FEMS Microbiology Ecology Advance Access published May 15, 2015

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Effects of annual and inter-annual environmental variability on soil fungi associated with

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an old-growth, temperate hardwood forest.

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Running title: Environmental variability and forest soil fungi

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David J. Burke1,2*

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The Holden Arboretum, 9500 Sperry Road, Kirtland, OH, USA.

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The Department of Biology, Case Western Reserve University, Cleveland, OH 44106, USA

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Corresponding author: *The Holden Arboretum, 9500 Sperry Road, Kirtland, OH, USA, e-

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mail: [email protected], phone: 440-602-3858, fax: 440-602-8005.

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Tables: 5

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Figures: 4

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Supplementary tables:

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Supplementary figures:

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Abstract

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Seasonal and inter-annual variability in temperature, precipitation and chemical resources may

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regulate fungal community structure in forests but the effect of such variability is still poorly

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understood. In this study, I examined changes in fungal communities over two years and how

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these changes were correlated to natural variation in soil conditions. Soil cores were collected

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every month for three years from permanent plots established in an old-growth hardwood forest

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and molecular methods were used to detect fungal species. Species richness and diversity were

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not consistent between years with richness and diversity significantly affected by season in one

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year but significantly affected by depth in the other year. These differences were associated with

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variation in late winter snow cover. Fungal communities significantly varied by plot location,

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season and depth and differences were consistent between years but fungal species within the

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community were not consistent in their seasonality or in their preference for certain soil depths.

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Some fungal species, however, were found to be consistently correlated with soil chemistry

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across sampled years. These results suggest that fungal community changes reflect the behavior

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of the individual species within the community pool and how those species respond to local

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resource availability.

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Key words: climatic variability, ectomycorrhizal fungi, hardwood forest, soil nutrients, TRFLP,

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snow cover

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Introduction Soil fungi are important components of temperate forests and play an integral role in

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organic matter decomposition, carbon (C) and nutrient cycling, and serve as an important food

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resource for soil organisms (Linderman, 1988; Rayner & Boddy, 1988; Smith & Read, 2008). In

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addition, soil fungi can affect the distribution of important microbial functional groups, such as

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those involved in N cycling (Bomberg et al., 2003; Burke et al., 2012). One important group of

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soil fungi are the mycorrhizal fungi that form mutually beneficial relationships with many forest

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plants. These fungi colonize plant roots, receive carbon from the plant in the form of sugars and

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organic acids, and directly benefit the plant by increasing overall nutrient gain as well as plant

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growth (Smith & Read, 2008). Although a number of studies have explored the environmental

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factors that govern fungal community composition, diversity and species occurrence in forests

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(Goodman & Trofymow, 1998; Dickie et al., 2002; Lilleskov et al., 2003; Burke et al., 2009) we

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still know relatively little about how soil fungi may be affected by global climate change.

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It may be expected that greater variability in temperature and precipitation associated

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with global climate change could lead to altered seasonal and inter-annual variability in

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microbial community composition (Monson et al., 2006), with consequences for the timing and

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magnitude of many ecosystem processes (Sulkava & Huhta, 2003). Seasonal and inter-annual

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variability in temperature and precipitation play an important role in regulating nutrient cycling,

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plant growth, and soil microbial community structure in forests (Groffman et al., 2001b, Tierney

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et al., 2001; Aerts et al., 2006; Buckeridge & Grogan, 2008). Temperature and precipitation are

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major controls on microbial growth and reproduction, and may be expected to regulate

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community structure and species distributions (McArthur, 2006). In addition, temperature and

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precipitation can affect functional activity of microbes; for example, soil moisture frequently

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controls production of extracellular enzymes and thus the rate of organic matter decomposition

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and nutrient cycling (Buckeridge & Grogan, 2008; Steinweg et al., 2012; A’Bear et al., 2014).

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Alteration in winter temperatures and especially greater soil freezing associated with reduced

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snow cover can increase nutrient loss from soil (Fitzhugh et al., 2001) but greater soil freezing

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could also increase plant growth, possibly through increases in nutrient availability following

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death of soil microbes.

