NITROGEN INPUTS BY BIOLOGICAL SOIL CRUSTS IN GRASSLANDS OF
INTERIOR BRITISH COLUMBIA
by
Kasia Caputa
B.Sc. Honours, University of Northern British Columbia, 2008

THESIS SUBMITTED IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
IN
NATURAL RESOURCES AND ENVIRONMENTAL STUDIES
(ENVIRONMENTAL SCIENCE)

UNIVERSITY OF NORTHERN BRITISH COLUMBIA
March 2013
© Kasia Caputa 2013

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ABSTRACT
The relationship between climate and inputs of nitrogen and carbon by biological soil
crusts (BSCs) was examined in British Columbia’s Chilcotin and Thompson River grasslands.
Variation in nitrogen and carbon of BSCs and associated soils was examined in relation to
gradients in temperature and moisture availability. Patterns of N-fixation activity by BSCs were
also examined in relation to climate, and used to model N- fixation rates throughout the year.
BSCs were found to significantly contribute to soil carbon and nitrogen, particularly in the labile
N fraction. Two peak periods were observed for N-fixation - one during the spring snowmelt, and
one during midsummer rainy periods. N-inputs appeared to be sensitive to changes in precipitation
patterns. Annual estimates of N-inputs for BSCs in this ecosystem could be as high as 83 kg N ha'
1but the fate of this N (i.e. plant uptake, denitrification or volatization) is still unknown.

TABLE OF CONTENTS
ABSTRACT................................................................................................................................... ii
LIST OF TABLES......................................................................................................................... vi
LIST OF FIGURES...................................................................................................................... vii
ACKNOWLEDGEMENTS............................................................................................................ix
BACKGROUND............................................................................................................................. 1
Grassland ecosystems of Interior British Columbia..................................................................1
Biological Soil Crusts............................................................................................................... 4
Carbon and Nitrogen Cycling in Grassland and Shrub-steppe Soils.........................................7
Biological N 2 Fixation............................................................................................................ 12
Biological Soil Crusts and Climate Change in Grasslands and Shrub-steppe........................15
RESEARCH OBJECTIVES.......................................................................................................... 19
CHAPTER 1:

NUTRIENT COMPOSITION OF BIOLOGICAL SOIL CRUSTS AND

ASSOCIATED SOILS ACROSS AN ELEVATION GRADIENT IN LAC DU BOIS
GRASSLANDS, BRITISH COLUMBIA.....................................................................................21
INTRODUCTION.....................................................................................................................21
METHODS................................................................................................................................23
Study Site...............................................................................................................................23
Field Methods.........................................................................................................................25
Data Analysis.........................................................................................................................26
RESULTS..................................................................................................................................26

29

DISCUSSION
CHAPTER 2:

SEASONAL PATTERNS OF NITROGEN FIXATION IN BIOLOGICAL

SOIL CRUSTS FROM BRITISH COLUMBIA’S CHILCOTIN REGION.................................. 34
INTRODUCTION.....................................................................................................................34
METHODS............................................................................................................................... 36
Study Site.............................................................................................................................. 36
Microclimate Measurements.................................................................................................. 37
Acetylene Reduction Analysis............................................................................................... 40
1^-Incubations......................................................................................................................42
Modeling BSC N-fixation activity under field conditions.....................................................43
RESULTS................................................................................................................................. 45
Farwell Canyon BSC Communities....................................................................................... 45
Acetylene Reduction Assays..................................................................................................47
BSC Microclimate Data.........................................................................................................47
Predicted BSC N-fixation....................................................................................................... 51
DISCUSSION............................................................................................................................57
Response of nitrogenase activity to microclimate and seasonal patterns............................... 57
Potential landscape-level nitrogen inputs............................................................................... 60
Seasonal patterns of nitrogen inputs by Chilcotin BSC communities.................................... 62
SUMMARY AND CONCLUSIONS............................................................................................64

REFERENCES

LIST OF TABLES
Table 1.1 Summary of climate normals at Kamloops Airport (Environment Canada 2012) and
climate estimates at different elevations in Lac du bois Grasslands, British Columbia*.............. 24
Table 1.2: Selected Chemical Properties of Biological Soil Crusts and underlying mineral soils at
Lac du Bois Grasslands, British Columbia. The mean differences reported are significantly
different based on a paired two-sample t-test (P<0.05).................................................................27
Table 2.1. Climatic characteristics of the very hot dry bunchgrass biogeoclimatic subzone
(BGxh3) for the study site (Steen and Coupe 1997)......................................................................36
Table 2.2. Summary of climate normals at Williams Lake Airport (from Environment Canada
2012) and at Farwell Canyon, British Columbia (estimated from Climate B.C. modelling using
Wang et al. (2012)..........................................................................................................................37
Table 2.3. Summary of conversion ratio for ethylene production to 15N-incorporation (±SE) by
biological soil crusts incubated for 48 hours at 21°C and 200 pmol PAR m * V . n=10................43
Table 2.4. Species identified in BSC samples from Farwell Canyon study site. Species marked
with an "X" include those found in the dark BSC communities sampled for ARA incubations. 46
Table 2.5. Summary of estimated seasonal N-inputs per hectare by BSCs in a shrub-steppe in
Farwell Canyon, B.C. The estimates are based on conversion from modeled C2 H2 inputs using
both an experimentally derived C2 H2 :N2 conversion ratio of 0.06 and a commonly used
conversion ratio of 3. Landscape level N-input estimates were determined using a BSC cover
estimate of 52.7%...........................................................................................................................57

LIST OF FIGURES
Figure 1.1 Views of the upper elevation grasslands (top left), lower elevation shrub-steppe
(bottom left) and the representative biological soil crust (right) at the Lac du Bois Grasslands,
British Columbia (Photos - P. Sanborn)........................................................................................25
Figure 1.2: Total Carbon, Total Nitrogen and Mineralizable Nitrogen in the biological soil crusts
(BSC) and underlying soil (1-1 Ocm) across an elevation gradient in Lac du Bois Grasslands,
British Columbia. Error bars represent standard error and letters denote significant differences
(p<0.05) across soil type and elevation class................................................................................. 28
Figure 1.3: Ratio of Total C:N and 1SN natural abundance in the biological soil crusts (BSC) and
underlying soil (1-1 Ocm) across an elevation gradient in Lac du Bois Grasslands, British
Columbia. Error bars represent standard error and letters denote significant differences (p<0.05)
across soil type and elevation class................................................................................................ 29
Figure 2.1 View of the vegetation and landscape representative of the study site, at Farwell
Canyon, British Columbia..............................................................................................................38
Figure 2.2. Calibration curve for mean relative impedance response (n=8) and gravimetric
moisture content (MC) of field collected BSC (n=8-12). Error bars represent standard error...... 39
Figure 2.3. Temperature and Light Response of acetylene reduction assay (ARA) fitted with
multivariate curves by BSCs collected in (A) March and (B) July, n =10 for each datapoint.....48
Figure 2.4. (A) Relative ARA response to moisture content for BSCs pre-treated at optimum
conditions and incubated at 21°C and 200 pmol PAR m 'V 1 (B) Relative ARA response over
time starting at 2 hours after hydration to 30% moisture content.................................................. 48
Figure 2.5. Summary of BSC microclimate from 11 November 2009 to 1 October 2011 at
Farwell Canyon, British Columbia.................................................................................................49
Figure 2.6. View of biological soil crusts melting out from under snow cover in early March at
the Farwell Canyon study site. The width of the crust segment in the center of the image is ca. 9
cm.................................................................................................................................................. 50
Figure 2.7. BSC microclimate on south-facing aspect from 27 Feb. to 4 March 2011. From top:
predicted rates of acetylene reduction (ARA) from microclimate model, air and BSC surface
temperature, and incident photosynthetically active radiation (PAR) (pmol PAR m-2s-l ) at the
BSC surface................................................................................................................................... 51
Figure 2.8.(A-D) Time (in hours) spent by hydrated (> 7.5% MC w/w) BSCs and (E-H) the
estimated amount of C2 H2 fixed (in mmol of C2 H2 -m‘2) by hydrated BSCs at temperatures plotted
in 5°C class intervals for winter (October to April) and summer (May to September), for the
periods November 2009- September 2010 (Year 1) and October 2010 to September 2011 (Year
2). Calculations exclude periods of missing data (from 1 Nov - 11 Nov Year 1 and 9 Mar -13
Apr Year 2).................................................................................................................................... 52

Figure 2.9. Predicted ARA rates based on BSC microclimate measurements during a spring
snowmelt event at Farwell Canyon in March 2011. The symbols represent ARA rates measured
in situ with standard error (Y-error bars) and incubation time (X-error bars)................................53
Figure 2.10. Predicted ARA rates based on BSC microclimate measurements during a rain event
at Farwell Canyon in May 2011. The symbols represent ARA rates measured in situ with
standard error (Y-error bars) and incubation time (X-error bars).................................................. 54
Figure 2.11. Predicted ARA rates based on BSC microclimate measurements during a rain event
at Farwell Canyon in July 2011. The symbols represent ARA rates measured in situ with standard
error (Y-error bars) and incubation time (X-error bars).................................................................55
Figure 2.12. Monthly summary of BSC hydration time and estimated ARA activity at a shrubsteppe and shrub-steppe in Farwell Canyon, B.C. Asterisks denote time periods with missing data.
...................................................................................................................................................... 56

ACKNOWLEDGEMENTS
Financial support for this study was provided by the Provincial Government of British Columbia
as part of the Future Forests Ecosystems Scientific Council Grant. Thanks goes out to Ray Coupe
and Rick Tucker at the Williams Lake Range Branch, for assistance in site selection, and to Riske
Creek Ranch, for allowing us to work on their property.
I would like to thank Burkhard Budel, and Curtis Bjork for identification of biological soil crust
cyanobacteria and lichen species. Myles Stocki for providing 15N analysis and Clive Dawson for
the remaining soil chemical analyses. Thank you to Chuck Bulmer for providing a digital
elevation model for Lac du Bois park. Many thanks Katherine Stewart, Sarah Campbell, Kaela
Perry and Chris Fetterly for assistance in the field and/or laboratory.
None of this would have been possible without my co-advisors Darwyn Coxson and Paul
Sanborn, for providing endless support, knowledge and advice, both in the field and during the
analysis and writing stages. Also thank you to Lauchlan Fraser, for assistance and advice as a
Committee member.
Special thanks to my husband, family and friends, for your support and companionship on this
great journey.

BACKGROUND
Grassland ecosystems of Interior British Columbia
The grasslands and shrub-steppe in the interior plateau region of British Columbia are
unique ecosystems with high ecological and recreational value and a critical resource for the
Province's ranching industry. Roughly 90% of grasslands in British Columbia are used for
grazing domestic livestock (GCC 2004) and have been important to British Columbia's
ranching industry since the mid-1800s (Van Ryswyk and McLean 1989).
Grasslands and shrub-steppe occupy less than 1% of the total land base of the
province, yet provide a number of important ecosystem services, including water storage and
filtration, carbon sequestration and habitat to almost 30% of threatened and/or endangered
species in B.C. (GCC 2004). Most of the these areas in British Columbia seriously
deteriorated through intensive use for livestock grazing during the 19th and early 20th
centuries (Tisdale 1947, Wikeem and Wikeem 2004). Restoration efforts and improved range
management beginning in the mid 20th century have led to the slow recovery of some
grassland areas, but many are still highly threatened by conversion to urban and agricultural
land, encroachment by forest and invasive non-native species and overuse by poorly managed
grazing and recreational activities (GCC 2004). Climate change may introduce additional
impacts to these sensitive ecosystems and intensive study is needed to gain an understanding
of these impacts.
Physiography, Climate and Soils
The interior plateau of central British Columbia is located between the Coast Ranges
and Columbia Mountains. The topography is rugged and deeply dissected by the Fraser and
Thompson Rivers and their tributaries, which have steep-sided valleys 600 to 900 meters

below the plateau (Valentine 1978). Conditions are generally warm to hot and dry in the
summer, and moderately cold and dry in the winter. Most of the precipitation occurs in
December to January as either snow or rain, with a second peak of rainfall in June, while the
driest months are generally March and April (Meidinger and Pojar 1991). The climatic
influence by the rainshadow effect of the mountain ranges to the west is most intense in the
deeper valleys (Meidinger and Pojar 1991). The grasslands and shrub-steppe of the interior
plateau occur mainly in these valleys, where forest establishment is restricted by drought.
The soils found in these grasslands range from Brown to Black Chernozems; this
variation in soil type is based on the degree of moisture effectiveness (estimated
evapotranspiration minus precipitation). The Brown Chernozems occur at low elevations
where evapotranspiration is highest, and have formed predominately on lacustrine silts and
fluvial sands and gravels found in valley bottoms (Valentine 1978). These soils are
characterized by a thin A horizon with an average carbon content of 1.3% (Fenger 1982). The
Dark Brown and Black Chemozemic soils occur in subsequently drier and cooler areas,
usually at higher elevations, where most of the parent material is glacial till composed of a
variety of rock, including basalt lava, igneous granites, sedimentary shale, sandstone and
limestone (Van Ryswyk and McLean 1989). Dark Brown and Black Chernozems are
characterized by thicker and better expressed A horizons and increasing carbon content
compared to the Brown Chemozoms. A common feature to most of the soils in this region is a
silty aeolian veneer, on which most of the A horizons have formed (Fenger 1982). The
gradient of moisture effectiveness found across elevations also has an effect on a number of
soil physical and chemical properties. Some differences in soil properties with increasing
elevation include increased cation exchange capacity (CEC), increased potassium (K),

