Introduction
Knowledge about the spatial distribution and activity patterns of soil
microbial communities is essential to understand ecosystem functioning as
soil microbes play a fundamental role in biogeochemical cycling and drive
productivity in terrestrial ecosystems (van de Heijden et al., 2008). Soil
microbial diversity in the Arctic is comparable to that in other biomes (Chu
et al., 2010), and the spatiotemporal variability in microbial community
composition is large (Lipson, 2007; Blaud et al., 2015; Ferrari et al.,
2016). However, it is still uncertain which environmental factors drive the
heterogeneity of soil microbial properties in the Arctic.
Location of the three transects investigated (Tr1–Tr3) and
automated weather stations (AWSs) in Petunia Bay (Petuniabukta),
Billefjorden, central Svalbard. Map source: map sheet C7, Svalbard 1:100000, Norwegian Polar Institute 2008.
Altitudinal transects offer a great opportunity to study the distribution of
microbial communities adapted to local habitats and explain patterns through
natural gradients of soil conditions, presence or absence of vegetation and
different climate regimes within short spatial distances (Ma et al., 2004;
Körner et al., 2007). Climate change will further affect environmental
conditions in the Arctic (Collins et al., 2013), including the expected
upward migration of vegetation and increasing plant cover (Vuorinen et al.,
2017; Yu et al., 2017). Therefore, the knowledge of current microbial
distribution and activity patterns along the altitudinal gradients together
with identifying their controlling factors can help to predict the future
development of ecosystems in this region. However, such studies are scarce
despite the fact that the arctic tundra comprises 5 % of the land on
Earth's land surface (Nemergut et al., 2005) and that most coastal areas in
the northern circumpolar region have a mountainous character. To date, only a
few studies assessing altitudinal trends in soil microbial properties have
been conducted in the Scandinavian Arctic (Löffler et al., 2008;
Männistö et al., 2007). Research on spatial variation in microbial
community composition and activity in polar regions was conducted mainly
within a narrow elevation range (Oberbauer et al., 2007; Trevors et al.,
2010; Björk et al., 2008; Chu et al., 2010; Van Horn et al., 2013; Blaud
et al., 2015; Tytgat et al., 2016) or was focused on initial soil development
following glacier retreat (Bekku et al., 2004; Yoshitake et al., 2007;
Schütte et al., 2010). The majority of studies on elevational patterns in
microbial community structure (MCS) and activity have been carried out in
mountain regions of the lower latitudes from the tropics to the temperate
zones. The studies commonly show that microbial activity decreases with
increasing elevation (Schinner, 1982 – Alps; Niklińska and Klimek, 2007
– Polish Carpathians; Margesin et al., 2009 – Alps), while there are no
general altitudinal patterns in soil microbial diversity and community
structure. For example, the microbial community composition did not change
along elevational gradients in the Swiss Alps (Lazzaro et al., 2015), while
other studies have documented decreasing bacterial (Ma et al., 2004 –
western China; Lipson, 2007 – Rocky Mountains; Shen et al., 2013 –
northeastern China) and fungal (Schinner and Gstraunthaler, 1981 – Alps)
diversity with increasing altitude. Several studies reported a
mid-altitudinal peak in microbial diversity (Fierer et al., 2011 – Peru;
Singh et al., 2012 – Mt. Fuji, Japan; Meng et al., 2013 – central China).
In addition to the fungal and bacterial diversity, the relative abundance of these
main microbial functional groups is also variable. For example, Djukic et
al. (2010 – Alps), Xu et al. (2014 – Himalayas) and Hu et al. (2016 –
Himalayas) found a decreasing ratio of fungi to bacteria (F/B) with
increasing elevation, while Margesin et al. (2009 – Alps) reported the
opposite trend in the Central Alps.
Research focusing on environmental controls over microbial communities in
polar and alpine regions has recognized many significant factors, including
vegetation, litter C / N stoichiometry, organic carbon content, soil pH,
nutrient availability, microclimatic conditions and bedrock chemistry.
However, the effect of these variables was site and scale specific (Van Horn
et al., 2013; Blaud et al., 2015; Ferrari et al., 2016), which highlights the
need for further research on environmental controls of microbial community
size, activity and structure on local and regional scales. To extend our
knowledge about microbial ecology and soil functioning in arctic ecosystems,
we conducted a study aiming to assess the activity, biomass and structure of
soil microbial communities and to determine their controlling environmental
factors along three altitudinal transects located in central Svalbard. These
transects spanned from vegetated tundra habitats in narrow areas at sea level
to unvegetated soils at the top of the coastal mountains. The specific
objectives of our study were (i) to describe gradients of microclimatic and
geochemical soil properties; (ii) to assess microbial activity (soil
respiration) and the abundance of main microbial groups (fungi, Gram-negative
and Gram-positive bacteria, Actinobacteria, phototrophic microorganisms)
using phospholipid fatty acid (PLFA) analysis; and (iii) to identify
environmental factors explaining any patterns in soil microbial parameters
along these altitudinal gradients.
Materials and methods
Study area and soil sampling
Petunia Bay (Billefjorden; 78∘40′ N, 16∘35′ E) is
located in the center of Svalbard archipelago (Norway) and represents a
typical High Arctic ecosystem in the northern circumpolar region. The mean,
minimum and maximum air temperatures recorded in the area at 25 m above the
sea level (a.s.l.) in the period of 2013–2015 were -3.7, -28.3 and
17 ∘C, respectively. The temperatures stayed permanently below
0 ∘C for 8 months a year (Ambrožová and Láska,
2017). The mean annual precipitation in the central Svalbard area is only
191 mm (Svalbard Airport, Longyearbyen, 1981–2010) and is equally
distributed throughout the year (Førland et al., 2011).