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However, most studies that have examined climate change effects on forests have not

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explicitly examined soil fungal communities, but rather have focused on functional activities or

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changes in biomass (Campbell et al., 2005; Buckeridge & Grogan, 2008) with mycorrhizal

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community studies often focused on the effects of increased CO2 (Rygiewicz et al., 2000 and

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Klamer et al., 2002). Yet, recent observational studies of arctic soil fungi suggest that these fungi

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may be highly sensitive to changes in their environment including changes associated with

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climate and soil pH (Timling et al., 2014). Loss of winter snow cover associated with climate

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warming can alter soil freeze thaw dynamics and affect belowground processes (Tierney et al.,

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2001) and studies have indicated that distinct microbial communities persist under snow cover,

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and removal of snow cover may change these communities (Monson et al., 2006). Work

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examining typical annual variation in mycorrhizal communities has found that some species are

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stable, with little variation between seasons, while other species are more seasonally abundant

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(e.g. summer or winter)(Izzo et al., 2005; Koide et al., 2007; Richard et al., 2011). Precipitation

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has also been found to strongly determine fungal biomass in forest soil (Okada et al., 2011). But

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additional studies on the effects of inter-annual changes in temperature and precipitation,

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including variation in snow cover, are necessary to better predict the effects of changing climate

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and snow cover on forest fungal communities.

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Of additional interest is how changes in precipitation and temperature with variation in

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snow cover could affect fungal distribution at different depths within the soil profile. Previous

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studies have found that soil at deeper depths within the profile can have more consistent and

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potentially higher temperatures during the winter months than soil at more shallow depths

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(Henry 2007). This could affect soil freeze thaw cycles, and potentially alter fungal distribution

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if fungal taxa are sensitive to temperature. A number of studies have found that fungal taxa vary

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with depth within the soil profile, with some fungi more common in mineral as compared to

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shallow organic layers (Rosling et al., 2003; Shahin et al., 2013). Nonetheless, whether fungal

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distribution with soil depth is related to soil temperature dynamics has not been well examined.

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In this study I hypothesized that 1) annual variability in soil moisture and temperature

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would affect the abundance and distribution of soil fungi; 2) inter-annual variability in snow

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cover would alter species distributions between years and 3) communities at deeper depths

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within the soil profile would be less responsive to annual and inter-annual changes as a result of

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reduced seasonal variability in moisture and temperature. The way that fungi respond to

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variability in moisture and temperature could enhance our understanding of how fungi may

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respond to a changing climate that alters those patterns of variability. I collected soil cores every

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month for three years from permanent plots established in an old-growth hardwood forest in

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northeastern Ohio. Soil was subdivided into three depths and used to measure chemistry and

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determine fungal species distribution using terminal restriction fragment length polymorphism

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(TRFLP) coupled to a site specific database of fungal sequences. Soil temperature, moisture and

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snow cover were monitored during the three years of sampling.

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Materials and Methods

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Site description and soil sampling

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In 2006 six long-term sampling plots were established within Stebbins Gulch, a 360

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hectare mature, mixed-mesophytic forest located within The Holden Arboretum in northeastern

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Ohio, USA (41°36′N and 81°16′W). The study site was established in an approximately 80

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hectare, old growth, beech-maple forest dominated by American beech (Fagus grandifolia-

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~75% of canopy coverage) and sugar maple (Acer saccharum- ~15% of canopy coverage) with a

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herbaceous understory dominated by Allium tricoccum and Dicentra canadensis. The site has

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moderately drained silt loam soils with total precipitation averaging 116-cm per year, which

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includes approximately 287-cm of snowfall. For more detailed information about the field site,

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see Burke et al. (2009, 2012). Soils are acidic, with pH ranging from 3.5 to 5.6 with a mean pH

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of 4.0  0.1.