calcium (Ca), and magnesium (Mg), increased organic carbon, decreased C/N ratios, and
decreased soil bulk density (Van Ryswyk et al. 1966).
Ecology
The bunchgrass (BG) zone in British Columbia occurs from valley bottoms up to
approximately 700 - 1000 m in parts of the Okanagan valley, Similkameen River valley,
Thompson River valley, Nicola River valley, and the middle Fraser and lower Chilcotin River
valleys (Meidinger and Pojar 1991). This zone occurs mainly as isolated pockets within these
deeply dissected valleys, but existed up to 1,300 to 1,800 m a.s.l. during the early Holocene
(10,000 to 8,000 years ago). The bunchgrass zone represents the northern extent of the
grassland and shrub-steppe that occupies the northern part of the intermountain region
between the Rocky Mountains to the east and the Coast and Cascade Mountain to the west
(Wikeem and Wikeem 2004). Pseudoroegneria spicata ((Pursh) A. Love) and Artemisia
tridentata (Nutt.) are common throughout the region. These grasslands and shrub-steppe
developed with little influence from grazing by large ungulates, and are much more sensitive
to grazing impacts than those east of the Rocky Mountains (Mack and Thompson 1982).
The British Columbia Ecological Classification system describes two Bunchgrass
subzones that are found at two distinct elevational bands. Below 700 m, the lower grassland,
or BGxh (Very Dry Hot subzone), is a shrub-steppe ecosystem dominated by P. spicata, A.
tridentata and a well-developed biological soil crust community. Poa secunda (J. Presl),
Ericameria nauseosa (Pall, ex Pursh) and Artemisia frigida (Willd.) are also common. At
approximately 700 to 1000 m in elevation, the BGxw (Very Dry Warm subzone), or the
middle grassland, is dominated by P. spicata, and characterized by the absence of A.
tridentata. Other common species include P. Secunda, E. nauseosa, A. frigida, and

Koeleria macraniha ((Ledeb.) Schult.). The upper grasslands, found 900-1000 m in elevation,
are incorporated in the Interior Douglas-fir (IDF) zone, and are dominated by P. spicata,
together with Festuca scabrella (Torr. ex Hook. var. major Vasey), in the central parts of the
zone, and Stipa richardsonii ((Link) Barkworth) and Festuca saximontana (Rydb.) in the
Chilcotin (Meidinger and Pojar 1991). Van Ryswyk et al. (1966) described these changes in
vegetation zones as abrupt, and found that they were related to sharp differences in the ratio of
precipitation to temperature.
Biological Soil Crusts
Biological soil crusts (BSCs - also referred to as cryptogamic, microbiotic, or
biocrusts) are communities of lichens, bryophytes, free-living cyanobacteria and algae, micro­
fungi, and heterotrophic bacteria closely associated with the soil surface (Belnap 2003). BSC
occur in arid and semi-arid ecosystems throughout the world, achieving greatest cover in areas
with little vegetation. Much of the knowledge of BSCs in grasslands and shrub-steppe comes
from studies in the western United States, while BSC communities in British Columbia have
received relatively little attention (Marsh et al. 2006).
A number of important ecological functions in arid and semi-arid environments are
attributed to BSCs. Some of these functions include promoting soil stability and preventing
soil erosion by wind and water (Neff et al. 2005, Malam Issa et al. 2007), improving soil water
infiltration and/or storage (Belnap 2006), moderating fluctuations in soil climate (Veluci et al.
2006), influencing nutrient influx via dust entrapment (Williams et al., 2012) and contributing
to nutrient cycling and soil fertility (Evans and Lange 2001, Elbert et al. 2012). BSCs have
also been found to increase the uptake of certain trace minerals in nearby plants, creating
higher quality forage for herbivores (Harper and Belnap 2001) and plants growing on BSCs
4

have been found to have increased height, phytomass and nitrogen content (Langhans et al.
2009). These ecological functions often depend on the species composition and physical
structure of the BSC, which are predominately controlled by climate and disturbance history.
The diversity of BSCs is generally positively correlated with ecosystem function, but this can
depend on the function being studied (Bowker et al. 2010, Maestre et al. 2012). The
contribution of BSCs to ecosystem functions is also generally highest in relatively undisturbed
crusts with high biomass (Belnap 2003).
Biological soil crusts around the world are often dominated by similar species,
including the cyanobacteria Microcoleus vaginatus (Vaucher) Gomont, Nos toe commune
Vaucher Ex Bomet and Flahault, Schizothrix calcicola (C. A. Agardh) Gomont; the lichens
Collema tenax (Sw.)Ach., Fulgensia desertorum (Tomin) Poelt, Psora decipiens (Hedwig)
Hofftn, Placidium squamulosum (Ach.) Breuss; and the mosses Syntrichia ruralis (Hedwig),
Pterygoneurum ovatum Hedw. Dixon and Bryum spp. (Evans and Johansen 1999). The
dominant species composition of BSCs is often determined by the stage of succession. BSC
formation is initiated by non-heterocystous filamentous cyanobacteria, often Microcoleus spp.
These cyanobacteria initiate the process of stabilization through a cyclic process of binding
coarse soil particles and trapping fine atmospheric particles to form alternate layers of fine and
coarse sediments (Williams et al. 2012). In addition to stabilizing the soil, this process
improves the water holding capacity of the soil and prevents leaching of nutrients, creating a
suitable habitat for other BSC organisms to colonize (Yeager et al. 2004). These organisms
include free-living cyanobacteria Nostoc spp., Scytonema spp. and cyanolichens Collema spp.
that are capable of fixing nitrogen (ie. converting atmospheric nitrogen (N2) into ammonium
(NH4+)). Much of this fixed nitrogen is released into the soil, which can be the major source of
5

N in arid and semi-arid environments (Evans and Ehleringer 1993, Evans and Belnap 1999).
Frequent or intense disturbances can revert and keep BSCs in earlier successional stages, and
the rate of recovery to later successional stages has been found to vary by climate. Other
factors that can affect the species composition of BSCs in a given region include soil
chemistry, climate and vegetation (Ponzetti and McCune 2001, Root and McCune 2012).
The BSC occurring in grasslands and shrub-steppe of interior British Columbia
consists of lichens, bryophytes, free-living algae and cyanobacteria. The diversity of lichens
and bryophytes along an elevation gradient in the Chilcotin region of central interior British
Columbia is described in Marsh et al. (2006). Upper elevation sites have the highest number
of species, followed by lower, then middle sites. These differences in species richness may be
related to different disturbance histories rather than climatic variables. Grassland sites were
also characterized by different BSC species than shrub-steppe sites.
Differences in climate, species composition and age affect the microtopography of the
BSC, which influence its appearance, as well as the movement and infiltration of materials
such as dust, water and seeds (Belnap 2003). Early succession BSCs and those found in hot,
hyper-arid deserts, are dominated by cyanobacteria, which create a veiy smooth surface
structure. In regions with lower potential evapotranspiration, the dominant species shift to
lichens and mosses, which give the BSC a rougher surface. These differences in surface relief
can be explained by a greater affinity for dust trapping and accumulation by these organisms
compared to cyanobacteria alone (Williams et al. 2012). Repeated expansion and contraction
of the BSC surface caused by wet-dry cycles creates an increasingly complex surface
topography over time by creating cracks and pores that enhance dust capture, and building
pinnacles, towers and pedestals (Williams et al. 2012). The occurence of frost may also affect
6

the microtopography of BSCs, creating a pinnacled surface in areas dominated by
cyanobacteria and a rolling surface in areas dominated by lichens and mosses. The grasslands
and shrub-steppe of British Columbia are considered a cold, semi-arid environment, and the
BSC surface is primarily rolling. This type of surface promotes trapping of seeds and nutrientrich dust particles, soil porosity, formation of soil aggregates, abundance of soil invertebrates
and localized water infiltration and retention (Belnap 2003, 2006).
BSC organisms span a range of trophic levels, which allows them to contribute to a
wide selection of ecosystem functions. Also, unlike many plant communities, BSCs are able
to maintain many of these functions during periods of drought. Despite this resilience, BSCs
are highly sensitive to surface disturbance caused by grazers and human activity (Eldridge
1998). Such disturbances, particularly when they occur when the soil is dry, can destroy the
cohesive structure of the BSC layer. Changes in fire cycles and introduction of annual grasses
and other invasive species can also negatively impact BSC abundance, composition and
structure (Belnap et al. 2006), which in turn affect the ability of BSCs to perform ecosystem
services (Belnap 2003). Full recovery of BSC structure and function can take over a century in
cooler regions, and multiple centuries in hotter regions. This combination of high ecological
function and sensitivity to disturbance makes BSCs key candidates for studying the overall
health of arid and semi-arid ecosystems (Belnap 1996, Ponzetti and McCune 2001).
Carbon and Nitrogen Cycling in Grassland and Shrub-steppe Soils
The biogeochemical cycling of N is often of interest in terrestrial ecosystems because
it is a limiting nutrient for plant productivity (West 1991). Carbon inputs fuel soil food webs
and provide the energy needed for many biogeochemical processes. Carbon in the form of soil
organic carbon (SOC) also provides many benefits to soil structure. Soil carbon fluxes are
7

important for measuring the ability of different ecosystems to take up and sequester
atmospheric CO2 . Grassland soils may be of particular importance for carbon sequestration
due to their ability to store large amounts of SOC in the A horizons (Pennock et al. 2011).
BSCs have been found to be major contributors to both carbon and nitrogen cycling in arid
and semi-arid environments throughout the world (Elbert et al. 2012). In North America,
BSCs are responsible for up to 10% of the total fixed carbon and 80% of the total biologically
fixed nitrogen in terrestrial systems (Elbert et al. 2012).
Fluxes o f Soil C and N
The primary inputs of soil carbon in grasslands and shrub-steppe vary with climate,
seasonality, and the dominant vegetation. In more mesic and higher elevation environments
where grasses dominate, carbon input into the soil occurs via the breakdown of below-ground
tissues of grasses, thus creating a soil profile with little to no organic layer and a fairly deep A
horizon enriched in SOC (Pennock et al. 2011). In shrub-steppe ecosystems, soil carbon is
also added through the breakdown of leaf litter on the surface of the soil. The relative
contribution of BSCs to soil carbon increases in more xeric environments as plant cover
decreases. In areas that are too hot and dry to support a dense cover of grasses and shrubs,
BSCs become the dominant cover and the primary contributor of fixed carbon (Belnap et al.
2001). At times when plants cannot, BSCs are able to fix carbon due to their ability to rapidly
regain physiological function upon hydration after desiccation or freezing. The carbon gain
via net photosynthesis by BSC organisms is necessary for fueling nitrogen fixation in BSCs.
Carbon fixed by BSC organisms can enter the soil via leaching or decaying processes (Lange
2001). The soil carbon released by BSCs is an important step in the process of BSC
development and structure, and is also utilized by many heterotrophic organisms in the soil.
8

Losses of soil carbon can occur when respiration exceeds carbon gain via photosynthesis. Soil
carbon can also be lost by burning or directly via soil erosion. Increased temperatures tend to
lead to losses in soil carbon due to higher respiration rates and a decline in photosynthetic
activity if temperatures exceed the optimum for photosynthesis.
In arid and semi-arid environments, nitrogen is the most important factor limiting
biological activity after carbon and moisture availability (West 1991).The primary inputs of
nitrogen into soils are via atmospheric deposition, biological nitrogen fixation, and
decomposition of plant and animal residues. N-fixation by BSCs has been found to be the
dominant source of N in a number of arid and semi-arid ecosystems throughout the world
(Evans and Lange 2001). In B.C. grasslands and shrub-steppe, plants capable of symbiotic Nfixation are not common, thus it is expected that BSCs are also a major source of N in this
system. As much as 70% of the nitrogen fixed by BSCs is released to the surrounding soil,
mainly in the form of NCb* and small quantities of NH4 +, amides, peptides and free amino
acids, becoming available to plants and soil microbes (Belnap 2001). Nitrogen that enters the
soil can either be taken up by plants, leached down, lost by soil erosion, or returned to the
atmosphere via nitrification, denitrification and ammonia volatilization. Gaseous losses appear
to be the most prevalent in BSC associated soils, particularly the production of N 2 , N 2 O and
NO2 during nitrification and denitrification, which can account for as much as 80% of fixed N.
BSCs can provide ideal environments for these processes following precipitation events due to
the high availability of labile nitrogen and carbon caused by cell leakage (Evans and Johansen
1999). Conversely, BSC associated soils in the Colorado plateau have been found to have
negligible rates of denitrification due to extremely minimal presence of denitrifiers (Johnson
et al. 2007).
9