In August of 2012, we collected soils from three altitudinal transects
(Tr1–Tr3) on the east coast of Petunia Bay. Each transect was characterized
by four sampling sites at altitudes of 25, 275, 525 and 765 m a.s.l.
(±5 m). The transects were located on slopes with similar exposition
(Tr1 W–E, Tr2 WNW–ESE, Tr3 WSW–ENE; Fig. 1) and lithostratigraphy. Soils
at the lowest elevations developed from Holocene slope (Tr1 and Tr3) or
marine shore deposits (Tr2), while the bedrock at the more elevated sites
consisted of dolomite and limestone with units of basal calcareous sandstone
(Dallmann et al., 2004). The soils were classified as Leptic Cryosols (Jones
et al., 2010) with loamy texture and clay content increasing with altitude
(Table 2). Their depth ranged from 0.15 to 0.2 m to only a few centimeters at 25 and
765 m a.s.l., respectively. A poorly developed organic horizon was present
only at the lowest elevation. The sampling locations were selected in
geomorphologically stable areas with similar slopes (20 ± 5∘).
At each sampling site, nine soil cores (4 cm deep, 5.6 cm in diameter) were
collected and then combined, three at a time, into three different mixed
samples. Each sample was made up from one soil core taken from the edge of
the vegetation tussocks (if vegetation was present) and two cores taken at
increasing distance from the vegetation to maintain the consistency with
respect to the heterogeneity of vegetation cover and soil surface. The
triplicates were collected approximately 5 m apart. Immediately after
sampling, the soil was sieved (2 mm) to remove larger rocks and roots,
sealed in plastic bags, and kept frozen at -20 ∘C till further
processing. Soil subsamples for biomarker analysis were freeze-dried as soon
as possible and stored at -80 ∘C until extraction.
The transects represented climosequences from High Arctic tundra to
unvegetated bare soil. Vegetation at the two lowest sites was dominated by
Dryas octopetala, with significant contribution of Saxifraga oppositifolia, and variable contributions of Cassiope tetragona,
Salix polaris and sedges (Carex nardina, C. rupestris, C. misandra; Prach et al., 2012; personal
observations by Petr Kotas, 2012). The
vascular plants formed scattered patches at 525 m a.s.l. with Salix polaris and Saxifraga oppositifolia being the most abundant
species. The soils at the most elevated sites were covered mainly by soil
crusts, with scarce occurrence of Saxifraga oppositifolia and
Papaver dahlianum (personal observations by Petr Kotas, 2012). The percentage cover of the main surface
types (i.e., stones, bare soil, vegetation, crusts and mosses) was estimated
at each sampling site in an area of approximately 1 m2 in close
vicinity to the coring sites (Table S1, Fig. S6 in the Supplement).
Climatic variables; temperatures given in ∘C.
Site
Means
Means
Means
Min daily
Max daily
Mean daily
Max daily
Number of days
Number of days
Positive soil
altitude
summer
winter
year
means winter
means summer
amplitude
amplitude
with daily
with daily
surface energy
(m a.s.l.)
summer
summer
mean > 0 ∘C
mean > 5 ∘C
balance
25
5.8
-3.6
-0.8
-7.0
11.2
5.2
10.9
110
62
615
280
7.1
-5.7
-2.7
-10.3
14.5
8.5
18.2
96
54
571
520
5.8
-8.9
-4.9
-15.8
14.7
8.1
17.7
91
40
480
765
5.3
-9.5
-6.6
-17.1
11.6
5.5
14.0
51
11
290
Monitoring of microclimatic characteristics
To describe the soil microclimatic conditions along the altitudinal
transects, we continuously measured soil temperature at -5 cm from 2012 to
2013 directly at the sampling sites of Tr1 using data-loggers (Minikin Ti
Slim, EMS Brno, CZ). The soil water content at the time of sampling was
determined in soil subsamples by drying to constant weight at
105 ∘C. The temperature regimes at the respective altitudinal levels
were characterized by 10 climatic variables (Table 1). The period of
above-zero daily mean ground temperatures is referred to as the summer season
throughout the text. We also measured the number of days with daily mean
ground temperatures above 5 ∘C, which characterizes the period
suitable for vascular plant growth (Kleidon and Mooney, 2000). The soil
surface energy balance was estimated as the sum of daily mean summer
temperatures. The records from 3 years (2011–2013) of continuous
measurements at two automated weather stations located at 25 and
455 m a.s.l., approximately 3 km from the transects (hereafter referred to as
AWS25 and AWS455, respectively; Fig. 1; see Ambrožová and
Láska, 2017, for a detailed description), were used to evaluate the
seasonal variations in soil temperature and moisture regimes (Figs. S2, S3,
respectively) and coupling of soil and atmospheric temperatures (measured at
-5 cm and 2 m above surface, respectively; Fig. S2). Even though we were
not able to continuously measure soil moisture directly at the sampling
sites, we regarded data from both automated weather station locations as representative for the
evaluation of seasonal moisture regimes.
Soil characteristics
The particle size distribution was assessed using the aerometric method
(Lovelland and Whalley, 2001). Soil type was classified according to the US
Department of Agriculture. The soil pH was determined in a soil–water mixture
(1:5, w/v) using a glass electrode. The cation exchange capacity (CEC)
was considered to be equal to the sum of the soil exchangeable base cations
Mg2+, Ca2+, Na+ and K+ extracted with 1 M NH4Cl
(Richter et al., 1992). The contribution of H+ and Al3+ ions was
neglected due to the high soil pH. Base cations accessible for plant and
microbial uptake (Mg2+, Ca2+, Na+, K+) were extracted
with Mehlich 3 reagent (Zbíral and Němec, 2000). Cations were
measured using atomic absorption spectroscopy (AA240FS, Agilent Technologies,
USA). Total soil organic carbon (TOC) and nitrogen (TN) contents were
measured in HCl fumigated samples (Harris et al., 2001) using an elemental
analyzer (vario MICRO cube, Elementar, Germany).
