The Impact of Moss Species and Biomass on the Growth of Pinus sylvestris Tree Seedlings at Different Precipitation Frequencies

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1 Forests 214, 5, ; doi:1.339/f Article OPEN ACCESS forests ISSN The Impct of Moss Species nd Biomss on the Growth of Pinus sylvestris Tree Seedlings t Different Precipittion Frequencies Bs M. Stuiver *, Dvid A. Wrdle, Michel J. Gundle nd Mrie-Chrlotte Nilsson Deprtment of Forest Ecology nd Mngement, Swedish University of Agriculturl Sciences, SE Umeå, Sweden; E-Mils: Dvid.Wrdle@slu.se (D.A.W.); Michel.Gundle@slu.se (M.J.G.); Mrie-Chrlotte.Nilsson@slu.se (M.-C.N.) * Author to whom correspondence should e ddressed; E-Mil: Bs.Stuiver@slu.se; Tel.: ; Fx: Received: 9 My 214; in revised form: 13 July 214 / Accepted: 21 July 214 / Pulished: 6 August 214 Astrct: Borel forests re chrcterized y n extensive moss lyer, which my hve oth competitive nd fcilittive effects on forest regenertion. We conducted greenhouse experiment to investigte how vrition in moss species nd iomss, in comintion with precipittion frequency, ffect Pinus sylvestris seedling growth. We found tht moss species differed in their effects on seedling growth, nd moss iomss hd negtive effects on seedlings, primrily when it reched mximl levels. When moss iomss ws mximl, seedling iomss decresed, wheres height nd ove- reltive to elow-ground mss incresed, due to competition for light. The effect tht moss iomss hd on seedling performnce differed mong the moss species. Hylocomium splendens nd Polytrichum commune reduced seedling growth the most, likely ecuse of their tller growth form. Seedlings were not dversely ffected y Sphgnum girgensohnii nd Pleurozium schreeri, possily ecuse they were not tll enough to compete for light nd improved soil resource vilility. Reduced precipittion frequency decresed the growth of ll moss species, except P. commune, while it impired the growth of seedlings only when they were grown with P. commune. Our findings suggest tht chnges in moss species nd iomss, which cn e ltered y disturnce or climte chnge, cn influence forest regenertion.

2 Forests 214, Keywords: orel forest; ryophyte; climte chnge; competition; drought; fcilittion; forest regenertion; interctions; moss depth; Scots pine 1. Introduction Successful forest regenertion is crucil for the long-term sustinle mngement of orel forest resources. A significnt proportion of the forest floor in orel forests is covered y mosses [1,2], nd therefore, the rejuvention of the stnd is likely to e influenced y the properties of the moss lyer. Rejuvention of coniferous seedlings ppers prticulrly difficult in forests with n undnt moss presence [3,4], ecuse mosses cn outcompete seedlings for resources, such s light, spce [5,6] nd nutrients [7,8]. Previous work hs lso shown tht these negtive effects of mosses on seedlings cn vry depending on vriety of externl fctors, such s the degree of iotic stress [6,9] nd other site chrcteristics (e.g., the type of growth sustrte eneth the mosses) [5,1]. Mosses my lso positively ffect tree seedlings indirectly y regulting moisture vilility [11,12], reducing temperture fluctutions [6,13], providing nutrients through enhncing litter decomposition rtes [2,14] nd potentilly contriuting to enhnced soil nitrogen levels vi symioses with N 2 -fixing cynocteri [15,16]. Given tht mosses cn hve oth positive nd negtive effects on tree seedlings, there is need for n improved understnding of when these different effects emerge. Even though mosses re widely undnt in orel forests, the moss lyer cn e highly vrile due to differences in moss species composition [17,18]. Moss species hve mny different morphologicl [6,19] nd physiologicl chrcteristics [19,2] tht cn exert differentil effects on seedlings. Moss species tht re dpted to shdy conditions with low evportion rtes generlly hve lrge shoots, which could potentilly llow these species to e more effective light competitors, while lso mking these species more sensitive to desicction [21]. In ddition, moss species vry sustntilly in their wter retention cpcity [21,22], which cn determine their desicction rte nd their recovery following drought [23,24]. Differences in wter retention cpcity mong species my determine how severely the moss lyer is impcted y drought, nd this could, in turn, hve consequences for seedling performnce [25,26]. Further, due to their morphologicl nd physiologicl chrcteristics, moss species differ in their cpcity to intercept nd retin nitrogen [2], s well s in their cpcity to sor phosphte [27]; however, it is unknown whether the impct of moss species on nutrient cycling is eneficil or detrimentl to tree seedlings. Despite the lrge sptil nd temporl vrition in moss species composition tht cn occur in orel forests, reltively few controlled experiments hve explicitly evluted how different moss species impct tree seedling growth. Most studies to dte hve een performed in nturl environments [4,5] or hve used dense trnsplnted moss cushions [2] nd hve, therefore, not specificlly controlled for moss iomss nd environmentl fctors. Borel moss lyers lso differ in moss iomss within the sme species, which is determined y such properties s moss depth nd shoot density. Moss iomss cn y itself, nd independently of moss species identity, lso hve effects on ecologicl processes tht my hve consequences for tree seedling growth nd lloction ptterns [28 3]. For instnce, moss cushions of high iomss re