The natural abundance of the rare stable nitrogen isotope, 15N, can be used to study
nitrogen dynamics in an ecosystem (Robinson 2001). This approach takes advantage of the
fact that the isotopic composition (81SN) of N in the atmosphere is 0%o (per mil, or part per
thousand), and organisms and soil that receive most of their N from fixation will have 815N
near 0%o. Measurements of 8I5N have been used in this way to track the different pathways of
N-input into a desert ecosystem (Russow et al. 2004). Chemical reactions involving N result
in fractionation, as I5N is more energetically expensive to break or form chemical bonds with
than 14N. Thus soils where more N losses occur over time than N inputs will have a higher
S15N, which has been found on disturbed soils in arid and semi-arid envrionments (Evans and
Ehleringer 1993, Frank and Evans 1997, Evans and Belnap 1999, Aranibar et al. 2003).Values
of ,5N natural abundance that are within the range of -2%o to 2%o have been associated with
significant atmospheric inputs ofN (Evans and Lange 2001).
Distribution o f Soil C and N
The spatial distribution of nutrient availability and cycling in semi-arid grasslands and
shrub-steppe can be influenced by several interacting factors including landscape position
(Schimel et al. 1985, Verchot et al. 2002), vegetation type, microclimate and the physical
properties of the soil (Hook and Burke 2000). These interacting factors can be quite complex
and are drawn from studies in many different environments. Here I review how these factors
relate to the grasslands and shrub-steppe associated with biological soil crusts in our study
area.
Aird and semi-arid envrionments that are largely limited by moisture availability tend
to have a discontinuous aboveground cover of vegetation. This discontinuous cover has been
found to lead to the formation of resource islands, where nutrients and soil material
10

accumulate around plants, creating a heterogeneous distribution of soil nutrients. Shrubs and
grasses assist in resource island formation by the accumulation of litter from their above and
belowground tissues, and by acting as deposition sites for physical material redistributed by
erosion from plant interspaces. The resource island effect can be greatly counteracted by the
presence of well-developed BSCs (Housman et al. 2007). Biological soil crusts containing
later successional species of lichen and cyanobacteria allow a more homogenous distribution
of nutrients by directly fixing C and N, preventing soil loss and subsequent nutrient depletion
in the interspaces, and by creating habitat for soil fauna. The presence of BSCs can also
influence soil texture by trapping and holding particles in the silt/clay fractions through a
combination of surface roughness and the sticky cyanobacterial sheath (Belnap et al. 2001,
Williams et al. 2012). The negatively-charged silt/clay particles bind to plant essential
nutrients, increasing their availability relative to uncrusted soil.
The presence of grazers and the timing and density of grazing can also have a strong
effect on biogeochemical cycles. In the inter-mountain west, shrub-steppe is often
characterized by a clumped distribution of shrubs and grasses and a well-developed cover of
BSCs in the interspaces between plants (Mack and Thompson 1982). The evolutionary history
of this shrub-steppe includes very limited grazing by smaller ungulates (i.e. deer, sheep),
mostly during the winter. Livestock grazing in these ecosystems generally reduces C and N
input through changes in the species composition, cover and physiological activity of BSCs.
The decline in BSC cover also leads to increased losses of soil C, N and other nutrients due to
an increase in soil erosion by wind (Neff et al. 2005) and a decline in the physiological
activity of BSC organisms coupled with increased gaseous losses of N (Evans and Belnap
1999).
11

Biological N2 Fixation
The biological fixation of nitrogen is facilitated by the nitrogenase enzyme complex,
which is only found in certain prokaryotes. In biological soil crusts, most of the nitrogen
fixing organisms are free-living and lichenized cyanobacteria, including the genera Nostoc
and Scytonema (Belnap 2001). Nitrogenase is highly sensitive to oxygen and N-fixing
cyanobacteria must contain the enzyme within specialized cells called heterocysts that shelter
the reaction from O2 . Other N-fixing associations in BSCs include cyanobacteria living
epiphytically on mosses and chlorolichens (Belnap 2001).
The rate of nitrogen fixation can be measured directly by using I5N2, or indirectly
using acetylene reduction analysis (ARA). ARA measures the rate at which the nitrogen fixing
enzyme nitrogenase reduces acetylene (C2H2) gas to ethylene (C2H4 ), and provides an index of
nitrogenase activity (Stewart et al. 1967). ARA is preferred rather than measures of direct N2
input due to its ease of use and low cost. ARA provides a good measure of relative
nitrogenase activity, but must be calibrated to rates of direct 15N2 incorporation in order to
accurately estimate actual N-inputs (Evans and Johansen 1999, Belnap 2002). The theoretical
conversion ratio of ethylene production to N2 incorporation is 3:1, but has been found to vary
from 0.02 to as high as 56 (Belnap 2001). Conversion ratios can vary by species associations,
incubation temperatures and seasonality (Liengen 1999a).
Factors influencing N-fixation rates
It is important to understand the primary factors that influence rates of nitrogen
fixation in BSCs, as these rates have been found to vary both spatially and temporally. A
greater understanding of these factors can help explain variation and more accurately estimate
N inputs across the landscape. The dominant organisms present in BSCs influence the rates of
12

nitrogen fixation at optimal conditions (Belnap 2002). Light cyanobacterial crusts dominated
by Microcoleus, have the lowest rates of nitrogen fixation. Dark cyanobacteria crusts
dominated by Nostoc and Scytonema have intermediate rates of nitrogen fixation, while BSCs
dominated by Collema cyanolichens have the highest rates. The increase in nitrogenase
activity found between early and late successional stages of BSCs can be explained by an
increase in biomass of Nostoc and Scytonema (Housman et al. 2006).
Nitrogenase activity in biological soil crusts has been found to be highly dependent on
a number of abiotic factors, including temperature and moisture levels, and availability of C
and N (Evans and Johansen 1999, Belnap 2001, 2002, Hartley and Schlesinger 2002). These
controlling factors are hierarchical, with moisture being the ultimate determinant of
nitrogenase activity. BSCs are adapted to long periods of drought, and are inactive when dry,
but able to quickly resume physiological activity upon hydration (Evans and Johansen 1999).
The effects of moisture content on nitrogenase activity is highly dependent on species, habitat
and pre-experimental conditions (Belnap 2002). Long periods of desiccation can increase the
recovery time for nitrogenase activity. Prolonged hydration may also reduce N-fixation rates,
possibly due to losses of glucose that eventually lead to a depletion of energy reserves.
Alternating periods of wet and dry soils may lead to greater overall rates of nitrogen fixation
than prolonged periods of hydration (Belnap 2002). Different species also have different
moisture requirements for activation of physiological activity (moisture compensation point),
with cyanolichens having much higher moisture compensation points than cyanobacteria
(Lange 2001).
The secondary factor controlling nitrogenase activity is the amount and accessibility of
photosynthetic products (Belnap 2002). Nitrogen fixation is very energetically expensive; any
13

factors that affect recent and current photosynthetic rates will also affect potential nitrogenase
activity. Conditions that result in high respiration rates relative to photosynthesis can deplete
available carbon stores and increase the recovery time for nitrogenase activity (Coxson and
Kershaw 1983a). Moderate temperatures and long periods of moisture in the crust allow
maximum nitrogenase activity due mostly to high carbon stores from prolonged
photosynthetic activity (Belnap 2002). Some carbon stores are initially lost upon rehydration
and there is a lag time for photosynthesis to catch up with respiratory carbon losses. As light
intensity can affect rates of photosynthetic activity, it in turn can affect rates of nitrogenase
activity. The effect of light intensity on nitrogenase activity may depend on species and
habitat, as reported levels of light saturation vary. In general, most BSCs reach light saturation
at relatively low light levels, and do not show any decline with increasing light (Belnap 2001).
Optimal temperatures for nitrogenase activity are between 20-30°C for BSCs from
temperate, tropical and polar regions (Coxson and Kershaw 1983b, Liengen and Olsen 1997,
Belnap 2002). Regional differences in temperature optima for physiological activity may
reflect the timing and temperature conditions of moisture availability. Lower temperature
optima for N-fixation have been reported for polar environments (Liengen 1999b, Stewart et
al. 201 lb) where high soil moisture coincides with cooler spring and summer temperatures,
and higher optima have been reported for temperate regions in which most of the precipitation
falls in the summer and coincides with higher temperatures (Zhao et al. 2010). Temperatures
above the optimum tend to lead to a rapid decline in nitrogenase activity. The minimum
temperature for nitrogenase activity for most BSCs studied is 0°C, however, nitrogenase
activity has been recorded as low as -7.6°C (Belnap 2001). Recovery of nitrogenase activity

14

after freezing depends on conditions before and after freezing, and a lag time occurs after
freezing. Light exposure before freezing reduces recovery time.
Surface disturbances have been found to inhibit nitrogen fixation even without a
corresponding loss of BSC biomass. In light cyanobacterial (Microcoleus dominated) crusts,
this may occur due to a disruption in soil aggregates that results in a loss of anaerobic micro­
sites for non-heterocystic N-fixation to occur (Belnap 1996). N-fixation by dark
cyanobacterial (Scytonema and Nostoc dominated) and lichen dominated crusts may be less
sensitive to surface disturbance because they are not as reliant on aggregation for nitrogenase
activity. Surface disturbances do disrupt N-fixation over the long-term in these crusts by
promoting burial and wind erosion of the existing BSC surface. In the longer term, grazing
under even very light densities was found to reduce nitrogenase activity of Collema and dark
cyanobacteria in BSCs in a Mongolian steppe (Liu et al. 2009). The recovery of BSC function
following grazing can take decades, as depressed nitrogenase activity was found in BSCs 30
years after the cessation of grazing. Heavier grazing intensities were found to reduce overall N
input through a combination of decreased abundance of N-fixing lichens and cyanobacteria,
and decreased nitrogenase activity of these organisms (Ponzetti and McCune 2001, Housman
et al. 2006).
Biological Soil Crusts and Climate Change in Grasslands and Shrub-steppe
Across Canada, temperatures have increased by 0.5°C to 1.5°C over the last century,
while precipitation has also increased, particularly during the winter months (Zhang et al.
2000). Mean annual temperatures in western North America are expected to increase by
another 2-6°C over the next 100 years, accompanied by a general trend of increased annual

15

precipitation in the north (IPCC W GI2007). Seasonal precipitation patterns are also expected
to change, with less precipitation in the summer months, and more in the winter months.
Historical weather data show that mean annual temperatures in the Cariboo-Chilcotin
region have increased by approximately 1°C over the last century, and the rate of warming has
increased over the latter part of the century (Dawson et al. 2008). Mean winter temperatures
are expected to be the most impacted by climate change in this region, with a projected
increase of 2.5°C to 3.5°C over the next 50 years. Patterns of precipitation are expected to
change with predictions of increased in mean winter precipitation, but less snow accumulation
and earlier snowmelt, as well as decreased summer precipitation and increased evaporative
demand (Dawson et al. 2008, Spittlehouse 2008). Potential species ranges may expand or be
reduced northward and upward in elevation, and grasslands in particular may expand as
climate becomes less favorable for tree regeneration. The potential range of the bunchgrass
zone has been predicted to expand northward and upwards in elevation by over 400% in area
by 2055, while the hottest and driest parts of the bunchgrass zone will have climates more
similar to shrub-steppe in the northwest United States (Hamann and Wang 2006). The actual
response of ecosystems to these shifts in climate may be quite complex, particularly in a
region as topographically and climatically variable as British Columbia (Kimmins and
Lavender 1992). Changes in climate are also expected to result in increased frequency of
extreme events such as drought, heat waves and heavy precipitation (Walker and Sydneysmith
2008).
Sustainable management of ecosystems must include an understanding o f how systems
respond to change (Nitschke and Innes 2008). Grasslands and shrub-steppe in B.C. are
managed for multiple values, including rangeland and wildlife habitat. Climate change poses
16

risks to these values, such as increased risk of fires, and reduced ability for regeneration post­
disturbance. As BSC are an important component of the bunchgrass zone, understanding their
response to climate change is needed for assessing potentail future risks and vulnerabilities.
The bunchgrass zones of B.C. represent the northern range of shrub-steppe and grassland of
intermountain western North America and climate change is expected to have the greatest
impact at higher latitudes and altitudes, thus it is especially important to increase the
knowledge of climate vulnerabilities in this region.
Increased temperatures are expected to lead to declines in soil carbon and C:N ratios
due to increases in microbial activity and decomposition (Rosenzweig and Hillel 2000). Both
nitrification and denitrification rates may also increase, leading to greater losses of soil
nitrogen. Decreased soil moisture may also lead to declines in primary productivity and
nitrogen fixation, leading to further net losses of soil C and N. A reciprocal transplant
experiment in a Washington shrub-steppe found that a shift to a wanner and drier climate
reduced total soil carbon by 32% and nitrogen by 40% over a period of five years (Link et al.
2003). The majority of these losses were from the particulate organic matter fraction,
suggesting that climate change may also negatively impact soil structure and aggregate
stability, further accelerating soil nutrient losses.
The effects of increased temperature on biological soil crusts is not clear in the
literature. The presence and abundance of Collema has been found to shift dynamically
between years, and was negatively correlated with summer maximum temperatures and spring
minimum temperatures (Belnap et al. 2006). Conversely, an experimental 2-3°C increase in
soil temperature over a period of 2 years found little change in BSC cover, composition and
function with warming alone (Johnson et al. 2012, Zelikova et al. 2012).
17

