Sphagnum Solute transport in Sphagnum

Wou

ter Patberg Solute tran

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Sphagnum

dom

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bogs T

he ecop

hysiological eff

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Wouter Patberg

Solute transport in Sphagnum dominated bogsThe ecophysiological effects of

mixing by convective flow



Wouter Patberg

Colophon

Graphic design – Mirjam Patberg

Pictures – Wouter Patberg

Cover: Diepveen, Dwingelderveld

page 116: Veerles Veen, Dwingelderveld

Printing – Grafimedia, Facilitair bedrijf RuG

The research reported in this thesis was carried out at the Laboratory

of Plant Physiology, which is part of the Centre of Ecological and

Evolutionary Studies (CEES) of the University of Groningen,

P.O. Box 11103, 9700 CC Groningen, The Netherlands.

This research was financially supported by ALW grant 815-02-014.

ALW (Earth and life sciences) is part of NWO, the Netherlands

Organization for Scientific Research.

This thesis was printed with financial support from the University

of Groningen.

ISBN book: 978-90-367-5241-1

ISBN print: 978-90-367-5242-8

Solute transport in Sphagnum dominated bogsThe ecophysiological effects of mixing by convective flow

Proefschrift

ter verkrijging van het doctoraat in de

Wiskunde en Natuurwetenschappen

aan de Rijksuniversiteit Groningen

op gezag van de

Rector Magnificus, dr. E. Sterken,

in het openbaar te verdedigen op

vrijdag 16 december 2011

om 11:00 uur

door

Wouter Patberg

geboren op 12 april 1976

te Hoorn

RIJKSUNIVERSITEIT GRONINGEN

Promotores Prof. dr. J.T.M. Elzenga

Prof. dr. A.P. Grootjans

Copromotor Dr. A. J. P. Smolders

Beoordelingscommissie Prof. dr. R. van Diggelen

Prof. dr. J.G.M. Roelofs

Prof. dr. H. Joosten

Contents

Chapter 1 General introduction 7

Chapter 2 The transport of solutes by buoyancy-driven water flow 17

in a water-saturated Sphagnum layer; laboratory and field evidence

Wouter Patberg, Gert Jan Baaijens, Christian Fritz, Ab Grootjans,

Rodolpho Iturraspe, Alfons Smolders and Theo Elzenga

Chapter 3 Field characteristics of buoyancy-driven water flow 33

and its global occurrence

Wouter Patberg, Erwin Adema, Myra Boers, Gert Jan Baaijens, Christian Fritz,

Ab Grootjans, Rodolfo Iturraspe, Alfons Smolders and Theo Elzenga

Chapter 4 Physiological evidence for internal acropetal transport of nitrogen 45

in Sphagnum cuspidatum and S. fallax

Wouter Patberg, Bikila Warkineh Dullo, Alfons Smolders,

Ab Grootjans and Theo Elzenga

Chapter 5 The importance of groundwater carbon dioxide 57

in the restoration of Sphagnum bogs

Wouter Patberg, Gert Jan Baaijens, Alfons Smolders,

Ab Grootjans and Theo Elzenga

Chapter 6 Photosynthesis of three Sphagnum species after acclimatization 75

to high and low carbon dioxide availability

Wouter Patberg, Jan Erik van der Heide and Theo Elzenga

Chapter 7 Summary and synthesis 91

References 105

Samenvatting 119

Dankwoord 127

Chapter 1

General introduction

General introduction – 9

Background

Bog ecosystems

Bogs are wet, acidic, peat forming ecosystems which generally have a low cover of vascular plants

and are dominated by mosses of the genus Sphagnum (peat moss or ‘veenmos’ in Dutch; Rydin

& Jeglum, 2006). Sphagnum mosses play an important role in creating their own environment,

thereby gaining competitive advantage over other plant species (Kilham, 1982; Malmer et al., 1994;

Van Breemen, 1995).

Sphagnum mosses consist of densely clustered developing and expanding branches at the top

of the plant, the so-called capitulum (plural: capitula), and the stem with fully developed branch-

es. During growth, the stem elongates from the capitulum and the branches become distributed

along the new stem. The lower portion of Sphagnum plants gradually die and will form peat. The

capitulum is the part of the plant where the main production of biomass takes place. Also, the

highest metabolic activity and nutrient uptake in Sphagnum mosses was measured in the capitu-

lum (Aldous, 2002a; Johansson & Linder, 1980; Malmer, 1988; Malmer et al., 1994; Robroek et al.,

2009; Rydin & Jeglum, 2006). For example, the contribution of photosynthetic activity of the ca-

pitulum in dense Sphagnum hummocks and lawns has been estimated to be 98% in Sphagnum

fuscum and 60% in S. balticum (Johansson & Linder, 1980).

