Reindeer grazing affects plants

and mycelia in alpine treeline

and subarctic ecosystems

Frida Lindwall

Uppsats för avläggande av naturvetenskaplig masterexamen i Miljövetenskap 60 hp Institutionen för biologi och miljövetenskaper, Göteborgs universitet Januari 2013

 

Reindeer  grazing  affect  plants  and  mycelia  in  alpine  treeline  and  

subarctic  ecosystems    

 

This Master thesis consist of an introductory chapter followed by two papers:

Reindeer grazing has contrasting effect on species traits in Vaccinium vitis-idaea L. and Bistorta vivipara (L.) Gray. Frida Lindwall, Tage Vowles, Alf Ekblad and Robert G. Björk. (Submitted to Acta Oecologica)

Reindeer grazing affect the production of ectomycorrhizal mycelia in alpine tree-line ecosystems. Frida Lindwall, Tage Vowles, Alf Ekblad and Robert G. Björk (manuscript)

Contact info: Frida Lindwall fridalindwall@gmail.com +46725518559

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Table of Content

Reindeer  grazing  affect  plants  and  mycelia  in  alpine  and  arctic  ecosystems   1  

Introductory  chapter  for  master  thesis   4  

Introducerande  kapitel  för  examensarbete   7  

References   9  

Reindeer  grazing  has  contrasting  effect  on  species  traits  in  Vaccinium  vitis-­‐idaea  L.  and  Bistorta  vivipara  (L.)  Gray   11  

Abstract   12  

Introduction   13  

Materials  and  methods   14  

Study  site   14  

Sampling  and  processing   15  

Statistic  analyses   15  

Results  and  discussion   16  

Concluding  remarks   19  

Acknowledgements   19  

References   20  

Figure 1 24

Table 1 25

Table 2 26

Reindeer  grazing  affects  the  production  of  ectomycorrhizal  mycelia  in  alpine  treeline  ecosystems   27  

Abstract   28  

Introduction   29  

Material  and  method   31  

Sites  and  study  design   31  

3

Biomass  and  stable  isotope  analyses   33  

PLFA,  NLFA  and  ergosterol   33  

Data  analyses   34  

Results   34  

Growing  season  EMM  production   34  

Ergosterol   34  

Isotopic  compositions   35  

Discussion   35  

EMM  production   35  

Isotopic  signature   38  

Ergosterol   39  

Concluding  remarks   39  

Acknowledgment   40  

References   41  

Table 1 46

Table 2 47

Figure 1 48

Figure 2 49

Figure 3 50

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Introductory chapter for master thesis

Arctic species are adapted to exceptionally cold and harsh conditions; small changes in the

surrounding may cause dramatic changes in the distribution and composition of the

components of the system (Callaghan et al., 2007; Gottfried et al., 2012). The plants in an

ecosystem play an important role in the transport of substances between soil and atmosphere.

Plants assimilate carbon that is transported, via litter deposition, mycorrhiza and root exudates

to the soil, and nutrients are transported to plants via fungi. The climate change more rapidly

in the arctic than in any other biome in the world (McBean et al., 2005). Currently, the tree

line is advancing (e.g. Kullman and Öberg (2009)) and shrub cover is expanding (e.g. Sturm

et al., 2001; Tape et al., 2006); Elmendorf et al., 2012b) in the sub- and low arctic tundra.

These changes together with a potential decrease in lichen abundance, as a long-term response

to warming (Elmendorf et al., 2012a), will have large effects on the tundra and alpine treeline

ecosystem functioning (Callaghan et al., 2007; Elmendorf et al., 2012a; Olofsson et al., 2009).

To truly understand the carbon (C) and nitrogen (N) dynamics in a changing environment it is

vital to address the pools and flows of these elements and identify the key factors that may

affect them.

The native grazers in arctic and alpine tundra play a central role in structuring the vegetation

(Oksanen, 1990) by shaping the tree line (e.g. Van Bogaert et al. 2011) and influencing the

density of plants, species composition, and species richness (Eskelinen & Oksanen, 2006;

Manseau et al., 1996; Ravolainen et al., 2011; van der Wal et al., 2001; Virtanen, 2000). It has

been shown that grazing is the main environmental variable restricting birch sample growth in

alpine areas, even more important than warming (Hofgaard et al., 2010). Thus, since both the

climate (Callaghan et al., 2007; Elmendorf et al., 2012b) and herbivores (Oksanen, 1990;

Olofsson et al., 2009) affect the ecosystem structure and functioning, it is important to address

how individual species respond to environmental drivers in order to gain a deeper insight into

the processes that may shape our future landscapes. The first part of my thesis is a study on

how reindeer grazing affect the C and N allocation patterns in plants. The fieldwork was

conducted the year of 2011 in a sub arctic region, in the northern part of Sweden. Above- and

belowground parts of two plant species, Bistorta vivipara and Vaccinium vitis-idaea, were

sampled, using a 17-years old exclosure experiment. Biomass, C and N content and isotopic

signature were analysed. A lower C:N ratio were found in B. vivipara than V. vitis-idaea

suggesting a higher grazing pressure on that species. Reindeer grazing did not affect the R:S

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ratio appreciably nor the total biomass. However, difference in isotopic signature was found

between the species, indicating species-specific fractionation patterns and a difference in

isotopic signature between roots and shoots highlights the importance of analysing below- and

aboveground parts separately.

About 80% of all land plants live in symbiosis with fungi, forming mycorrhiza (Gehring &

Whitham, 1994). What is important to point out is that aboveground herbivores and fungi

share the same source of carbon, the plant. The link between herbivores, plants and fungi is

poorly understood and this interaction is important to address when trying to understand the

carbon and nitrogen dynamics in a system. An increase of mycelia production may decrease

the carbon flow from the biosphere to the atmosphere, and thereby affect the climate

(Treseder & Allen, 2000). In an ecosystem where nitrogen is limited and plants rely on the

symbiont for nutrient supply, as in the Fennoscandian Mountains, the interaction between

aboveground herbivores and fungi may be pronounced. There is a great need of knowledge on

how fungi are affected by their surrounding. In the second part of my thesis the effect of

reindeer grazing on mycelia production in alpine treeline ecosystems is addressed. The

fieldwork was done, the summer of 2011, on three different sites with two different vegetation

types along the Swedish mountain chain using 17-years old exclosures for studying the effects

of grazing. Sand filled mesh bags, allowing hypha but not roots to grow in (Wallander et al.,

2001), were placed in soil in early season, and collected in the end of the vegetation season.

Half of the sand was used for extracting mycelia by hand and 10 g was used for extracting the

fungal biomarker, ergosterol. The extracted mycelia was analysed for C and N content and

isotope signature. Reindeer grazing do, as hypothesised, affect the production of EMM due to

loss of photosynthetic plant parts and changes in cover and allocation patterns in plants. The

effect is inconsistent among vegetation types, depending on the dominant plant species and

the plant species reaction to grazing. This study suggests that herbivore affect the soil C

budget by decrease the abundance of shrubs and by changing their internal C allocation

patterns and thereby the production of EMM. The 15N signature indicates that N derived from

faeces is an important source for the fungi, and consequently also for plants. Moose grazing is

suggested to have larger impact on the 13C signature in the birch forest, as the EMM in

exclosures is more depleted. However, more mechanistic studies (i.e. stable isotope labelling

studies) are needed to separate the processes that affect the fungal biomass production.

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The overall conclusion is that reindeer grazing restrain the shrub cover and affect the

allocation patterns in plants and thereby also the soli C budget. However, the effect of grazing

on plants is not universal and may vary among vegetation types. The studies highlight the

importance of taking herbivores into account when trying to address the impact of climate

warming in arctic and alpine ecosystem.

7

Introducerande kapitel för examensarbete

Arktiska växter är anpassade för att klara av mycket tuffa och kalla förhållanden. Små

förändringar i omgivningen kan orsaka stora skillnader i utbredning och sammansättning av

ett känsligt ekosystems komponenter. Växterna i ekosystemet spelar en viktig roll när det

gäller transport av ämnen mellan mark och atmosfär. Växter assimilerar kol som sedan

transporteras till marken via förna, svamp och utsöndringar från rötter till marken. Kväve

transporteras till växter via svampar. Klimatet förändras mycket snabbt i arktiska miljöer,

mycket snabbare än i något annat biom (McBean et al., 2005). Trädgränsen förskjuts uppåt

(Kullman & Öberg, 2009) och busktäcket expanderar (Elmendorf et al., 2012b; Sturm et al.,

2001; Tape et al., 2006). Dessa förändringar tillsammans med en potentiell minskning av

lavtäcket som en effekt av en längre tids uppvärmning (Elmendorf et al., 2012a) kommer att

ha stora effekter på tundra samt alpina miljöer och funktioner (Callaghan et al., 2007;

Elmendorf et al., 2012a; Olofsson et al., 2009). För att verkligen förstå dynamiken hos kol

och kväve i ett föränderligt ekosystem, krävs ökad kunskap om dess flöden och pooler samt

om de nyckelfaktorer som kan påverka dem.