Warming in combination with increased precipitation frequency led to significant
declines in cyanobacterial abundance and biomass (Johnson et al. 2012) and a decline in
photosynthetic performance of lichens, particularly Collema (Zelikova et al. 2012). Belnap et
al. (2004) found that a shift to shorter, more frequent precipitation events led to a decline in
photosynthetic performance, nitrogenase activity and the ability to produce protective
pigments. Shorter hydration times can cause a net carbon loss in BSC organisms as
physiological activity takes time to overcome the losses caused by re-wetting. Prolonged
carbon losses lead to an inability to produce protective pigments, which can lead to increased
mortality of BSC organisms. Lichens and dark cyanobacterial BSCs are more sensitive to both
carbon losses from short precipitation events as well as damage by solar insolation than are
light Microcoleus dominated BSCs. This suggests that any changes in climate that lead to
shorter periods of BSC hydration can cause a shift in BSC organisms from late successional
lichen and dark cyanobacteria to more early successional Microcoleus (Belnap et al. 2004).
One effect of climate change that may be particularly important in B.C. grasslands and
shrub-steppe are changes that occur during winter months. Reduced snow cover as a result of
higher winter temperatures was found to increase the photosynthetic activity of Collema
(Zelikova et al. 2012). Physiological activity of soil cyanobacteria can resume rapidly in the
day during freeze-thaw cycles if sufficient light is available for photosynthetic activity
(Coxson and Kershaw 1983b). Thus, a decline in snow-cover in combination with increased
precipitation and warmer temperatures in the winter months may lead to an increase in BSC
activity and abundance. What is not known is whether these gains in the winter period could
offset the losses caused by a hotter and drier summer period.

18

RESEARCH OBJECTIVES

The purpose of this research is to develop a more comprehensive understanding o f the
relationship between climate and nitrogen inputs by biological soil crusts in British Columbia
grasslands and shrub-steppe. In chapter one, we will investigate variation in nitrogen and
carbon of BSCs and associated soils in relationship to gradients in temperature and moisture
availability that occur across an elevation gradient in Lac du Bois Grasslands, B.C. (see
objective 1). In chapter two, we will examine patterns o f nitrogen fixation activity in relation
to changes in moisture, light and temperature availability, and model nitrogen fixation rates
throughout the year at the microclimate scale in BSCs from Farwell Canyon, B.C (see
objective 2).
Objective 1:
a) Compare the availability of nitrogen, carbon, and the isotopic composition of
nitrogen in the BSC to that of the underlying soil in order to determine the
importance of BSCs to soil nitrogen and carbon inputs.
b) Examine spatial patterns of changes in nitrogen and carbon content in the BSC and
underlying soil across a gradient of temperature and moisture availability related to
elevation.
Objective 2:
a) Evaluate the response of ^.fixation by biological soil crusts from a typical shrubsteppe community to changes in moisture, temperature and light availability.

19

b) Model N2-fixation rates throughout the year based on the above response using
measurements of BSC- level moisture, temperature and light, and use these rates to
estimate landscape-level N inputs.
c) Evaluate the potential effects of future climate change on N-inputs by BSCs in British
Columbia shrub-steppe.

20

CHAPTER 1: NUTRIENT COMPOSITION OF BIOLOGICAL SOIL CRUSTS AND
ASSOCIATED SOILS ACROSS AN ELEVATION GRADIENT IN LAC DU BOIS
GRASSLANDS, BRITISH COLUMBIA

INTRODUCTION
Grassland and shrub-steppe ecosystems in British Columbia occupy less than 1% of
the province, but are home to an exceptional diversity of flora and fauna, including over 30%
of threatened and/or endangered species (GCC 2004). These ecosystems have greatly
decreased over the last century and are currently threatened by forest encroachment,
agricultural and urban expansion, and overuse by livestock grazing and recreational activities.
Due to their importance and threatened status, there is a growing need to better understand
the impacts of climate change on grasslands and shrub-steppe in B.C.
Temperature and precipitation in Western Canada have significantly increased over the
last century, particularly in the winter and spring (Zhang et al. 2000, Vincent and Mekis 2006).
Mean annual temperatures in western North America are expected to increase by another 26°C over the next 100 years, accompanied by a trend of increased annual precipitation in the
north (IPCC 2007). Seasonal precipitation patterns are also expected to change, with less
precipitation in the summer months, and more in the winter months. Potential impacts of
climate change on grassland and shrub-steppe ecosystems in B.C. range from expansion into
currently forested areas, changes in soil nutrient cycling leading to declines in soil carbon and
nitrogen, and environments that are more favorable for establishment of invasive exotic
grasses (Spittlehouse 2008). Understanding the potential ecological response and vulnerability
to climate change can help remove uncertainty when managing ecosystems (Nitschke and
Innes 2008)
21

An important component of many grassland and shrub-steppe ecosystems are
biological soil crusts (BSCs) - communities of lichens, mosses, free-living cyanobacteria and
algae intimately associated with the soil surface. BSCs are important sources of soil carbon
and nitrogen in many arid and semi-arid ecosystems (Evans and Johansen 1999, Elbert et al.
2012) and also play roles in improving soil stability and structure, preventing erosion by wind
and water, and improving soil water infiltration and retention (Belnap 2003). BSC
communities are useful model systems for studying soil ecological processes due to their
multiple ecosystem roles and high sensitivity to disturbance (Bowker et al. 2010). BSCs are
important components of grassland and shrub-steppe of Western Canada, but relatively little
research has been conducted on BSC communities in this area (Marsh et al. 2006).
The natural abundance of 1SN (81SN) is a useful tool for assessing ecosystem nitrogen
dynamics, and has been used in a number of ecosystem studies with BSCs (Evans and
Ehleringer 1993, Evans and Belnap 1999, Aranibar et al. 2003, Russow et al. 2004). Systems
that rely solely on atmospheric fixation for nitrogen will have 815N values close to that of
atmospheric (<0%o); values of 8iSN ranging from -2 to 2%o are indicative of significant input
via N-fixation (Evans and Lange 2001).
The physiography of Lac du Bois grasslands, located near Kamloops, B.C., where
grass and shrub communities occur across an elevation range of approximately 300-1200 m,
provides a unique opportunity to study the contribution of BSCs to soil nutrient cycling
across a climatic gradient of temperature and precipitation. The use of elevation gradients as
a proxy for climate change has been applied in a number ecological studies, including several
grassland systems (Dahlgren et al. 1997, Smith et al. 2002, Link et al. 2003). The purpose o f
this study was to (1) compare the availability of nitrogen, carbon, and the isotopic
22

composition of nitrogen in the BSC to that of the underlying soil in order to determine the
importance of BSCs to soil nitrogen and carbon inputs; and (2) examine spatial patterns of
changes in nitrogen and carbon content in the BSC and underlying soil across a gradient of
temperature and moisture availability related to elevation.
METHODS
Study Site
The study area is located 6 km northwest of Kamloops, British Columbia, Canada,
within Lac Du Bois Grassland Provincial Park (50.75° N, 120.4° W). The park is
approximately 15,000 hectares in size and supports shrub-steppe, grassland and interior
Douglas-fir forest on gently sloping, south-facing terrain. The climate in the area is semi-arid
and mild continental, with a mean annual temperature of 8.9°C and mean annual
precipitation of 279 mm. Average monthly temperatures at the Kamloops airport (345 m
a.s.l.) range from -4.2°C in January to 21°C in July (Environment Canada 2012). Most of the
summer precipitation falls during thunderstorms throughout June and August, and most of
the winter precipitation falls as snow in December and January (Tisdale 1947). The driest
months are March and April. Climate becomes cooler and wetter with increasing elevation
(Table 1.1).
The study site can be classified in three distinct vegetation zones, which are related to
sharp differences in the ratio of precipitation to temperature with elevation (Van Ryswyk et
al. 1966). Below 700 m, the BGxh (Very Dry Hot subzone), is a shrub-steppe dominated by
P. spicata, A. tridentata and a well-developed biological soil crust. Although this zone is a
shrub-steppe, it is commonly referred to as the lower grassland, as described in Van Ryswyk

23

et al. (1966). At approximately 700 to 1000 m in elevation, the BGxw (Very Dry Warm
subzone), or the middle grassland, is dominated by P. spicata, and characterized by the
absence of A. tridentata. Other common species include P. Secunda, E. nauseosa, A. frigida,
and K. macrantha. The upper grasslands, found 900-1000 m in elevation, are incorporated in
the Interior Douglas-fir Very Dry Hot variant (IDFxh2), and are by dominated P. spicata and
F. scabrella (Meidinger and Pojar 1991). The respective soils associated with these
grasslands are Brown, Dark Brown, and Black Chernozems that developed on parent
material composed of glacial till. A common feature to most of the soils in this region is a
silty aeolian veneer, on which most of the Ah horizons have formed (Fenger 1982).
Table 1.1 Summary of climate normals at Kamloops Airport (Environment Canada 2012) and climate
estimates at different elevations in Lac du bois Grasslands, British Columbia1".
Location
Elev.
Temperature (°C)
Site (Period)
Precipitation (mm)
(m)
Mean Mean Mean Mean Precip. as
(lat., long.)
annual Jan.
July
annual snow (cm)
345
8.9
-4.2
21
279
Kamloops
50°42'08.00" N,
75.5
120°26'31.00" W
Airport
(1971-2000)
380
7.6
^.8
19.2
291
54
Lac du Bois
50°43'03.07" N,
120o26'46.72"W
(1971-2000)
50°45T6.09"N,
722
6.5
-5.4
18
369
78
120o27'16.56" W
50°47'22.56" N,
16.4
392
917
5.1
-6.3
103
120°26'49.27" W
•Temperature and precipitation normals for 1971 - 2000 from ClimateWNA modelling using Wang et al.
(2012; see http://www.genetics.forestry.ubc.ca/cfcg/ClimateWNA/ClimateWNA.html).

24

Figure 1.1 Views of the upper elevation grasslands (top left), lower elevation shrub-steppe (bottom left)
and the representative biological soil crust (right) at the Lac du Bois Grasslands, British Columbia
(Photos - P. Sanborn).

Field Methods
A digital elevation model of the park (C. Bulmer, B.C. Ministry of Forests and Range,
Research Branch) was used to randomly select 50 sites along south-east to south-west facing
slopes within the park boundaries. Sites were limited to grass dominated biogeoclimatic
zones (BGxh, BGxw, IDFxh2) and slopes less than 30%. Sampling was conducted over a
period of two days in June 2010. Sites ranged from an elevation of 380 m to 917 m. A

25

sample of the BSC and 10 cm of underlying mineral soil, consisting of the Ah horizon, were
collected from each site, and the elevation, slope, aspect, dominant vegetation and
approximate crust cover were also recorded. At each site, BSCs exhibiting the least amount
of surface disturbance were selected from interspaces between vegetation.
Samples were air-dried, sieved (<2 mm) and sent for analysis of total nitrogen, total
carbon and mineralizable nitrogen (Ministry of Forests and Range, Analytical Chemistry
Section, Victoria, B.C., Canada). A small amount (~30 g) from each sample was finely
ground with a ball mill and sent for analysis of 15N natural abundance (Stable Isotope
Facilities, University of Saskatchewan, Saskatoon, SK, Canada).
Data Analysis
Differences in chemical parameters between the crust and underlying soil were
assessed using a paired t-test. The sampling points were grouped based on the divisions
between lower, middle and upper grasslands described in Van Ryswyk et al. (1966). A twoway ANOVA was conducted to detect significant (p<0.05) differences in measured
parameters across elevation and between the BSC and soil. Pair-wise multiple comparisons
were conducted using the Holm-Sidak method. Statistical analyses were conducted in
SigmaPlot 11.0 (Systat Software, Inc., San Jose CA, USA).
RESULTS
The BSC layer had significantly (p<0.05) higher total carbon, total nitrogen and
mineralizable nitrogen than the underlying (l-10cm) mineral soil (Table 1.2). The 8I5N
natural abundance in the BSC ranged from -0.38 to 4.2596o and was significantly lower
compared to the underlying soil. Total carbon and total nitrogen were approximately twofold

26

higher, while mineralizable nitrogen was almost 6 times higher in the BSC relative to the
mineral soil.
Table 1.2: Selected Chemical Properties of Biological Soil Crusts and underlying mineral soils at Lac
du Bois Grasslands, British Columbia. The mean differences reported are significantly different based
on a paired two-sample t-test (P<0.05)
Diff
P
Parameter
Soil
BSC
(±SE)
mean
(±SE)
mean
(±SE)
mean
(0.70)
2.23
2.73
(0.332) <0.0001
Total C (%)
4.97
(0.15)
(0.027) 0.186
(0.018) <0.0001
Total N(%)
0.361
(0.012) 0.175
(0.240) <0.0001
C:N
13.25 (0.27)
1.32
11.93
(0.13)
Mineralizable N (mg/kg)
266.6 (18.6)
47.55
219.04 (16.22) <0.0001
(4.03)
(0.16)
2.91
(1.65)
5ISN Natural abundance (%o) 2.24
5.15
(0.20)
<0.0001

Total carbon, nitrogen and mineralizable nitrogen were also found to increase
significantly (P<0.05) with elevation in both the BSC and the underlying mineral soil (Figure
1.2). The C:N ratios were consistent across elevations, and were significantly lower in the
soil compared to the crust, except at the lowest elevations (Figure 1.2). The 5,5N natural
abundance was not found to be related to elevation in either the BSC or the soil (Figure 1.2).
The proportion of mineralizable N within the total N pool was significantly higher (P<0.001)
in the BSC compared to the soil across all elevations, and also increased significantly
(PO.OOl) with decreasing elevation in the BSC.

