Increased air humidity and understory composition shape short root traits and the colonizing ectomycorrhizal fungal community in silver birch stands

Forest Ecology and Management 310 (2013) 720–728

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Increased air humidity and understory composition shape short root traits and the colonizing ectomycorrhizal fungal community in silver birch stands Kaarin Parts ⇑, Leho Tedersoo, Krista Lõhmus, Priit Kupper, Katrin Rosenvald, Anu Sõber, Ivika Ostonen Institute of Ecology and Earth Sciences, University of Tartu, Vanemuise 46, 51014 Tartu, Estonia

a r t i c l e

i n f o

Article history: Received 21 July 2013 Received in revised form 9 September 2013 Accepted 11 September 2013 Available online 6 October 2013 Keywords: Free Air Humidity Manipulation (FAHM) Climate change Ectomycorrhiza Hydrophobicity Root morphology Silver birch

a b s t r a c t Climate change is predicted to bring about a rise in precipitation and air humidity at northern latitudes, which could have considerable impact on forest management. This paper investigates the effect of increased air humidity and understory composition on short root morphology and on the relative abundance of colonizing ectomycorrhizal (EcM) fungal associates in silver birch (Betula pendula Roth.) stands. Short root morphological traits of silver birch were analyzed at increased humidity and ambient conditions for two different understories (early-successional grasses and diverse ‘‘forest’’ understory) in three consecutive years (2009–2011). The fungal community was determined in 2010 (after three seasons of misting) using molecular methods. The study was conducted on the Free Air Humidity Manipulation (FAHM) experimental facility established in Estonia. Silver birches responded to the rise in air humidity by forming longer and thinner short roots, which can be interpreted as a morphological adaptation leading to an increase in the absorptive area. The response was stronger when humidification concurred with the species-poor understory of pioneer grasses. The inter- and intra-treatment variation in short root morphological parameters decreased by the third year. Using molecular methods, overall 64 EcM taxonomic units were distinguished. Hydrophilic fungal morphotypes dominated significantly in humidified plots, hydrophobic morphotypes in control plots. Our results suggest that rising air humidity causes a morphological stress response in EcM short roots. Young trees show the ability to adapt to climate change with great plasticity by modifying short root length, diameter and specific root length (SRL). Humidification leads to a shift in the fungal colonizers towards the dominance of hydrophilic taxa, which may alter ecosystem functioning. Ó 2013 Elsevier B.V. All rights reserved.

1. Introduction Climate change scenarios for the year 2100 predict an increase in air temperature (by 2.3–4.5 °C) and precipitation (by 5–30%) in Northern Europe including the Baltic region (IPCC, 2007; Kont

Abbreviations: D, short root diameter (mm); SRA, specific root area (m2 kg 1); RTD, root tissue density (kg m 3); SRL, specific root length (m g 1); L, short root length (mm); M, short root mass (mg); RTFL, root tip frequency per unit of length (no mm 1); TF, throughfall precipitation (mm); SWP, soil water potential at the depth of 15 cm (kPa); ST, soil temperature at the depth of 15 cm (°C); AT, air temperature (°C); RH, relative air humidity (%); SOM, soil organic matter (%); SoilN, soil nitrogen concentration (%); ESG, early-successional grasses; F, diverse ‘‘forest’’ understory; H, humidification, misting; C, control. ⇑ Corresponding author. Tel.: +372 55 656 986; fax: +372 7375 825. E-mail addresses: [email protected] (K. Parts), [email protected] (L. Tedersoo), [email protected] (K. Lõhmus), [email protected] (P. Kupper), [email protected] (K. Rosenvald), [email protected] (A. Sõber), ivika.ostonen@ ut.ee (I. Ostonen). 0378-1127/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.foreco.2013.09.017

et al., 2003). Because the increase in precipitation is generally manifested through increased cloud cover and frequency of wet days, air humidity will rise too . A unique Free Air Humidity Manipulation (FAHM) facility was established in Estonia to investigate the effect of increased air humidity on the forest ecosystem (Kupper et al., 2011). The silver birch (Betula pendula Roth.) was chosen as the object of investigation in the FAHM experiment, as it is commercially the most important broadleaved species in Northern Europe, an excellent pioneer and improver of soil fertility on abandoned agricultural and mining areas or otherwise unproductive sites (Rosenvald et al., 2011a). It has been shown that air humidification, both directly and through a decline in leaf temperature, decreases the humidity gradient between leaf and air, which reduces the average diurnal stem sap flux density per unit of projected leaf area (Kupper et al., 2011). This impedes the mass flow of soluble minerals in the soil and nutrient uptake of roots (Cramer et al., 2009), potentially leading to decreased growth and productivity in fast-growing tree species.