De inhemska herbivorerna i arktiska och alpina miljöer formar vegetationen (Oksanen, 1990)

genom påverkan på trädgränsen, densiteten av växter, artsammansättning och artrikedom

(Eskelinen & Oksanen, 2006; Manseau et al., 1996; Ravolainen et al., 2011; Van Bogaert et

al., 2011; van der Wal et al., 2001; Virtanen, 2000). Det har visats att herbivorer är den mest

begränsande variabeln för björktillväxt i alpina områden, till och med viktigare än

klimatförändringar (Hofgaard et al., 2010). För att få djupare insikt i processer som kan

komma att forma landskapet är det viktigt att förstå hur individuella arter reagerar på

miljömässiga drivkrafter. Den första delen i detta examensarbete behandlar renbetets

påverkan på individuella växters kol- och kväveallokering mellan de under- och ovanjordiska

delarna. Fältarbetet utfördes i på en subarktisk rished i norra Sverige, under sommaren 2011.

Ovan- och underjordiska växtdelar, från de två arterna Bistorta vivipara och Vaccinium vitis-

idaea, samlades in och analyserades för biomassa, C och N innehåll samt 13C och 15N-

signatur. Renstängsel från ett 17 år gammalt experiment användes för att se renbetets effekt på

växterna. En lägre C:N kvot hos B. vivipara indikerar på ett högre betestryck hos den arten än

hos V. vitis-idaea. Inga skillnaderna mellan total biomassa och R:S förhållandet hittades, men

studien visar att det finns en skillnad mellan arterna i uppsättning och fraktionering av

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isotoper. Studien belyser vikten av att analysera ovan- och underjordiska växtdelar separat för

att få en sann bild av växternas isotopsammansättning.

Ungefär 80 % av alla landlevande växter lever i symbios med svamp, de formar mykorrhiza,

(Gehring & Whitham, 1994). Betande djur och svamp delar samma källa till kol, nämligen

växten, men denna interaktion är relativt outforskad. För att förstå kol- och kvävedynamiken i

ett ekosystem är det viktigt att behandla interaktionen mellan dessa parter. En ökning av

mycelproduktion kan minska mängden kol som flödar från biosfären till atmosfären, och

därmed påverkar klimatet (Treseder & Allen, 2000). Det finns ett stort behov av kunskap om

hur svamp reagerar på olika faktorer i sin omgivning. Den andra delen av examensarbetet

behandlar hur mycelproduktion påverkas av renbete. Studien är gjord vid trädgränsen i tre

olika alpina områden med två olika vegetationstyper. Fältarbete genomfördes under

sommaren 2011 på tre olika fjäll, där 17 år gamla stängsel användes för att undersöka effekten

av att stänga ute renar och därmed förändra betestrycket. Sandfyllda nätpåsar, som tillåter

hyfer men inte rötter att växa in (Wallander et al., 2001) användes för insamling av mycel

under en växtsäsong. Från hälften av sanden extraherades mycel för hand, och från 10 g

extraherades biomarkören ergosterol. Det extraherade mycelet analyserades för C och N

innehåll samt 13C och 15N-signatur. Studien visar att renbete påverkar produktionen av mycel

på grund av förlorad fotosyntetiska växtdelar samt förändring i täckning och

allokeringsmönster hos växter. Effekten av bete är motstridig mellan de båda

vegetationstyperna, vilket beror på att olika växtarter dominerar och dess respons på betet.

Studien föreslår att bete påverkar C budgeten i marken expansionen av busktäcket minskar

och för att den interna C allokeringen hos växter förändras och därmed produktionen av

mycel. Studien visar även att bete av älg påverkar 13C signaturen i björkskogen, eftersom

mycelet innanför stängslen är mer urlakat. Fler mekanistiska studier (t ex märkning med

stabila isotoper) behövs för att separera de processer som påverkar produktionen av svamp.

Den övergripande slutsatsen är att renbete påverkar markens C innehåll eftersom expansionen

av busktäcket minskar och växter allokeringsmönster förändras. Men renbetets påverkan på

växter är mångsidigt och varierar mellan olika vegetationstyper. Studierna belyser vikten av

att ta hänsyn till betet när man försöker förutsäga klimatförändringarnas påverkan i arktiska

och alpina miljöer.

9

References

Callaghan, T. V., Björn, L., Chapin, F. S., Chemov, Y., Christensen, T. R., Huntley, B., . . . Shaver, G. R. (2007).

Arctic tundra and polars desert ecosystems Arctic Climate Impact Assessment scientific report (pp. 243-352).

University of Alaska Fairbanks: Cambridge University Press.

Elmendorf, S. C., Henry, G. H., Hollister, R. D., Björk, R. G., Bjorkman, A. D., Callaghan, T. V., . . . Wookey,

P. A. (2012a). Global assessment of experimental climate warming on tundra vegetation: heterogeneity over

space and time. [Research Support, Non-U.S. Gov't]. Ecol Lett, 15(2), 164-175. doi: 10.1111/j.1461-

0248.2011.01716.x

Elmendorf, S. C., Henry, G. H. R., Hollister, R. D., Björk, R. G., Boulanger-Lapointe, N., Cooper, E. J., . . .

Wipf, S. (2012b). Plot-scale evidence of tundra vegetation change and links to recent summer warming. Nature

Climate Change, 2(6), 453-457. doi: 10.1038/nclimate1465

Eskelinen, A., & Oksanen, J. (2006). Changes in the abundance, composition and species richness of mountain

vegetation in relation to summer grazing by reindeer Journal of vegetation science, 17, 245-254.

Gehring, C. A., & Whitham, T. G. (1994). Interactions between aboveground berbivores and the mycorrhizal

mutualists of plants. TREE, 9(7), 251-255.

Gottfried, M., Pauli, H., Futschik, A., Akhalkatsi, M., Barancok, P., Alonso, J. L. B., . . . Grabherr, G. (2012).

Continent-wide response of mountain vegetation to climate change. [Article]. Nature Climate Change, 2(2), 111-

115. doi: 10.1038/nclimate1329

Hofgaard, A., Lokken, J. O., Dalen, L., & Hytteborn, H. (2010). Comparing warming and grazing effects on

birch growth in an alpine environment - a 10-year experiment. Plant Ecology & Diversity, 3(1), 19-27. doi:

10.1080/17550871003717016

Kullman, L., & Öberg, L. (2009). Post-Little Ice Age tree line rise and climate warming in the Swedish Scandes:

a landscape ecological perspective. journal of ecology, 97(3), 415-429. doi: 10.1111/j.1365-2745.2009.01488.x

Manseau, M., Huot, J., & Crete, M. (1996). Effects of summer grazing by caribou on composition and

productivity of vegetation: Community and landscape level. journal of ecology, 84(4), 503-513.

McBean, G., Alekseev, G., Chen, D., Førland, E., Fyfe, J., Groisman, P. Y., . . . Whitfield, P. h. (2005). Arctic

climate: past and present. In C. Symon, L. Arris & B. Heal (Eds.), Arctic Climate Impact Assessment scientific

report (pp. 21-60). Camebridge: Cambridge University Press.

Oksanen, L. (1990). Predation, herbivory, and plant strategies along gradients of primary production. In J. B.

Grace & D. Tilman (Eds.), Perspective on plant competition (pp. 445-474): Academic press.

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Olofsson, J., Oksanen, L., Callaghan, T., Hulme, P. E., Oksanen, T., & Suominen, O. (2009). Herbivores inhibit

climate-driven shrub expansion on the tundra. Global Change Biology, 15(11), 2681-2693. doi: 10.1111/j.1365-

2486.2009.01935.x

Ravolainen, V. T., Brathen, K. A., Ims, R. A., Yoccoz, N. G., Henden, J.-A., & Killengreen, S. T. (2011). Rapid,

landscape scale responses in riparian tundra vegetation to exclusion of small and large mammalian herbivores.

Basic and Applied Ecology, 12(8), 643-653. doi: 10.1016/j.baae.2011.09.009

Sturm, M., Racine, C., & Tape, K. (2001). Climate change - Increasing shrub abundance in the Arctic. [Article].

Nature, 411(6837), 546-547. doi: 10.1038/35079180

Tape, K. E. N., Sturm, M., & Racine, C. (2006). The evidence for shrub expansion in Northern Alaska and the

Pan-Arctic. Global Change Biology, 12(4), 686-702. doi: 10.1111/j.1365-2486.2006.01128.x

Treseder, K. K., & Allen, M. F. (2000). Mycorrhizal fungi have a potential role in soil carbon storage under

elevated CO2 and nitrogen deposition. New Phytologist, 147(1), 189-200. doi: 10.1046/j.1469-

8137.2000.00690.x

Van Bogaert, R., Haneca, K., Hoogesteger, J., Jonasson, C., De Dapper, M., & Callaghan, T. V. (2011). A

century of tree line changes in sub-Arctic Sweden shows local and regional variability and only a minor

influence of 20th century climate warming. Journal of Biogeography, 38(5), 907-921. doi: 10.1111/j.1365-

2699.2010.02453.x

Van der Wal, R., van Lieshout, S. M. J., & Loonen, M. (2001). Herbivore impact on moss depth, soil

temperature and arctic plant growth. Polar Biology, 24(1), 29-32. doi: 10.1007/s003000000170

Virtanen, R. (2000). Effects of grazing on above-ground biomass on a mountain snowbed, NW Finland. OIkOS,

90(2), 295-300. doi: 10.1034/j.1600-0706.2000.900209.x

Wallander, H., Nilsson, L., Ola., Hagerberg, D., & Bååth, E. (2001). Estimation of the biomass and seasonal

growth of external mycelium of ectomycorrhizal fungi in the field. New Phytologist, 151, 753-760.