3 Forests 214, likely to serve s stronger resource competitors with seedlings, therey potentilly diminishing seedling performnce [31,32]. In ddition to potentil negtive effects (i.e., competition), mosses my hve severl positive effects on tree seedlings tht vry with depth nd shoot density. As such, high moss iomss will retin wter etter [11,21,33], which my reduce seedling desicction in dry periods. The moist environment within mosses hs lso een shown to support N 2 -fixtion nd N minerliztion processes [14,15] tht could potentilly increse nutrient vilility for seedlings. Those processes tht cn potentilly stimulte tree seedlings cn e negtively impcted y drought, especilly when moss iomss is impired to level where the moss is less le to retin moisture. Understnding how drought impcts the moss lyer nd, thus, tree seedling regenertion is currently relevnt in the orel region, ecuse it is predicted tht climte wrming will result in longer nd more intense summer drought periods in the future [34 36]. There is therefore need to understnd whether climte chnge might cuse shift in the positive nd negtive impcts of mosses on tree seedling performnce nd the implictions of this for future forest regenertion. We investigted the effects of moss species, moss iomss nd precipittion frequency on Pinus sylvestris seedlings in order to help explin vrition in tree seedling performnce in nturl moss-dominted communities in Swedish orel forests. The four moss species we considered were Pleurozium schreeri, Hylocomium splendens, Polytrichum commune nd Sphgnum girgensohnii, which ll occur commonly in these forests, ut which differ in moisture retention [22,37], ssocitive N 2 -fixtion rtes [15,38], morphology nd growth form [39 41]. This study ws conducted s greenhouse mesocosm experiment tht llowed us to test three hypotheses. First, we hypothesized tht P. sylvestris seedling growth will e differentilly ffected y the four moss species, even when the iomss of the different mosses is the sme, s consequence of differences in the fundmentl chrcteristics tht my impct on seedling performnce. Specificlly, moss species with high wter retining cpcity or ssocitive N 2 -fixtion rte re more likely to hve positive effects on seedling growth due to their ility to increse moisture retention nd soil N vilility. Further, mosses with more detched growth form re expected to e less competitive ginst P. sylvestris seedlings for light nd spce. Second, we hypothesized tht within ech moss species, intermedite moss iomss on per re sis will mximize seedling performnce. This is ecuse t intermedite iomss, there will e eneficil effects of moss in enhncing moisture nd N input reltive to when the moss is sent, ut less competition reltive to when the moss iomss is low. Third, we hypothesized tht the presence of mosses will medite the dverse effects of reduced precipittion frequency on the growth of P. sylvestris seedlings, ut tht this effect of mosses will vry ccording to moss species nd the moss iomss present. Through exploring these three hypotheses, we will e le to etter understnd how the performnce of P. sylvestris seedlings cn e ffected y moss species nd iomss under contrsting moisture regimes nd, therefore, the role tht mosses my ply in mediting tree seedling regenertion under contrsting precipittion regimes in the orel forest.

4 Forests 214, Experimentl Section 2.1. Moss Communities Shoots of P. schreeri, H. splendens, P. commune nd S. girgensohnii were collected from three mixed P. sylvestris nd Pice ies forest stnds tht were t lest 8 yers old t Vksliden ( N, E), Kuläcksliden ( N E) nd Sävr ( N E) in northern Sweden during Septemer, 211. Within these forest stnds, we locted dense monoculture ptches of ech moss species, from which we collected totl of thirty cm sections, to e used in our greenhouse experiment. These sections were returned to the l, nd litter nd non-trget moss species were removed from ech section. We destructively hrvested suset of the sections to compre their dry weights per unit re, which showed tht the verge moss iomss per unit re for ll species ws 618 g dry weight m 2 nd did not differ significntly etween species (ANOVA F 3,13 =.832, p =.54, SuppLementry Informtion) Experimentl Design In order to test our hypotheses, we set-up greenhouse mesocosm experiment consisting of 24 freely-drined lck plstic pots (11 cm 11 cm 11 cm), consisting of 12 moss free control pots, nd the reminder covered with one of the four different moss species (i.e., 48 pots per species). The pots were lined with filter pper nd filled with 3 cm of qurtz snd nd on top of this 5 cm of sieved humus collected t Kuläcksliden. The highest initil moss iomss used in the pots ws 5 g dry weight m 2 for ech of the four species, which corresponds to pproximtely 8% of the iomss mesured in monospecific moss cushions in the field when verged cross ll species (see the Supplementry Informtion). We chose 8% to llow mosses to grow up to their mximum vlue (i.e., the mesured field iomss) during the experiment. We estimted the dry:fresh weight rtio for ech species (see Supplementry Informtion), enling us to clculte the mount of fresh weight needed to e dded to ech pot to provide the desired dry weight. The experiment ws rrnged s full-fctoril split-plot design rrnged in 12 locks. Ech lock contined one control pot with % moss cover nd 16 pots representing ll comintions of the four moss species nd ech of the following four moss iomss clsses (MBC); 8% (i.e., 5 g m 2 ), 4% (25 g m 2 ), 2% (125 g m 2 ) nd 8% (5 g m 2 ) of the reference moss iomss vlue. The moss cushions were reconstructed within ech pot y plcing the necessry numer of shoots, required to chieve the desired iomss levels, in ech pot. Becuse the morphology of ech moss species differed, the shoot density nd height differed etween the mosses for ny given MBC. After pots were constructed in lte Novemer erly Decemer, 211, they were plced in the greenhouse to cclimtize for 12 dys efore newly germinted seeds were plnted. Growing conditions in the greenhouse were set t temperture of 16 ± 2 C, 12 µmol s 1 m 2 rdition nd n 18-hour dy length, s done in previous experiment focused on mosses collected from this region [14]. Reltive humidity ws kept t 7% ± 1%, which is comprle to those vlues tht occur in the field [42], with fine spry system. After 12 dys, ech pot (n = 24) ws plnted with ten newly germinted P. sylvestris seeds, which were crefully plced on top of the humus lyer, mening tht the seedlings strted to grow elow the moss lyer when present. After 6 weeks (i.e., Jnury, 212), the lest