K. Parts et al. / Forest Ecology and Management 310 (2013) 720–728

Indeed, lower leaf nitrogen (N) and phosphorus (P) contents have initially been witnessed in silver birches growing in conditions of elevated air humidity (Sellin et al., 2013). Decreased transpiration also causes a rise in soil humidity (Hansen et al., 2013), which directly influences the growth environment of plant roots and soil microbes. The forests´ response to climate change depends highly on the acclimation ability of plant fine root systems and their microbial and fungal partners in the rhizosphere. Short roots (the distal roots with primary structure) constitute the most active part of the fine roots and are responsible for water and nutrient uptake. In boreal forests, short roots are predominantly colonized by EcM fungi, which increase the absorbing surface up to 60 times (Simard et al., 2002). For example, over 95% of the short roots of silver birch may form ectomycorrhizae (Uri et al., 2007). Plants react to changes in environmental conditions (a rise in atmospheric [CO2] and temperature, nutrient limitation, shifts in soil moisture, etc.) through alterations in short root morphology, biomass, and turnover or stimulation of microorganisms adjacent to roots – shifts in the mycorhizosphere (Lõhmus et al., 2006a; Phillips and Fahey, 2006). For example, it has been shown that CO2 enrichment and long-term warming induce an increase in high biomass fungi with proteolytic capacity and a decrease in fungi that utilize labile N (Deslippe et al., 2011; Godbold et al., 1997). These alterations can significantly influence the nutrient uptake and vitality of plants (Fransson et al., 2005; Gorissen and Kuyper, 2000), thus the productivity of ecosystems, their potential in sequestering carbon, and consequently atmospheric [CO2] and global climatic conditions. While the effects of CO2 enrichment on fine roots and the EcM fungal community have been well documented (Cudlin et al., 2007; Lukac et al., 2009; Simard and Austin, 2010), the effects of increased air humidity remain poorly understood. Short roots can be characterized by morphological parameters such as specific root length (SRL), specific root area (SRA) and root tissue density (RTD). Presuming that nutrient acquisition is proportional to the length or surface area, and the cost of forming and maintaining roots is proportional to mass (Eissenstat and Yanai, 1997; Ostonen et al., 2007), SRA and SRL are characteristics of root economy and foraging efficiency (Lõhmus et al., 1989; Ostonen et al., 2007). SRA and SRL depend on the diameter, length and RTD of the root (Ostonen et al., 2007), which may react to changes in the environment in opposite ways and must be taken into consideration when analyzing the functional morphological parameters. High SRL suggests fast growth and intensive soil exploration and is important for sufficient uptake of P (Silberbush and Barber, 1983). In stress conditions, trees have been shown to apply an intensive foraging strategy, i.e. increasing the absorptive surface per unit of mass (SRA, SRL and branching frequency) (Rosenvald et al., 2011a,b; Ryser, 2006), along with an extensive foraging strategy of increasing the biomass of the fine root system (Lõhmus et al., 2006b; Ostonen et al., 2011). Fast-growing species in productive habitats, where rapid acquisition of nutrients is essential to withstand competition, also exhibit high SRL and SRA (Kupper et al., 2012; Ostonen et al., 1999). In forest ecosystems, understory vegetation has considerable effects on the carbon cycle (Borja et al., 2008), nutrient availability to trees (Picon-Cochard et al., 2006), and thus, stand regeneration through resource competition. Previous studies have shown that grass vegetation shows higher water and NO3- capture capacity than tree seedlings (Picon-Cochard et al., 2006) and has greatest effect on early-successional tree species through competition for both resources and soil space (Messier et al., 2009). Early-successional tree seedlings responded to the fine root competition of grasses with higher fine root biomass and SRL in unoccupied soil space, indicating a strategy of root avoidance when possible (Messier

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et al., 2009). Understory plants have also been shown to influence the rhizosphere through differences in soil microbial community and the particular plant-soil effect may depend on plant species, functional group (e.g. grasses vs. forbs) and site-specific soil properties (Bezemer et al., 2006). So far, little is known about the possible morphological reaction of tree short roots to the varying growing conditions induced by different understory types. Besides the above-mentioned environmental factors, morphological parameters of EcM short roots are highly dependent on the age of the plant and the colonizing fungi (Ostonen et al., 2009; Rosenvald et al., 2013; Ryser, 2006). Younger trees have higher SRL and SRA, due to lower root diameter, mass and RTD (Rosenvald et al., 2011a, 2013). The identity of the fungal colonizer is the main factor determining EcM short root diameter, length and mass. Thereby, it also significantly influences SRA and SRL (Makita et al., 2012; Ostonen et al., 2009; Sun et al., 2010). EcM fungal taxa with contrasting foraging strategies also differ in their capacities of enzymatic activities, nutrient uptake and translocation (Courty et al., 2010; Lilleskov et al., 2002, 2011; Tedersoo et al., 2012). Therefore, EcM partners have major impact on tree nutrition both directly and through changes in morphological traits. The different ecological strategies of EcM fungi have been associated with exploration type and hydrophobicity (Lilleskov et al., 2011; Unestam and Sun, 1995). Hydrophilic morphotypes (prevalently concurring with contact-, short-distance and medium-distance smooth exploration types) have lower proteolytic capabilities and depend on the availability of labile nitrogen forms, thus representing the exploiting ruderal strategy (Hobbie and Agerer, 2010; Lilleskov et al., 2011; Tedersoo et al., 2012). These morphotypes prosper in humid environments and have been shown to tolerate waterlogging and oxygen deficiency better (Bakker et al., 2006; Stenström, 1991). Because of lower investment in proteolytic enzymes and extramatrical mycelium, these morphotypes are assumed to be less costly to the host in terms of carbon expenditure. On the other hand, high extramatrical biomass and rhizomorph forming hydrophobic morphotypes characterize environments, where labile N is scarce and insoluble organic N-sources are widely dispersed and spatially concentrated. Hydrophobic rhizomorphs facilitate effective long-distance water transport, prevent leakage of solutes and represent stress tolerant species in cases of drought and consequent nutrient limitation (Hobbie and Agerer, 2010). In such conditions, the costly formation of high extramatrical biomass and exudation of extracellular enzymes, capable of decomposing complex organic substrates, is advantageous. Producing extensive hydrophobic hyphal mats may also drive out other microorganisms and thus render a competitive quality (Unestam and Sun, 1995). Undoubtedly the effectiveness of a morphotype, whether low biomass forming and hydrophilic or high extramatrical biomass forming and hydrophobic, depends on the specific conditions and limiting resources. Our objective was to investigate the interactive effects of increased air humidity and understory composition on the morphology and colonizing fungal community of EcM short roots of silver birch. In order to comprehend the dynamics in root morphology, samples were taken before the treatment commenced and in three consecutive years starting after two seasons of misting. The colonizing EcM fungal community was identified after three years of humidification. This paper aims to contribute to understanding how much silver birches rely on the intensive strategy of modifying short root morphology as an alternative to the extensive strategy of expanding their root systems in response to environmental stress. On the basis of morphological plasticity we aim to assess short root acclimation ability of the silver birch in relation to the increase in air humidity, predicted to co-occur with climate change at higher latitudes. Knowledge about the effect of