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Submitted to Acta Oecologica

Text pages: 12, Tables: 2, Figures: 1

Reindeer  grazing  has  contrasting  effect  on  species  traits  in  Vaccinium  

vitis-­‐idaea  L.  and  Bistorta  vivipara  (L.)  Gray  

Frida Lindwalla, Tage Vowlesa, Alf Ekbladb, and Robert G. Björkc, 1

aDepartment of Biological and Environmental Sciences, University of Gothenburg, P.O. Box 461, SE-405 30

Gothenburg, Sweden. bSchool of Science and Technology, Örebro University, SE-701 82 Örebro, Sweden cDepartment of Earth

Sciences, University of Gothenburg, Gothenburg, Sweden

1 Corresponding author: Department of Earth Sciences, University of Gothenburg, P.O. Box

460, SE-405 30 Gothenburg, Sweden. E-mail: robert.bjork@gu.se. Telephone: +46 31 786

2835. Fax: +46 31 786 1986.

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Abstract

That reindeer grazing can have large effects on plant communities is well known, but how reindeer

grazing is affecting plant traits and plant carbon (C) and nitrogen (N) allocation has not been studied in

the same extent. This study was conducted in a sub-arctic dry heath in northern Sweden. 17-year-old

reindeer exclosures were used to test whether reindeer grazing affects the C:N ratio (a plant quality

index), and the δ13C and δ15N (indicators of changes in C and N dynamics) as well as the C and N content

of above- and below ground parts of the evergreen dwarf shrub Vaccinium vitis-idaea L. and the perennial

forb Bistorta vivipara (L.) Gray. A lower C:N ratio found in B. vivipara compared to V. vitis-idaea

suggesting a higher grazing pressure on that species. We found that grazing reduced the total C content,

by 26%, and increased the δ15N, by 1‰, in the leaves of Bistorta vivipara, while no changes were

observed in V. vitis-idaea. Fine roots of B. vivipara had higher δ13C (1‰) and δ15N (2.5‰) than the

leaves, while such differences were not found in V. vitis-idaea. We concluded that reindeer grazing has

contrasting effects, both direct and indirect, on individual plant species. The results also highlight the

importance of analysing both above- and belowground plant parts when interpreting natural variations in

δ13C and δ15N.

Keywords: biomass allocation, carbon, grazing, nitrogen, stabile isotopes, tundra

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Introduction

The native grazers on the tundra play a central role in structuring the vegetation (Oksanen,

1990) by shaping the tree line (Van Bogaert et al., 2011) and influencing the density of plants,

species composition, and species richness (Eskelinen and Oksanen, 2006; Manseau et al.,

1996; Ravolainen et al., 2011; Suominen and Olofsson, 2000; van der Wal et al., 2001;

Virtanen, 2000). Since tundra species also are adapted to exceptionally cold and harsh

conditions, rapid climate warming is likely to cause dramatic changes in the distribution of

plants and ecosystems (Callaghan et al., 2007; Elmendorf et al., 2012; Gottfried et al., 2012).

In order to gain a deeper insight into the processes that may shape our future landscapes it is

therefore important to understand how individual species respond to changes in different

environmental drivers.

There are three mechanisms by which plants are affected by grazing – defoliation,

fertilization and trampling (Sørensen et al., 2009) – and there is ample evidence to suggest

that these mechanisms, on their own or in combination, can have a profound impact on

vegetation (van der Wal, 2006). Reindeer (Rangifer tarandus L.) feed on different plant

species depending on season. Grasses, forbs and leaves of deciduous shrubs and trees

dominate their diet during the summer while during winter they mostly eat lichens (Cetraria

spp., Sterocaulon spp., and Cladonia sp.) but also tissue from evergreen dwarf shrubs such as

Empetrum nigrum ssp. hermaphroditum (Hagerup) Böcher and Vaccinium spp. (Eriksson et

al., 2007). Many tundra ecosystems are nitrogen (N) limited due to suppressed N

mineralization in the cold environment. The direct impact on vegetation caused by trampling

and browsing by herbivores may have immediate effects on the ecosystem N dynamics, but

today’s research is not conclusive on the grazing effects on N pools and availability (Cargill

and Jefferies, 1984; Gauthier et al., 1995; Johnson and Matchett, 2001; McNaughton, 1979;

Mulder, 1999; Olofsson et al., 2004; Pastor et al., 1993; Ritchie et al., 1998; Stark and

Grellman, 2002; Zhu et al., 2008). To determine plants’ internal allocation patterns of N and

C, stable isotopes analyses have frequently been used (Brooks et al., 1997; Brüggemann et al.,

2011). The heavier stable isotopes of C and N (13C and 15N) have almost exactly the same

chemical and physical properties as the lighter ones (12C and 14N), except when considering

mass. Diffusion rate is slower and chemical bonds are stronger for the heavier isotope, and

therefore heavier isotopes are discriminated against in transportation and chemical reactions;

an isotopic fractionation occurs (Brand and Coplen, 2012). Isotopes can give indications on

14

the origin of nutrients (Michelsen et al., 1996), the availability of N in soil (Craine et al.,

2009) and how nutrients and C are transported and allocated in plants (Brüggemann et al.,

2011). The C to N (C:N) ratio of the plant tissue determines the palatability of the plants

(White, 1978). It has been found that the C:N ratio in plant tissue tends to be lower when

herbivores are present, suggesting that herbivores improve plant tissue quality (Sjögersten et

al., 2011). It has also been found that plants with a high R:S ratio increase in abundance with

increased grazing pressure (Evju et al., 2009), suggesting that high R:S ratio is a tolerance

strategy against herbivores. However, no study exists on how reindeer grazing affects

individual plant species’ internal allocation of C and N in the arctic and sub-arctic region,

which is N limited (Shaver et al., 1992).

Therefore, the aim of this study was to investigate how traits of two plant species in a

sub-arctic dry heath were affected by the exclusion of reindeer. Utilising seventeen-year old

reindeer exclosures, the perennial forb Bistorta vivipara (L.) Gray and the perennial evergreen

dwarf shrub Vaccinium vitis-idaea L. were examined for responses to grazing. The traits

investigated were biomass allocation (above- and below ground), C and N content of fine

roots and leaves and their isotopic composition (δ15N and δ13C). Due to the chemical defence

in evergreen plants (Bryant et al., 1991), the higher digestibility of forbs than evergreens

(Cornelissen et al., 2004) and that reindeer seem to prefer forbs in favour of other plants due

to higher N content (Trudell and White, 1981) we hypothesise that the effect of exclosure

differs between the two species. Furthermore, we hypothesise that B. vivipara increases its

belowground biomass allocation when grazed, thus increasing the R:S ratio, whereas V. vitis-

idaea does not respond to grazing since it should be a less preferred food resource. The C and

N isotope signature in plants is hypothesised to differ between exclosures and ambient plots

due to the changes in carbon allocation patterns and nitrogen availability brought forth by

reindeer.

Materials and methods

Study site

The study site is a dry heath located at 840 m above sea level in northern Sweden (N67°46.5’

E17°32.1’). The site is situated in an all year grazing area for reindeer with an annual mean air

temperature of -1.0 °C and a precipitation of 460 mm per year. There is no information

available on the exact number of reindeer visiting the site each year. However, reindeer

droppings were counted at the site in 1997 and again in 2011. The number of droppings found

15

in 1997 (± standard deviation) was 2 076 ± 1 415 per hectare (Eriksson et al., 2007), and in

2011 it was 1667 ± 1414 (T Vowles, unpublished data). During this time the number of

reindeer owned by the adjacent Sami village, Unna Tjerusj, has fluctuated between about

4000 and 6000 (around 6000 at the time of sampling) and was even higher prior to 1997.

Thus, the grazing pressure has been fairly high and fairly constant over at least the past 15

years. Dominating plant species in the field layer are the dwarf shrubs Empetrum nigrum ssp.

hermaphroditum, Vaccinium myrtillus L. and V. vitis-idaea, the graminoids Deshampsia

flexuosa L., Calamagrostis lapponica Wahlenb. and Carex bigelowii Torr. ex Schwein.

Dominating forbs were B. vivipara, Solidago virgaurea L. and Hieracium alpina L. The

bottom layer consisted of several moss species of the genera Dicranum and Polytricum and

lichens of the genera Cladonia, Cetraria and Stereocaulon.

Sampling and processing

Six plots, 25×25 m, were established in 1996 (Eriksson et al., 2007). To exclude reindeer

three plots were surrounded by 1.7 m high fences (hereafter called exclosures) and three were

left unprotected (ambient plots). The sampling was done in mid-August 2011, where three B.

vivipara and three V. vitis-idaea were randomly collected from each plot. To standardize the

sample volume, a 125 cm3 soil cube (5×5×5 cm) were cut out from the soil and stored

separately in plastic bags and frozen (within a few hours of collection) before further

processing. After thawing, the root system was cleaned from soil (see Björk et al. (2007))

after which the above- and belowground parts of the plant were separated. The dry mass was

determined after drying for 24 h at 70°C. After weighing the above- and belowground

biomass, the leaves and the fine roots (diameter < 2 mm) were collected and weighed

separately. To get a sufficient amount of sample, the fine roots from the three samples from

each plot were put together, and ground for C and N analyses. The C and N concentrations

and isotopic composition (δ13C or δ15N = ((Rsample – Rstandard)/Rstandard) × 1000 (‰), where R is

the molar ratio 13C/12C or 15N/14N) were determined from the milled materials as described by

Boström et al. (2007).