Microbial respiration
As we were not able to measure soil respiration on site or immediately after
soil collection, we measured the potential respiratory activity (soil
CO2 production) in a laboratory incubation experiment. We stored and
transported the soils in a frozen state because it was previously
demonstrated that freezing–thawing has a weaker effect on microbial activity
than long-term refrigeration (Stenberg et al., 1998), comparable to that of
drying–rewetting (Clein and Schimel, 1994). We then measured microbial
respiration in slowly melted field-moist soils twice during the adaptation
period (days 4 and 12 of the incubation), which allowed the microbial activity
to stabilize after respiratory flushes following sieving pretreatment
(Thomson et al., 2010) and freeze–thaw events (Schimel and Clein, 1996), and
on day 13, when we expected microbial activity to have settled. Briefly, on
day 1, soil subsamples (10 g) were incubated in 100 mL flasks at
6 ∘C, which corresponds to the mean summer soil temperature of all
sites along Tr1. On days 4, 12 and 13, the cumulative CO2 production
from the soils was measured using an Agilent 6850 GC system (Agilent
Technologies, CA, USA). The flasks were then thoroughly ventilated and sealed
again. Due to the high soil pH, the total amount of produced CO2 was
corrected for its dissolution and dissociation in soil solution according to
the Henderson–Hasselbach equation (Sparling and West, 1990) and expressed as
the microbial respiration rate per day.
Microbial biomass and community structure
The soil microbial community structure was determined using PLFA analysis
according to a modified protocol of Frostegård et al. (1993). Briefly,
1–3 g (according to TOC content) of freeze-dried soil was extracted twice
with a single-phase extraction mixture consisting of chloroform, methanol and
citrate buffer. After phase separation overnight, achieved by adding more
chloroform and buffer, the organic phase was purified on silica columns
(LC-Si SPE Supelclean 250 mg/3 mL; Supelco®,
PA, USA) using chloroform, acetone and methanol. The polar fraction was
trans-esterified to fatty acid methyl esters (FAMEs) (Bossio and Scow, 1998).
All FAMEs were quantified using methyl-nonadecanoate (19:0) as an internal
standard. To identify the FAMEs, retention times and mass spectra were
compared with those obtained from standards (Bacterial Acid Methyl Esters
standard, 37-component FAME Mix, PUFA-2 and PUFA-3; Supelco, USA). An ISQ
mass spectrometer (MS) equipped with a Focus gas chromatograph (GC) (Thermo
Fisher Scientific, USA) was used for chromatographic separation and
detection.
Only specific PLFAs were used to assess the microbial community structure:
a14:0, i15:0, a15:0, i16:0, i17:0 and a17:0 were used as markers
of Gram-positive bacteria (G+); 16:1ω9, 16:1ω5, cy17:0,
18:1ω11, 18:1ω7 and cy19:0 as markers of Gram-negative
bacteria (G-); 10Me16:0 and 10Me18:0 as markers of Actinobacteria
(Kroppenstedt, 1985); 18:1ω9 and 18:2ω6,9 as fungal markers
(Frostegård and Bååth, 1996); and the polyunsaturated fatty acids
18:4ω3 and 20:5ω3 were used as markers of phototrophic
microorganisms (Hardison et al., 2013; Khotimchenko et al., 2002). The sum of
Actinobacterial markers, PLFAs specific to G+ and G- bacteria, and
general bacterial markers 15:0, 17:0 and 18:1ω5 were used to
calculate bacterial biomass and ratio of fungi to bacteria (F/B). The sum
of all lipid markers mentioned above and nonspecific PLFAs 14:0, 16:0,
18:0 and 16:1ω7 was used as a proxy for microbial biomass
(PLFAtot).
Sterol analyses
The β-sitosterol and brassicasterol were used as biomarkers of plant
(Sinsabaugh et al., 1997) and microalgal (Volkman, 1986, 2003) residues in
organic matter (OM), respectively. Both sterols were determined
simultaneously using microwave-assisted extraction adapted from Montgomery et
al. (2000) and GC/MS analysis (ISQ MS equipped with Focus GC, Thermo Fisher
Scientific, USA). Briefly, 0.5 g of freeze-dried soil was treated with 6 mL
of methanol and 2 mL of 2 M NaOH. Vials were heated twice at the center of
a microwave oven (2450 MHz and 540 W output) for 25 s. After cooling, the
contents were neutralized with 1 M HCl, treated with 3 mL of methanol and
extracted with hexane (3 × 4 mL). Extracts were spiked with an
internal standard (cholesterol), evaporated and derivatized by adding
pyridine and 1 % BSTFA at 60 ∘C for 30 min prior to analysis.
The sterols were quantified using an internal standard calibration procedure.
Statistical analyses
All data were checked for normality and homoscedasticity, and log-transformed
if necessary. The relative PLFA data (mol %) were log-transformed in all
statistical tests. The significance of environmental gradients and
corresponding shifts in MCS (mol % of summed PLFA specific for fungi,
G- and G+ bacteria, Actinobacteria, and soil phototrophic microorganisms)
in the horizontal (i.e., effect of transect) and vertical directions (i.e.,
effect of altitude) was tested using partial redundancy analyses (RDAs) with
covariates. Variation partitioning was subsequently performed to quantify the
unique and shared effects of transect and altitude on variability in MCS. A
forward selection procedure was used to identify the soil geochemical
parameters best explaining shifts in MCS. During the forward selection
procedure, only P values adjusted with Holms correction were considered.