27

Soil (1-10 cm)

N

200

2

100

<600 m

600-800 m

800-1000 m

Elevation

Figure 1.2: Total Carbon, Total Nitrogen and Mineralizable Nitrogen in the biological soil crusts
(BSC) and underlying soil (l-10cm) across an elevation gradient in Lac du Bois Grasslands, British
Columbia. Error bars represent standard error and letters denote significant differences (p<0.05)
across soil type and elevation class.

28

Soil (1-10 cm)

<600 m

600-800 m

800-1000 m

Elevation

Figure 1.3: Ratio of Total C:N and I5N natural abundance in the biological soil crusts (BSC) and
underlying soil (l-10cm) across an elevation gradient in Lac du Bois Grasslands, British Columbia.
Error bars represent standard error and letters denote significant differences (p<0.05) across soil type
and elevation class.

DISCUSSION
The average total C, total N and mineralizable N in both the crust and underlying soil
were all within the range of that reported for Chilcotin grassland and shrub-steppe ecosystems
some 200 km north of the present study site (Marsh et al. 2006). The twofold enrichment of
nitrogen in the BSC suggests that that the cyanobacteria and cyanolichen components
contribute to soil nitrogen content. Many BSC species have been found to release N29

compounds, particularly in the form of nitrate and ammonia into the surrounding soil (Belnap
et al. 2001). In addition to their ability to fix nitrogen, BSCs may also be able to capture and
immobilize exogenous nitrogen that could otherwise be lost from the ecosystem (Hawkes
2003). Similarly, BSC organisms can contribute to soil carbon through the release of
extracellular carbon compounds, which can represent up to 50% of the total fixed carbon
(Belnap et al. 2001).
The much higher concentrations of mineralizable nitrogen in the BSC relative to the
underlying soil also suggests that substantial release of labile nitrogen is occurring in these
ecosystems. Furthermore, the mineralizable N comprised 8.1% of the total N pool in the BSC
compared to only 2.4% in the underlying soil. This suggests that a higher proportion of
nitrogen within biological crusts is in an active form that is readily available for plant and
microbial uptake. Also, while BSCs at the lower elevation grasslands had smaller nitrogen
pools than at higher elevation grasslands, they had the highest proportion of mineralizable
nitrogen within that pool (~10.8%) indicating that the relative proportion of biologically active
nitrogen increases with decreasing elevation. This change in availability only occurs in the
BSC, not in the underlying soil.
Values of 815N ranging from -2 to 2%o are indicative of significant input via Nfixation (Evans and Lange 2001). The average 5,SN value for the BSCs in Lac du Bois
Grasslands (2.2496o) is slightly higher than this range, similar to that reported for BSCs in the
Chilcotin region of British Columbia (Marsh et al. 2006) and for disturbed cyanobacterial
crusts in Utah (Evans and Belnap 1999). This suggests that N-input via fixation is important,
but may not be the only source of N for BSCs in this ecosystem. The higher 8,SN in some
sites may also be due to higher rates of N losses compared to inputs via fixation. Processes
30

such as ammonia volatilization and denitrification are subject to fractionation, meaning that
,4N is favoured over l5N during the transformation. This results in an enrichment of 15N in
the remaining N-pool, and higher than expected 5,5N.
The increase in soil nitrogen and carbon with elevation is likely related to climatic
gradients of temperature and precipitation (Van Ryswyk et al. 1966, Smith et al. 2002).
Lower elevation sites have lower annual precipitation and higher average temperature,
resulting in overall higher potential evapotranspiration. Lower soil carbon at lower elevations
can be attributed to increased soil respiration and soil carbon turnover due to higher
temperature, or to decreased allocation to below-ground biomass in plants (Link et al. 2003).
Lower soil N at lower elevations may be due to higher rates of gaseous losses caused by
precipitation on dry soil, as well as a higher N turnover in the litter (Smith et al. 2002).
Lower crust and soil N may also be due to lower rates of N-fixation in the BSC as higher
temperatures can lead to the decline of cyanolichens and heterocystous cyanobacteria
(Belnap et al. 2004, 2006).
The 51SN showed large variability in both the BSC (-0.38 to 4.25%o) and soil (0.20 to
7.23%o), but we were unable to explain this variability using elevation or any of the other

measured variables. Factors unrelated to climate may affect the I5N natural abundance of the
BSC and soil. Cattle are grazed at a low density throughout Lac du Bois Grassland park. The
presence of grazers may increase soil 1SN abundance by facilitating N-losses via
volatilization and nitrification in urine and dung patches (Frank and Evans 1997).
Furthermore, soil trampling by grazers can stimulate N-mineralization, increasing losses of
inorganic N by leaching, ammonia volatilization, and/or denitrification. Another factor that
may affect 8,SN in the BSCs among sites is the influence of plant litter in areas with dense

grass or shrub cover. BSCs growing under shrubs have been found to higher 815N due to an
increased incorporation of N from leaf litter rather than from N-fixation (Billings et al. 2003).
In order to better understand this spatial variation in 8ISN future studies of BSC nutrient
cycling in this area should take into account present and historical disturbance, as well as the
influence of surrounding vegetation.
Our results suggest that biological soil crusts contribute to soil nitrogen and carbon
across all elevations in this ecosystem. While soil C and N within BSCs is greater at higher
elevations, their overall contribution to soil nutrients is much lower, due to lower crust cover
relative to vegetation. A recent analysis of vegetation cover at Lac du Bois grasslands found
that soil lichen cover declined from an average of 25.1% (±17.51) at lower elevations to
2.18% (±2.81) in the upper grasslands (Lee 2011). Cyanobacterial crusts were not measured,
but bare soil cover (part of which may be cyanobacterial crusts) declined with increasing
elevation from 17.59% (±7.87) to 0.93% (±1.19).
It is also important to note that although our upper elevation sample sites were
selected within the biogeoclimatic zone representing the upper grasslands (IDFxh), we only
selected sites with a southern aspect, which are warmer and drier than sites representative of
this zone. Thus the results of this study may not fully apply to conditions typical of the upper
grasslands.
Current climate models predict an increase in temperature in this region of at least
2°C over the next 70 years. This increase in temperature would lead to an increase in
evaporative demand as summer precipitation is also expected to decrease (Spittlehouse 2008).
The difference in mean annual temperature between our lowest and highest sites is 2.5°C.

32

This suggests that the soils, vegetation and BSC communities in the upper sites could
eventually resemble those in the lower sites. Similar to studies o f other elevation transects in
grasslands and shrub-steppe, we may expect both soil C and N to decrease as respiration and
denitrification increase and productivity decreases (Smith et al. 2002, Link et al. 2003). At
the lower elevations, species composition may shift to a greater abundance cyanobacteriadominated BSCs, as lichens, particularly the cyanolichen Collema have been found to be the
most sensitive to increases in temperature and declines in precipitation (Belnap et al. 2004,
Belnap 2006).

33

CHAPTER 2: SEASONAL PATTERNS OF NITROGEN FIXATION IN
BIOLOGICAL SOIL CRUSTS FROM BRITISH COLUMBIA’S CHILCOTIN
REGION.

INTRODUCTION
Biological soil crusts (BSC) are communities of lichens, mosses, free-living
cyanobacteria and algae found intimately associated with the soil surface in arid and semiarid ecosystems throughout the world. BSCs have been found to improve soil stability and
structure and prevent erosion by wind and water (Neff et al. 2005, Malam Issa et al. 2007),
improve soil water infiltration and retention (Belnap 2006) and are important contributors to
soil carbon and nitrogen cycles (Evans and Johansen 1999, Elbert et al. 2012). Certain
cyanobacteria (i.e. Nostoc, Scytonema) and lichens (i.e. Collema spp.) found in BSC
communities are also able to fix atmospheric nitrogen. Much of this fixed nitrogen is
released into the surrounding soil and can be the dominant source of nitrogen in many arid
and semi-arid ecosystems (Evans and Ehleringer 1993, Evans and Belnap 1999). Seasonal
differences in nitrogen fixation by BSCs can be attributed to differences in temperature,
moisture and light availability. The most important factor controlling nitrogen fixation is
moisture availability, as physiological activity ceases when moisture is not available. Upon
hydration, most BSC organisms are able to rapidly resume physiological function. The rate
of recovery of nitrogen fixation is largely dependent on the availability of carbon stores,
which are related to the environmental conditions prior to desiccation as well as the
conditions dining the recovery period (Coxson and Kershaw 1983a). Rates of nitrogenase
activity have been found to be positively related to light intensity, but have been found to
achieve maximum rates at relatively low light levels (Belnap 2001). After moisture and
34

carbon availability, the next most important factor controlling rates of nitrogenase activity is
temperature. Optimum nitrogenase activity generally occurs between 20 and 30°C, and will
cease when temperatures are too low or too high (Evans and Johansen 1999).
Most of the BSC studies in North America have focused on the shrub-steppe and
grasslands of the Columbia and inter-mountain basins. The grasslands and shrub-steppe of
British Columbia represent the northern extent of those ecosystems further south and show
many similarities in community composition of BSCs (Marsh et al. 2006). There are
however, some differences in climate and precipitation patterns within these regions (Tisdale
1947). Thus, BSCs in British Columbia may have different estimates and seasonal patterns of
nitrogen fixation than those already established for North America.
The acetylene reduction assay (ARA) is a commonly used method for measuring
nitrogen fixation potential due to its relatively low cost and ease of use compared to the more
direct measurement using 15N incorporation (Evans and Johansen 1999, Belnap 2001). It
works on the principle that nitrogenase will readily accept acetylene and convert it to
ethylene. The theoretical conversion of 3 mol of acetylene assimilation to 1 mol of N 2
incorporation is often used to estimate nitrogen inputs from ARA. The true conversion ratio
however, has been found to vary among species and environmental conditions, and
conversion factors specific to the study should be determined by comparing ARA rates to
rates of 15N incorporation under the same conditions (Stewart et al. 1967).
The purpose of this study was to measure the response of nitrogen fixation activity by
BSCs to changes in temperature, light and moisture. This response was then used to estimate
nitrogen inputs by BSC communities based on a two-year dataset of BSC microclimate in a

35

shrub-steppe in the Chilcotin region of British Columbia, Canada. Estimates of nitrogen
inputs can be used to determine the relative importance of BSCs to nitrogen cycling in these
ecosystems and help develop management strategies under a changing climate regime.
METHODS
Study Site
The main study site was located at Farwell Canyon (51°49'27.35" N, 122°32'46.00"
W), a shrub-steppe located along the Chilcotin River approximately 50 km southwest of
Williams Lake, British Columbia. This site was located within the very hot dry subzone of
the British Columbia Ecological Classification bunchgrass zone (BGxh) (Steen and Coupe
1997). The climate in this part of the bunchgrass zone is characterized by warm to hot, dry
summers and moderately cold winters with most of the precipitation occurring in December
to January and in June (Table 2.1). The nearest long-term climate data set is from the
Williams Lake airport, 40 km northeast of the study site. Climate B.C. modelling (Wang et
al. 2012) suggests that the Farwell Canyon site is drier (348 mm) than Williams Lake (450
mm) and warmer (Table 2.2).
Table 2.1. Climatic characteristics of the very hot dry bunchgrass biogeoclimatic subzone (BGxh3)
for the study site (Steen and Coupd 1997).
Precipitation (mm)
Temperature (°C)
Mean annual
Wannest month
Coldest Month
Frost free days

5.9
19
-10.6
182

Mean annual
Mean summer
Mean winter
Precipitation as snow
(cm)

330
177
153
71

Study site topography consists of a series of terraces leading down towards the river,
dissected by steep valley slopes. Soils at the site are Brown Chernozems that have formed on
36

sandy fluvial and glaciofluvial parent materials covered with an aeolian veneer (10-50 cm
thick) of silt and fine sand (Steen and Coupe 1997). The area selected for BSC sampling and
microclimate measurements was on a lower terrace (515m elevation) with a slight southfacing aspect. Although the study site was located on privately owned rangeland, the area has
seen relatively little physical disturbance, as most grazing by cattle occurs on the upper
terraces.
Table 2.2. Summary of climate normals at Williams Lake Airport (from Environment Canada 2012)
and at Farwell Canyon, British Columbia (estimated from Climate B.C. modelling using Wang et al.
(2012).