Statistic analyses

To investigate grazing effects on R:S ratios, aboveground-, belowground- and total biomass,

each species was analysed separately using a nested ANOVA. In this analysis, treatment was

a fixed factor and plot and replications were random factors within a hierarchical design.

Because of the limited amount of fine root and leaf biomass, the three replicates were pooled

16

together to plot samples to ensure a large enough sample for C and N analysis. Thus, to

investigate treatment effects on C and N traits in fine root and leaf biomass, C:N ratio, δ13C

and δ15N, a MANOVA was used with treatment as fix factor and plot as random factor.

Furthermore, the differences in C:N ratio, C and N content, δ13C, and δ15N between species

and fine roots and leaves were determined using a nested ANOVA with species and treatment

as fixed factors and plot as random factor. However, the limited amount of fine roots of V.

vitis-idaea made it impossible to test for differences between species and grazing. All data

were, after addition of a constant, log-transformed and concomitantly scaled to unit variance

to achieve a normal distribution and to eliminate skewness and ensure homogeneity of

variances according to (Økland et al., 2001). To increase the statistical power and to reduce

the risk of making Type II errors an α = 0.10 was used to test for significant differences.

Results and discussion

The exclusion of reindeer made the leaves of B. vivipara more depleted in δ15N (P = 0.044),

whereas the leaves of V. vitis-idaea were unaffected (Fig. 1). It also seems possible that the

fine roots of V. vitis idaea were more depleted when excluding reindeer. However, the fine

roots of V. vitis-idaea was not statistically tested since we just got one pooled sample from

each treatment, but the effect size of the treatment is in the same range as for B. vivipara

leaves, 1‰. There are three possible explanations for the overall pattern of 15N enrichment in

the grazed plots. First, reindeer faeces, which are only deposited in grazed plots, are likely to

be enriched in 15N (Finstad and Kielland, 2011). Enrichment of faeces has also been found in

other organisms: 1-4 ‰ in goose faeces (Sjögersten et al., 2010), about 6‰ in faeces from

small mammals (Hwang et al., 2007), about 3‰ in goat faeces (Codron et al., 2011) and 3‰

in sheep faeces (Wittmer et al., 2010). But while faeces are enriched in 15N urine is depleted

compared to the diet. Therefore, if urine and faeces are circulated back to the soil-plant

system, our data suggest that faeces would be a central N source and also much more

important than urine for the plants targeted in this study. However, this N source is allocated

differently in the two species, since when reindeer were excluded the greatest depletion of 15N

in B. vivipara was in the leaves whereas in V. vitis-ideae it was in the roots. A second

possibility may be differences in mycorrhiza association. Hobbie and Hobbie (2006) found

that plants were more 15N depleted in symbiosis with fungi, i.e. mycorrhiza, than if not. The

transport of N through hyphae towards the plant discriminates against 15N. As a consequence,

the N remaining in the fungus is enriched in 15N, while the N transferred to the plant is 15N

depleted. Due to the lower grazing pressure on plants in exclosures it is feasible to believe that

17

more C is available to fungi inside the exclosures. With more C available, mycorrhiza

colonization may increase (Gehring and Whitham, 1991) and, consequently, more 15N

depleted N would be transported to the plants in exclosures compared to plants in the ambient

plots. However, when comparing extramatrical mycelia production at other sites in

Fennoscandia this trend was not conclusive among vegetation types (F. Lindwall, unpublished

data) and the effect of aboveground herbivores on mycorrhizal colonization and mycelia

production is still unclear (Gehring and Whitham, 2002). Thirdly, Schulze et al. (1994) found

that the δ15N increases with the soil depth, and since the whole root system was not examined

in this study, it is possible that plants in the ambient plots had deeper root systems than in the

exclosures. However, the δ15N signature follows the same pattern between treatments for both

species but the belowground biomass does not. Furthermore, earlier studies (Björk et al.,

2007; Michelsen et al., 1996) have found that the majority of the root system of arctic plants

is in the organic layer, usually only a few centimetres thick in dry tundra heaths, making the

third explanation unlikely. Thus, we conclude that the most likely explanation for the 15N

enriched plants in grazed plots is the addition of 15N enriched droppings into the system, but

changes in ectomycorrhizal mycelia production or community change cannot be ruled out.

Furthermore, we found a higher (P = 0.099) amount of C in the leaves of B. vivipara in

exclosures than in ambient plots (Tab. 2), suggesting that the plants protected from grazers are

able to allocate more C to the leaves. The R:S ratio in B. vivipara tends (P = 0.16) to be

higher in ambient plots, and as Evju et al. (2009) argue, an increase of C to the roots is a

tolerance strategy against herbivory. If plants are protected against herbivory, C may instead

be allocated to the leaves. If there also is a sufficient amount of N, the productivity inside the

exclosures will increase (Larcher, 2003). However in contrast with earlier studies that showed

an increased abundance and height of forbs in exclosures (Bråthen and Oksanen, 2001;

Oksanen and Moen, 1994; Pajunen et al., 2008), we found no effect on total, above ground or

below ground biomass or on total abundance (T Vowles, unpublished data) in either of the

species (Tab. 1), and also, no significant grazing effect on total C and N content in V. vitis-

idaea leaves. The effect of reindeer on primary production and soil nutrient availability is not

clear and contrasting results from earlier studies show both negative (Stark and Grellman,

2002) and positive effects (Johnson and Matchett, 2001) of grazing on the available N. The

lemming population peak this year, was one of the largest for decades and had a large impact

on the alpine areas in Sweden. This may explain the lack of treatment effect on biomass, and

even though we found treatment effects in C and N traits in B. vivipara, the differences may

18

have been larger if lemmings also were excluded (Olofsson et al., 2012). Despite potential

interfering lemming effects, our results, as hypothesized, show that individual plant species

respond differently to reindeer grazing, which also will have implications on how species will

respond to environmental change in sub-arctic ecosystems.

Moreover, there was a clear difference in δ15N, δ13C, C:N ratio, and C, N and biomass

allocation patterns between the species, under ambient conditions (see Tab. 2 and Fig. 1). The

C:N ratio, which is about twice as high in V. vitis-idaea than in B. vivipara (P < 0.001),

indicates that B. vivipara is, as hypothesised, of higher quality as forage. The C:N ratio is in

general higher in evergreens than deciduous plants because evergreens have higher

concentrations of lignin and other secondary C substances (often used as defense substance

against grazing) than plants with shorter leaf lifespan (Aerts, 1995). The δ15N was

significantly lower (P < 0.001) for B. vivipara than for V. vitis-idaea, especially in the leaves.

The δ15N in leaves of both plants were more depleted than the fine roots, which was also

found by Emmerton et al. (2001), and may be explained by an internal discrimination of 15N

that occurs between roots and shoots (Brüggemann et al., 2011). Thus, above- and

belowground plant parts should both be taken into account when considering plant isotope

composition. There is also a difference in δ13C in the leaves between the two species, where

B. vivipara is more depleted (P = 0.057), which is consistent with results shown by Brooks et

al. (1997). They explained the differences between plant functional types as an effect of

different photosynthetic rates and different degrees of discrimination against 13C in the

assimilation of carbon dioxide (CO2). Differences in boundary layer and stomatal- and

internal conductance (Brüggemann et al., 2011; Warren, 2007) may occur between the two

species and there are also different responses between plant functional groups due to

environmental factors (Brooks et al., 1997; Warren, 2007). The difference in δ13C and δ15N

between fine roots and leaves is larger for B. vivipara than V. vitis-idaea. This pattern is

probably a result of differences in rate and amount of assimilated C between deciduous plants

and evergreens (Warren, 2007), but also species-specific internal fractioning of isotopes

between shoot and roots (Brüggemann et al., 2011; Dawson et al., 2002). Thus, the different

δ13C and δ 15N signatures between species highlight the species specificity in the fractionation

process occurring in both photosynthesis and N transfer between the plant and its fungal

symbiont. The lower C:N ratio found in B. vivipara supports the hypothesis that reindeer

should prefer to eat B. vivipara over V. vitis-idaea, although V. vitis-idaea is also affected by

the reindeers, which may indicate a rather high grazing pressure in this area.

19

Concluding remarks

The herbivore population has important effects on plant carbon and nitrogen dynamics. The

higher δ15N signatures in the ambient plots are probably an indication that faeces, which are 15N enriched, are an important N source for plants in tundra ecosystems. The C:N ratio

supports the idea that B. vivipara is a food resource of higher quality than V. vitis-idaea.

There was a larger difference in the isotope signature between roots and shoots in B. vivipara

than V. vitis-idaea, which is probably a result of differences in internal isotopic fractionation

and photosynthetic rate between plant functional types. Clearly, reindeer do affect plant traits

either directly, by removing and destroying biomass, or indirectly, by addition of nitrogen via

faeces. For deeper insights into how reindeer are affecting nutrient dynamics in tundra

ecosystems, more mechanistic studies (e.g. isotopic labelling studies) are needed to separate

the processes behind the species-specific differences in allocation patterns found in this study.

Acknowledgements

The authors thank Mathias Molau and Paloma Alvarez Blanco for their assistance in the field.

The Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning

(grant no 214-2010-1411 to RGB) supported this work and is gratefully acknowledged. The

work was also conducted with financial support from the strategic research area BECC

(Biodiversity and Ecosystems in a Changing Climate; www.cec.lu.se/research/becc).

20

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24

Figure 1. δ13C (a) and δ15N (b) in leaves and fine roots in Vaccinium vitis-idaea and Bistorta vivipara (n = 3)

exposed (Ambient) or not exposed (Exclosures) to grazing by reindeer. The error bars represent standard error.

** P < 0.05 indicates significant difference between treatments.

25

Table 1. Total biomass, above- and belowground biomass and root: shoot (R:S) ratio for the species Bistorta

vivipara and Vaccinium vitis-idaea in ambient plots (exposed to grazing by reindeer) and exclosures (not

exposed reindeer). Values show mean and standard error (±SE) for n = 3 plots

 

Bistorta  vivipara   Vaccinium  vitis-­‐idaea  

Ambient   Exclosure   Ambient   Exclosure  

Total  biomass  (mg)     160.8  (22.8)   207.7  (31.3)   104.0  (20.7)   138.2  (41.2)  

Aboveground  biomass  (mg)     39.5  (5.5)   51.2  (8.1)   80.3  (16.6)   118.5  (40.3)  

Belowground  biomass  (mg)     121.3  (18.8)   156.5  (26.7)   23.7  (5.2)   19.7  (4.1)  

R:S     4.29  (1.2)   2.86  (0.3)   0.39  (0.1)   0.27  (0.1)  

26

Table 2. The C and N content and the C:N ratio for leaves and fine roots of the two species Bistorta vivipara and

Vaccinium vitis-idaea in ambient plots (exposed to grazing by reindeer) and in exclosures (not exposed to

reindeer). Values show mean and standard error (±SE) for n = 3 plots. Significant treatment effects are shown

with * (P < 0.1).

 

Bistorta  vivipara   Vaccinium  vitis-­‐idaea  

Leaf   Fine  root   Leaf   Fine  roota  

Ambient   Exclosure   Ambient   Exclosure   Ambient   Exclosure   Ambient   Exclosure  

C  (g  C  plant-­‐1)   8.8  (0.9)   11.9  (1.2)*   7.9  (0.9)   9.3  (1.2)   38.1  (7.5)   47.2  (12.4)   0.8   0.5  

N  (g  N  plant-­‐1)   0.4  (0.03)   0.5  (0.04)   0.2  (0.03)   0.3  (0.06)   0.9  (0.14)   1.1  (0.3)   0.01   0.01  

C/N   24.2  (0.8)   21.8  (1.2)   33.1  (1.5)   35.0  (0.8)   42.5  (1.7)   42.4  (1.6)   54.5   63.8  

a. The fine root samples of V. vitis-idaea were, because of the limited amount of sample, merged into one

pooled sample, making it impossible to test for differences between species and grazing.

27

Manuscript

Text pages: 18, Tables: 2, Figures: 3

Reindeer  grazing  affects  the  production  of  ectomycorrhizal  mycelia  in  

alpine  treeline  ecosystems  

Frida Lindwalla, Tage Vowlesa, Alf Ekbladb, Robert G. Björkc,2

aDepartment of Biological and Environmental Sciences, University of Gothenburg, P.O. Box 461, SE-405 30

Gothenburg, Sweden. bSchool of Science and Technology, Örebro University, SE-701 82 Örebro, Sweden. cDepartment of Earth Sciences, University of Gothenburg, Gothenburg, Sweden

2 Corresponding author: Department of Earth Sciences, University of Gothenburg, P.O. Box

460, SE-405 30 Gothenburg, Sweden. E-mail: robert.bjork@gu.se. Telephone: +46 31 786

2835. Fax: +46 31 786 1986.

28

Abstract

Fungi play an important role in the soil carbon (C) dynamics as they contribute to the soil organic matter

pool. Herbivores and mycorrhizal fungi share the same source of C, i.e. plants. Thus, it is likely that

herbivory indirectly can affect the production of mycorrhizal mycelia. This study addresses the impact of

reindeer grazing on the production of extramatrical mycelia (EMM) in the Scandinavian mountains, an

unexplored area of research. The study was conducted, using a 17-year old exclosure experiment, at two

different vegetation types, birch forest heath type with mosses and alpine dry heath at three different sites in

the mountain range. The sampling was done using ingrowth mesh bags allowing hyphae but not roots to grow

in. Mycelia and rhizomorphs were extracted by hand, weighed and analysed for C content (to estimate EMM

production) and δ13C and δ15N (indicators of changes in C and nitrogen (N) dynamics). Also, the

biomarker ergosterol was extracted for estimation of the production but also to compare the different

approaches. There was a significant larger EMM production in the birch forest than on the dry heath, due to

less grazing pressure and overall higher production in the birch forest. The EMM productions were nearly 2

and 4 times larger in the exclosures in the birch forest on Långfjället and Fulufjället, respectively. The EMM

production, on the dry heath on Långfjället, was 3 times larger in ambient plots than exclosures, which is

explained by a decreased abundance and an increased C allocation to the roots of dwarf birch (Betula nana)

when reindeer are present. Mycelia was 1.0‰ more 13C depleted in exclosures in the birch forest on

Fulufjället (no reindeer grazing exists), indicating that grazing by moose may be important for determining

the isotopic signature in the birch forests with high moose abundance. At the dry heaths, the 15N showed

contrasting response in the exclosures, with 1.7‰ depletion and 1.5‰ enrichment compared to ambient plots

in Fulufjället and Långfjället, respectively. However, several factors determine the 15N signature and none

can be ruled out. This study shows that reindeer restrain the expanding shrub cover and affect the C

allocation in plants, and thus the EMM production and soil C budget.

Key words: Alpine, biomass production, carbon, ectomycorhizae, extramatrical mycelia, grazing, reindeer,

stable isotopes, treeline ecosystem

29

Introduction

About 80% of all land plants are in symbiosis with fungi, forming mycorrhizae, and the

symbiosis is found in almost all terrestrial ecosystems (Gehring & Whitham, 2002). In

exchange for carbon (C), the fungi, due to the extramatrical mycelia (EMM) (i.e. an increased

nutrient absorbing surface area) (Rousseau et al., 1994), enhance the plant’s ability to take up

nutrients (Allen et al., 2003; Smith & Read, 2008) and they also protect the plant from

pathogens and improve plant water status (Cairney, 1999). There are two general types of

mycorrhiza (Endo- and ectomycorrhiza) classified after how they associate with the plant root

(Ricklefs, 2008). Endomycorrhizal fungi connect intracellularly (i.e. the hypha penetrates the

cell), while ectomycorrhizal (EcM) fungi form a sheet on the outside of the roots and

penetrate the root between the epidermal and cortical cell (Finlay, 2008). Depending on the

type of mycorrhiza, 10-50% of the photosynthate is transported to, and thus support, the fungi

(see review by Simard et al. (2002)). Fungi have an important role in the C cycling in an

ecosystem, it has been estimated that more than 60% of all C in a young poplar forest that

enters the soil organic matter (SOM) pool, originates from extramatrical mycelia (EMM) and

is thereby the dominant pathway (Godbold et al., 2006). Thus, an increase of EMM

production may increase C sequestration in soil and thereby counteract the release of carbon

to the atmosphere (Godbold et al., 2006; Treseder & Allen, 2000). However, the EMM may

also contribute to the decomposition of SOM and, thus, the release of CO2 to the atmosphere

(Talbot et al., 2008). The plant together with its mycorrhizal symbiont forms a direct link

between the atmosphere and the soil, and further knowledge about the processes affecting the

growth of EMM is needed to understand their role in ecosystem C dynamics.

Since herbivores and fungi share the same carbon source, i.e. the host plant, it is likely

that interaction between them occurs. However, both positive and negative effects of grazing

on mycorrhiza have been found (reviewed by Gehring and Whitham (1994). Gehring and

Whitham (1994) concluded that most mycorrhizal fungal species (both AM and EcM) were

negatively affected by herbivory, due to a lack of photosynthate, but some species increased

their colonization when host plants were grazed. Possibly, this increased colonization is due to

an increase of C allocation to the roots (and belowground), which is suggested as a strategy to

endure herbivory (Evju et al., 2009). In a review paper, Treseder and Allen (2000) reported

that an increased root biomass tends to increase the hyphal biomass, but they also highlight

that this is not a universal relationship and may vary among ecosystems. However, the lack of

C due to defoliation is maybe the most obvious negative impact on the ectomycorrhizal

30

community, but also an addition of nitrogen (e.g. via faeces) may negatively affect the fungi.

For instance, Lilleskov et al. (2002) have found that the ectomycorrhizal (EcM) diversity

declined when the N deposition increased, and Parrent et al. (2006) have reported a decline in

EcM richness with increased net mineralization. In an ecosystem where nitrogen is limited

and plants rely on the symbiont for nutrient supply, as in mountain ecosystems, the interaction

between mammal herbivores and fungi may be pronounced. Reindeer (Rangifer tarandus L.)

are selective in the choice of forage, which may have a profound effect on the plant species

composition (Bråthen & Oksanen, 2001). Also many fungal species are selective in their

choice of host plant and the herbivores may, thereby, indirectly affect the fungal species

composition by structuring the plant community (Gehring & Whitham, 2002). Different plant

species also respond differently to grazing (Bråthen & Oksanen, 2001; Evju et al., 2009;

Manseau et al., 1996) and the effect of grazing on mycelia production may therefore vary

among vegetation types. There are several studies treating the effect of herbivores on

mycorrhizal colonization (Allen, 1991; Gange et al., 2002; Ruotsalainen & Eskelinen, 2011),

but there are few, if any, studies about the effect on EMM production in an alpine or arctic

biome. Knowledge about the EMM production, and the factors affecting it, is needed to truly

understand C dynamics in this sensitive environment.