This procedure is slightly less conservative than the often recommended
Bonferroni correction, but it is a sequential procedure and takes into
account that the candidate predictors with stronger effects are selected
first (Holm, 1979). The multivariate tests were performed without
standardization by samples, but with centering and standardization by
variables (because the variables were not always measured on the same scale;
see Šmilauer and Lepš, 2014) and a Monte Carlo test with 1999
permutations. Only the adjusted explained variation is referred to throughout
the text. As the triplicate samples cannot be considered as independent
observations due to the relatively small distance between samples (otherwise
there would be nine independent transects), only the sampling sites were freely
permuted, while the individual samples were exchangeable only within the
sampling sites. The differences in particular soil and microbial parameters
between the respective transects and altitudes were addressed with ANOVA
complemented with Tukey HSD post hoc tests. Pearson correlation coefficients
were used to assess how tightly different variables were related to each
other. All statistical tests were considered significant at P < 0.05. Multivariate statistical analyses were performed with
CANOCO for Windows version 5.0 (Ter Braak and Šmilauer, 2012). For ANOVA,
Tukey HSD tests and correlations between soil and/or microbial parameters,
Statistica 13 was used (StatSoft, USA).
Results
Altitudinal changes in soil microclimate
The soil microclimate at the sites studied was characterized by two distinct
periods reflecting air temperature dynamics (compare Fig. S2a with b). The
winter period typically lasted from the middle of September to early June.
The winter soil temperatures were stratified according to elevation and mean
temperatures decreased from -4 ∘C at 25 m a.s.l. to
-10 ∘C at 765 m a.s.l. (Table 1, Fig. S2). By contrast, the
short summer period was characterized by a significant diurnal fluctuation of
soil temperatures and weak altitudinal temperature stratification (Fig. S2).
The length of the summer season more than doubled at the lowest elevations
compared with the most elevated study sites. The period with daily mean soil
temperatures above 5 ∘C was shorter by a factor of almost 4 at
the highest elevation. The positive surface energy balance gradually
decreased with increasing altitude (Table 1). The maximum daily mean
temperatures and diurnal temperature fluctuations were highest at the
mid-elevation sites; the highest mean summer soil temperatures were observed
at 275 m a.s.l. By contrast, the least and most elevated sites experienced
lower summer maximum daily means and soil temperature amplitudes (Table 1).
The effect of altitude on soil moisture was significant along Tr1 and Tr3 (P < 0.001 and 0.01, F=22.76 and 7.39, respectively), with soil
moisture content decreasing with increasing elevation, but nonsignificant
along Tr2. Continual volumetric measurements of soil water content at
AWS25 and AWS455 showed that soil moisture was relatively stable
during the summer season, and no desiccation events occurred during the summer
periods of 2011–2013 (for more information, see Fig. S3).
Geochemical characteristics of soils along three altitudinal
transects (Tr1–Tr3). Means ± SD (n=3) are given in the upper
part of the table. Results of two-way ANOVA (F values) of the effects of
transect (Tr), altitude (Alt) and their interaction (Tr × Alt) are presented
in the lower part of the table.
Transect
Altitude
Soil type
Soil moisture
pH
CEC
Ca2+
Mg2+
K+
Na+
(m a.s.l.)
(%)
(meq/100 g-1)
(mg g-1)
(mg g-1)
(µg g-1)
(µg g-1)
Tr1
25
sandy loam
a28.4 ± 2.5
b 7.8 ± 0.1
a35.8 ± 0.4
b4.9 ± 0.2
c0.50 ± 0.03
b104 ± 2.3
a16.0 ± 1.4
275
sandy loam-loam
b18.0 ± 0.5
b7.9 ± 0.2
b27.4 ± 2.3
b5.2 ± 0.6
c0.55 ± 0.08
b81 ± 8.8
bc8.4 ± 1.3
525
loam
b18.6 ± 2.5
b8.1 ± 0.1
b30.3 ± 0.7
b4.3 ± 0.4
b0.85 ± 0.04
a160 ± 18.1
b11.3 ± 1.1
765
clay-loam
c12.1 ± 1.8
a9 ± 0.0
b26.8 ± 2.3
a19.8 ± 1.0
a1.25 ± 0.06
c11 ± 2.7
c7.3 ± 0.0
Tr2
25
sandy loam
a21.1 ± 2.4
c7.8 ± 0.1
b25.6 ± 2.7
b14.7 ± 2.6
c0.19 ± 0.01
ab52 ± 4.0
a13.2 ± 1.7
275
sandy loam-loam
a21.1 ± 2.4
c7.9 ± 0.1
b30.3 ± 1.7
ab16.5 ± 1.1
b0.26 ± 0.01
a59 ± 4.3
ab10.1 ± 1.7
525
sandy loam-loam
a21.7 ± 5.3
b8.4 ± 0.1
b30.8 ± 1.1
c7.8 ± 1.6
a0.34 ± 0.01
a69 ± 3.3
ab9.6 ± 1.8
765
loam
a22.5 ± 1.7
a8.8 ± 0.1
a45.1 ± 0.5
a27.9 ± 9.3
b0.25 ± 0.01
b41 ± 8.8
b8.1 ± 1.4
Tr3
25
sandy loam
a39.5 ± 1.4
b8.1 ± 0.1
a49.4 ± 2.1
c7.7 ± 0.3
a0.20 ± 0.03
b52 ± 5.3
a17.1 ± 1.1
275
sandy loam-loam
ab31.9 ± 2.9
b8.1 ± 0.1
b39.2 ± 5.4
b10.8 ± 0.6
a0.21 ± 0.01
ab59 ± 1.9
a18.5 ± 0.5
525
loam
ab28.2 ± 6.5
b8 ± 0.1
b34.9 ± 3.0
ab13.0 ± 4.6
a0.22 ± 0.00
a66 ± 6.6
a18.4 ± 3.1
765
loam
b22.5 ± 1.7
a8.8 ± 0.1
b30.6 ± 3.9
a14.2 ± 0.1
b0.16 ± 0.00
b52 ± 1.6
b9.9 ± 0.2
d.f.