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understory type and about changes in the EcM community can be applied to work out efficient afforestation and reforestation practices, which anticipate the changing climate. We postulated the following hypotheses: (1) increased air humidity causes a morphological stress response of increased SRL, SRA or branching frequency, reflecting rapid acclimation of EcM short roots to the changed environment; (2) diverse ‘‘forest’’ understory relative to grass understory will have the ability to mitigate the effect of humidification; (3) increased air humidity and understory type cause a shift in the EcM fungal community composition.

information on the growing conditions. Overall, humidified plots exhibited significantly higher values of soil water potential (SWP) during the three summers than control plots, mainly due to decreased transpiration (Kupper et al., 2011; Hansen et al., 2013). The SWP of humidified plots in October 2010 and 2011 was lower than that of control plots, which could be explained by postponed leaf senescence in humidified plots, enabling trees to transpire up to a month longer (P. Kupper, personal communication). Also worth noting is the relatively low level of precipitation in July 2010 and 2011 (Table 1), which affected the SWP of humidified plots less severely. There were no substantial differences in soil temperature between the treatments during the whole study period (Table 1). In addition, the pH of humidified plots was significantly higher in 2009 (means: 4.41 ± 0.08 for H and 4.15 ± 0.04 for C; paired t-test, p = 0.05) and 2011 (means: 4.48 ± 0.04 for H and 4.29 ± 0.04 for C; paired t-test, p < 0.01). Concurrent leaf nitrogen measurements revealed significantly lower values in humidified plots in 2009 (2.4 ± 0.11% compared to 2.7 ± 0.14% in control; Sellin et al., 2013), but by 2011 the situation had reversed – the leaves in humidified plots contained significantly more nitrogen (3.1 ± 0.07% compared to 2.7 ± 0.08% in control). However, the height of humidified silver birches remained significantly lower throughout the study period. In 2009, the height of silver birches in H plots was 3.00 ± 0.06 m compared to 3.51 ± 0.05 m in C plots (Sellin et al., 2013). By 2011, the height of silver birches in H plots had risen to 5.06 ± 0.08 m compared to 5.44 ± 0.08 m in control (Hansen et al., submitted). The concentrations of Ca, K, Mg, N and P (mg kg 1) in soil samples were determined by inductively coupled Plasma Mass Spectrometry (ICP-MS) in the laboratory of the Finnish Forest Research Institute in 2008. Soil pH, soil organic matter (%) and nitrogen concentration (%) were measured each study year. To determine soil nitrogen concentrations (Kjeldahl), Tecator ASN 3313 was used. The analyses were carried out at the Biochemistry Laboratory of the Estonian University of Life Sciences. To investigate the impact of soil biota and understory species composition on ecosystem functioning, two different types of ground vegetation were established in the plots, representing either disturbed forest vegetation (F), such as that usually recorded in recent clear-cut areas, or early-successional vegetation (ESG – early-successional grasses) with low diversity and a strong dominance of a few grass species, such as that in abandoned arable

2. Materials and methods 2.1. Description of the experimental site The study was carried out in silver birch (Betula pendula Roth.) plots growing on the FAHM (Free Air Humidity Manipulation) experimental site, which is situated at Rõka village, Järvselja Experimental Forest District (58°24´N, 27°18´E, altitude 40–48 m), in south-eastern Estonia. The average annual precipitation is 650 mm; the average temperature is 17.0° in July and 6.7° in January. The growing season lasts from mid-April to October. The study site has been established on former agricultural land. The soil is a fertile Endogenic Mollic Planosol (WRB) with the A-horizon being 27 cm thick. Soil nitrogen content varied from 0.10% to 0.17%, C/N ratio 11.8 and pH 4.0–4.7. One-year-old silver birch seedlings were planted in the experimental area in spring 2006. Stand density in the experimental plots is 10 000 trees ha-1 and the distance between trees is 1 m. The site contains humidified (H) and control (C) plots (samples were taken from two H and two C plots). Humidification started on 1 June 2008 and has been carried out daily throughout all following growing seasons. Misting took place if the ambient relative air humidity (RH) dropped <75% and wind speed was <4 ms 1. The FAHM system enabled an average increase in RH of 7% (maximum 18%) over the ambient level during misting. For a more detailed description of the study site and the humidification system, see Kupper et al. (2011). The monthly means of meteorological parameters (precipitation, soil water potential, soil temperature, air temperature, and relative humidity) are presented in Table 1 for background

Table 1 Mean monthly values of meteorological parameters throughout the vegetation periods of the three study years (2009–2011). Abbreviations: throughfall precipitation (TF; mm), soil water potential at the depth of 15 cm (SWP; kPa), soil temperature at the depth of 15 cm (ST; °C), air temperature (AT; °C) and relative humidity (RH;%); C – control, H – humidification. 2009

