DOI: 10.5061/dryad.98sf7m0n1
title: Physiological responses of fen mosses along a nitrogen gradient point to competition restricting their fundamental niches

description: Nutrient availability determines vegetation patterns and ecological
 functioning of intact groundwater-fed peatlands (fens). Bryophytes,
 commonly referred to as ‘brown mosses’, dominate calcareous fens (rich
 fens), are an integral part of their unique biodiversity and contribute
 significantly to peat formation and carbon sequestration. Brown mosses are
 replaced by vascular plants as nutrient availability increases. The
 decline of brown mosses may either be due to their physiological
 intolerance of high nutrient levels or to them being outcompeted by
 vascular plants. We aimed to distinguish between these two hypotheses by
 investigating whether the ecological optima reflect the physiological
 optima of brown mosses. Eight brown moss species, common in calcareous
 fens of the northern hemisphere, were grown under a gradient of nitrogen
 and phosphorus availability. Biomass increment, chlorophyll content and
 biomass nutrient concentration were measured. All brown moss species
 studied achieved the highest growth rates and chlorophyll contents when
 grown under conditions of nitrogen and phosphorus concentrations higher
 than those where they occur naturally at the highest frequency. Two of the
 species showed no growth saturation even at the highest levels of nutrient
 availability, while the others appeared potassium-limited at the highest N
 and P levels. Brown mosses dominate natural fens at the lower edge of
 their physiological optimum in terms of nutrient availability, i.e., their
 realized niche is much narrower than the fundamental one. Based on the
 literature, we argue that it is the competition for light with vascular
 plants which limits the occurrence of brown mosses in nutrient-rich
 habitats and prevents them from occupying their entire fundamental niche.
description: Analysed species
 We
 chose eight brown moss species that dominate calcareous fens in temperate Europe: Aulacomnium palustre
 (Hedw.) Schwägr., Calliergon giganteum (Schimp.) Kindb., Calliergonella
 cuspidata (Hedw.) Loeske, Campylium stellatum (Hedw.)
 Lange & C. E. O.
 Jensen, Climacium
 dendroides (Hedw.) F. Weber
 & D. Mohr, Hamatocaulis vernicosus (Mitt.) Hedenäs, Scorpidium cossonii (Schimp.) L.E.Anderson, H.A.Crum & W.R.Buck
 and Tomentypnum nitens (Hedw.) Loeske. These species occupy different microhabitats
 along gradients of wetness, nutrient availability and productivity under field conditions (de Mars et al.1996, Štechová et al. 2008,
 Jabłońska et al. 2011, Kooijman 2012, Pawlikowski et al. 2013, Table
 1.). Sampling site
 Bryophyte samples were
 collected in December 2017 from calcareous fens of the upper Biebrza valley, north-eastern
 Poland (between Ostrowie 53.71979 °N 23.30223 °E and Szuszalewo 53.71355
 °N 23.35722 °E). The vascular plant annual production at the sampling
 sites was 120 – 315 g m-2 and total vascular plant cover was ca. 60%. The
 moss annual production was 372 – 440 g m-2 y-1 and exceeded the production
 of vascular plants (own unpublished data). Total moss cover was relatively
 high (c. 95%). Bryophytes were accompanied by small sedges such as Carex
 panicea, C. limosa, C. rostrata, and C. lasiocarpa, some dicotyledonous
 plants, such as Menyanthes trifoliata and Potentilla palustris, and shrub
 birch Betula humilis. The average water level measured by automatic
 loggers (logging interval 1 hour) in 9 piezometers located in the
 proximity of the collection sites between 15/09/2017 and 15/09/2018 was
 1.1 cm below the surface (minimum 12 cm below the surface, maximum 6.9 cm
 above the surface) (own unpublished data). Mean concentrations of the main
 elements in water samples collected in summer 2017 at a depth of 0 – 5 cm below the moss carpet were (mg·L-1): 0.05 N-NH4+, 0.025 N-NO3-, 0.5
 P-PO43-, 0.6 K+, 7.9 S-SO42-, 55.8 Ca2+, 13.5 Mg2+, 7.3 total
 Fe, 6.1 Na2+ and pH was 6.5 – 6.7.