Tr
2
31.4***
0.10
22.1***
6.43**
634***
51.7***
36.2***
Alt
3
11.1***
98***
4.61*
14.1***
66.9***
74.9***
18.7***
Tr × Alt
6
5.07**
5.6***
20.5***
0.83
60.6***
31.6***
3.94**
Different letters indicate significant differences between
sampling sites along particular transects (P < 0.05; upper part
of the table). Statistically significant differences are indicated by
* P < 0.05, ** P < 0.01 and *** P < 0.001 (lower part of the table).
Total soil carbon (TOC) and nitrogen (TN) contents, their molar
ratios, contents of sitosterol in TOC, and sitosterol / brassicasterol
ratios in soils along three altitudinal transects (Tr1–Tr3).
Means ± SD (n=3) are given in the upper part of the table. Results
of two-way ANOVA (F values) of the effects of transect (Tr), altitude
(Alt) and their interaction (Tr × Alt) are presented in the lower part of the
table.
Transect
Altitude
TOC
TN
TOC / TN
Sitosterol
Sitosterol / brassicasterol
(m a.s.l.)
(mg g-1)
(mg g-1)
(µg g-1 TOC)
Tr1
25
c70.6 ± 13.4
b5.0 ± 1.01
b12.1 ± 0.2
c534 ± 62.8
b5.5 ± 0.4
275
b21.1 ± 1.9
a2.0 ± 0.29
ab9.0 ± 0.7
bc521 ± 140
b5.3 ± 0.8
525
b18.5 ± 4.2
a1.8 ± 0.31
ab8.8 ± 0.7
ab293 ± 66.5
b4.7 ± 1.0
765
a4.4 ± 1.5
a0.5 ± 0.07
a7.9 ± 2.6
a81.1 ± 2.7
a2.3 ± 0.4
Tr2
25
ab30.6 ± 4.8
a1.9 ± 0.40
c13.7 ± 0.9
bc515 ± 44.9
b6.7 ± 0.7
275
b37.2 ± 5.0
a3.0 ± 0.26
b10.7 ± 0.7
c616 ± 143
b5.6 ± 1.2
525
a24.4 ± 7.8
a1.9 ± 0.64
b9.8 ± 1.2
ab299 ± 73.3
a2.9 ± 0.4
765
a21.6 ± 3.6
a2.8 ± 0.20
a6.7 ± 0.6
a161 ± 36.9
a2.7 ± 0.7
Tr3
25
c81.1 ± 8.7
b6.1 ± 0.38
b11.5 ± 0.7
b587 ± 144
b6.4 ± 2.1
275
b62.2 ± 9.1
ab4.8 ± 0.32
b11 ± 0.7
ab370 ± 42.9
a4.2 ± 0.7
525
ab39.6 ± 11.4
a4.8 ± 0.32
b10.6 ± 0.6
a270 ± 112
a3.3 ± 1.0
765
a23.1 ± 3.9
a2.5 ± 0.37
a7.9 ± 0.2
a151 ± 37.8
a3.1 ± 0.9
d.f.
Tr
2
27.8***
31.5***
1.57
0.79
1.04
Alt
3
42.4***
26.4***
23.6***
28.4***
14.4***
Tr × Alt
6
8.33***
11.3***
1.96
1.34
2.17
Different letters indicate significant differences between sampling sites
along particular transects (P < 0.05; upper part of the table).
Statistically significant differences are indicated by * P < 0.05, ** P < 0.01 and
*** P < 0.001 (lower part of the table).
The soil PLFA contents (a) and potential respiration rates measured
at day 13 (b) and at day 4 (c) in soils from different elevations along
three altitudinal transects (Tr1–Tr3). Error bars indicate mean ± SD
(n=3). Lowercase letters denote significant differences between
altitudes within particular transects (P < 0.05; one-way ANOVA
combined with Tukey post hoc test).
Gradients of soil geochemical properties and surface vegetation
cover
Both factors, transect and altitude, significantly affected soil geochemical
properties (partial RDA, pseudo–F=8.3, P < 0.001) and
explained 61 % of the total variation in soil characteristics. The RDA
ascribed most of the explained variability (73 %) to vertical zonation.
Accordingly, altitude had a significant effect on all soil parameters (Tables 2, 3,
Fig. S4), but a significant interactive effect of transect and altitude
indicated that the elevational trends were in most cases specific for
particular transects (Tables 2, 3). Especially CEC and the availabilities of
Ca2+, Mg2+, K+ and Na+ were spatially variable,
reflecting the complicated geology of the Petunia Bay area. The soils along
Tr1 were significantly richer in available Mg2+ and K+ than soils
from the other two transects (Table 2). Mg2+ availability also
significantly increased with increasing elevation along Tr1 (Table 2). Other
soil properties showed more systematic altitudinal patterns. The mean soil pH
ranged from 7.8 to 9.0 and increased with altitude along all transects
(Table 2, Fig. S4). By contrast, the soil TOC and TN contents declined
towards higher elevations along all transects, the exception being the lowest
site of Tr2, with lower soil OM content compared with the respective sites
from Tr1 and Tr3. The OM-poorest soil occurred at the highest site of Tr1
(Table 3). The soil C / N ratio, sitosterol content in TOC and the ratio
between plant-derived sitosterol and brassicasterol of algal origin were
solely affected by the altitude. Their values systematically decreased with
an increasing elevation irrespective of the soil OM content (Table 3),
indicating an altitudinal shift in the OM quality and origin. The percentage
of plant cover also continuously decreased with an increasing elevation along
Tr1 and Tr3 but was comparable at the three lower sites along Tr2 (Fig. S5),
which significantly reflected the trends in soil OM content (r=0.53; P=0.001). Lichenized soil crusts were the predominant type of soil surface
cover at all sites, while mosses covered very small proportions of the
surface area. Bare surface without any vegetation (bare soil) occurred only
at the two most elevated sites of all transects (Fig. S5, Table S1).