May C

June H

C

July H

C

August H

C

September H

C

October H

C

H

TF SWP ST AT RH

14.4 30.0 9.1 11.5 67.0

23.7 9.8 9.5 11.2 68.4

107.0 20.5 12.1 14.1 78.6

105.0 12.0 13.0 14.0 78.9

71.4 85.9 15.3 17.0 82.1

97.4 12.0 15.7 16.6 83.7

35.2 206.2 14.9 15.2 83.3

42.5 3.8 15.4 14.7 84.6

56.5 137.1 13.2 12.7 86.1

63.8 5.7 13.4 12.2 88.2

70.8 15.0 7.1 4.0 90.7

67.7 1.5 7.0 3.8 91.3

2010 TF SWP ST AT RH

38.0 8.9 10.6 12.5 76.0

45.0 13.6 11.4 12.5 77.3

93.7 21.9 13.0 14.8 77.0

85.1 36.0 13.7 14.9 77.1

33.4 168.9 17.7 22.1 73.7

25.6 132.4 18.5 21.7 75.7

103.4 147.5 17.7 18.2 81.6

104.8 97.0 17.7 17.9 84.1

93.9 14.2 12.5 10.9 86.8

108.0 17.2 12.2 10.7 88.9

41.5 3.4 6.9 3.9 85.7

41.4 12.6 6.5 3.8 86.2

2011 TF SWP ST AT RH

39.2 13.2 9.8 11.2 69.2

38.0 20.8 9.8 11.4 69.6

28.3 137.4 14.1 17.3 73.4

24.6 104.1 14.5 17.1 75.4

17.7 212.2 17.2 20.7 77.7

15.3 154.4 17.4 20.4 80.5

38.6 185.3 16.0 16.7 79.7

37.9 185.6 15.9 16.3 82.3

32.8 180.4 13.1 12.4 85.7

29.5 189.5 13.1 12.4 87.2

42.9 59.2 8.6 7.0 86.5

45.8 92.8 8.6 6.9 87.1

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fields (Kupper et al., 2011). The experimental ‘‘forest community’’ was created by means of sodding in order to inoculate seedlings with mature forest soil biota and thus enhance the positive effect of the rhizosphere. Its understory was dominated by Ranunculus repens L., Lathyrus pratensis L., Festuca rubra L., and Veronica chamaedrys L. and consisted of 67 species. The early-successional grass (ESG) vegetation was established by sowing the seeds of Phleum pretense L. onto the agricultural soil with other early-successional species emerging spontaneously. It comprised only 31 species and exhibited strong dominance of Elymus repens (L.) Gould accompanied by Aegopodium podagraria L. The layout of the experimental plot is illustrated in Fig. 1. 2.2. Morphological studies: sampling and measurements Silver birch short root (first and second order root) samples for morphological studies were dug up by spade from two humidified (H1 and H4; eight samples from each) and two control plots (C1 and C4) in October 2009, 2010 and 2011. Samples were also taken in 2007, before the humidification treatment began, to indentify the initial state. Each plot comprised two quarters of different understory; four samples were collected from both quarters. Roots were traced from random silver birch tree trunks and samples taken from the depth of 0–20 cm. The roots of understory vegetation were removed and samples were divided into 10–20 subsamples to facilitate work under the microscope. All morphological parameters were averaged on the subsample level (comprising 15–20 root tips), i.e. n = 10–20 per quarter. During the three years, respectively 2589, 2266, and 3013 silver birch root tips were analyzed after separating them from long roots, cleaning and counting under microscope. The length (LS), diameter (D), and surface area (SA) of the short roots were measured by the WinRHIZO™ Pro 2003b image analysis system (Regent Instruments Inc., 2003). The airdry roots were dried at 70 °C for two to three hours to constant weight and weighed to 0.05 mg accuracy. Root tissue density (RTD, kg m 3), specific root area (SRA, m2 kg 1), and specific root length (SRL, m g 1) were calculated as MS/VS, SA/MS, and LS/MS, respectively, where VS is the volume, LS is the length and MS is the dry mass of all the root tips in a subsample (Ostonen et al., 2007). Root tip frequency was expressed as number of root tips per unit of length (RTFL). Mean short root mass (M, mg) was calculated as the dry mass of all the short roots in a subsample divided by the respective number of root tips. Similarly, mean short root length (L, mm) was calculated as the length of all the short roots in a subsample divided by the number of root tips (Ostonen et al., 1999).

Fig. 1. The layout of the FAHM experimental plot. Abbreviations: F – diverse ‘‘forest’’ understory, ESG – early-successional grass understory. Samples were taken only from the quarters with silver birches. A modified figure from Kupper et al. (2011).

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2.3. EcM fungal community analysis: sampling and molecular techniques To determine the composition of the EcM fungal community, altogether 32 root samples were collected from two humidified and two control plots (four root samples per quarter as described in the morphology section) in October 2010. The roots of silver birch were separated from the roots of understory vegetation, cut into 5 cm fragments, and two or three fragments from each sample were subjected to morphotyping on the basis of mantle color, texture, and the presence of emanating hyphae, rhizomorphs, and cystidia. The relative abundance of each morphotype was estimated and all the morphotypes were classified as either hydrophilic or hydrophobic. The criteria for grouping morphotypes hydrophobic were silvery appearance of mycorrhizae due to air bubbles trapped between hyphae and floating on water; if mycorrhizae sunk into water and had no silvery appearance, they were considered hydrophilic. Categorization on the basis of hydrophobicity was verified, if possible, using relevant literature (Agerer, 2006; the DEEMY information system). At least two individual root tips of each morphotype per sample were immersed into CTAB lysis buffer [100 mM Tris-HCI (pH 8.0), 1.4 M NaCl, 20 mM EDTA, 2% cetyl-trimethyl-ammonium-bromide] and maintained at room temperature until molecular analyses. One or two representative root tips of each morphotype per sample were subjected to DNA analysis. Fungal taxa were identified by use of sequence analysis of the nuclear rDNA Internal Transcriber Spacer (ITS) region. The DNA was extracted using a Qiagen DNeasy 96 Plant Kit (Qiagen, Crawley, UK) according to the manufacturer’s instructions. Primers, PCR conditions, product purification, sequencing, and sequence processing are described in Tedersoo et al. (2010). Sequences were assigned to operational taxonomic units (OTUs) based on a 97.0% ITS barcoding threshold (Tedersoo et al., 2003); but 99.0% was used for the genera Laccaria and Hebeloma, which display little divergence in the ITS region. In order to identify the EcM fungi and possible contaminants, representative sequences of each species were subjected to a megablast search against International Nucleotide Sequence Databases (INSD) and UNITE (Abarenkov et al., 2010).