 Preparation of bryophyte
 material We sampled 10 – 15 moss
 patches, each about 10 cm
 in diameter, from every species studied. Samples were transported to the laboratory,
 watered with distilled water and
 <br>stored in glass
 bowls in a cooling room under constant light conditions,
 with a photosynthetic
 photon flux density of around 2 μmol m−2s−1, at about 4 °C for around 2 months.
 <br>Before onset of the experiment, we moved mosses
 to the greenhouse for 14 days to allow them to acclimate
 to the new conditions. After the
 acclimatisation period, the plants did <br>not show any symptoms of
 stress, such as tip drying or losing colour. We washed the mosses with distilled water and
 cut off the top 3 cm of their shoots. We weighed the fresh mass of each
 sample after blotting it twice with paper towels in a standardized way and
 recalculated it to dry mass (see Supplement S1 for calibration of the dry
 mass of bryophytes at the starting point). The experimental units were
 colony fragments corresponding to about 3 g dry mass per single species
 placed in stainless-steel baskets (3 cm x 3 cm x 4.5 cm). The density of
 shoots in the basket was similar to that under natural conditions (based
 on visual assessments). Each basket was placed in a jar (540 ml,
 Supplement S2) filled with an experimental nutrient solution. At the
 beginning of the experiment, the mosses were submerged in the solution up
 to about 1 cm below their tip. Experimental design
 The experimental N and P
 gradient ranged from low concentrations, typical for natural fens
 dominated by brown mosses (Jabłońska et al. 2011), to high values,
 corresponding to substantial N and P enrichment due to eutrophication in
 agricultural landscapes (Table 2.). A. palustre, H. vernicosus, S.
 cossoni, T. nitens were cultivated in nine levels of nutrient
 availability, C. cuspidata, C. stellatum, C. dendroides in eight levels
 (without the 9th level), whereas C. giganteum was cultivated in four
 nutrient levels (2nd, 4th, 6th, and 8th) because of shortage of plant
 material for these species. Each experimental unit contained one basket in
 one jar with one moss species in one type of nutrient treatment. We placed
 experimental units of all species in a box (the experimental block). There
 were five blocks for each nutrient level as replicates. We covered the plastic boxes with glass to
 avoid fungal contamination and to assure constant high moisture, but we
 left a 1 cm gap to enable airflow (Supplement S2). 
 As a nutrient solution, we
 used bottled mineral water (Muszyna Cechini), which is
 naturally rich in
 bicarbonates – 1314 mg·L-1. The water is medium saturated with CO2
 (1500 – 4000 mg·L-1). The concentrations of the main elements, after
 mixing 1 part of Muszyna water with 3 parts of distilled water,
 corresponded to the average element concentrations measured in the
 groundwater of well-preserved calcareous fens. The concentrations of elements in
 solution were (mg·L-1): K 1.24, S 4.56, Ca 56.25, Mg 13.35, Na 11,05, Fe
 0.14, Mn 0.05, B 0.03, Zn 0.0003, Cu 0.0003, Mo 0.00005,
 and Co 0.00008. Measurements were performed with Inductively Coupled
 Plasma Mass Spectrometer ICP MS (Perkin Elmer NexION
 300D,
 USA). We did not add extra K to the solution because
 K availability is usually
 stable along natural and anthropogenic nutrient gradients in calcareous
 fens, as it is rather related to the local climate or hydrogeology (Venterink et al.
 2002). Thus, K concentration was constant in all NP treatments.
 N and P were added as
 NH4NO3 and NH4H2PO4 taking into account the initial N concentration (0.075
 mg N·L-1) present in the solution at each level (from ammonium (N-NH4+)
 and nitrates (N-NO3) dissolved in the mineral water; measured − with a
 SAN++ continuous flow analyser (Skalar, Netherlands)).