Correlation between the abundance of the main microbial groups
(bold italic) and the soil geochemical parameters that were retained by
forward selection from all explanatory variables collected. The altitude of
sampling sites was used as a supplementary variable. Arrows indicate the direction
in which the respective parameter value increases, solid lines indicate microbial
groups, dotted lines indicate environmental variables retained by the forward
selection procedure. Upright triangles are centroids of sites with corresponding elevation (n=9) and numbers indicate elevation (m a.s.l.). The thin solid line encases
sites along Transect 1 (Tr1), the dashed line encases sites along Transect 2
(Tr2) and the dotted line encases sites along Transect 3 (Tr3). The downward triangles represent
sites at 25 m a.s.l., the circles represent sites at 275 m a.s.l., the squares represent sites at 525 m a.s.l.,
and the crosses represent sites at 765 m a.s.l. The numbers
in parentheses are the portions of the variation explained by each axis.
Relative abundance of specific PLFAs within the microbial community
(a) and fungi to bacteria (F/B) ratios (b) along
altitudinal transects (Tr1–Tr3). Error bars indicate mean ± SD (n=3). Lower case letters denote significant differences between altitudes
within particular transects (P < 0.05; one-way ANOVA combined
with Tukey post hoc test).
Soil microbial biomass and activity
Soil PLFA content, used here as a measure of soil microbial biomass, was
significantly positively correlated with soil TOC and TN contents (r=0.773 and 0.719, respectively; both P < 0.0001) and soil
moisture (r = 0.772; P < 0.0001). It was negatively
affected by Mg2+ availability (r=-0.775; P < 0.0001).
Despite these relations, soil PLFA content did not show any altitudinal
pattern. The amounts of soil PLFA were comparable among the differently
elevated sites along particular transects (Fig. 2a). Only the most elevated
site of Tr1 had significantly lower soil PLFA content than other sites, which
corresponded with its very low stock of OM (Table 3). Similarly, neither the
flush of microbial respiration measured after soil thawing (day 4 of
incubation) nor the respiration measured after stabilization (day 12, not
shown, and day 13) showed any systematic altitudinal pattern (Fig. 2b, c).
Generally, the flush respiration rate was closely related (r=0.74,
P < 0.0001, n=36) to microbial respiration after
stabilization and 2.3 ± 0.3 times higher than the latter, showing a
similar freeze–thaw effect on the whole set of samples independent of
altitude and transect. The microbial respiration rates measured between
days 4 and 12 and after stabilization (day 13) did not
significantly differ in any soil samples. The three lower sites along each
transect (from 25 to 525 m a.s.l.) had comparable microbial respiration
rates after stabilization. However, the most elevated site of Tr1 had a
significantly lower microbial respiration rate, whilst the most elevated
sites of Tr2 and Tr3 produced markedly more CO2 than the other sites
along these transects (Fig. 2b). The respiration rate was neither related to
PLFA nor to TOC contents, but significant positive correlations with soil
Ca2+ availability and F/B ratio, and a negative correlation with
Mg2+ availability (r=0.489, 0.661 and -0.545; P=0.003,
< 0.001 and 0.001, respectively) was observed.
Microbial community structure
The factors altitude and transect explained 51 % of the total variation
in the MCS, with 66 % of the explained variability ascribed to altitude,
26 % to transect and 8 % shared by both factors. In fact, the
partial RDA revealed a significant interactive effect of both factors on MCS
(pseudo–F= 4.8, P < 0.001). The soil geochemical variables
explained 72 % of the variation in the MCS (pseudo–F= 7.1; P < 0.001), indicating that the separate and interactive effect of
altitude and transect on MCS was largely driven by vertical and horizontal
variability in soil properties. The forward selection of explanatory
variables retained four geochemical parameters: Mg2+ availability, pH,
moisture and TOC content, all together accounting for 55 % of variation
in the data (pseudo–F= 11.6, P < 0.001). The most
pronounced shift in the MCS was caused by different altitudinal preferences
of bacteria and fungi. Bacteria were consistently more abundant in the soils
from lower elevations, having lower pH and higher TOC and moisture contents
(Fig. 3). In general, PLFAs specific to G- bacteria were more abundant than
PLFAs of G+ bacteria (Fig. 4a; mean G-/G+ ratio
± SD = 1.76 ± 0.17; n=36). By contrast, the fungal
contribution to the microbial community increased with increasing altitude,
at the sites with TOC poorer soils and higher pH (Fig. 3). Therefore, the
F/B ratio gradually increased with increasing altitude along all three
transects (Fig. 4b). The significant interactive effect of altitude and
transect on MCS was mainly connected to a strong effect of soil Mg2+
availability, which was higher along the whole Tr1 and differentiated its
microbial communities from the sites of Tr2 and Tr3, where microbial
communities of corresponding sites were more similar. The differences in MCS
among the respective sites along Tr1 on the one hand and the other two
transects on the other hand further increased towards higher elevations, together
with increasing soil Mg2+ availability along Tr1 (Fig. 3). As a result,
the soil with the poorest TOC content and highest Mg2+ concentration at
the highest site on Tr1 had the most distinct MCS of all sites. Its microbial
community was characterized by higher abundance of Actinobacteria and PLFAs
of phototrophic microorganisms and a much lower contribution of G- bacteria
compared with the communities at all other sampling sites (Figs. 3, 4a).