5 Forests 214, vigorous individuls were removed from ech pot, leving the three most vigorous individuls remining. At the sme time, we strted two precipittion frequency tretments, i.e., mient (wtering every second dy) nd reduced (wtering every sixth dy). The tretments were ssigned y rndomly ssigning hlf of the 12 locks to ech of the two precipittion frequency tretments nd were imposed for 12 weeks etween Jnury nd April, 212. Ech precipittion frequency tretment received the sme totl quntity of wter over time (i.e., 726 ml deionized wter per month, equivlent to 6 mm precipittion), so tht the less frequent tretment ws sujected to fewer, hevier precipittion events. The mient frequency nd quntity of wter dded were sed on oservtions etween 27 nd 21 t meteorologicl sttion of the Swedish Meteorologicl nd Hydrologicl Institute (SMHI) in the Arjeplog re; see lso [14]. The reduced precipittion frequency tretment simultes longer periods etween rinfll events nd hevier individul rinfll events tht re expected to occur in the orel forests of northern Sweden s result of climte chnge [34 36]. Eighteen weeks fter initil plnting (i.e., April, 212), ll pots were destructively hrvested, nd the height of ech seedling ws mesured. The shoots nd roots of the seedlings were oven dried t 7 C for 48 hours nd weighed seprtely. Before the destructive hrvest, moss depth ws lso mesured. To ssess moss growth during the experiment, the oven dry weight (7 C, 48 hours) of the moss present in ech pot t the end of the experiment ws lso mesured nd compred to the estimted iomss of the moss dded to the pot t the strt of the experiment N 2 -Fixtion nd Aville Nutrients In ech of the 12 locks, we lso set up n dditionl five pots, one for ech of the four moss species, which hd 8% MBC nd one pot tht hd no mosses (% MBC), resulting in totl of 6 pots. As descried for the pots ove, six of the 12 locks received the mient precipittion frequency nd the other six the reduced precipittion frequency. These pots were not plnted with seedlings nd were insted used to study iologicl N 2 -fixtion rtes nd soil nutrient fluxes to ssess possile feedcks of the moss species on the nutrient sttus of the pots. At the end of the experiment (i.e., fter 18 weeks), we nlyzed the cynocteril N 2 -fixtion for the moss in ech of these pots using the cetylene reduction method, s descried in detil in previous studies focused on N 2 -fixtion in mosses [1,14]. This involved rndom smpling of 3 shoots for P. schreeri or P. commune or 15 shoots for H. splendens or S. girgensohnii (which hve lrger shoots) from ech pot. The smples from ech pot were divided into three seprte 22-mL glss vils nd were fully hydrted with 3 ml deionized wter, fter which they were injected with 1% hedspce of cetylene. The smples were incuted in greenhouse conditions for 24 hours, fter which the smples were nlyzed for ethylene concentrtions on gs chromtogrph using ethylene stndrds (Clrus 5 GC, PerkinElmer Inc. Wlthm, Msschusetts). These concentrtions were trnsformed to ethylene mss (µmol g 1 dy 1 ) using the universl gs lw. We used the 3 mol cetylene mol N 1 rtio s descried in DeLuc et l. [43] to convert the mesured ethylene ppm to N 2 -fixtion rte. After N 2 -fixtion mesurements were mde, shoots were dried t 7 C for 48 hours nd weighed, so tht N 2 -fixtion rtes could e reported on per grm of moss sis. We used the sme pots s for determining N 2 -fixtion rtes (i.e., without seedlings) to ssess soil nutrient vilility. This ws done y plcing resin cpsule (PST1 cpsule, Uniest Bozemn,

6 Forests 214, Bozemn, MT, USA) contining pproximtely 1 g of mixed ed ionic resins in the humus lyer of ech pot t the eginning of the experiment [44]. These cpsules were removed fter 18 weeks nd lter extrcted with three seprte extrctions of 1 ml 1 M KCl, nd run for NH 4 +, NO 3 nd PO 4 3 on n Autonlyzer 3 (SEAL Anlyticl) Sttisticl Anlysis We nlyzed most of the response vriles (i.e., seedling height, shoot, root nd totl seedling iomss, shoot-to-root rtio nd moss growth) using three-wy ANOVAs, with moss species, MBC nd precipittion frequency serving s fixed fctors, nd we included rndom lock fctor. In order to meet the ssumptions of normlity nd homogeneity of vrinces, seedling shoot, root nd totl iomss were squre root trnsformed, wheres the shoot-to-root rtio ws log trnsformed. The moss growth dt ws trnsformed with squre root +1 trnsformtion. We compred the effects of moss species nd precipittion frequency on N 2 -fixtion rtes nd soil nutrients using two-wy ANOVAs, including rndom lock fctor. When significnt differences were detected, we performed Tukey HSD post hoc tests. All sttisticl tests were performed in R v from R Core Tem (212), except t-tests, which were performed in IBM SPSS Sttistics 19, nd the figures were compiled with GrphPd Prism Results nd Discussion 3.1. Effect of Moss Species We found tht seedling height, shoot, root nd totl iomss, s well s the shoot-to-root rtio differed mong the moss species present in the pots (Tle 1). The presence of three of the four moss species (i.e., P. schreeri, H. splendens nd S. girgensohnii) significntly incresed the height growth of the seedlings (Figure 1). Seedling iomss ws most negtively ffected y H. splendens nd most positively y S. girgensohnii. However, the iomss of seedlings in pots contining ny of the four moss species did not differ from tht of seedlings grown in the moss-free control pots. Specificlly, seedlings growing in S. girgensohnii chieved significntly more shoot iomss thn they did in either H. splendens or P. commune (Figure 1). The root iomss of the seedlings in the S. girgensohnii pots ws lso significntly lrger thn the root iomss of the seedlings grown in the other moss covered pots (Tle 1, Figure 1c). The root iomss of the seedlings in pots with P. schreeri nd P. commune ws, in turn, significntly lrger thn those grown with H. splendens (Figure 1c). Totl seedling iomss ws highest when seedlings were grown in pots with P. schreeri nd S. girgensohnii nd lowest in pots covered with H. splendens nd P. commune (Figure 1d). The shoot-to-root rtio of seedlings in H. splendens pots ws lmost sttisticlly significntly lrger thn for seedlings in S. girgensohnii pots (p =.62, Figure 1e).