One of the most used methods to study mycelia production is to use ingrowth bags (or

core), but several different approaches have been used to estimate the EMM content in the

mesh bags. The determination of the chemical marker ergosterol is frequently used to estimate

the fungal biomass in soil (Djajakirana et al., 1996; Möttönen et al., 1999; Bååth, 2001;

Wallander et al., 2001; Hagerberg et al., 2003; Zhao et al., 2005; Clemmensen et al. 2006;

Högberg, 2006; Karliński et al., 2010). Ergosterol is a sterol almost exclusively found in cell

membranes of living fungi, and not in plant or animal cells (Wallander et al., 2012). However,

variation in ergosterol content has been reported (Djajakirana et al., 1996; Montgomery et al.,

2000; Olsson et al., 2003; Zhao et al., 2005) and problems occur when converting ergosterol

content to fungal biomass. An average value, of 3 µg/mg fungi, for ergosterol content in fungi

is often used (Clemmensen et al., 2006; Salmanowicz & Nylund, 1988; Wallander et al.,

2001). Thus, the conversion may be misleading, since the amount of ergosterol in fungi may

not be constant over time or other abiotic factors may affect the sterol content in the fungi

(Wallander et al., 2012). Another way is to collect mycelia by hand from the sand and weigh

and analyse the mycelia for C, a rapid and inexpensive method that avoids conversion factors

(Wallander et al., 2012). However, the method assumes that all extracted material is of fungal

31

origin and there is a risk that parts of the mycelia are left behind. A comparison between the

two different approaches for EMM production estimation has never been done, and is needed

for trustful and accurate comparisons between studies.

The aim of this study is twofold. First, we want to examine the effect of grazing on the

mycelia production, using sand filled ingrowth mesh bags (Wallander et al., 2001), in

exclosures, where reindeer were excluded, and ambient plots, where reindeer had free access.

Since herbivores affect the plant by removing aboveground tissue, we hypothesis that the

amount of C transported to the fungi would differ between grazed and ungrazed plots, either

by a decreased ability for the plant to support the fungi with C or by a increased root

production as a tolerance strategy against herbivory, and thereby increases the amount of C

transported below ground. To gain deeper insight into the C and N dynamics the natural

abundance of 13C and 15N was analysed. Since herbivores affect the photosynthetic apparatus

and the nitrogen composition in soil the stable isotope natural abundance is expected to differ

between treatments. Second, we also wanted to compare the different EMM production

estimates used; the biomarker ergosterol and extracting mycelia by hand from the sand for

weighing and analysis of carbon content.

Material and method

Sites and study design

The study was conducted over the growing season of 2011, on three mountains, Fulufjället

(FU), Långfjället (LO) and Poullanvare (PO), in the Scandinavian mountain range (see Tab. 1

for detailed information). On each mountain, two vegetation types were investigated – a dry

heath and a mountain birch (Betula pubescens Ehrh. ssp. czerepanovii (N. I. Orlova) Hämet-

Ahti) forest heath type with mosses. In 1995, six plots (25×25 m) were established on each

mountain and vegetation type (Eriksson et al., 2007) and the same plots were used in the

present study. Fences (1.7 m), used for reindeer protection, surrounded three of the six plots.

These plots are hereafter called exclosures and the mycelia production in these plots was

compared to the mycelia production in ambient plots, where reindeer had free access. In total,

36 plots were used for sampling. In summer 2011 a plant species inventory was also

conducted and soil temperature has been measured at a depth of 2 cm in each plot since spring

2011, using Tinytag loggers.

32

Fulufjället is the southernmost located field site in this study. The dry heath, located on a

north-facing slope, is dominated by Betula nana L., Calluna vulgaris (L.) Hull, Vaccinium

vitis-idaea L., Vaccinium myrtillus L., Empetrum nigrum ssp. hermaphroditum L.,

Deschampsia flexuosa L., Hieracium alpine L. and Trientalis europaea L. and the bottom

layer is dominated by Cladonia spp and Cetraria islandica (L.) Ach., The birch forest heath

type with mosses is a low forest dominated by B. pubescens L. with undergrowth of dwarf

shrubs, Vaccinium sp., and E. nigrum and grasses, D. flexuosa and Nardus stricta as well as

herbs, T. europea, Linnea borealis L. and Solidago virgaurea L. In the bottom layer mosses,

such as Pleurozium schreberi L. and Barbilophozia lycopodioides (Wall.) Loeske, are more

common than lichens. Since there are no Sami villages nearby Fulufjället, reindeer from

Sweden never visit this site (Eriksson et al., 2007). However, on rare occasion small groups

of reindeers from Norway cross the border and visit the site, but the grazing pressure from

reindeer is negligible. Nevertheless, the moose population on Fulufjället is, according to the

Swedish Environmental Protection Agency, very large (Naturvårdsverket, 2002), and the

grazing pressure in the birch forest, a preferred forage species (Wam & Hjeljord, 2010) is

therefore assumed to be large.

Långfjället is also located in the southern part of the mountain range, just 400 km north of

Fulufjället. The vegetation is similar to Fulufjället although the birch forest also contains

Pinus sylvestris L. In summer time reindeer frequently graze this site. The abundance of

Betula nana, the utterly dominant ectomycorrhizal plant in the field at this site, was on the dry

heath higher in exclosures compared to ambient plots. The dry heath is located on an east-

facing slope.

Poullanvare is the northernmost sampling site located about 50 km north of the town

Kiruna. The dry heath is situated in a north-westerly slope and the vegetation is similar to the

other two mountains with the main difference that C. vulgaris does not grow here but the

grass Calamagrostis lapponica (Wahlenb.) Hartm. is common. Unfortunately, due to sabotage

of the fences, reindeer were not properly excluded at this site. Instead the herd was enclosed

and the grazing pressure inside the exclosures was extremely large early in the season.

In each plot, four ingrowths bags (mesh size 50 µm; length 10 cm and diameter 2 cm)

(also used and explained in detail by Wallander et al. (2001)), were, in early season (see table

1 for details) placed in the soil (0-10 cm soil depth). The bags contained 40 g pure sand (with

no nutrients), burned in 550°C for 24 hours to ensure that no carbon remained, allowing only

33

mycorrhizal mycelia to grow. In fall 2011 the bags were collected and frozen within a few

hours before further processing.

Biomass and stable isotope analyses

To obtain an even distribution the sand from the four ingrowths bags was mixed and 80 g, of

totally 160 g, was randomly collected. The sand was extracted in water and mycelia and

rhizomorphs floating in the water were collected with tweezers and freeze-dried. To get the

water content of the sand, the remaining sand was freeze-dried and then weighed for

compensation in the biomass estimation (see below). When dry, the extracted mycelia were

further rinsed from sand grains before analysed for C concentrations and C and N isotopic

composition as described by Boström et al. (2007). The isotopic composition is expressed in

the standard notation (δ13C or δ15N) in parts per thousand (‰) relative to the international

standards, N in air or C in Vienna Pee Dee Belemnite (δ13C or δ15N = ((Rsample –

Rstandard)/Rstandard) × 1000 (‰), where R is the molar ratio 13C/12C or 15N/14N) (Högberg et al.,

1999). The production (g C m-2) of extramatrical mycelia (EMM) was calculated as

𝐸𝑀𝑀 =!!""  ×  !%  ×  

!!"!!!"#! !!"#∗!%

! (Eq. 1)

where mEMM is the EMM dry mass in g, C% is the carbon content in % of EMM dry weight,

mtot is the total weight in g of the sampled sand (160g in this study), mext is the weight of the

extracted wet sand (i.e. 80 g in this study), w% is the sand water content in % and A is the

tube area (i.e. 0.00063 m2 in this study).

PLFA, NLFA and ergosterol

Ten grams of freeze-dried sand from each sample was used for PLFAs analysis. To compare

the PLFA and ergosterol content in the mycelia extracted sand with the sand not extracted

from mycelia, both types were analysed. Also totally new, unused sand was examined. Thirty-

six mycelia extracted samples, 36 non-extracted samples and 5 pure sand samples were

analysed, making a total of 77 samples. Lipids were extracted using the method described by

Frostegård et al. (1991) with some small modifications. In each 10 g sample di-nonadecanoyl

phosphatidylcholine was added as an internal standard. Extracted lipids were fractionated

using a silicic column. The first, chloroform, fraction, was further analysed for NLFA 16:1ω5

(biomarker for arbuscular fungi) and ergosterol while the third, methanol fraction was further

34

analysed for PLFA 18:1ω9 and 18:2ω6,9 (biomarker for fungi). The phospholipids were

transesterified with 0.5 M sodium methoxide, which was analysed on a gas chromatograph

(GC) with a 30 m DB-5 column and a flame ionization detector.