Discussion
Our measurements showed that soils along an elevation gradient from 25 to
765 m a.s.l. face different microclimatic regimes. During the winter
period, soil temperatures were relatively stable but significantly stratified
according to altitude (Figs. S1, S2a). The significant decrease in mean
winter soil temperatures with increasing elevation (Table 1) can strongly
reduce winter soil microbial activity at high altitudes compared with the
least elevated sites (Drotz et al., 2010; Nikrad et al., 2016). The
comparison of summer temperatures and their fluctuations indicated that both
the lowest and highest sites experienced on average a colder but more stable
soil microclimate during summer compared with the mid-elevation sites. The
significantly longer summer season, increasing number of days with a mean
temperature above 5 ∘C and a rising positive surface energy balance
with decreasing elevation (Table 1) positively affected the occurrence and
proliferation of vascular plants (Kleidon and Mooney, 2000; Klimeš and
Doležal, 2010), which had strong implications for the change in edaphic
conditions along transects. The pronouncedly greater plant cover at lower
elevations (Table S1) resulted in increased litter inputs and greater stocks
of soil OM with a higher C / N ratio (Table 3). Plant growth was also
associated with decreasing soil pH via root respiration, cation uptake, and
release of H+ and organic acids from roots (van Breemen at al., 1984).
The increasing soil OM content was further positively related to soil
moisture (Fig. 3).
The altitudinal shifts in soil edaphic properties were not significantly
reflected in soil microbial biomass and potential microbial respiration. Soil
PLFA contents were generally comparable between all sites along any
particular elevation transect, with the exception of the very low soil PLFA
concentration at the highest site of the Tr1 (Fig. 2a). There are no other
studies from High Arctic ecosystems reporting an altitude effect on soil
microbial biomass. However, other studies conducted on alpine gradients in
the temperate and boreal zones documented weak or absent altitudinal trends
in the microbial biomass (Djukic et al., 2010; Xu et al., 2014, using PLFA;
Löffler et al., 2008, using cell counts) but also a negative effect of
elevation in the Alps (Margesin et al., 2009) and northern Finland (Väre
et al., 1997). Importantly, none of the studies considered unvegetated
habitats and all of them were conducted in soils with acidic or neutral soil
pH.
Microbial respiration did not change systematically with increasing
elevation. The three lowest sites along each transect always had comparable
soil microbial respiration rates (Fig. 2b), while soil microbial activities
at the highest sites differed. The most elevated site on Tr1 showed
significantly lower respiration rates than the lower sites on this transect,
which was in line with the lowest OM content and soil PLFA content. However,
the soils from the highest sites on both Tr2 and Tr3 respired significantly
more than the soils from lower sites on these transects, irrespective of the
relatively stable microbial biomass along these transects. This is in
contrast to other studies, which reported decreasing microbial activity with
increasing elevation (Schinner, 1982 – Alps; Väre et al., 1997 –
northern Finland; Niklińska and Klimek, 2007 – Polish Carpathians).
However, these studies were conducted at lower latitudes and the altitudinal
gradients studied did not include unvegetated habitats.
Microbial activities in this study were measured in sieved freeze-stored and
not intact fresh samples (see Sect. 2.3 for details). The respiration
rates measured after thawing were necessarily affected by sample handling and
show the potential activity of soil microbial communities in the soils. The
in situ microbial respiration could differ from those values as sieving and
freezing–thawing has been shown to affect soil C and N fluxes (Hassink et
al., 1992) and increase respiration rates (Thomson et al., 2010). The effect
of sieving on measured soil respiration could be stronger in samples from the
most elevated sites due to disruption of the vertical organization of the
biological soil crusts (Belnap and Lange, 2003) compared with the more
homogeneous soils from lower altitudes. However, the respiration rates in
three subsequent measurements (after flush – day 4, after adaptation –
day 12, and after stabilization – day 13) were positively correlated (r= 0.93 and 0.74, both P < 0.0001, n=36), the ratios between
the flush and stabilized respiration rates were comparable across all soils
(compare Fig. 2b and c), and the differences in microbial activities between
sites described above were consistent. Our data are also in agreement with
the study of Larsen et al. (2002), who found a comparable response of
microbial activity to freeze–thaw events in two different arctic ecosystem
types. We thus suggest that the soils responded similarly to the storage
treatments, independent of site location, and that the observed differences
in potential soil microbial activities are representative for the transects
studied. Therefore, the higher soil microbial respiration at the most
elevated sites points to a greater lability of the present OM (Lipson et al.,
2000 – Rocky Mountains; Uhlířová et al., 2007 – Siberian
tundra) and/or to a shift in microbial communities towards groups with higher
potential to mineralize the OM (Gavazov, 2010 – Alps; Djukic et al., 2013 –
Alps). Previous studies, considering either bare soil or vegetated habitats,
reported an increasing complexity of soil OM with elevation (Ley et al., 2004
– Rocky Mountains; Xu et al., 2014 – Himalayas). However, in this study the
bulk of OM and microbial biomass at the most elevated sites was associated
with biological soil crusts with high algal and cyanobacterial abundance
(Table S1, Fig. S5), known for their high microbial activity (Pushkareva et
al., 2017 – Svalbard; Bastida et al., 2014 – Spain). The high microbial
activity at the most elevated sites could be ascribed to a prevalence of
compounds of algal or cyanobacterial origin with very low proportion of complex
and slowly decomposable compounds and protective waxes (like cutin and
suberin) mainly derived from vascular plants. In accordance with this, the
sitosterol-to-brassicasterol ratio gradually decreases with increasing
elevation (Table 3) and the increasing sitosterol content in the TOC pool at
lower elevations points to a growing importance of microalgal sources of OM in
high-elevation habitats (Sinsabaugh et al., 1997; Rontani et al., 2012). Even
though both sterols can be found in higher plants and microalgae, the
changing ratio indicates a shift in the origin of OM (reviewed by Volkman,
1986, see also Volkman, 2003). Changes within microbial communities, which
can also help to explain higher soil microbial respiration at the most
elevated sites, are discussed below.