7 Forests 214, Tle 1. The effect of four moss species, moss iomss clss nd precipittion frequency nd their interction effects on Pinus sylvestris seedling nd moss growth; vlues represent the results (F-vlue) of three-wy ANOVA with replictes orgnized into locks (serving s rndom fctor) nd significnt levels (p-vlues) in prentheses; significnt effects re shown in old (p <.5). Response vrile Tree Seedling Height Shoot iomss Root iomss Totl iomss Shoot:root rtio Moss species (S) (<.1) (<.1) (<.1) (<.1) (.48) Moss iomss clss (MBC) (<.1) (.1) (<.1) (<.1) (<.1) Precipittion frequency (W) (.253).12 (.75) 1.38 (.99).491 (.485).122 (.728) S MBC 1.87 (.71) (.51) (.23) (.22).477 (.888) S W.93 (.448) (.114) 2.52 (.43) (.57).516 (.724) MBC W.94 (.423) (.99) 1.16 (.387) (.178).81 (.49) Moss growth (<.1) (<.1) (<.1) (.1) (.6) 1.88 (.148).541 S MBC W 1.17 (.429).765 (.649).783 (.632).798 (.619).66 (.744) (.843) Degrees of freedom (Df) for tree seedling nd moss growth (within prenthesis) re s follows. S: Df = 4(3); MBC: Df = 4(3); W: Df = 1(1); S MBC: Df = 16(9); S W: Df = 4(3); MBC W: Df = 4(3); S MBC W: Df = 16(9); residuls: Df = 149(155); 1 The moss iomss clss includes % (control), 8%, 2%, 4% nd 8% of reference iomss vlues of ech species. Figure 1. Response of Pinus sylvestris seedlings grown in pots contining different moss species, verged cross precipittion regimes nd ll moss iomss clsses (MBC), including moss-free control; rs show the mens + SE. Within ech supnel, different letters ove rs indicte significnt pirwise differences t p <.5 (Tukey s HSD); ANOVA results re presented in Tle 1. () 8 Seedlind height (mm) c c () Shoot iomss (mg) (c) Root iomss( m g ) c c (d) Totl iomss (mg) (e) Shoot : Root Control P. schreeri H. splendens P. commune S. girgensohnii Control P. schreeri H. splendens P. commune S. girgensohnii. Control P. schreeri H. splendens P. commune S. girgensohnii

8 Forests 214, Effect of Moss Biomss We found tht moss iomss clss (MBC) when verged cross ll moss species hd significnt impct on seedling growth (Tle 1). As such, seedlings growing in the 4% nd 8% MBC pots were significntly tller thn seedlings in the control nd 8% MBC pots (Figure 2). On verge, seedlings were tller thn the verge moss depth in the 8%, 2% nd 4% MBC, ut shorter thn moss depth in the 8% MBC (Figure S1). Conversely, seedling iomss ws negtively ffected y the 8% MBC compred to the other MBC, ut did not differ significntly from seedling iomss in the moss-free control pots. Specificlly, the shoot iomss of the seedlings ws significntly lower in the 8% MBC compred to the 2% MBC pots (Figure 2). The root nd totl iomss of the seedlings grown t 8% MBC were significntly lower compred to the root nd totl iomss of the seedlings in the 8%, 2% nd 4% MBC pots, ut not compred to the control (Figure 2c,d). The shoot-to-root rtio ws significntly higher in the 8% MBC pots reltive to ll other MBC tretments, ut not reltive to the control (Figure 2e). Figure 2. Response of Pinus sylvestris seedlings grown in pots contining different moss species of different moss iomss clss (MBC), including moss-free control, verged cross precipittion regimes nd ll moss species; rs show mens + SE; within ech supnel, different letters ove rs indicte significnt pirwise differences t p <.5 (Tukey s HSD); ANOVA results re presented in Tle 1. () 8 Seedling height (mm) c c c () 6 Shoot iomss (mg) 4 2 (c) 6 Root iomss (mg) (d) 12 Totl iomss (mg) 8 4 (e) Shoot Root MBC (%) MBC (%) MBC (%) 3.3. Effects of the Interction etween Moss Species nd Moss Biomss A significnt effect of the interction etween moss species nd MBC ws found for seedling root nd totl iomss (Tle 1). These interctions showed tht the effect of incresing moss iomss (i.e., MBC) on seedling growth ws not equl for ll moss species. For oth root nd totl seedling iomss, the significnt effect of the interction etween moss species nd MBC ws due to strong negtive effect of H. splendens in the 8% MBC pots (Figure 3). There ws lso significnt effect of MBC on oth vriles in S. girgensohnii pots ccording to ANOVA (Tle 2), ut the post hoc comprison (Tukey s HSD) did not show difference etween moss iomss clsses for this species (Figure 3). There ws no effect of MBC on root or totl seedling iomss in the P. schreeri nd P. commune pots

9 Forests 214, (Tle 2, Figure 3). Mesurements of moss depth in the 8% MBC pots were significntly higher for H. splendens thn for P. schreeri nd S. girgensohnii (Figure S1). Figure 3. Response of Pinus sylvestris () seedling root iomss nd () totl seedling iomss cross moss species nd ll moss iomss clsses (MBC); rs show mens + SE; significnt differences re shown y different letters t p <.5 (Tukey s HSD); ANOVA results re presented in Tles 1 nd 2. () 8 Root iomss (mg) c Moss iomss clss % 8% 2% 4% 8% () 15 Totl iomss (mg) 1 5 Control P. schreeri H.splendens P. commune S.girgensohnii Tle 2. The effect of moss iomss clss (MBC) nd precipittion frequency (W) on Pinus sylvestris seedling nd moss growth for ech of four moss species; there were no significnt interction effects etween MBC nd precipittion frequency for ny of the species nd response vriles (results not presented); the vlues show the results (F-vlue) nd significnt levels (p-vlues) in prentheses of two-wy ANOVA with replictes orgnized into locks (serving s rndom fctor); significnt effects re shown in old (p <.5). Tree Seedling Moss species Height P. schreeri MBC (.1) W.459 (.52) Shoot iomss 1.5 (.417).72 (.789) Root iomss (.63).81 (.777) Totl iomss (.182).99 (.754) Shoot:root rtio (.215).89 (.767) Moss growth (<.1) 6.81 (.2)