The ergosterol was extracted from the NLFA fraction, by adding pyridine and BSTFA

and incubated for 10 min in 60 °C. The NLFA fraction was further processed; sodium-

metoxide and heptan was added and the samples were sonicated for 30 min. HAc and water

was added and the upper phase was dried and dissolved in acetonitrile. All samples were

analysed in a GC with a 30 m DB-5 column and a flame ionization detector.

Data analyses

Significant differences between exclosures and ambient plots were tested with two-sample t-

test using R (R version 2.15.1). To achieve a normal distribution all data were log transformed

and to increase the statistical power and to reduce the risk of making Type II errors (i.e. to

accept the null hypothesis, even though it is false) an α = 0.10 was used to test for significant

differences.

Results

EMM production

In general, the EMM production was significantly higher (P = 0.042) in the birch forests (2.0

± 0.4 SE g C m-2) compared to the dry heaths (1.6 ± 0.3 g C m-2). The EMM production in the

birch forest on Fulufjället and Långfjället was significantly higher (P < 0.05) in the exclosures

compared to the ambient plots, 4 and 2 times, respectively. No treatment effect was found in

the birch forest on Poullanvare. On the dry heaths, only Långfjället showed a significant

difference (P = 0.065) in EMM production between the treatments. However, in contrast to

the birch forest the ambient plots had 3 times higher EMM production than exclosures. No

treatment effect was found on the dry heath on Fulufjället or Poullanvare.

Ergosterol

Not all samples were successfully extracted, resulting in a limited amount of data of

ergosterol content, and no PLFA or NLFA data. Since no replications were extracted from the

sites no statistical analysis was done. However, the results of ergosterol are shown in Fig. 3

35

and the average conversion factor between ergosterol and mycelia was 5.5 ± 1.25 SE mg/g C,

calculated from the extracted ergosterol in 10 g of sand and the weighed mycelia in 80 g sand.

Isotopic compositions

In general the EMM sampled in the ambient plots in the birch forests were 1.2 ‰ more 13C

depleted compared to the ambient plots on the dry heaths (P = 0.042). Furthermore, the EMM

were significantly 1.0‰ (P = 0.078) and 1.2‰ (P = 0.025) more 13C depleted in exclosures in

the birch forest on Fulufjället and Poullanvare dry heath, respectively, compared to the

ambient plots (Tab. 2). No treatment effect on 13C in EMM was found in the birch forest or

the dry heath at Långfjället.

There was no general difference in 15N-signature between the vegetation types and the

treatment effect varied between sites and vegetation types (Tab. 2). The EMM in dry heath

exclosures on Fulufjället were significantly 1.7‰ (P = 0.032) more 15N-depleted in exclosures

than ambient plots while the opposite was found on Långfjället where EMM in the dry heath

exclosures were significantly 1.5‰ (P = 0.080) more enriched compared to the ambient plots.

No treatment effect on 15N in EMM was found in the birch forest or the dry heath at

Poullanvare.

Discussion

EMM production

This study demonstrates, for the first time, that aboveground grazing has an effect on EMM

production in an alpine treeline ecosystem. The EMM production is overall very low, as

expected in this low productive environment, with a maximum EMM production of 6.1 g C

m-2 and an average of 2.0 g C m-2 in the birch forests and 1.6 g C m-2 on the dry heaths,

respectively. Wallander et al. (2001) report an hyphal production of 29.5 and 21 g C m-2 in a

Norway spruce forest and a mixed oak–spruce forest respectively and EMM production is

reported to vary between 8 and 50 g m-2 in a review by Ekblad et al. (2012). These findings

show a much higher production than seen in the alpine treeline ecosystem in the present

study. However, our results are in line with Clemmensen et al. (2006), who showed a biomass

production of 1.3 g m-2 on a dry heath in subarctic Sweden. The larger production of EMM in

the birch forest than on the dry heaths is most likely due to the different dominating plant

species and their ability to support the fungi with C. The ectomycorrhizal B. pubescens

36

dominate the forest while B. nana is the dominating ectomycorrhizal species in the dry heath.

B. pubescens can because of the higher production in forests than dry heaths (Hartley et al.,

2012) and because of lower reindeer grazing pressure due to the higher stature of trees than

shrubs, transport more C to the fungi than B. nana, which explains the higher production.

Within the birch forest an effect of reindeer exclusion was found on both Långfjället and

Fulufjället, where the EMM production was higher in exclosures. Since plants in exclosures

are protected from heavy grazing pressure, more of the assimilated C can be transported to the

fungi, and thereby increase the production of EMM and, thus, the nitrogen intake to the plant.

The loss of aboveground biomass due to grazing may reduce the amount of assimilated C

transported to the roots and fungi (Trent et al., 1988), which may explain the lower mycelia

production in ambient plots in the birch forest on Långfjället. However, the effect seen on

Fulufjället is, most likely, not an effect of reindeer grazing since reindeer rarely visit this site.

Instead, the pattern is probably due to a difference in the abundance of birch trees, with more

trees in exclosures, which simply is due to an unluckily plot selection and not an effect of

grazing. Also the effect may partly be due to grazing by moose (Alces alces) which

population has been reported, by the Swedish Environmental Protection Agency, to be very

large on Fulufjället (Naturvårdsverket, 2002). The EMM production did not differ between

treatments in the birch forest at Poullanvare, which probably is an effect of an autumnal moth

(Epirrita autumnata L.) outbreak in 1995 (Karlsson & Weih, 2003) and 2004 (Klemola et al.,

2008). The population of E. autumnata fluctuates with periods of 9-10 years and does serious

damage on the foliage of mountain birch in northern Fennoscandia (Tanhuanpää et al., 2002).

This outbreak often occurs in mature forests, leaving young trees and stands less damaged

(Ruohomaki et al., 1997; Tenow et al., 2004), changing the forest structure, making the effect

of reindeer grazing negligible. Between 1995 and 2011 the amount of mountain birch stems

increased in both exclosures and ambient plots in the birch forest on Poullanvare. However,

the average height of the trees decreased (Eriksson et al., 2007; T. Vowles, unpublished data),

suggesting a rejuvenation of the birch forest. Younger trees allow higher EMM production

than old trees (Wallander et al., 2010). Thus, the rejuvenation of the forest may explain the

somewhat higher EMM production in Poullanvare than Långfjället and Fulufjället (Tab. 2).

Also, no damage on vegetation due to reindeer grazing was observed and the dropping count

was low at this site (T. Vowles, unpublished data). Thus, the lack of effect by excluding

reindeer may partly be explained by a fairly low grazing pressure.

37

The situation is the opposite on the dry heath, were the dominated EcM plant species is B.

nana. The EMM production was, as seen in Långfjället, positively affected by grazing. There

is a trend that B. nana increases in abundance and height when protected from grazing, (Fig. 1

and 2), which is in line with other exclosure studies (Eskelinen & Oksanen, 2006; Oksanen &

Moen, 1994; Pajunen et al., 2008). In ambient plots, where grazers suppress B. nana, C is

allocated to the roots as a tolerance strategy against herbivory (Evju et al., 2009) allowing

higher EMM production (Treseder & Allen, 2000). Hence, less EMM production in

exclosures is probably due to an investment of C in aboveground tissue, due to the release of

stress from grazing, and less to roots. On the other hand, on Fulufjället, where no reindeer

graze, the EMM production seems to increase with height of B. nana. Thus, our data suggest

that B. nana invest in roots when affected by herbivores, and when released from the stress C

is allocated to aboveground tissue until it reaches a certain height, where the investment is

directed to both above- and belowground plant parts.

Furthermore, the input of N, derived from faeces, in ambient plots may also affect the

production of EMM. For example, Nilsson and Wallander (2003) and Kjøller et al. (2012)

found that EMM production decreased with fertilization. This pattern was explained by a

change in EcM community structure but also as a decrease of fine root production when

plants are fertilized, and thereby a reduction in the mycorrhiza colonization and also the

production of mycelia. It has also been shown that nitrogen fertilization had a small effect on

the belowground EcM community (Deslippe et al., 2011; Treseder et al., 2007; Wallenda &

Kottke, 1998) and Clemmensen et al. (2006) found an increase of EMM production during

fertilization. Lilleskov et al. (2002) found that some EcM fungal species increased linearly

with net nitrification rate in soil, some species were found only at sites with high nitrification

rate and some species were nitrophobic, i.e. their abundance increased with decreasing

nitrification rate. Hence, it is very likely that the fungal taxa and abundance differ between the

treatments and a fungal species analysis is required for accurate conclusions about the effect

of added N.

To conclude the above-discussed results this study has shown that EMM production has

responded differently to grazing at the different sites. This variance comes from different host

plant species inhabiting these areas that respond differently to grazing and with different

supporting abilities. Our results, as hypothesised, show that grazing has an impact on the

production of mycelia, because it affects the allocation patterns of C in plants and the N

38

availability. The patterns are not consistent throughout the different vegetation types and we

conclude that the surrounding environment and the host plant species is important for the

response in fungi. Also, there are 5000-6000 different EcM fungal species (Agerer, 2006) and

it is not very likely that all of those species respond in the same way to changes in their

surroundings (Deslippe et al., 2011). Thus, studies covering the response of treatment effects

on fungal species composition are needed to truly understand the link between aboveground

herbivores and the fungi.