While the soil PLFA content did not change along the elevation transects
studied, we found a systematic altitudinal shift in PLFA composition,
resulting in a significantly increasing F/B ratio towards higher
elevations. This shift was best explained by a decreasing soil OM content and
soil moisture and increasing pH (Fig. 3). Reports about soil F/B ratios and
their altitudinal changes from the High Arctic are missing, but studies from
lower latitudes showed either a similar trend of increasing F/B ratio with altitude in the Alps (Margesin et al., 2009) or the opposite altitudinal
effect in the Alps (Djukic et al., 2010) and Himalayas (Xu et al., 2014; Hu
et al., 2016). Such divergent results indicate that altitude alone is not the
key driving factor of the soil F/B ratio. In contrast to our observations,
these studies reported very low soil F/B ratios of 0.05–0.2, which may
indicate an important role of fungi in the functioning of arctic habitats.
Soil pH has previously been identified as the main driver of fungal vs.
bacterial dominance in the soil (Bååth and Anderson, 2003; Högberg et
al., 2007; Rousk et al., 2009; Siles and Margesin, 2016). Fungi have been
found to be more acid tolerant than bacteria, leading to a higher F/B ratio
in acidic soils (Högberg et al., 2007; Rousk et al., 2009; reviewed by
Strickland and Rousk, 2010). However, here we report high F/B ratios in
alkaline soils (pH 7.8–9.0) and an increase in F/B ratios with increasing
soil pH. A similar trend was reported by Hu et al. (2016), but the authors
found F/B ratios 1 order of magnitude lower than in our study. A possible
explanation of the generally high fungal abundance and increasing F/B ratio
with elevation could be a greater competitiveness of fungi compared to
bacteria at sites with highly alkaline soil pH and severe winter microclimate
due to their wider pH (Wheeler et al., 1991) and lower temperature (Margesin
et al., 2003) growth optima. We further found that the increasing F/B ratio
was significantly coupled with an increasing soil respiration (r=0.649;
P < 0.001). Indeed, such a relationship may be related to a
greater fungal ability either to grow in the soil conditions at the most
elevated sites or to utilize available C sources more efficiently (Ley et
al., 2004; Bardgett et al., 2005; Nemergut et al., 2005; van der Heijden et
al., 2008). In turn, the higher bacterial contribution at lower elevations
may be associated with a bacterial preference for utilization of labile root
exudates released by vascular plants (Lipson et al., 1999, 2002) and more
benign soil conditions represented by higher moisture contents and less
alkaline soil pH. However, direct linking of potential activities measured in
the incubation experiment with in situ abundance of bacteria and fungi may be
problematic as the handling of sample could alter the original MCS (Petersen
and Klug, 1994). As the projected warming in the Arctic (Collins et al.,
2013) will likely cause an upward migration of the vegetation and increasing
plant cover to the detriment of lichens and biological soil crusts (Vuorinen
et al., 2017; Yu et al., 2017; de Mesquita et al., 2017), the soil microbial
communities could respond by decreasing their F/B ratios at higher
elevations.
Apart from the systematic altitudinal shift in the F/B ratio connected
mostly with the plant occurrence and its effect on soil edaphic conditions,
we observed a strong shift in the bacterial composition, which differentiated
the altitudinal trends in the soil MCS along Tr1 from trends along Tr2 and
Tr3. This difference between transects increased towards higher elevations
and was best explained by Mg2+ availability. The character of the parent
substrate thus mostly controlled soil microbial properties at the most
elevated sites, which generally had low OM content (Fig. 3). The soils from
Tr1, except at the lowest site, had lower G--to-G+ bacterial ratios
within microbial communities than soils from the other two transects.
Further, the microbial community of the most elevated site of Tr1 showed
greater abundance of actinobacteria and phototrophic microorganisms than all
other sites (Figs. 3, 4a). This site was the most extreme habitat among all
the sites studied, with the highest proportion of bare, unvegetated soil
surface (Fig. S5), the lowest OM and moisture contents, the highest soil pH
and Mg2+ availability, and consequently also the most distinct microbial
characteristics (Figs. 2, 3). It is known that high Mg2+ availability
inhibits growth of many soil bacterial species. The inhibitive Mg2+
levels observed were 5 and 50 ppm for G- and G+ bacteria, respectively
(Webb, 1949), indicating that these bacterial groups significantly differ in
their tolerance for enhanced Mg2+ levels. Assuming that half of the
available Mg2+ was in soil solution and the average soil moisture
content was 20 %, the Mg2+ concentrations would have ranged
from approximately 16 to 140 ppm, which could explain the decreased
abundance of G- bacteria at sites with high Mg2+ availability. This
inhibitive Mg2+ effect is also in accordance with the negative
correlations between Mg2+ availability and soil microbial biomass and
respiration found in our study and could explain the lower microbial biomass
and respiration in the soils from Tr1. Our data thus indicate that, in
addition to
the traditionally identified drivers of microbial activity and MCS such as
soil OM content, moisture and pH, Mg2+ availability is an important
factor in shaping the microbial environment along arctic altitudinal
transects on dolomitic parent materials.