Site (Period)

Location
(lat., long.)

Elev.
(m)

Farwell Canyon
(1971-2000)

51°49'27.35" N,
122°32,46.00" W

515

Temperature (°C)
Precipitation (mm)
Mean
Mean Mean
Mean
Precip. as
annual winter summer annual snow
(cm)
5.2
-1.0
348
14.0
72

Williams Lake
(1971-2000)
(2009-2010)
(2010-2011)

52°10'59.00" N,
122°03'15.00 W

940

4.2

-1.9

12.7

450

193

4.9
3.7

-0.6
-2.5

12.6
12.3

348
526

117
162

The site vegetation was dominated by A. tridentata, P. spicata, and a well-developed
dark-colored biological soil crust community on open soil surfaces between vegetation
patches. Samples were randomly collected from BSC communities that were representative
of the study site.
Microclimate Measurements
Soil surface microclimate of dark BSC communities at the Farwell Canyon site was
monitored from November 2009 to October 2011. Sensors were placed in two adjacent BSC

37

patches on the lower terrace. Each patch was ca. 4 m2 in area, located in gaps between
matured, tridentata plants. Crust-level parameters measured were temperature, moisture and
light intensity.

Figure 2.1 View of the vegetation and landscape representative of the study site, at Farwell Canyon,
British Columbia.

BSC surface temperature (°C) was measured with fme-wire (Type T, copperconstantan) thermocouples (n=6) connected to a multiplexer (AM25T, Campbell Scientific
Inc). Additional fine-wire (unshielded) thermocouples (n-2) were placed approximately 1 m
above ground to measure ambient air temperature. Light intensity, as incident
photosynthetically active radiation (pmol PAR m 'V ), was measured with LI-COR Quantum
38

sensors (n=4) (Li-Cor, Lincoln NE, USA) placed on the crust surface. Volumetric soil
moisture was measured with ECH2 O EC5 moisture sensors («=4) (Decagon Devices,
Pullman WA, USA), buried 2 cm below the BSC surface.
50

40

30

20

10

0

0.0

0.2

0.4

0.6

0.8

1.0

Relative Impedence Response

Figure 2.2. Calibration curve for mean relative impedance response (n=8) and gravimetric moisture
content (MC) of field collected BSC (w=8-12). Error bars represent standard error.

BSC hydration status was determined using impedance clips (n=8) inserted 2 mm
apart at the crust surface (after Coxson 1991). Values from the impedance clips were
standardized to a relative response of 100% during known wetting events. This was done
every few months as the impedance response was found to diminish over time. The mean
relative impedance response across all the sensors was calibrated against the gravimetric
moisture (w/w) content of crust samples collected during different periods throughout the
two years (Figure 2.2). An exponential curve fitted against this plot
(f=2.7797*exp(2.7797*x); R2=0.9551) was then used to convert the impedance values to %
moisture of the BSC. The impedance response method does not provide accurate
39

measurements at sub-zero temperatures. We therefore made the assumption that the moisture
content of BSC remained constant after temperatures fell below 0°C until the next thaw
period. This assumption may be accurate for crusts under a snowpack; however, exposed
crusts often continue to dry from sublimation at sub-zero temperatures, an error that is not
corrected in the present data set.
Measurements for each sensor were taken every 30 seconds and summaries of
average, maximum and minimum measurements were recorded at 10 minute intervals using
a Campbell Scientific CR23X datalogger. The microclimate dataset is continuous with the
exception of the period March 10-April 19,2010. A B.C. Ministry of Transportation weather
station at Riske Creek (11 km northeast of study site at 1111 m a.s.l.) recorded two days of
precipitation with a total of 3.2 mm during this period.
An additional datalogger (Campbell Scientific CR10 and CR10X) was set up in a
secondary area of south-facing BSC communities using the same types of sensors from
Februaiy to May 2011, providing additional spot observations during the snow melt period.
Acetylene Reduction Analysis
Measurements of nitrogenase activity were conducted using the acetylene reduction
assay (Stewart et al. 1967). BSC samples were collected as disks 2.5 cm in diameter and 1
cm deep. These were stored dry in growth chambers; summer collections were held at
10/15°C with an 18/6 hour light-dark photoperiod, while winter collections were held at
5/10°C with an 8/16 hour light-dark photoperiod. The size of the samples approximated that
of mounds that characterize the BSC surface in this ecosystem, which generally range from 2
to 10 cm in diameter. Samples were hydrated to ~30% moisture content (w/w) for 24 hours
40

prior to ARA treatment. ARA incubations were conducted in 100 mL glass cuvettes
equipped with rubber stoppers and sealed to glass plates with high-vacuum grease. The air
within the cuvettes was replaced with acetylene gas equal to 10% of the chamber volume.
The acetylene was generated on-site with CaC2 and water. A second control sample
containing a BSC sample was fitted with a fine-wire thermocouple to monitor BSC surface
temperature within cuvettes. Samples were incubated for 4-6 hours with 10 replicates for
each set of incubations.
A subsample (0.5 mL) of the air within the cuvettes was injected into a gas
chromatograph (SRI 8610A, Wennick Scientific Corporation, Ottawa, ON, Canada) fitted
with a Porapak column (Alltech Canada, Guelph, ON, Canada) and a flame ionization
detector, to measure the concentration of ethylene produced. A calibration gas containing
101 pi l 1ethylene (Linde Canada Ltd., Edmonton, Alta) was used to calibrate the gas
chromatograph for each incubation. Control cuvettes containing (1) BSCs without acetylene
and (2) bare soil (no BSC) injected with acetylene, did not show any production of ethylene.
In order to determine the effect of thallus moisture content on nitrogenase activity,
summer collected BSC were hydrated to 30% moisture content (w/w) and pre-treated for 48
hours, after which time ARA incubations were conducted at 21°C and 200 pmol PAR m 'V 1,
allowing the crusts to dry down to 20,15, 10, 7.5, and 5% MC (w/w) between sequential
incubations. In order to determine the effect of length of prior desiccation exposure, three
sets (n=10) of summer collected BSC samples were collected and pre-treated as described
above for 48 hours, then incubated in order to determine baseline ARA rates. Each set of
samples was held dry for a period of 1 week, 3 weeks, or 6 weeks. The samples were then
hydrated and incubated after 2,24,48, 72 and 96 hours since hydration.
41

ARA incubations were conducted in situ at the study site during several precipitation
and snowmelt episodes over the course of the study. Crusts were collected in the same
manner as described above and immediately incubated for 4-6 hours with 10% (v/v) of
acetylene, with cuvettes placed in-situ on the soil surface. Temperature within the cuvettes
was kept within 3°C of ambient crust temperatures using ice packed around the cuvettes to
cool the incubation chambers when necessary. A sample of the air within the incubation
chambers was collected using vacutainers (Becton-Dickson, Franklin Lakes, NJ) and were
brought back to the lab for analysis with the gas chromatograph. Reference containers were
tested for ethylene contamination.
In order to determine the effect of temperature and light intensity on nitrogenase
activity incubations were conducted under the following temperature and light combinations:
14,21, 28°C and 100, 300, 600,1000 pmol PAR m ' V 1 for BSCs collected in the summer.
Winter-collected BSCs were incubated at 0, 7, 14, 21°C and 100, 300,600 pmol PAR m ' V 1.
Dark incubations (0 pmol PAR m ' V 1) were conducted at 10°C for both winter and summer
collected BSCs. Rates of nitrogenase activity were calculated as micromoles of ethylene
reduced per hour per m2 based on the incubation time and the area of the crust samples.
lsN-Incubations
Summer collected BSC samples were collected, stored and pre-incubated in the same
manner as for the ARA incubations. An incubation time of 48 hours was used in order to
ensure detection of 15N enrichment. Two sets of 10 replicates were incubated at 14°C and
21°C respectively, under 200 pmol PAR m ' V 1. The air within the cuvettes (10% v/v) was
replaced with 10% (v/v) 15N2 gas (Cambridge Isotope Laboratories Inc., Andover, MA, USA,
15N2 , 98%+). Each cuvette was also injected with 5% (v/v) CO2 in order to minimize CO2
42

limitation during the long incubation period. After each incubation, samples were
immediately dried at 105°C, ground in a ball mill and sent for 15N and total N analysis
(Stable Isotope Facilities, University of Saskatchewan, Saskatoon, SK, Canada). Control
samples (n=1 0 ) were incubated at the same time but with C2 H2 were used to determine the
natural abundance ,5N and the acetylene reduction rate. The rate o f nitrogen uptake was
calculated as per Liengen (1999a) using the equation:
Y =

( atom % 15J W e„ \
V

100

J

/ to ta l Nsampie x 109\ /

\

t x 28

/

100

\

\% 1 5 n a ir )

^ '

where Y (nmol N-gdw"l-h_1) is the rate of N 2 uptake, atom% 15N«cess is the difference
between atom% 1^sample and atom% 1^control, total N is the total amount of nitrogen in the
sample (g- 1 0 0 gdw-1), t is the incubation time, 28 is the molecular weight of N 2 (g/mol), and
%15N air is the percentage of 1SN out of the total amount of N gas in each incubation
chamber (12.81%). Conversion ratios for ,SN uptake to ARA were calculated from the
pooled averages of the samples incubated with 15N and the samples incubated with C 2 H2 for
each temperature (Table 2.3).
Table 2.3. Summary of conversion ratio for ethylene production to 15N-incorporation (±SE) by
biological soil crusts incubated for 48 hours at 21°C and 200 pmol PAR m 'V 1. n=10.
Total N (fi/gdw)
0.126
(0.011)

Atom %,SN
excess
0.02703
(0.0042)

,5N2 fixed
(nmol/cm2/h)
124.64
(21.45)

C2H4 Produced
(nmol/cmz/h)
7.38
(0.238)

C1H4/N2
0.059

Modeling BSC N-fixation activity under field conditions
The data obtained from the ARA temperature x light matrices for winter and summer
collected BSCs were fitted to a 3-dimensional Gaussian curve in SigmaPlot 11.0. Individual
43

curves were obtained for each of the winter and summer periods (R2=0.9648, p<0.0001;
R2=0.9634, p<0.0001). These curves were subsequently used to estimate optimal ARA in
field BSC communities based on measured field microclimate conditions within 10 minute
blocks in the dataset. Model assumptions include saturation of ARA above 600 pmol PAR
m 'V 1in winter and above 1000 pmol PAR-m'2-s'1in summer and continued ARA activity
during periods of darkness under snow cover. ARA activity was set at zero when
temperatures were below -5°C. The dates of May 1st to September 30th were used to
differentiate between winter and summer in the data, which roughly corresponded to the
frost-free period in the dataset. The calculated ARA estimates (which assumed optimal BSC
moisture content and optimal pre-treatment hydration conditions) were corrected for actual
moisture content and prior desiccation exposure using polynomial curves fitted to relative
ARA responses for these variables. Crusts were assumed to be physiologically active at
moisture contents above 7.5% (w/w), corresponding to the lowest moisture content at which
ARA activity was detectable during laboratory experiments.
Visual estimates of percent cover of dark BSCs at the Farwell Canyon study site were
taken within 30 cm x 30 cm quadrats placed at 5 m intervals along N-S oriented 50 m
transect lines. Lines were placed 10 m apart, with four groups of three lines dispersed within
an approximately 1 km2 area around the study site.

44

RESULTS
Farwell Canyon BSC Communities
BSC communities on the lower Chilcotin River valley terraces at Farwell Canyon
showed little evidence of past grazing; crust communities were largely intact, with mean
cover of dark crusts averaging 52.7% (+ or - 2.73% S.E., n=120). The BSC communities
contained several rare species, including Gypsophila macrophylla, and what appears to be a
new species of Endocarpon (C. Bjork, Personal Communication). Collema coccophorum and
C. tenax were common nitrogen fixing lichen constituents of the dark BSC communities,
while Nostoc sp. and Scytonema cf. intertextum were abundant free-living constituents
(Table 2.4).