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Long-term sampling plots were established between 50 and 100-m apart, and measured 4

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x 4 meters in size. Plots were marked in the center by a 2-m steel pole, driven into the ground,

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and to which was attached HOBO Pro Series air and relative humidity data loggers (Onset

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Computer Corporation, Bourne, MA) to record air temperature and humidity every 12 minutes

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throughout the year. Air temperature loggers were set 1.5 m above the ground. Stowaway Tidbit

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Temperature Loggers (Onset Computer Corporation, Bourne, MA) were used to record soil

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temperature and were buried 1-m from the center pole in each plot such that a logger was present

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between 1-3 cm depth (hereafter 2-cm soil depth), 5-7 cm depth (hereafter 6-cm soil depth) and

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9-11 cm (hereafter 10-cm soil depth). Stowaway data loggers recorded soil temperature every 8

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minutes throughout the year.

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Long-term plots were broken into 16 sampling quadrats, each measuring 1 x 1 meter in

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size. Two soil cores measuring 2-cm in diameter were collected to a depth of 15-cm every month

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(around the middle of each month) throughout the year with a metal soil corer from within one

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randomly selected quadrat. The cores were collected from the corners of each quadrat such that

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they were at least 50-cm apart (Supplemental Figure 1). These samples were subdivided into 4

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depths: surface litter layer (Oi), and the three soil depths corresponding to 2-, 6- and 10-cm as

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defined above. The subdivided samples from each core were composited in the field so that one

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set of four samples were retained for each plot (24 samples total per month, 6 plots x 4 depths).

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Samples were kept in a cooler on ice, and transported to the laboratory where they were stored at

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-70ºC until processing. Soil samples have been collected every month from these plots for the

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past 7 years, and I present data here from November 1 2006 until October 31 2009. The

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temperature time series presented here begins on November 1 2006 and continues until October

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31 2009. Soil samples (excluding litter samples) collected from each plot and depth for the entire

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2006-2007 year (November 2006-October 2007) and from the entire 2008-2009 year (November

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2008-October 2009) were analyzed for this study for soil chemistry and fungal community

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structure (6 plots by 3 soil depths by 12 months by 2 years = 432 samples examined). These

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years were examined because the winters varied in the depth and duration of snow cover, with

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the 2006-2007 year having abundant snow fall and continuous snow depth between early January

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and early April, and the 2008-2009 winter having variable snow cover, with snow cover absent

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at some times during the winter including during the sample events.

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Analysis of soil environment

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Frozen and stored soil samples were thawed and used to measure gravimetric water

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content which is expressed here as [(g water g FW soil-1) x 100%] (Jarrell et al., 1999) and is

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meant to represent field fresh soil. Soil for C and N was oven dried and pulverized in a Precellys

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homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France) and analyzed on an ECS

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4010 CHNSO elemental analyzer (Costech Analytical, Valencia, CA). Labile soil inorganic

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phosphorous (Pi; readily available) and organic phosphorous (Po; easily mineralizable) were

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extracted from pulverized oven dried soil by adding 0.5 M NaHCO3 (pH 8.5) and shaking at 100

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rpm on an orbital shaker (Lab-Line, Melrose Park, IL) for 30 min (Olsen et al., 1954). Pi was

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determined using the modified ascorbic acid method (Kuo, 1996) directly on the NaHCO3

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extracts. Total available P (Pt) was determined by further digestion of NaHCO3 extracts with 1.8

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N H2SO4 and (NH4)2S2O2 (EPA, 1971) while Po was the difference between Pt and Pi. A total of

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648 samples (6 plots by 3 soil depths by 12 months by 3 years) collected between November

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2006 and October 2009 were analyzed for soil chemistry. Temperature and relative humidity

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data were averaged over the course of the day, and daily averages are represented here. Visual

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observation of snow cover was also made throughout the winter months, and snow gauges

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attached to each of the long-term plot center poles were used to estimate the depth of snow cover

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at that sampling time.