10 Forests 214, Moss species Height H. splendens MBC (<.1) W 1.71 (.37) P. commune MBC (.32) W.877 (.355) S. girgensohnii MBC (<.1) Tle 2. Cont. Shoot iomss (<.1).188 (.667).65 (.662) (.33) (.95) Tree Seedling Root iomss (<.1).82 (.776) (.94) (.8) 3.85 (.26) Totl iomss (<.1).166 (.686) (.349) (.14) (.41) Shoot:root rtio (.67).44 (.528) (.24).12 (.731) (.8) Moss growth (<.1) (.1) 3.16 (.45).22 (.656) (<.1) W (.231) (.18) (.83) (.69) (.938) (.3) Degrees of freedom (Df) for tree seedling nd moss growth (within prenthesis) re s follows; MBC: Df = 4(3); W: Df = 1(1); residuls: Df = 45(35); 1 Moss iomss clss includes % (control), 8%, 2%, 4% nd 8% of reference iomss vlues of ech species Precipittion Effect We did not find ny min effect of the precipittion frequency on seedling iomss (Tle 1); however, there ws significnt nd mrginlly non-significnt interctive effect t p =.5 of precipittion tretment nd species on seedling root iomss nd totl iomss, respectively (Tle 1). These interctive effects were minly driven y significntly higher shoot, root nd totl seedling iomss in the mient precipittion frequency compred to the reduced precipittion frequency for P. commune, ut not for the other species (Tle 2). Specificlly, when verged cross ll MBC tretments for P. commune, the shoot, root nd totl seedling iomss (men ± SE) ws 49.1 mg ± 3.9, 51.9 mg ± 3. nd 11. mg ± 6.8, respectively, under the mient precipittion frequency nd 4.8 mg ± 2.1, 44.3 mg ± 2.8 nd 85.1 mg ± 4. t the reduced precipittion frequency. When testing specificlly for the precipittion effect on seedling growth in the moss-free control pots, we did not find significnt differences in seedling performnce etween precipittion frequencies (t-test vlues (p-vlue) were: seedling shoot iomss:.553 (.592); root iomss:.459 (.656); totl iomss:.11 (.915); shoot:root rtio: (.114); nd seedling height:.293 (.776)) Moss Growth Over the course of the experiment, the growth of the mosses differed cross species with S. girgensohnii showing the lrgest moss iomss growth nd P. commune the lowest (Tle 1, Figure 4). The lrgest moss iomss growth per pot ws chieved in the pots with the highest MBC (Tles 1 nd 2, Figure 4). This pttern ws consistent for ll moss species, even though the mgnitude differed etween species (i.e., no significnt differences within the P. commune pots), which ws reflected in the interctive effect of moss species nd MBC (Tle 1, Figure 4). Across ll

11 Forests 214, pots, the moss iomss incresed y 43% during the 18 weeks of the experiment. However, the reltive moss iomss growth ws the lrgest in the pots with the lowest MBC present for which the moss iomss on verge incresed y 78% (dt not shown). During the experiment, the moss iomss in the 8% MBC tretment for P. schreeri nd H. splendens incresed y 1.4 ±.2 nd 1.6 ±.3 g dry weight pot 1 (men ± SE), respectively. This mens tht over this time, the iomss for these two species ecme similr to the 1% reference iomss vlue, s moss growth of 1.5 g dry weight pot 1 would e needed for reference iomss vlues to e chieved (Figure 4). Menwhile, for S. girgensohnii, growth in the 8% MBC tretment ws 2.8 ±.3 g dry weight pot 1, mening tht the finl iomss gretly exceeded the 1% reference iomss vlue. There ws significnt overll effect of precipittion frequency on moss growth (Tle 1), with mosses growing etter under mient thn reduced frequency. When species were considered seprtely, this effect ws sttisticlly significnt for ll moss species, except P. commune (Tle 2, Figure 4). Moss depth showed n overll negtive response to reduced precipittion frequency (ANOVA F = 4.962, p =.27), ut no significnt effects on precipittion frequency within ech species were found (Tukey s HSD, dt not shown). Figure 4. Moss iomss growth (dry weight) per pot of ech of the four moss species during the course of the experiment: () verged cross precipittion regimes nd expressed for ech initil moss iomss clss (MBC); nd () verged cross ll moss iomss clsses (MBC) nd expressed in reltion to precipittion frequency (mient nd reduced); ll moss species hd the sme strting dry weight for ech iomss clss; different cpitl letters show significnt differences t p <.5 (Tukey s HSD) mong moss species (), nd different lower cse letters show significnt differences t p <.5 (Tukey s HSD) mong moss iomss clsses () nd precipittion frequency tretments (); rs show mens + SE. ANOVA results re presented in Tles 1 nd 2. () Moss growth (g pot -1 ) hij B defg fghi cde B c cdef efg ghi j C ij A cd efgh ij ghij Moss iomss clss 8% 2% 4% 8% () Moss growth (g pot -1 ) 2 1 Precipittion frequency mient reduced P. schreeri H. splendens P. commune S. girgensohnii

12 Forests 214, Effect of Moss Species on N 2 -Fixtion nd Nutrient Avilility The mount of N 2 fixed y cynocteri tht live in symiosis with mosses differed etween moss species (Tle 3); the highest N 2 -fixtion rtes occurred in the Pleurozium schreeri nd H. splendens pots, while in the P. commune pots, no N 2 -fixtion took plce (Tle 3, Figure 5). Additionlly, the moss species differed significntly in their effects on soil NO 3 -N nd PO 4 3 -P, ut not NH 4 + -N mesured in the resin cpsules (Tle 3). The presence of three of the moss species (i.e., P. schreeri, P. commune nd S. girgensohnii) hd positive effect on the mount of resin sored NO 3 -N reltive to the moss free control (Figure 5). Further, significntly lower mounts of resin sored PO 4 3 -P were found in pots with P. commune nd S. girgensohnii present compred to pots with P. schreeri (Figure 5c), ut not compred to the control. There were no effects of precipittion frequency, or interctive effects of frequency nd species, on N 2 fixtion or on sorption of ny of the nutrients y resin cpsules (Tle 3). Tle 3. The effect of moss species, precipittion frequency nd the moss species precipittion frequency interction on N 2 fixtion rtes nd sored soil nutrients (NH 4 + -N, NO 3 -N nd PO 4 3 -P) y resin cpsules; the vlues show the results (F-vlue) nd significnt levels (p-vlues) in prentheses of two-wy ANOVA with the lock s rndom fctor; significnt effects re shown in old (p <.5). Response Vrile N 2 Fixtion NH + 4 -N NO 3 -N PO 3 4 -P Moss species (S) (<.1) 1.97 (.371) 6.34 (<.1) (.1) Precipittion frequency (W).772 (.387).368 (.548).68 (.795).138 (.711) S W 1.93 (.151).312 (.868).348 (.844).213 (.93) N 2 fixtion: S: Df = 3; W: Df =1; S W: Df = 3; residuls Df = 35; NH + 4 -N, NO 3 -N, PO 3 4 -P: S: Df = 4; W: Df = 1; S W: Df = 4; residuls Df = 45. Figure 5. Biogeochemicl properties ssocited with the four moss species t the highest moss iomss clss nd moss-free control: () iologicl N 2 -fixtion; () resin sored soil NO 3 -N; nd (c) resin sored soil PO 4 3 -P. Brs show mens + SE; mens within ech pnel topped with different letters show significnt differences etween the moss species (Tukey s HSD, p <.5); ANOVA results re presented in Tle 3. () N2 fixed (ug g mos s -1 d -1 ) P.schreeri c H. splendens P. commune S. girgensohnii ().5 NO3 - -N (mg c psu le -1 ) Control P. schreeri H. splendend P. commune S. girgensohniii (c) PO4 3- -P(mgcpsule -1 ) Control P. schreeri H. splendens P. commune S. girgensohnii