 A logarithmic scale of N and P
 concentrations was applied. N and P concentrations at the first and second
 levels corresponded to those measured in groundwater of well-preserved
 calcareous fens in north-eastern Poland
 (mean
 concentrations in Biebrza and Rospuda
 fens were (mg·L-1): 0.05 N-NH4+, 0.025 N-NO3-, 0.5 P-PO43-, 0.6 K+; own data and Jabłońska et al. (2011)), where
 the bryophyte species studied occur with high
 abundances. Subsequent
 levels of nutrient concentrations corresponded to more eutrophic
 calcareous fens (e.g. Lenz and Wild 2001, Kieckbusch and
 Schrautzer 2007) up to almost 25 mg N L-1 in the solution at the 9th level
 typical for highly nutrient-enriched wetlands (Emsens et al. 2016). The
 ratio between N and P concentrations in the water was kept constant at
 about 1.6 (between 1.59 and 1.67) because such a ratio is a median for
 well-preserved calcareous fens in north-eastern Poland (own data) and indicates N-limitation.
 The solution with added N and P was changed every 3 days during the
 experiment and mosses were additionally sprayed with distilled water to avoid shoot drying. We mixed
 the solution in a 20 L canister and poured it into jars. The pH ranged
 from 6.5 at the beginning to 7.9 after each 3-day period, because the
 solution with Muszyna water contained additional free
 CO2, which was gradually released from the water.
 By replacing the solutions, we avoided the problem of calcium
 precipitation (c.f. Vicherová et al. 2015). We avoided possible systematic environmental
 effects within each block by moving the blocks every 3 days during
 the experimental
 period. The experiment lasted 54 days from 12 February
 2018 to 06 April 2018. The average greenhouse temperature was about 10 °C
 in February and about 15 °C in March and April. The mosses grew in
 daylight supported by additional illumination with white LED lamps in
 16h/8h light/dark cycles with a photosynthetic photon flux density of 196
 μmol m−2s−1, which is
 recommended light regime for bryophyte growth (Duckett et al.
 2004). Growth measurement
 At the end of the experiment,
 we oven-dried each sample to constant weight (65 °C, 24 h) and measured
 the dry mass on an analytical balance (KERN 410-11 Electronic Analytical Balance 41/
 resolution: 0.0001g). We
 calculated the relative biomass increment (RBI) of each moss sample by
 dividing the dry mass gain at the end of the experiment (dry mass at the
 end of the experiment minus the initial dry mass of the sample) by the
 initial dry mass (recalculated from the fresh mass as described
 in Supplement S1). Any positive number indicates growth, any negative
 number indicates a loss of biomass over time.  Chlorophyll content 
 Using a CCM-300 chlorophyll
 meter (Opti-Sciences, Inc., Hudson, NH, USA), we measured the ratio of
 chlorophyll fluorescence at 735 nm (far-red) and that at 700 nm (red)
 (chlorophyll fluorescence ratio F735/F700) which correlated to the
 chlorophyll content in vascular plants (Gitelson et al. 1999) and
 used it as a proxy for chlorophyll content in moss tissue.
 N, P, K in bryophyte
 biomass From each bryophyte sample, we cut off tops that
 grew during the experiment (i.e. above the steel basket edge), froze the
 samples in liquid nitrogen and ground them in a mortar to a homogenous
 powder. N content was
 determined by the dry combustion method (Thermo
 Scientific Flash2000 CHNS/O Analyzer, USA). For P and K analysis, samples
 were digested in 65% HNO3 using a Speedwave 4 apparatus (Berghof,
 Germany), and then P content was measured using a SAN++ continuous flow
 analyzer (Skalar, Nederland), and K content was measured using a flame
 atomic absorption spectrometer (ContrAA700, Analytik Jena AG, Germany).
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