Isotopic signature

In general the EMM in the dry heaths were more 13C-enriched than in the birch forests, which

most likely is due to drier soils at the dry heaths. Drier conditions impacts the stomatal

conductance, as stomata closes, changing the isotope signature toward the 13C value of air; i.e.

drought is making the assimilated C less depleted (Brüggemann et al., 2011). Drier conditions

may also decrease photosynthesis, which leads to more internal carbon being used and a

change in the isotopic signature to less 13C depleted occurring. It is also possible that the more 13C-depleted EMM found in exclosures in the birch forest on Fulufjället is an effect of grazing

by moose (Alces alces L.), which prefer birch as forage (Wam & Hjeljord, 2010). The moose

population on Fulufjället is very large (Naturvårdsverket, 2002) and the grazing pressure in

the birch forest is assumed to be high. As the photosynthetic capacity in each individual birch

tree declines with an increased grazing pressure, the isotopic signature of C in the tree may

change. When the input of C highly enriched in 12C decreases, due to grazing, the plant will

become less depleted in 13C with time. Thus, mycelia forming mycorrhiza with plants affected

by grazing will receive less depleted C, as in the ambient plots (Brüggemann et al., 2011).

This pattern was not found in the forest on Långfjället, suggesting that moose grazing may be

influential on the isotopic signature in mountain birch forests.

Furthermore, excluding grazing did not consistently alter the 15N signature of the mycelia

across sites. In the dry heaths, the EMM was more 15N-depleted in exclosures on Fulufjället,

while on Långfjället exclosures were more 15N-enriched. Several factors can affect the isotope

signature; the fungal species composition (Clemmensen et al., 2006; Emmerton et al., 2001;

Hobbie & Colpaert, 2003), soil microbial activity (Clemmensen et al., 2008), availability of

different N-forms in the soil (Emmerton et al., 2001; Hobbie & Colpaert, 2003), degree of

mycorrhizal colonization and magnitude of C flux to fungi (Hobbie & Colpaert, 2003). Since

39

none of them can solely explain the 15N pattern in this study it is likely that several of them

are interacting in different ways to create the pattern found in our study.

Ergosterol

Varying ergosterol content was found at the different sites, and it does not correspond very

well to the weighed mycelia biomass (Fig. 3). The conversion factor of 5.5 mg/g C in EMM is

slightly lower than found and used elsewhere (Clemmensen et al., 2006; Salmanowicz &

Nylund, 1988; Wallander et al., 2001) and there is a very large variance in the data. Thus, the

interpretation of these results should be made with caution. It has been shown that different

results are obtained if using a GC-MS or a high performance liquid chromatographer (HPLC)

(Olsson et al., 2003). But also, if using the same instrument the results may vary a lot, even

within the same species; Frey et al. (1994) found an ergosterol content of 0.063 mg/g mycelia

of Glomus intraradices, while Fontaine et al. (2001) studying the same species did not find

any ergosterol at all. Also, as Olsson et al. (2003) concludes; there are always taxonomic

differences in the substances used as biomarkers. Since it is very likely that the fungal species

compositions will change with shifting environmental conditions, and if the variation in

ergosterol content between species is not addressed, it may not be trustful to use as a

biomarker for comparison between treatments. Furthermore, some samples where the mycelia

had already been extracted, contained fairly high amounts of ergosterol indicating that some

mycelia was left behind when handpicked from sand. Hence, both methods have

shortcomings.

Concluding remarks

This study confirms that grazing affects the production of EMM and, consequently, has an

impact on the carbon budget in soil. As reported by Hartley et al. (2012) there is a potential

loss of C associated with an altered amount of trees and shrubs in the arctic, due to less carbon

storage and fast turnover. Our data suggest that reindeer restrain the otherwise expanding

cover of shrubs (Sturm et al., 2001), as also reported by Olofsson et al. (2009), but also affect

the internal C allocation patterns leading to an increase of the EMM production in the dry

heaths. Thus, as the mycelium is the dominant pathway of C to the SOM pool (Godbold et al.,

2006), reindeer grazing could, due to an increased EMM production, mitigate the increased

CO2 concentration in the atmosphere. This study highlights the importance of taking

herbivores into account when trying to address the impact of climate change on alpine treeline

40

ecosystem. Questions that remain unanswered are how the fungal species composition is

affected by aboveground grazing, if the fungal species composition affects the efficiency of

the symbiosis and also how different fungal species respond to enhance nitrogen availability

due to grazing. These questions are fundamental when trying to understand the carbon and

nitrogen dynamics and transports between herbivores, plants and fungi, and require further

research. A DNA analyses together with more mechanistic studies (i.e. isotope labeling

studies) are needed for an accurate conclusion on the response to grazing by the fungi.

Acknowledgment

The authors thank Kjell Vowles for his assistance in the field. The Swedish Research Council

for Environment, Agricultural Sciences and Spatial Planning (grant no 214-2010-1411 to

RGB) supported this work and is gratefully acknowledged. The work was also conducted with

financial support from the strategic research area BECC (Biodiversity and Ecosystems in a

Changing Climate; www.cec.lu.se/research/becc).

41

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46

Table 1. Coordinates, meters above sea level (m.a.s.l), Temperature measured at 2 cm depth during the time the

bags were in the soil, annual precipitation (Precip.) and days the sand bags were in the soil are given for the six

sites on three different mountains Fulufjället (FU), Långfjället (LO) and Poullanvare (PO) and two different

vegetation types, birch forest heath with mosses (BM) and dry heath (DH).

Site   Coordinates   m.a.s.l   Temp  (C°)   Precip.  (mm)   Days  in  soil  

FU-­‐BM   N  61°38'42.2"  E012°35'24.5"  

880   9.1  834   148  

FU-­‐DH   N  61°38'08.1"  E012°38'18.9"  

930   8.5  

LO-­‐BM   N  62°03'55.5"  E012°14'45.9"  

800   8.6  697   148  

LO-­‐DH   N  62°06'49.8"  E012°16'20.1"  

840   8.4  

PO-­‐BM   N  68°20'13.2"  E021°19'15.6"   460   7.6  

444   112  PO-­‐DH  

N  68°20'23.7"  E021°10'41.0"   580   -

47

Table 2. The average EMM production (g C m-2), δ15N and δ13C in ambient plots (Amb.) and exclosures (Exc.)

on the three mountains, Fulufjället (FU), Långfjället (LO) and Poullanvare (PO), and two vegetation types, birch

forest heath type with mosses (BM) and dry heath (DH). The bold numbers show a significant difference

between treatments. Standard error is given in brackets. * P < 0.1 and ** P < 0.05

   EMM  (g  C  m-­‐2)   δ15N   δ13C  

FU-­‐BM   Amb.   0.94  (0.38)**   -­‐1.32  (0.39)   -­‐26.58  (0.40)*  Exc.   4.44  (1.11)   -­‐0.61  (0.54)   -­‐27.60  (0.13)  

FU-­‐DH   Amb.   0.99  (0.57)   -­‐0.98  (0.26)**   -­‐27.23  (0.30)  Exc.   1.25  (0.18)   -­‐2.64  (0.44)   -­‐26.79  (0.35)  

LO-­‐BM   Amb.   1.03  (0.22)**   -­‐1.49  (0.84)   -­‐28.36  (0.29)  Exc.   2.00  (0.23)   -­‐2.08  (0.32)   -­‐27.74  (0.22)  

LO-­‐DH   Amb.   4.74  (0.57)*   -­‐2.39  (0.11)*   -­‐27.02  (0.16)  Exc.   1.62  (0.69)   -­‐0.92  (0.61)   -­‐26.49  (0.57)  

PO-­‐BM   Amb.   1.99  (0.58)   -­‐0.91  (0.52)   -­‐28.73  (1.00)  Exc.   1.72  (0.61)   -­‐0.43  (0.23)   -­‐28.28  (0.69)  

PO-­‐DH  Amb.   0.46  (0.18)   -­‐1.06  (1.15)   -­‐25.74  (0.20)**  Exc.   0.32  (0.12)   -­‐1.11  (0.50)   -­‐26.90  (0.28)  

48

Figure 1. The figure shows the relationship between the cover of Betula nana, the most important EcM plant

species, and mycelia production (g C m-2) at the dry heath on Fulufjället, Långfjället and Poullanvare in ambient

plots (circles) and exclosures (triangles). The shaded are is 95% confidence interval.

0

2

4

6

0 10 20 30 40Betula nana frequency

Myc

elia

bio

mas

s

Treatment

Ambient

Exclosures

Site

FUDH

LODH

PODH

49

Figure 2. The figure shows the relationship between the height (cm) of Betula nana and mycelia production (g C

m-2) at the dry heath on Fulufjället, Långfjället and Poullanvare in ambient plots (circles) and exclosures

(triangles). The shaded are is 95% confidence interval.

−5

0

5

33 36 39Betula nana height

Myc

elia

bio

mas

s

Treatment

Ambient

Exclosures

Site

FUDH

LODH

PODH

50

Figure 3. Correlation between the ergosterol (mg) extracted from 10 g sand and the mycelia mass (g C)

converted from mycelia weighed in 80 g sand. Plots are from both birch forest and dry heath both exclosures and

ambient plots on Fulufjället, Långfjället and Poullanvare.

y  =  0,0183x  +  0,1985  R²  =  8,6E-­‐05  

0  

0,1  

0,2  

0,3  

0,4  

0,5  

0,6  

0,7  

0   0,05   0,1   0,15   0,2   0,25  

Ergosterol  (m

g)  

Mycelia  mass  (g  C)