13 Forests 214, Discussion Even though reserch on how mosses influence vsculr plnts hs ttrcted incresing ttention during the lst decde, much is still unknown of the driving processes ehind these effects [2]. The im of this study ws to improve our understnding of the influence tht mosses my hve on the growth of P. sylvestris seedlings nd to determine how this influence vries mong different moss species, levels of moss iomss nd precipittion frequencies. We found tht P. sylvestris seedling growth could e either reduced or promoted depending on the moss species nd the iomss of the moss lyer in which the seedlings grew. Even though moss growth ws decresed y reduced precipittion frequency for three of the four moss species, our study provided little evidence tht this, in turn, modifies seedling growth. In greement with our first hypothesis, we found tht moss species differed in their effects on P. sylvestris seedling growth. Three of the four moss species cused seedlings to grow significntly tller compred to when no mosses were present. These results re consistent with previous study on other species, where Pice gluc seedlings grew tller with Polytrichum spp. compred to when the moss lyer ws sent [3]. These findings suggest tht the seedlings experience limittion in light vilility induced y the moss lyer, wherey seedlings increse their height growth to void light competition [45,46]. When totl seedling iomss ws considered, we found the lowest iomss when seedlings were grown with H. splendens nd P. commune nd highest when grown with S. girgensohnii nd P. schreeri, even when moss iomss ws kept constnt cross species. This mens tht the differences cross moss species in their effects were due to differences in moss species ttriutes tht re independent of moss iomss. The morphology of oth H. splendens nd P. commune differs from tht of the other two species in tht they hve stronger support system nd tller growth form [5,39]. This tller growth form would hve cused lower light vilility, resulting in lower iomss production of the seedlings. The negtive effect of H. splendens nd P. commune reltive to the other two species could not e explined in terms of the modifiction of soil nutrient vilility for the seedlings, ecuse ville levels of soil nutrients were never impired y the presence of mosses. However, unlike for the other three species, tmospheric N 2 -fixtion ws sent in the P. commune pots, nd this species lso hs the lowest wter retention cpcity [21,22]; oth of these fctors could hve contriuted to its negtive effect on seedling growth. In contrst, the positive effect of S. girgensohnii nd P. schreeri on seedling iomss reltive to the other two species my hve een due to comintion of fctors. For S. girgensohnii, its high wter retention cpcity provided y its hyline cell structure my hve incresed the ccessiility of moisture or nutrients to seedlings [21,47]. Likewise, oth of these species enhnced soil NO 3 -N vilility nd supported N 2 -fixtion nd were less likely to cuse light limittion of the seedlings ecuse of their shorter stture. The contrsting effect of the moss species used in this study is prtly in greement with previous reserch, which showed tht Sphgnum spp. enhnced the performnce of P. ies seedlings, while H. splendens impired them [5]. However, most studies hve found tht P. schreeri ffected seedling emergence negtively [4,2], which contrdicts our findings. Contrry to the predictions of our second hypothesis, we found no evidence of enhnced seedling performnce in the intermedite moss iomss clsses; insted, we found tht seedling iomss ws reduced y the highest iomss level, ut did not differ significntly mong the other levels. Our

14 Forests 214, results lso showed tht seedling height nd shoot-to-root rtios were highest in the highest moss iomss clss. This suggests tht moss cushions with high field iomss cuse seedlings to grow tller slender shoots tht contin less iomss nd llocte more C oveground thn elowground. Our finding is in greement with previous work showing tht the presence of mosses cn increse seedling oveground iomss lloction nd height nd decrese stem dimeter [29,3]; our results extend those findings y showing tht these types of chnges occur primrily when moss iomss is t or ner the mximum iomss likely to e found in nturl or field conditions. The fct tht seedlings llocte more resources to shoots reltive to roots is most likely driven y shding y the mosses nd is imed t enling seedlings to void light competition [45]. This effect ws gretest in the highest moss iomss clss, ecuse oth moss depth nd shoot density were highest in this tretment nd ecuse this tretment showed the gretest increse in moss iomss during the experiment, which should mintin these competitive effects through time. In ddition to the decline in seedling growth in the highest MBC cross ll species, the dt lso showed severl species-specific responses to the MBC tretments, s reveled y n effect of the interction etween moss species nd MBC on root nd totl iomss. This interction effect ws lrgely driven y the strong negtive response of seedling root nd totl iomss to the highest moss iomss (8% MBC) tretment for H. splendens, ut not for the other three moss species. The prticulrly negtive effect of H. splendens in the highest MBC ws explined y the greter depth of the moss lyer for this species nd the size of its mture shoot segments, which hve een shown to increse in size when density is high [48]. The horizontl growth form of H. splendens leves on nnul segments likely further contriutes to effectiveness in intercepting light [21,39]. Even though the moss lyer should cuse decrese in light vilility, seedlings re often le to use resources from their seeds for initil growth [49,5], which llows them to grow ove the moss lyer to mximize photosynthesis. Our study showed tht the criticl moss depth t which mosses competitively suppress P. sylvestris seedlings is pproximtely 7 mm. However, it is importnt to recognize tht under nturl conditions, mosses increse their oveground iomss until lte summer or erly utumn, while seedling shoot growth stops erlier in the summer [5,51], mening tht mosses re le to overgrow the seedlings when they re inctive. This is lso supported y findings tht the mortlity of seedlings is minly due to smothering [5,52]. Consequently, our results suggest tht moss depth rther thn moss iomss is the most importnt ttriute in explining seedling performnce. This is supported y severl studies where thinner moss lyers hve een shown to e ssocited with improved seedling estlishment reltive to thicker moss lyers [53,54]. We found limited support for our third hypothesis tht seedlings would e less sensitive to reductions in precipittion frequency when grown in pots with mosses compred to without. Further, we found no evidence tht reduced precipittion frequency impcted seedlings more when less moss iomss ws present. We expected tht higher moss iomss would provide more fvorle moisture conditions to the seedlings, s suggested y previous studies [48,55]; however, our dt showed tht seedling growth ws not limited y the moisture (i.e., precipittion frequency) tretments in our experiment. Although seedlings were unffected y precipittion frequency, moss growth of every species, except P. commune, ws negtively impcted y the reduced precipittion frequency, showing tht this tretment ws effective t reducing moisture vilility in the moss lyer. This reduced growth ws expected, given tht most mosses re poikilohydric nd tht their wter content should