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DNA purification and amplification

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DNA was extracted from soil using a bead beating protocol. Soil with visible root tissue

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removed (up to 200-mg fresh weight) was placed in a 1.5-ml bead beating tube containing 500-

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mg of 400 μM glass beads (VWR, West Chester, Pennsylvania, USA) and 750 µl 2% CTAB

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(cetyltrimethyl-ammonium bromide). Samples were then beaten for 40s in a Precellys

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homogenizer at 6500 rpm and purified by phenol/chloroform extraction and precipitation with

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20% polyethylene glycol 8000 in 2.5 M NaCl with incubation at 37oC. DNA was suspended in

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200 µl TE buffer and stored at –20oC. To amplify soil fungi, the internal transcribed spacer 2

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region (ITS2) was targeted using primers 58A2F and NLB4 (Martin & Rygiewicz, 2005; Burke

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et al., 2005). From previous work, these ITS2 primers have been found to more consistently

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amplify some fungal species as compared to ITS1 primers, and the region also provided the best

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discrimination between species when used in conjunction with terminal restriction fragment

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length polymorphism (TRFLP) typing of fungal communities (Burke et al., 2009). Primers were

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labeled either with the fluorescent dye 6FAM (58A2F) or HEX (NLB4) and PCR was carried out

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in 50μl reaction volumes using 1-µl of purified DNA (approximately 100 ng), 0.2 µm of primers,

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2.0 mM MgCl, 0.2 mM dNTP, 0.15 mg ml-1 Bovine Serum Albumin, and 1.0 units FastStart Taq

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DNA polymerase (Roche Diagnostics Corporation, Indianapolis, USA) on an PTC 100 Thermal

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Cycler (MJ Research, Boston, USA). An initial denaturation step of 5 minutes at 94 C was

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followed by amplification for 35 cycles at the following conditions: 30 seconds at 94 C, 60

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seconds at 60 C, 90 seconds at 72 C. A final 5-minute extension at 72 C completed the

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protocol. Although the goal was to target all soil fungi, for the purposes of this study I assume

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that soil patterns also reflect root tip colonization patterns for ECM fungi.

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Identification of fungal species in soil samples

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Labeled PCR product was cut with endonucleases AluI and HaeIII (Promega, Madison

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WI, USA) and TRFLPs were completed through the Cornell Bioresource Center using an

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Applied BioSystems 3730xl DNA Analyzer. TRFLPs were analyzed with PeakScanner software

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version 1 (http://www.appliedbiosystems.com). For each core, 3 useful TRFLP profiles were

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generated (there is a conserved AluI restriction site in the 28S rDNA that generates little

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variability for TRFLP), and these profiles were used to identify fungal species using the program

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Fragsort (Sciarini & Michel, 2002; http://www.oardc.ohio-state.edu/trflpfragsort/index.php)

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which compared the patterns to a site specific database as previously described and detailed

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(Burke et al., 2009). The database we used for identifying fungal species for this study contained

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398 identified fungal isolates separated from sporocarps, mycorrhizal root tips, and soil clones

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representing 187 species. Use of this database is skewed toward Basidiomycetes and may under

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represent Ascomycetes present within soil samples. Although plants associated with arbuscular

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mycorrhizal fungi are present at the field site, the database does not contain arbuscular

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mycorrhizal taxa and cannot be used to identify them in the soil samples. The identification of

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fungal species then is limited by the database itself, and is not intended to include all fungi

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present within soil samples. The strength of the approach however is that we can identify soil

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fungi to species in most instances, permitting a more precise examination of changes in fungal

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community structure and fungal distribution.