45

Table 2.4. Species identified in BSC samples from Farwell Canyon study site. Species marked with
an "X" include those found in the dark BSC communities sampled for ARA incubations.
Species
Comments
Cyanobacteria
Nostoc sp. *
x
Scytonema cf. intertextum*
x
Chroococcidiopsis sp.
x
Lichens
Acarospora schleicheri
Caloplaca atroalba
Caloplaca cerina
Caloplaca tominii
x
Candelariella aggregate
x
Cladonia pyxidata
x
rare species
Collema coccophorum*
x
Collema tenax*
x
Diploschistes muscorum
Endocarpon sp. nov.
x
rare species
Endocarpon pusillum
x
Fulgensia bracteata
x
rare species
Gypsoplaca macrophylla
rare species
Lecanora hagenii group
Megaspora verrucosa?
x
Peltigera ponojensis*
Physconia muscigena
Placidium lachneum
x
Placidium lacinulatum
x
Placidium squamulosum
Polychidium muscicola*
Psora decipiens
Psora globifera
Psora Montana
Rinodina terrestris
x
Toninia sedifolia
Caloplaca ammiospila?
^Indicates N-fixing species

46

Acetylene Reduction Assays
The temperature-light response of ARA under optimal hydration shows an increase in
ARA activity with increasing temperature and light for both winter- and summer-collected
BSC (Figure 2.3). Winter-collected crusts showed lower ARA activity than summer
collected crusts at similar temperature and light conditions with a maximum rate of 116
pmol C2 H2 -m'2 h'1 at 21°C and 600 pmol PAR m ’V 1 compared to 173 pmol C2 H2 -m'2 h'1 at
the same conditions for summer collected crusts. There was little difference in ARA rates
between BSC incubated at 21 and 28°C. ARA activity increased with increasing light
intensity, though this pattern appeared less pronounced at lower temperatures. ARA rates
declined slightly between 30% and 15% MC and then exhibited a marked drop below 15%
MC (Figure 2.3). The recovery curve of ARA activity over time after hydration showed that
peak ARA activity occurs after BSCs are held hydrated for between 24 and 48 hours (Figure
2.4). ARA rates were higher for summer collected material when incubated at 21 °C, but were
not significantly different (t-test; p>0.05) when incubated at 14°C. There were no significant
differences (ANOVA, P>0.05) between the recovery curves of BSCs held dry for different
time periods.
BSC Microclimate Data
A comparison of temperature and precipitation recorded at the Williams Lake Airport
(Environment Canada 2012) during the study period with the 30 year average (1971-2000)
reveals that 2009-2010 was a dry year compared to normal, while 2010-2011 was a wet year,
particularly in the summer. Winter temperatures were warmer than normal in 2009-2010 and
cooler than normal in 2010-2011, while summer temperatures were fairly close to normal for
both years (Table 2.2).
47

A. Writer

B. Summer

180

250

140

200

• C 120
150
100
3 > 801

$

401

1000
800

20

800

'C\

400
200

-10

Figure 2.3. Temperature and Light Response of acetylene reduction assay (ARA) fitted with
multivariate curves by BSCs collected in (A) March and (B) July, n =10 for each data point.

120

R = 0.98

R2 = 0.9492
100

80 60
40 20

0

10

20

0

30

Moisture Content (% w/w)

20

40

60

80

100

120

140

Time Since Hydration (hours)

Figure 2.4. (A) Relative ARA response to moisture content for BSCs pre-treated at optimum
conditions and incubated at 21°C and 200 pmol PAR m 'V 1 (B) Relative ARA response over time
starting at 2 hours after hydration to 30% moisture content.

48

V 500

| 200

Feb

May

Aug

Nov

Figure 2.5. Summary of BSC microclimate from 11 November 2009 to 1 October 2011 at Farwell
Canyon, British Columbia.

As snow-melt pockets develop during the late winter period (Figure 2.6) extreme diel
temperature fluctuations can be experienced by hydrated BSCs. A representative data set
from snow melt pockets under late-winter conditions (Figure 2.7) shows daily freeze-thaw
cycles, with night-time BSC temperatures reaching -20°C, followed by crust surface
temperatures of over 10°C under full sun conditions during the following midday period.

49

Figure 2.6. View of biological soil crusts melting out from under snow cover in early March at the
Farwell Canyon study site. The width of the crust segment in the center of the image is ca. 9 cm.
Hydration episodes at the BSC surface due to rainfall were on average 18 hours long,
with a maximum hydration episode of 115 hours. Time between wetting events lasted an
average of 2 days with a maximum of 16 days. The pattern of hydration events varied
between seasons and between the two study years. Hydrated BSCs in the summer spent a
majority of time in the light (65% in 2010 and 56% in 2011), while in the winter, hydrated
BSCs were in the dark for the majority of time (70% in 2009-2010 and 56% in 2010-2011)
(Figure 2.8). Thallus temperatures for hydrated BSCs were predominately between -10 and
0°C in the winter and between 5 and 15°C in the summer.

50

Predicted BSC N-fixation
Although BSC communities experience extreme daily temperature fluctuations
during the late winter period, model values for ARA on a south-facing aspect in late
February and early March (Figure 2.7) predict substantial recovery during midday thaw
periods.

Air
BSC

-10

-20

1500
1000
500

12

24

12

24

12

24

12

24

12

24

12

24

Hours

27

28

1

February

2

3

4

March

Figure 2.7. BSC microclimate on south-facing aspect from 27 Feb. to 4 March 2011. From top:
predicted rates of acetylene reduction (ARA) from microclimate model, air and BSC surface
temperature, and incident photosynthetically active radiation (PAR) (pmol PAR m-2s-l ) at the BSC
surface.
51

Verification of late-winter ARA recovery was obtained in mid-March at the Farwell
Canyon site (Figure 2.9). Field measurements of ARA activity showed peak rates in the midaftemoon and low but measurable rates overnight (Figure 2.9). ARA rates measured in the
field were similar both days, even though temperature and light levels were lower during the
second day of incubations. This resulted in predicted ARA rates being lower than actual rates
during the second day of incubations.

C. Sum m er Y ear 1

A. W inter Year 1*
Dark
Light

■
■

800
600
400

TJ

&

200 -5
o

X

D. Sum m er Y ear 2

3eo

X

F. Sum m er Y ear 1

H. Sum m er Y ear 2

G. W inter Y ear 2

Temperature Class (°C)

Figure 2.8.(A-D) Time (in hours) spent by hydrated (> 7.5% MC w/w) BSCs and (E-H) the estimated
amount of C2H2fixed (in mmol of C2H2-m'2) by hydrated BSCs at temperatures plotted in 5°C class
intervals for winter (October to April) and summer (May to September), for the periods November
2009- September 2010 (Year 1) and October 2010 to September 2011 (Year 2). Calculations exclude
periods of missing data (from 1 Nov - 11 Nov Year 1 and 9 Mar - 13 Apr Year 2).

52

-

BSC
Air

-10

EC 400 O

200 -

20 -

co 100

•

-

ARA - Predicted
ARA - Field

40 -

20 Mar 16

6

12

6 Mar 17

6

12

6 Mar 18

6

12

6 M ar 19

6

12

6 M ar 20

Figure 2.9. Predicted ARA rates based on BSC microclimate measurements during a spring snowmelt
event at Farwell Canyon in March 2011. The symbols represent ARA rates measured in situ with
standard error (Y-error bars) and incubation time (X-error bars).

Field measurements of ARA during spring-time rainfall events also found that
predicted ARA was sometimes lower than actual rates during field measurements (Figure
2.10). Field ARA measured 30 hours after wetting (on May 17) was higher than predicted by

53

the model, as were rates measured the following night (Figure 2.10). During the following
period of full sun exposure as crusts started to dry ARA rates were variable, but were close to
predicted rates.

40 -

BSC
Air

30 ■
20 -

> V v -r " ' v - 'v

1200 CL 800 ■

400 ■
g>

25 20 15 -

10 -

ARA-Field
ARA - Predicted

200 -

May 16

6

12

6 May 17

6

12

6 May 18

6

12

6 M ay 19

6

12

6 M ay 20

Figure 2.10. Predicted ARA rates based on BSC microclimate measurements during a rain event at
Farwell Canyon in May 2011. The symbols represent ARA rates measured in situ with standard error
(Y-error bars) and incubation time (X-error bars).

54

BSC
Air

30 -

1600 VC 1200 -

2:

400 •

^

300 -

o

100 -

_g>

Jul 10

•

A R A -F ield
ARA - Predicted

8

12

6

12

8

1

,

Ju l 12

e

12

8

Jul 13

8

12

8

Jul 14

Figure 2.11. Predicted ARA rates based on BSC microclimate measurements during a rain event at
Farwell Canyon in July 2011. The symbols represent ARA rates measured in situ with standard error
(Y-error bars) and incubation time (X-error bars).
Observed ARA activity during a rain event in July shows a pattern of physiological
recovery after wetting (Figure 2.11). ARA rates of 14 pm olm 2 h'1were measured in the
mid-morning, shortly after wetting, and rose to 116 pmol m2 h'’ in the late afternoon,
approximately 12 hours after initial hydration. Maximum ARA activity was reached during
the following morning, with average rates of nearly 200 pmol m2 hT. The model
55

underestimated ARA rates during this recovery period, but fitted more closely with the field
data after 24 hours from initial hydration. A drop in ARA rates during periods of high solar
insolation immediately prior to drying was observed was observed during both summer field
incubation sets (Figure 2.10 and Figure 2.11).
Based on the model, the majority of N-fixation activity in the summer occurred under
light conditions between the temperatures of 10 and 20°C (Figure 2.12). Only 15% of
fixation activity during the summer occurred in the dark, although hydrated BSCs were in the
dark approximately 40% of the time. The distribution of summer ARA activity across
temperature and light classes was similar for both years, but Year 2 had higher ARA activity
overall. A greater amount of ARA activity occurred in the light and at higher temperatures in
Year 2 compared to Year 1.

800
■Q 600 -

>> 400 -

2

200 -

-

O

o

c

n

Uf

(V

w

>. c

W
9 - IS

g
—3

at a. ft

5^

>

o

w
.®
“ oW f wf

cz jo

*= t

nw s ® ^

>, c

Q- ® 3

2010-2011

2009 - 2010

Figure 2.12. Monthly summary of BSC hydration time and estimated ARA activity at a shrub-steppe
and shrub-steppe in Farwell Canyon, B.C. Asterisks denote time periods with missing data.
56

The monthly patterns of ARA activity and BSC hydration differed between the two
study years (Figure 2.8). February had the highest monthly ARA activity in Year 1, followed
by January and March. In Year 2, July had the highest monthly ARA activity, followed by
March and May.
Predicted landscape-level nitrogen inputs based on a 3:1 C 2 H2 :N2 conversion ratio,
were between 0.7 and 0.8 kg N-ha'1(36 to 40 kg N-ha'1with a 0.06 conversion ratio) in the
winter period (Table 2.5). Predicted nitrogen inputs in the summer were between 0.57 and
0.96 kg N-ha'1(28 to 48 kg N-ha'1 with a 0.06 conversion ratio).
Table 2.5. Summary of estimated seasonal N-inputs per hectare by BSCs in a shrub-steppe in Farwell
Canyon, B.C. The estimates are based on conversion from modeled C2H2 inputs using both an
experimentally derived C2H2:N2 conversion ratio of 0.06 and a commonly used conversion ratio of 3.
Landscape level N-input estimates were determined using a BSC cover estimate of 52.7%.
Estimated N-input
Estimated N-input
Est. C2H2
Time
(0.06:1
C2H2:N2ratio)
(3:1
C2H2:N2
ratio)
Hydrated
fixed
(mmol-m'2) (mg-m'2)
(kg-ha'1)
(days)
(kg-ha'1)
(mg-m'2)
Year 1 (2009-2010)
Winter*
119.2
152
0.80
40
16.3
7621
Summer
22.5
11.5
0.57
108
5381
28
Year 2 (2010-2011)
14.8
Winter
142.5
138
0.73
6981
36
9087
Summer
29.6
19.5
182
0.96
48