15 Forests 214, therefore e strongly influenced y the surrounding environment [24,39]. The lck of response of P. commune to precipittion frequency could e ecuse it ws less wter limited thn the other species. Of the four species, this is the species with the lowest wter retention cpcity [21,22], ut it is lso the only one with n underground rhizome through which it cn trnsport wter nd nutrients up from the soil into its wter conducting tissue inside the stem [27,39]. These specific chrcteristics could e the reson tht we found significnt effect of the precipittion tretment when seedlings were grown with P. commune. It is possile tht the seedlings hd to compete directly with P. commune, ut not with the other three species, for soil moisture. Even though the experiment ws conducted in greenhouse environment, the moss iomss incresed in ll MBC ctegories, indicting tht the mosses remined helthy during the experiment nd cn therefore e considered s comprle to moss cushions in nturl forest conditions. Although P. commune showed visul signs of desicction stress, which is proly due to the impct of trnsplnttion, it lso incresed in iomss nd grew new shoots. In nturl conditions, the negtive effect of trnsplnttions would e sent nd might led to P. commune hving stronger impct on seedling growth thn our dt indicted. Our dt suggests tht seedlings in stnds with undnt P. commune cover my e prticulrly impcted y extended periods without rinfll, which is predicted to ecome more common in the orel region s result of glol climte chnge [34]. Our results showed tht moss species hd vrying effects on seedling growth reltive to the moss-free control, in contrst to mny studies tht hve primrily reported negtive impcts of mosses [4,5,32]. However, there re lso studies which hve shown tht some site conditions which exert negtive effects on seed germintion re lso those tht re the most suitle for seedling estlishment [2,56]. Since our study used seedlings from lredy germinted seeds, it remins uncler whether the fctors we focused on would hve the sme impct on the germintion process, nd this would require further exmintion. Previous studies hve lso shown other mechnism y which mosses nd vsculr plnts interct. For exmple, under nturl conditions in orel nd rctic regions, the moss lyer influences soil temperture fluctutions [13,54], which cn, in turn, impct soil iot nd nutrient minerliztion rtes [26]. These effects hve potentil consequences for seedling estlishment [13], s well s for the undnce of other vsculr plnts tht my serve s competitors for estlishing tree seedlings [32,53]. As such, there re likely to e multiple wys through which mosses nd their interction with the environment my impct on estlishing tree seedlings nd potentilly ffect the success rtes of forest regenertion, in ddition to the pthwys ddressed in our study. 4. Conclusions The effect of mosses on P. sylvestris seedlings is dependent on which moss species nd in wht moss iomss they grow. These effects my e negtive s consequence of competition y mosses for light nd positive s consequence of severl fctors, including the ility of mosses to supply seedlings with resources nd to modify their iotic environment. Our dt showed tht seedlings responded to vrition in moss iomss (nd prticulrly moss depth), through incresed height nd lloction of resources oveground reltive to elowground. The competitive effects of mosses were gretest when mosses were t their mximum iomss, ut this effect vried mong moss species nd

16 Forests 214, ws strongest for seedlings surrounded y H. splendens, ecuse this species hd greter depth of the moss lyer thn the other species t this iomss. Our findings re relevnt for understnding forest regenertion in orel forests, since it is known tht chnges in oth moss species composition nd iomss occur during forest succession fter oth nturl nd nthropogenic disturnces [18,57]. The vlues we used in the 8% moss iomss clsses (5 g/m 2 ) were comprle to moss iomss vlues of P. schreeri nd H. splendens mesured in 14 y-old uncut forest stnds in Estern Finlnd, while moss iomss during the first seven yers fter cler-cutting ws out 5% of this vlue [18]. It is lso known tht mosses cn e strongly ffected y glol chnge fctors, such s tmospheric N deposition [44,58], climtic wrming nd chnges in precipittion regimes [2,26]. As we hve shown in this study, chnges in precipittion cn drsticlly ffect moss growth nd the mount of moss iomss present in the forest. Consequently, our findings regrding the response of seedlings to vriety of moss conditions hve importnt implictions for understnding future forest regenertion in orel forests under chnging glol environment. Supplementry Informtion Prior to experimentl set-up, we first estlished reference vlues to chieve relistic weight estimte to represent the iomss per unit re of nturl dense monoculture moss cushions. From these reference vlues, we determined the clsses of the initil moss iomss to e used in the pots. These reference smples were lso used to estlish the reltionship etween the fresh weight (sturted conditions) nd dry weight of individul moss cushions (Tle S1). The mosses were therefore clened from litter nd ny other moss species present, sturted y sumerging in wter for t lest 3 minutes, spun with 2 pulses for pproximtely one minute to relese externl wter ccording to Fenton et l. [59], weighed (to otin the fresh weight) nd then oven dried (7 C, 48 hours) nd weighed gin to get the oven-dry weight. Tle S1. Nturl stnding iomss (men ± SE) of monospecific moss cushions collected from the field nd the dry: fresh weight rtio of these cushions for ech of the four moss species used in the experiment; the nturl stnding iomss ws used to determine the highest moss iomss clss to e used for ech species when the experiment ws set up, which ws set t 5 g m 2 equl, which is pproximtely 8% of the nturl stnding iomss verged cross ll species (see the Methods section); the dry:fresh weight rtios were used during the experimentl set-up to determine the mount of moss wet weight tht corresponded to given dry weight. Species Nturl Monoculture Moss Cushion Biomss Cushion Dry/ (DW g m 2 ) Fresh Weight Rtio Pleurozium schreeri ± ±.4 Hylocomium splendens ± ±.1 Polytrichum commune ± ±.13 Sphgnum girgensohnii ± ±.1 Men 618 8% of men weight n = 4; 2 n = 3.