2.4. Statistical analyses Statistical analyses were carried out using STATISTICA 7, R and CANOCO programs. All analyses for morphology were done on the subsample level. Short root variables were checked for normality using Lilliefors and Kolmogorov–Smirnov tests. Repeated measures ANOVA was used to test the significance of the study year and the effect of humidification and understory type over all years. Twoway ANOVA was employed to test the effect of humidification and understory type in each separate year. The effect of understory type within both treatment groups was checked with the t-test. Redundancy analysis (RDA) (CANOCO program; ter Braak and Šmilauer, 2002) was used to detect and visualize relationships between short root morphological characteristics, the two treatments, understory types and environmental conditions throughout the three consecutive years. On the ordination plots, all the morphological samples from an understory quarter have been merged into a common centroid. The following meteorological factors and soil chemical components were analyzed for significance in explaining the variance in EcM root morphology (using RDA) and in fungal community composition (using CCA and adonis): TF (throughfall; mm), SWP (soil water potential, kPa), ST (soil temperature; °C), AT (air temperature; °C), RH (relative air humidity; %) from May to October; soil pH; soil organic matter (%); soil Ca, K, Mg, N and P concentrations (mg kg 1). The significance of RDA results was tested with the Monte Carlo permutation procedure.

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Table 2 The mean values and standard errors of short root morphological parameters before humidification in 2007 and at increased humidity (H) and ambient conditions (C) over the three study years (2009–2011). ⁄P < 0.05; ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001; the significance of humidification effect in each study year in bold (Two-way ANOVA; predictors: humidification (C/H) and understory type (ESG/F)). Abbreviations: D – short root diameter, SRA – specific root area, RTD – root tissue density, SRL – specific root length, L – short root length, M – short root mass, RTFL – root tip frequency per unit of length.

2007 2007 2009 2009 2010 2010 2011 2011

C (initial state) H (initial state) C H C H C H

D (mm)

SRA (m2 kg

1

0.212 ± 0.007 0.207 ± 0.009 0.216 ± 0.004 0.215 ± 0.003 0.273 ± 0.003⁄⁄⁄ 0.242 ± 0.003⁄⁄⁄ 0.303 ± 0.004⁄ 0.289 ± 0.004⁄

181.0 ± 18.6 212.0 ± 29.4 98.6 ± 2.5⁄⁄⁄ 131.5 ± 4.3⁄⁄⁄ 137.3 ± 3.0 140.3 ± 2.9 93.1 ± 2.4 92.1 ± 1.9

)

RTD (kg m

3

)

112.7 ± 11.8 103.9 ± 10.2 194.4 ± 4.1⁄⁄⁄ 152.0 ± 4.6⁄⁄⁄ 111.4 ± 2.5⁄⁄ 121.8 ± 2.2⁄⁄ 148.4 ± 3.0 155.6 ± 2.7

To evaluate the effect of humidification, understory type, different soil chemical components, and meteorological conditions on explaining the dissimilarities in the fungal community species composition (whether a species appears in a sample or not), the adonis function (vegan package in R) was used. To analyze and illustrate the dissimilarities in fungal communities (species relative abundance and distribution according to hydrophobicity), the canonical correspondence analysis (CCA) was employed. The Monte Carlo permutation test was used to decide whether soil and meteorological characteristics had a significant effect on EcM fungal species composition. The difference in colonization percentages of hydrophilic and hydrophobic morphotypes between the treatments and understory types was checked by Two-way ANOVA. The differences in the values of environmental factors (that obtained significance in CCA) between the treatments (H and C) were tested using the t-test. The value of a = 0.05 was used as the significance level for all the analyses.

SRL (m g

1

)

277.6 ± 32.5 337.0 ± 51.9 150.0 ± 5.9⁄⁄⁄ 199.0 ± 8.1⁄⁄⁄ 163.4 ± 4.6⁄⁄⁄ 188.3 ± 5.4⁄⁄⁄ 100.2 ± 3.2 103.8 ± 3.0

L (mm)

M (mg)

RTFL (no mm

1.95 ± 0.13 1.68 ± 0.08 1.48 ± 0.04 1.56 ± 0.04 1.51 ± 0.04⁄⁄ 1.67 ± 0.05⁄⁄ 1.32 ± 0.05 1.42 ± 0.03

0.0077 ± 0.001 0.0061 ± 0.0009 0.0106 ± 0.0005⁄⁄⁄ 0.0085 ± 0.0003⁄⁄⁄ 0.0096 ± 0.0003 0.0093 ± 0.0004 0.0135 ± 0.0004 0.0142 ± 0.0004

5.27 ± 0.34 6.09 ± 0.29 7.00 ± 0.18 6.66 ± 0.15 6.98 ± 0.20⁄ 6.40 ± 0.20⁄ 8.41 ± 0.31⁄⁄ 7.38 ± 0.19⁄⁄

1

)

3. Results 3.1. The effect of increased air humidity and understory type on EcM root morphology Humidification affected EcM root morphology significantly throughout the three study years (Table 2) (Repeated Measures ANOVA; p varied from 0.023 to 0.048). All EcM root morphological traits, except for L and RTFL, depended substantially on the study year (p varied from 0.000 to 0.001). Inside each study year, humidification had extensive influence in 2009 and 2010; however, the number of morphological parameters that the treatment affected decreased greatly by 2011. While in 2009 and 2010, according to Two-way ANOVA, five parameters out of eight had significant differences between the treatments, in 2011, only two parameters (D and RTFL) differed significantly (Table 2). The decrease in variance within and between the treatments and the persistent trend of forming thicker and heavier short roots as the stand develops is depicted on the ordination plot (Fig. 2A). The initial point of

Fig. 2. (A) Ordination biplot based on redundancy analysis (RDA) of EcM root morphological parameters of silver birch indicating the effect of humidification throughout three study years and (B) meteorological conditions that significantly correlate with these parameters (Monte Carlo permutation test, (A) p = 0.001; (B) p = 0.001). Open triangles depict the overall means of the study years, closed triangles represent the means of the treatments (C and H). In figure (A) the blue continuous circles group samples from humidified plots; the red dashed circles surround samples from control plots of each year. The means of each quarter (n = 214–404 analyzed short roots) are indicated by dots. Abbreviations: C – control, H – humidification, G –early-successional grass understory, F – diverse ‘‘forest’’ understory. The abbreviations for meteorological and morphological parameters have been listed at the beginning of the article as a footnote.

