Manuscript under review for journal Biogeosciences Discussion started: 20 November 2017 c Author(s) 2017.CC BY 4.0 License.perennial grass Achnatherum hymenoides (Roem & Schult) and native perennial 130 shrub Atriplex confertifolia (Torr.& Frém
Biogeosciences Discuss., https://doi.org/10.5194/bg-2017-433Manuscript under review for journal Biogeosciences Discussion started: 20 November 2017 c Author(s) 2017.CC BY 4.0 License.capacity due to clay content, or fungi capitalizing on the more abundant N form specific to a soil.More information is needed to identify the importance of N form 372 and the movement of organic N within fungal loops. 374 Biogeosciences Discuss., https://doi.org/10.5194/bg-2017-433Manuscript under review for journal Biogeosciences Discussion started: 20 November 2017 c Author(s) 2017.CC BY 4.0 License.ourPleosporales taxa were unidentifiable and potentially novel, suggesting that much 392 remains unknown about dark septates in deserts.3945.ConclusionBiocrusts, potentially, are interconnected in extensive biological networks.Dark 396 septate endophytes may act as conduits within the network by acting as both a sink and source for translocating resources.In light of the absence of N movement in 398 moss-dominated crusts, mosses potentially hindered fungal loops.No isotopic label entered A. hymenoides consistent with the fungal loop hypothesis that predicts plant 400 activity only after a larger rainfall event.Our results add to the indirect evidence of fungal loops, but more information is needed to quantify the direct translocation of N 402 through dark septate fungi, characterize the magnitude and directionality of resources within the endophytic relationship, and demonstrate the importance of a second larger 404 rainfall in structuring resource exchange.