DISCUSSION
Response of nitrogenase activity to microclimate and seasonal patterns
The general temperature, light and moisture response curves for nitrogenase activity
are similar to those of other BSCs and terrestrial cyanobacteria (Coxson and Kershaw 1983a,
Marsh et al. 2006, Su et al. 2011). Nitrogenase activity decreased with declining moisture
below 15% MC (w/w) and showed little response at higher moisture contents. It is difficult to
compare moisture response to other studies as different measures of moisture contents are
57

often used (Belnap 2001), but the shape of this response is similar to that reported for other
BSCs and terrestrial cyanobacteria. Cyanobacteria-dominated BSCs from the hilly Loess
Plateau in the northern Shaanxi province of China exhibited little change in nitrogenase
activity at field water capacities above 40%, which corresponded to 12-19% (w/w)
depending on the age of the crust (Zhao et al. 2010). Recovery of nitrogenase activity can
vary based on prior hydration conditions (Coxson and Kershaw 1983a, Belnap 2002). Full
recovery of nitrogenase activity occurred between 24 and 48 hours of continuous hydration.
Our field measurements suggest the recovery curve used in the model may overestimate
actual recovery time, as field ARA measurements within several hours after hydration were
typically higher than predicted rates.
The optimal thallus temperature for nitrogenase activity in this study was between 21
and 28°C. This is consistent with the optimal temperature for BSCs collected from other
cold-temperate environments (Liengen 1999b, Belnap 2002, Marsh et al. 2006). Nitrogenase
activity has been shown to sharply decline at temperatures above the optimum (Belnap
2002), but hydrated BSCs at our study site rarely experienced temperatures above 30°C.
Most of the literature reports nitrogenase activity to cease below 0°C, but nitrogenase
activity has been reported at temperatures as low as -7.5°C (Belnap 2001). Our laboratory
incubations detected low but measureable rates of nitrogenase activity at a thallus
temperature of 0°C, and our field data showed overnight ARA rates at temperatures down to
-3°C fit closely to modelled rates. In order to provide a conservative estimate of rates of
winter nitrogen fixation, our calculations assumed that nitrogenase activity ceased at
temperatures below -5°C. This temperature cut-off also minimizes bias in our ARA estimates
during long periods when BSCs were in the dark under snow pack at temperatures below 58

5°C (as we did not correct for the decline in nitrogen fixation during prolonged dark periods
under snowpack). It is reasonable to assume that very little ARA activity would occur during
long-term burial under snow pack, as was reported by Crittenden and Kershaw (1979) in
Stereocaulon mats.
Our microclimate measurements show repeated freeze-thaw cycles in the late winter
and early spring. Temperatures for hydrated BSCs during these periods could range from 10°C at night to over 20°C in the day. The response of nitrogenase activity to freeze-thaw
events in Nostoc commune was dependent on the temperature and light conditions during the
diurnal cycles (Coxson and Kershaw 1983b). Nitrogenase can take several days to recover
fully after long periods of inactivation during freezing, but recovery is rapid during freezethaw if carbon stores can be replenished by photosynthesis during the day (Coxson and
Kershaw 1983b). We did not incorporate recovery time between freeze-thaw events, and our
late-winter field incubation results suggest that the model fit closely and may even
underestimate nitrogenase activity after freezing.
In general, soil cyanobacteria and cyanolichens have been found to reach light
saturation at low light levels (Belnap 2001). Light saturation for BSCs in this study was
between 600 and 1000 pmol PAR m ' V for crusts collected in the summer and between 300
and 600 pmol PAR m 'V in the winter. Light saturation also appeared to be temperature
dependent, with rates stabilizing at lower light levels under lower temperatures. Reported
light saturation for BSC organisms is variable; nitrogenase activity in N. commune reached
saturations levels at above 900 pmol PAR m ' V (Coxson and Kershaw 1983b),
cyanobacteria from the low arctic showed little to no response to light intensity (Stewart et
al. 2011b).
59

BSCs can continue to fix nitrogen in darkness, depending on the amount of available
carbon stores and the rate of respiration. There were 30 consecutive days in the winter of
2009 and 74 days in the winter of 2010 where the light levels did not exceed 50 pmol PAR
m 'V

due to snow cover. BSC temperatures during these periods did not exceed -0.2°C.

Dark ARA incubations conducted at 1°C showed continued low rates of nitrogenase activity
over the course of 48 hours. It is unclear how long nitrogenase activity can persist during
extended periods of low light and low temperatures under snow cover.
Potential landscape-level nitrogen inputs
The landscape level estimates for nitrogen input in the Chilcotin shrub-steppe
ecosystem varied dramatically, depending on which ratio was used to convert from acetylene
reduction estimates to actual nitrogen fixed. If we used the ARA/Nis conversion ratio
obtained in this study our estimate of landscape level nitrogen fixation reach approximately
75 kg/ha/year. These estimates for annual nitrogen BSC fixation are high compared to most
recent studies. They are however, within the range of 10-100 kg/ha/year reported by West
and Skujins (1977) for cold desert ecosystems. There are several factors that may result in
this high estimate, as discussed below.
One factor responsible for these high estimates of nitrogen is our experimentally
derived conversion ratio of 0.06. This conversion ratio is well below the theoretical factor of
3, often used in studies which do not calibrate ARA to ,5N2 incorporation. Conversion ratios
reported in the literature for terrestrial habitats have been found to vary widely. Our
conversion ratio was determined using similar methodology to Liengen (1999a), who
reported conversion ratios ranging from 0.022 to 0.073 for Arctic cyanobacterial crusts.
Belnap (2002) also used Liengen’s (1999a) conversion ratio of 0.062 and suggested that a 3:1
60

ratio greatly underestimates N inputs in most BSC studies. Differences in conversion ratios
may be due to a suppression of acetylene reduction, the favoring of l5N2 incorporation, or a
combination of both (Liengen 1999a). Suppression of ARA has been found to occur during
extended incubations. ARA rates during the side-by-side incubations in our study were
similar to those calculated during shorter incubation times under the same temperature and
light conditions, thus this is not likely to be a contributing factor in our study. Another
possibility, as suggested by Liengen (1999a), is the differential diffusion of acetylene,
ethylene and dinitrogen through the soil and polysaccharide gels surrounding the BSCs. The
exchange of smaller dinitrogen molecules may be favoured over the larger acetylene and
ethylene molecules, resulting in a lower conversion ratio.
ARA rates vary widely among different studies, over a range of 5 orders of
magnitude (Su et al. 2011). Many factors can affect these differences in rates, including pre­
treatment conditions (Coxson and Kershaw 1983a, Belnap 2002), species composition
(Belnap 2002, Stewart et al. 2011a, Su et al. 2011), successional stage (Housman et al. 2006)
and disturbance history (Belnap 1996). The suppression of acetylene reduction due to
differential diffusion or other effects may also play a role, further stressing the need for sitespecific conversion ratios. The optimum ARA rates observed in this study were similar to the
maximum rates observed in Collema dominated BSCs from a cold desert in Moab, Utah
(Belnap 2002).
There are several other factors that may affect our landscape-level estimate of
nitrogen input. Our estimate of BSC cover was based only on surveys of gently sloped,
south-facing terraces within the study area. BSC cover would be expected to decrease with
increasing slope. Aspect can strongly affect nitrogenase activity, even at the microscale, due
61

to differences in surface temperature and drying times (Davidson et al. 2002). Species
composition can also affect potential nitrogenase activity of BSCs. Chlorolichen dominated
BSCs will have lower rates of nitrogenase activity than cyanobacteria dominated dark BSCs
(Wu et al. 2009, Su et al. 2011). Our estimates of cover and nitrogenase activity were based
on material collected from relatively undisturbed shrub-steppe. A number of studies have
noted declines in both BSC cover and nitrogenase activity with grazing and other human
activity (Evans and Ehleringer 1993, Belnap 1996, Evans and Belnap 1999, Housman et al.
2006). The nitrogen fixation potential in areas more heavily impacted by surface disturbance
would likely be lower than what is reported here. Seasonality of grazing may also be an
important factor to consider. Memmott et al. (1998), for instance, found that BSC cover
declined from 27% in control sites in cold desert ecosystems of the US Great Basin to 14%
in summer grazed paddocks and 10% in spring grazed paddocks. These are values are similar
to what we have observed anecdotally in more heavily grazed Chilcotin shrub-steppe.
Seasonal patterns of nitrogen inputs by Chilcotin BSC communities
Traditional consideration of rangeland productivity focuses primarily on the spring
and summer growing seasons, when soils are unfrozen and water is available for plant
growth (Webb et al. 1978, Morton 1979). However, BSC communities do not face a similar
constraint. They can be physiologically active at any time of year, whenever water
availability and temperatures allow. In our 2009-2010 field season, which was characterized
by a relatively dry summer period, we found that high rates of BSC physiological activity
occurred during the late winter and early spring period, when snow melt episodes were
triggered by intense solar insolation. In our second field season the highest rates of ARA
activity occurred during July wetting events, although late winter (March) melt episodes
62

were again highly significant. The results of the model indicate that differences in seasonal
precipitation patterns may have profound effects of the overall potential fixed nitrogen and
the composition of BSC communities, a point emphasized by Belnap et al. (2004), who
found that Collema dominated crusts showed a large decline after treatments simulating
greater summer precipitation frequency.
Nitrogen released by BSC communities during summer rainfall events is likely
available to higher plant communities, however it is unclear what is the fate of fixed nitrogen
released to a shallow active layer above frozen ground in late winter. In many respects the
seasonal dynamics of late winter nitrogen cycling in Chilcotin shrub-steppe may be closer to
that of BSC communities in polar ecosystems, where released nitrogen can be entrained in a
shallow active layer (Stewart et al. 201 la, Niederberger et al. 2012). Marsh et al. (2006)
documented enrichment in total and mineralizable N in soils immediately under Chilcotin
BSC communities, suggesting that BSCs may play a major role in enriching these soils.
Marsh et al. (2006) also found that 15N natural abundance values in soils under BSCs were
much closer to atmospheric values than to soil derived values, again supporting the
hypothesis of BSC-derived nitrogen in Chilcotin soils. However, further estimates of
nitrogen uptake by plants, nitrogen volatilization, denitrification and leaching, are needed for
this area, particularly during the critical late-winter and early-spring period.

63

SUMMARY AND CONCLUSIONS

Biological soil crusts have been found to play significant roles in soil nitrogen
cycling in arid and semi-arid throughout the world, and British Columbia grasslands and
shrub-steppe appear to be no exception. Nitrogen inputs by BSCs in this ecosystem are also
sensitive to changes in temperature and precipitation patterns, stressing the need to include
these communities in any climate change planning and management of grasslands and shrubsteppe in this province.
At Lac du Bois Grasslands, we compared the nitrogen and carbon content o f BSCs
and the underlying soil across an elevation gradient corresponding with increasing
precipitation and decreased temperature. BSCs had a higher nitrogen and carbon content with
increasing elevation and consistently higher carbon and nitrogen than the underlying soil.
Furthermore, the average nitrogen isotopic composition of 15N in the BSCs was 2.24%o,
indicating that crusts are net importers of atmospheric nitrogen, and likely provide available
nitrogen to nearby plants. The decline in carbon and nitrogen content at lower elevations may
be due to lower rates of productivity and higher rates of N-losses. This suggests that a shift to
a climate with higher potential evapotranspiration will lead to a decline in nitrogen and
carbon content in soils occupied by BSCs.
In the Chilcotin grasslands, we determined the response of nitrogen fixation potential
in BSCs from a lower elevation grassland to changes in moisture, temperature and light
availability. Based on this response, we were able to model nitrogen fixation potential over a
two year period and determine an annual landscape estimate of nitrogen input for this

64

ecosystem. This landscape estimate varied substantially depending on the conversion ratio
used to convert rates of acetylene converted to rates of nitrogen fixed. Using a theoretical
ratio of 3:1, estimates of average annual BSC nitrogen fixation were 1.7 kgN h a '\ However,
using a conversion ratio of 0.06, obtained from side-by-side measurements o f acetylene
reduction and ,5N uptake, average landscape level estimates of BSC nitrogen fixation are 83
kg N ha'1. Our estimates of nitrogen fixation also varied between years due to differences in
the magnitude of summer precipitation.
The results from the Lac du Bois grasslands were very similar to those previously
reported in the Chilcotin region. This suggests that soil nutrient inputs by BSCs are similar in
both of these areas, and may be consistent throughout similar grassland and shrub-steppe
ecosystems in British Columbia. Our estimates for nitrogen fixation rates could also
potentially be applied to similar BSC communities throughout British Columbia.
These two studies both provide insight into the potential effects of climate change on
BSCs, but at different scales. Our study of an elevational transect at the Lac du Bois
grasslands suggests some potential effects of long-term shifts towards a hotter and drier
climate, while our study of annual N-fixation rates in the Chilcotin shows differences in Nfixation potential under different patterns of temperature and precipitation. The results of
both studies are important to the management of grasslands and shrub-steppe for climate
change as our future climate is predicted to have both long term directional shifts as well as
more short-term seasonal shifts such as timing of precipitation and snowmelt.
The results presented in this thesis open up a variety of opportunities for further
study. While we were able to quantify nitrogen inputs for lower elevation grasslands, the fate

65

of this fixed nitrogen is still largely unknown. Estimates of denitrification, ammonia
volatilization, and plant uptake are needed to better understand the significance of BSCs to
overall N-cycling in these ecosystems. Estimates of N-fixation rates for different BSC
community types, including those found in higher elevation grasslands, can also help refine
our model. Furthermore, our results suggest that BSCs contribute to soil carbon in
grasslands, but the significance of BSCs to carbon cycling in this region is also yet to be
quantified. Given the growing interest in soil carbon sequestration as a tool for reducing
greenhouse gas concentrations in the atmosphere, this question has many practical
implications.

66

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