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Table 3 The mean values and standard errors of short root morphological traits at increased humidity and ambient conditions for two different types of understory in 2009. ⁄P < 0.05; ⁄⁄ P < 0.01, ⁄⁄⁄P < 0.001; the significance of understory effect within the treatment group in bold (t-test). Abbreviations: D – short root diameter, SRA – specific root area, RTD – root tissue density, SRL – specific root length, L – short root length, M – short root mass, RTFL – root tip frequency per unit of length, ESG – early-successional grasses.

Control ESG understory Control ‘‘Forest’’ understory Humidification ESG understory Humidification ‘‘Forest’’ understory

D (mm)

SRA (m2 kg

1

0.220 ± 0.006 0.212 ± 0.006 0.211 ± 0.003 0.219 ± 0.004

98.4 ± 3.3 98.7 ± 3.7 157.7 ± 6.1⁄⁄⁄ 111.7 ± 3.8⁄⁄⁄

)

RTD (kg m

3

)

190.8 ± 6.5 197.8 ± 5.0 124.6 ± 3.4⁄⁄⁄ 172.7 ± 5.9⁄⁄⁄

2007, when misting had not yet begun, is also presented on the ordination plot to better illustrate the dynamics of EcM root morphology. Humidified and control plots were merged in the initial point, since the morphological parameters did not differ significantly (Table 2). According to redundancy analysis, morphological parameters correlated significantly with air and soil temperatures, soil water potential, the precipitation and relative air humidity of July, soil organic matter, Ca, Mg, N concentration and soil pH (Fig. 2B). The concentrations of Ca and Mg were not included in the ordination biplot, since they exhibited weak correlation with the first two axes. The first axis correlated mainly with October SWP. Altogether, these factors accounted for 40.9% of the total variation in morphological root traits (Fig. 2B). Understory affected the majority of the root morphological traits significantly (except D) only in humidified plots and only in 2009, when the canopy had not yet closed completely (Table 3). A single exception to this was L, which was affected by understory type also in 2011. When understory effect was considered, differences between the two treatments (H and C) became more pronounced. For example, in 2009 the SRL in humidified plots was significantly higher than in control plots, being exceptionally high in humidified plots with ESG understory (Fig. 3 and Table 3). Understory composition also affected parameters (L and RTFL), where humidification had no significant effect in 2009. Roots growing in quarters with ESG understory had significantly longer root tips (p < 0.007) and less root tips per unit of length (p < 0.004). In the case of L the same was observed in 2011 (p < 0.014). 3.2. The dynamics of morphological root traits In 2009, the SRA of humidified plots was significantly higher (Fig. 2 and Table 2). This was achieved by significantly lower RTD

SRL (m g

1

)

145.7 ± 7.2 154.2 ± 9.4 243.2 ± 12.8⁄⁄⁄ 165.6 ± 6.7⁄⁄⁄

L (mm)

M (mg)

RTFL (no mm

1.53 ± 0.06 1.43 ± 0.05 1.66 ± 0.05⁄⁄ 1.48 ± 0.05⁄⁄

0.0112 ± 0.0008 0.0100 ± 0.0006 0.0073 ± 0.0004⁄⁄⁄ 0.0095 ± 0.0004⁄⁄⁄

6.80 ± 0.28 7.18 ± 0.22 6.14 ± 0.16⁄⁄ 7.06 ± 0.22⁄⁄

1

)

and M of H plots (especially in quarters of ESG understory; Table 3) in 2009. The effect of humidification on SRA disappeared in the following years, due to counterdirectional changes in RTD and D, which evened differences out (Table 2). The yearly dynamics of SRL were similar to that of the SRA. In 2009 and 2010, the SRL of H was significantly higher than C (Fig. 2 and Table 2), 33% and 15% respectively. By 2011, the significant effect of humidification on the three functional root parameters disappeared, and the overall absolute values of both SRL and SRA had fallen substantially (p < 0.01). In general, short roots were significantly heavier in 2011 than in the previous years (p < 0.001). Differences in short root diameter and length appeared in 2010 with significantly thinner and longer roots in humidified plots. In 2011, short roots of H plots were still thinner, but significant differences in short root length between C and H disappeared, although the consistent trend of forming longer short roots in humidified plots remained. RTFL, the reciprocal of L, was one of the two parameters that were still affected by humidity in 2011 – there were persistently less root tips per unit of length in humidified plots (Table 2). There was an overall increase in D for both treatments over the three years.

3.3. The effect of increased air humidity and understory type on the composition and structure of the EcM fungal community Sequence analysis revealed 64 fungal taxa (OTUs), of which 44 were singletons, (i.e. occurring in one sample). Among these 64 OTUs, species-level identification could be provided to 32 taxa from 16 lineages. The most frequent OTUs were Paxillus involutus, Tomentella cinerascens and Tomentella sublilacina. The most OTUrich lineage was /tomentella-thelephora, which tended to be more abundant in terms of colonization percentage in humidified plots.

Fig. 3. The effect of (A) humidification and (B) its interaction with understory type on specific root length (m g

1

) in 2009. ⁄P < 0.05; ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001; Two-way ANOVA.