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Statistical Analyses

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Differences in soil chemistry between plots, soil depth, and season were determined using

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3-Way ANOVA and data were transformed (log10 (moisture, C) or arcsinsqrt (N)) prior to

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ANOVA to maintain equality of variances. Soil N and water content data are presented as a

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percent, and both types of data were transformed prior to ANOVA. Chemistry data were

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analyzed using procedures in SigmaStat 3.5 (Systat Software Inc., CA, USA). To analyze

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seasonal affects on soil chemistry and fungal communities, samples were grouped into 4

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categories: winter (December/January/February), spring (March/April/May), summer

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(June/July/August) and autumn (September/October/November). Differences in fungal richness

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and Shannon diversity were determined through 3-Way ANOVA. Richness represents the fungal

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species positively identified in soil cores. Shannon diversity (H') was calculated for identified

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fungi, where the average TRF peak area was used as a measure of proportional abundance in that

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core (Burke et al., 2005, 2006). Evenness (E) is represented as Pielou’s J (H'/ln(richness). Both E

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and H' were calculated using procedures available through PC-ORD 4 (MjM Software, OR,

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USA). Permutation-based nonparametric MANOVA (PERMANOVA) was used to determine

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whether depth, season or plot location affected fungal communities and whether sample year

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affected these relationships. The year sampled was used as a covariate for PERMANOVA

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analysis which was completed in R using the vegan (v2.0-10) package (Oksanen et al., 2013).

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Since PERMANOVA found that fungal communities varied significantly between sampled

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years, we also conducted PERMANOVA on microbial communities separately for each year

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sampled. Non-metric multidimensional scaling (NMS) procedures available through PC-ORD 4

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were also used to determine whether fungal communities changed with depth, season or between

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plots and to visualize these relationships. The “autopilot” mode of PC-ORD was initially used

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and final results of the ordination confirmed manually. The Sørenson distance with a random

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starting configuration was used for these analyses. Ordination procedure included 250 runs with

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real data and 250 runs with randomized data and use of a Monte Carlo test to help select final

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dimensionality. Additional dimensions of the ordination that did not reduce stress by 5 or more

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were not considered useful for improving the ordination and the highest dimensionality that met

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this criterion was used for the final ordination. A total of 500 iterations were used for the final

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solution. Because the data set includes taxa at the species level, and many fungal species are

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infrequently encountered, the data set contains many zeros. This has the effect of increasing the

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stress of the final ordination, which could be reduced by eliminating rare species. However, rare

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species were included in the data set in order to offer as complete a picture of community

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structure as possible, even though this results in higher than desired final ordination stress. It

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should be noted however than NMS ordination generally confirmed PERMANOVA results and

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provides a useful visualization of fungal community structure despite the higher than desired

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stress levels. In addition, for NMS, PERMANOVA and diversity estimates the proportional

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abundance of all detected fungal species was used as an indicator of abundance within each

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sample (Burke et al., 2005, 2006) and all proportional abundance data were transformed before

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analysis. In some instances, samples failed to amplify with PCR. In those cases, we calculated

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the mean fungal abundance for that sample type and used it for the community analysis

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(PERMANOVA, NMS, diversity). For example, the 2-cm depth averaged across plots would be

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used for a missing 2-cm sample for that sample time point. Although most samples amplified

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adequately, 22 from the 2006-2007 year and 11 from the 2008-2009 year did not amplify

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successfully (out of 432 total samples) and the mean values were used for these samples during

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analysis. The mean scores were used to maintain a balanced design to meet PERMANOVA

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requirements. Indicator species analysis using procedures available through PC-ORD 4 was used

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to determine whether some fungal taxa displayed preference for depth, season or plot. This

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procedure included a Monte Carlo randomization test to determine whether the distribution

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pattern of fungal taxa were significant as compared to a randomized data set constructed from

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observed values. Pearson correlation coefficients were calculated for the named fungal taxa

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using all samples and time points (n=432; both sampled years) to determine whether significant

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correlations existed between fungal taxa and soil chemistry. Since intraspecific trait variation is

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expected to be low, fungal taxa should respond consistently to environmental variability across

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space and time, justifying using the combined data set to examine taxa correlations with

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environmental conditions. Proportional abundance was used for these tests with P

Effects of annual and interannual environmental variability on soil fungi associated with an old-growth, temperate hardwood forest.

Seasonal and interannual variability in temperature, precipitation and chemical resources may regulate fungal community structure in forests but the e...
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