17 Forests 214, Figure S1. () Pinus sylvestris seedling height (white rs) nd moss depth (lck rs) t the end of the experiment for ech moss iomss clss (MBC) cross ll moss species nd () for ech moss species t the 8% MBC; for oth pnels, different lower cse letters indicte significnt differences in seedling height t p <.5 nd different cpitl letters indicte significnt differences in moss depth t p <.5 ccording to Tukey s HSD test; ANOVA results for seedling height cross MBC re given in Tle 1; nd for moss species t 8% MBC F 3.39 =.68 (p =.6); for moss depth, cross MBC F = (p <.1); nd for moss species t 8% MBC F 3.39 = (p <.1); rs show mens + SE; the verticl xis scle refers to oth seedling height nd moss depth. () 8 Height (mm) c c D c C B A () 1 Height (mm) BC A AB C Seedling height Moss depth MBC (%) P. schreeri H. splendens P. commune S. girgensohnii Acknowledgments The reserch ws funded y TC4F (Trees nd Crops for the Future) grnt. We would like to thnk Kelley Gundle nd Mrgret Söderström for lortory ssistnce; nd Agnes Väppling, Mj Sndström nd Dvid Rehmerg for ssisting in the preprtions nd hrvesting of the experiment. Author Contriutions All uthors contriuted to the experimentl design, dt interprettion nd writing of this rticle. Bs Stuiver executed the experiment nd lso performed the sttisticl nlysis. Conflicts of Interest The uthors declre no conflict of interest. References nd Notes 1. Gundle, M.J.; Gustfsson, H.; Nilsson, M.-C. The sensitivity of nitrogen fixtion y fethermoss-cynocteri ssocition to litter nd moisture vriility in young nd old orel forests. Cn. J. For. Res. 29, 39, Turetsky, M.R.; Bond-Lmerty, B.; Euskirchen, E.; Tlot, J.; Frolking, S.; McGuire, A.D.; Tuittil, E.S. The resilience nd functionl role of moss in orel nd rctic ecosystems. New Phytol. 212, 196,

18 Forests 214, Hyppönen, M.; Hllikinen, V.; Niemelä, J.; Rutio, P. The contrdictory role of understory vegettion on the success of Scots pine regenertion. Silv Fenn. 213, 47, doi:org/ /sf Steijlen, I.; Nilsson, M.-C.; Zckrisson, O. Seed regenertion of Scots pine in orel forest stnds dominted y lichen nd fether moss. Cn. J. For. Res. 1995, 25, Hörnerg, G.; Ohlson, M.; Zckrisson, O. Influence of ryophytes nd microrelief conditions on Pice ies seed regenertion ptterns in orel old-growth swmp forests. Cn. J. For. Res. 1997, 27, Wheeler, J.A.; Hermnutz, L.; Mrino, P.M. Fethermoss seededs fcilitte lck spruce seedling recruitment in the forest tundr ecotone (Lrdor, Cnd). Oikos 211, 12, Zckrisson, O.; Dhlerg, A.; Norerg, G.; Nilsson, M.-C.; Jäderlund, A. Experiments on the effects of wter vilility nd exclusion of fungl hyphe on nutrient uptke nd estlishment of Pinus sylvestris seedlings in crpets of the moss Pleurozium schreeri. Ecoscience 1998, 5, Weer, M.G.; vn Cleve, K. Nitrogen trnsformtions in fether moss nd forest floor lyers of interior Alsk lck spruce ecosystems. Cn. J. For. Res. 1984, 14, Nilsson, M.-C.; Steijlen, I.; Zckrisson, O. Time-restricted seed regenertion of Scots pine in sites dominted y fether moss fter cler-cutting. Cn. J. For. Res. 1996, 26, Simrd, M.-J.; Bergeron, Y.; Sirois, L. Conifer seedling recruitment in southestern Cndin orel forest: The importnce of sustrte. J. Veg. Sci. 1998, 9, Blok, D.; Heijmns, M.M.P.D.; Schepmn-Stru, G.; vn Ruijven, J.; Prmentier, F.J.W.; Mximov, T.C.; Berendse, F. The cooling cpcity of mosses: Controls on wter nd energy fluxes in Sierin tundr site. Ecosystems 211, 14, Oleskog, G.; Shlen, K. Effects of seeded sustrte on moisture conditions nd germintion of Scots pine (Pinus sylvestris) seeds in mixed conifer stnd. New For. 2, 2, Soudzilovski, N.A.; Bodegom, P.M.; Cornelissen, J.H. Dominnt ryophyte control over high-ltitude soil temperture fluctutions predicted y het trnsfer trits, field moisture regime nd lws of therml insultion. Funct. Ecol. 213, 27, Jckson, B.G.; Mrtin, P.; Nilsson, M.-C.; Wrdle, D.A. Response of fether moss ssocited N 2 fixtion nd litter decomposition to vritions in simulted rinfll intensity nd frequency. Oikos 211, 12, By, G.; Nhr, N.; Oure, M.; Whitehouse, M.J.; Wrdle, D.A.; Zckrisson, O.; Nilsson, M.C.; Rsmussen, U. Borel fether mosses secrete chemicl signls to gin nitrogen. New Phytol. 213, 2, Berg, A.; Dnielsson, Å.; Svensson, B.H. Trnsfer of fixed-n from N 2 -fixing cynocteri ssocited with the moss Sphgnum riprium results in enhnced growth of the moss. Plnt Soil 213, 362, Schmlholz, M.; Hylnder, K. Succession of ryophyte ssemlges following cler-cut logging in orel spruce-dominted forests in south-centrl Sweden Does retrogressive succession occur? Cn. J. For. Res. 29, 39,