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Quarters with diverse ‘‘forest’’ understory exhibited a non-significant trend of having more OTUs identified. According to adonis, humidification treatment on its own didn´t affect the species composition of the fungal community significantly. However, humidification had significant effect on the relative abundance of hydrophobic and hydrophilic morphotypes; the latter dominated in humidified plots (p = 0.001; Two-way ANOVA; Fig. 4). The mean colonization percentages of the hydrophilic morphotypes were 33% in control plots and 72% in humidified plots. When all variables were analyzed conjointly, only soil pH (F1,26 = 2.705; p = 0.0015; adonis) and August SWP (F1,26 = 2.188; p = 0.0063; adonis) had significant impact on EcM fungal community composition. The structure of the fungal community in relation to the treatments, understory types and to the gradients of pH and Aug-SWP is depicted on the CCA ordination biplot (Fig. 4). Although according to CCA, the effect of pH on the fungal community composition was insignificant (p = 0.098), it was included in the ordination, since it had significant effect according to adonis. Humidification resulted in significantly higher pH values in soil in 2009 and 2011; in 2010, no significant difference was detected between the treatments.

4. Discussion 4.1. The effect of increased air humidity and the understory type on short root morphology Humidification led silver birches to grow persistently thinner and longer short roots. While the diameter retained significant differences up to the third study year, root length lost significant differences between the treatments, although the trend remained. Nevertheless, the effect of humidification on short root length is expressed through significantly lower root tip frequency per unit of length in H plots. Considering the understory, silver birches growing in quarters of ESG vegetation displayed a persistent trend of forming longer short roots (significant in 2009 and 2011), which was especially evident in humidified plots. Forming longer and thinner roots could be interpreted as a stress reaction (Ostonen et al., 2007) to limited tree nutrient acquisition due to decreased stem sap flux density (Kupper et al., 2011) that was amplified by the interaction of humidification and an understory of pioneer grasses. In 2009, leaf N concentrations were significantly lower in humidified plots than in control, indicating malnutrition. This coincided with significantly higher SRL and SRA and lower RTD of short roots growing in misted plots, the differences being mainly influenced by the values of the roots from the ESG quarters. Our observations are in good agreement with the results of Rosenvald et al. (2011b), who reported concurrence of high SRL and SRA values and low leaf N reflecting a shortage of mineral nutrients in the soil. Understory fine root and rhizome biomass formed approximately 90% of the total fine root biomass in 2009 with ESG understory having significantly greater fine root biomass than quarters with diverse ‘‘forest’’ understory in humidified plots (the means of September were respectively 1207 g m 2 and 601 g m 2), in control plots there was no difference between the two types of understory (I. Ostonen, unpublished). E. repens, the dominant species in ESG quarters, might have initially caused strong underground competition for the roots of silver birch and consequently additional stress. By 2010, after canopy closure, the biomass of understory vegetation dropped drastically, especially in the C plots, which were most disturbed by the drought, and the effect of understory on root morphology disappeared. Also the litter of grasses may have been of lower quality than the litter of forbs growing in quarters of diverse ‘‘forest’’ understory and may thus

Fig. 4. The species-environmental variables biplot of canonical correspondence analysis (CCA), which illustrates the shift in the EcM fungal community towards the dominance of hydrophilic morphotypes caused by humidification (Monte Carlo permutation test, p < 0.05). Blue font color indicates hydrophilic morphotypes, red font color – hydrophobic morphoypes; black species produced both hydrophilic and hydrophobic morphotypes in different samples. Large triangles depict the treatments and the two understory types (C – control, H – humidification, F – diverse ‘‘forest‘‘ understory, ESG – early-successional grass understory). Abbreviations: Cen geo – Cenococcum geophilum, Ent sin – Entoloma sinuatum, Ent nid – Entoloma nidorosum, Heb pus – Hebeloma pusillum, Heb sacc – Hebeloma sacchariolens, Heb sp – Hebeloma sp, Heb vel – Hebeloma velutipes, Ino curv – Inocybe curvipes, Lacc tort – Laccaria tortilis, Lact nec – Lactarius necator, Lact pub – Lactarius pubescens, Lec rig – Leccinum rigidipes, Pax inv – Paxillus involutus, Tom cin – Tomentella cinerascens, Tom ell – Tomentella ellisii, Tom subcl – Tomentella subclavigera, Tom subli – Tomentella sublilacina, Tom sp – Tomentella sp.

influence the soil conditions (Cheng et al., 2010; Semmartin et al., 2004). The year 2010 exhibited relatively high values of SRA and SRL, caused by very low values of RTD. Low RTD could be explained by a new generation of short roots formed after the relatively dry summer of 2010, whereas the growing season of 2009 was rainy and the mid-summer drought of July did not occur. Inversely to 2009, the RTD of control plots was lower than that of humidified plots after the dry summers of 2010 and 2011, indicating that humidification mitigated the impact of limited precipitation. Although the diameter of short roots in humidified plots remained smaller, changes in RTD cancelled out the differences in SRA and by 2011 in SRL. On the contrary, by the third year both SRA and SRL had decreased dramatically for both treatments, due to an overall increase in diameter (H roots were still thinner), and a relative rise in RTD and M compared to 2010. Similar dynamics (a rise in D, M and RTD and a decrease in SRA and SRL) have been reported in earlier studies on the morphology of silver birch and attributed to stand aging (Rosenvald et al., 2012). Also the intra-treatment variation of short root morphology decreased considerably in the course of the three years and the two treatment groups (H and C) began to increasingly overlap, as presented on the ordination plot (Fig. 2A). This implies that young fast-growing silver birches are much more reactive in their morphological response to environmental change and readily modify short root parameters. Along with morphological studies, measurements of fine root biomass have been carried out on the FAHM experimental site, the results of which are hereby described. Four years of misting

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resulted in significantly more fine root biomass of silver birch in humidified plots (I. Ostonen, unpublished), while in 2007, before humidification commenced, there were no differences between treatments (K. Lõhmus, unpublished). Leaf nitrogen concentrations had also risen to be significantly higher in humidified plots, compared to control plots. Our results suggest that initially saplings employ the intensive strategy of altering short root morphological parameters in response to a changed environment. However in longer term, trees growing in a humid climate rely mostly on the extensive strategy of expanding their fine root system. The diminished morphological response of short roots in humidified plots is balanced by an increase in fine root biomass per tree (I. Ostonen, unpublished), which seems to have eliminated the initial obstruction in nutrient uptake. 4.2. The effect of increased air humidity and understory type on the composition and structure of the EcM fungal community Three years of humidification caused a significant shift towards the dominance of hydrophilic morphotypes in the fungal community. Hydrophobic morphotypes formed the significant majority in control plots. Since the hydrophobicity of hyphae is related to the exploration type and the presence of rhizomorphs, the dominance of contact and short-distance exploration types can be expected in humidified plots (Hobbie and Agerer, 2010). These results agree with Bakker et al. (2006), who also reported the significantly larger proportion of hydrophilic contact exploration type in humid sites and the dominance of long and short distance exploration types in dry sites. The most abundant colonizers of humidified plots were representatives of the /tomentella-thelephora lineage, characterized by contact, short-distance, or medium-distance smooth exploration types and hydrophilic hyphae. This shift in community composition coincides with the notion of low extramatrical biomass producing hydrophilic morphotypes proliferating in humid environments and even preferring waterlogged conditions (Unestam and Sun, 1995) compared to hydrophobic fungi, whose complex extramatrical systems can be extremely disturbed by even brief drenching (Stenström, 1991). Humidification treatment affected the species composition of the EcM fungal community indirectly through changes in soil water potential and pH, but did not have a direct significant effect. Elevated air humidity increases soil moisture primarily through a reduction in water uptake by roots while moisture addition into soil as water vapor is negligible (Hansen et al., 2013; Kupper et al., 2011). The fact that August SWP had significance in determining fungal community composition coincides with the intensive production of new root tips after the mid-summer drought of 2010. The meteorological circumstances at that time, particularly soil moisture, may give a colonization advantage to hydrophilic morphotypes in humid habitats. It has been shown that soil acidity influences the colonization potential (Erland and Söderström, 1990), competitive strength (McAfee and Fortin, 1987) and fruit body production of EcM fungi (Agerer et al., 1998), thus causing shifts in the fungal community. Courty et al. (2005) have also described the varying enzymatic capabilities of different EcM species as largely dependent on soil horizon and pH. The activity profile of the same species in the same horizon may vary considerably within one square meter; therefore, the effect of specific pH values on the nutrient acquisition efficiency of different fungal species needs further investigation. Compared to quarters with sown early-successional grasses, there were more OTUs distinguished in diverse ’’forest‘‘ vegetation quarters, which may have created a more favorable growth environment and added to the strong understory effect of 2009. It has been argued that the identity of the EcM colonizers is more important in determining plant nutrition enhancement than EcM

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species richness, but certainly a larger number of species in the EcM community increases the probability of a fungal isolate being present that improves plant growth (Kipfer et al., 2012). Which of the following factors was the main cause of the understory effect on EcM short roots of the silver birch – higher diversity of fungal partners, the quality of different understory litter, or the strength of understory plants´ root competition – needs further analysis.

5. Conclusions These results suggest that the predicted rise in relative air humidity at higher latitudes may cause a considerable stress reaction on EcM short root level affecting tree mineral nutrient acquisition. Young silver birch trees showed the ability to acclimate with great plasticity by producing longer and thinner short roots, and initially increasing the specific area and length of their root systems. In addition, diverse ‘‘forest’’ understory relative to grass understory mitigated the morphological reaction of short roots to the increasingly humid growing conditions. Rising air humidity may also cause a shift in the fungal community towards the dominance of hydrophilic and generally low mycelial biomass forming taxa. The complex interactions between trees, their root symbionts, and understory vegetation must be taken into consideration in order to estimate the possible outcome of climate change on forest ecosystems and to work out flexible forestry practices for coping with these changes. With regard to forest management, our results indicate that a diverse understory of forbs should be preferred to pioneer grasses in the process of afforestation. When choosing inoculants to boost seedling establishment, hydrophobicity and related functional traits of ectomycorrhizae should be taken into account for the specific growing conditions.

Funding This work was supported by Estonian Science Foundation [Grant nos. 7792, 7452]; the European Regional Development Fund (Centre of Excellence ENVIRON – morphological analyses; Centre of Excellence FIBIR – fungal community analyses; Project no. 3.2.0802.11-0043 (BioAtmos) – soil chemical analyses); and the Ministry of Education and Research of the Republic of Estonia [projects SF0180025s12, IUT2-16]. The funding sources had no involvement in any stage of the study. Acknowledgements We thank Martin Zobel and Ülle Jõgar for background information on the species composition of understory vegetation, Arvo Tullus for background information on the heights of silver birches and Jaak Sõber for maintaining the FAHM facility. References Abarenkov, K., Nilsson, R.H., Larsson, K.H., et al., 2010. The UNITE database for molecular identification of fungi – recent updates and future perspectives. New Phytologist 186 (2), 281–285. Agerer, R., 2006. Fungal relationships and structural identity of their ectomycorrhizae. Mycological Progress 5, 67–107. Agerer, R., Taylor, A.F.S., Treu, R., 1998. Effects of acid irrigation and liming on the production of fruit bodies by ectomycorrhizal fungi. Plant and Soil 199, 83–89. Bakker, M.R., Augusto, L., Achat, D.L., 2006. Fine root distribution of trees and understory in mature stands of maritime pine (Pinus pinaster) on dry and humid sites. Plant and Soil 286, 37–51. Bezemer, M., Lawson, C.S., Hedlund, K., Edwards, A.R., Brook, A.J., Igual, J.M., Mortimer, S.R., Van der Putten, W.H., 2006. Plant species and functional group effects on abiotic and microbial soil properties and plant–soil feedback responses in two grasslands. Journal of Ecology 94, 893–904.

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