Looking at a cross section of a Sphagnum bog, it can be divided into two layers (Clymo, 1984;

Ingram, 1978). The upper 10 to 40 cm is called the acrotelm. This layer contains the living part of

the Sphagnum mosses and is a highly permeable layer where the groundwater table fluctuates. The

spongy acrotelm has a high hydraulic conductivity and the ability to retain water in dryer periods,

thus having a strong self-regulating effect on the depth of the water table (Ingram, 1978). The layer

below is called the catotelm, a slowly permeable, permanently water-saturated anaerobic layer

which contains most of the peat (Ingram, 1978).

Bog ecosystems are ombrotrophic, which means that they receive their nutrients solely by

atmospheric deposition and are therefore characterized by a low nutrient availability (Rydin

& Jeglum, 2006). The ability of Sphagnum mosses to deal with low nutrient availability is often

attributed to their high nutrient retention capacity. The bog surface appears as a layer of densely

packed Sphagnum capitula that efficiently intercept nutrients coming from the atmosphere (Aldous,

2002a; Woodin & Lee, 1987). Sphagnum mosses lack vascular tissue for water and nutrient uptake,

but they can take up water and nutrients over the entire surface of the plant because they lack a cu-

ticle (Brown, 1982; Brown & Bates, 1990). Sphagnum mosses are able to survive under low nitrogen

conditions due to their very efficient nitrogen utilization (Bridgham, 2002; Li & Vitt, 1997) ranging

from 50 to 90% (Aldous, 2002a; Li & Vitt, 1997). Woodin & Lee (1987) even measured a retention

of 100% of inorganic nitrogen at an unpolluted site, whereas chloride and sulphate were passing

freely through the moss mat. That the efficiency of nitrogen retention by Sphagnum results in a

competitive advantage of Sphagnum over vascular plants was shown by Aldous (2002a): vascular

plants received <1% of N recently added by wet deposition. Moreover, the ability of Sphagnum spe-

cies to grow in an environment with very low nutrient concentrations is often attributed to their

pronounced capacity to exchange hydrogen ions for mineral cations (Daniels & Eddy, 1985). By

doing so, releasing H+ ions in exchange for dissolved cations, Sphagnum species have the ability

to acidify their environment (Clymo, 1963; Clymo & Hayward, 1982). Another important feature

10 – Solute transport in Sphagnum dominated bogs

of Sphagnum to survive in nutrient poor habitats is the ability to recycle nutrients from senescent

Sphagnum tissue very efficiently (Aldous 2002a, 2002b; Malmer, 1988; Van Breemen, 1995).

In contrast to vascular plants, Sphagnum mosses lack stomata. Consequently, they cannot control

their water loss actively. Water lost by evaporation must be replaced by rain or by the water from

the peat below. Since there are no specialized cells for water transport in the stem, the upwards

water transport takes place externally by capillary movement facilitated by a network of spaces

between leaves, stems and branches (Rydin & Jeglum, 2006).

Sphagnum mosses are characterized by their high water-holding capacity (Clymo & Hayward,

1982). Much water can be retained in the hyaline cells, which are large, dead cells, which make up

about 80% of the plant’s volume. Hyaline cells can rapidly absorb water through their pores (with

have diameters from 5-20 μm), and water can be retained against suction pressures of 10-100 kPa

(Van Breemen, 1995). The hyaline cells are enclosed in a network of narrower chlorophyllose cells,

the cells that contain chlorophyll and enable the mosses to photosynthesize.

The microtopography of a Sphagnum bog is characterized by a diversity of wet depressions (pools

and hollows), relatively dry lawns and dry hummocks (Andrus et al., 1983). Each microhabitat is

occupied by a different group of Sphagnum mosses, broadly defined by its water retention capacity

(Andrus et al., 1983; Hayward & Clymo, 1982). Sphagnum species observed at increasing height

above the water table have an increasing capacity to conduct water by capillary action (Clymo &

Hayward, 1982).

Due to the wet, anoxic and acidic conditions in the catotelm, the production of Sphagnum

exceeds the decomposition of organic material, resulting in the accumulation of organic material

or peat (Clymo & Hayward, 1982; Clymo et al., 1998). Peatlands store more carbon than any other ter-

restrial ecosystem. It has been estimated that the accumulation of peat has led to a carbon pool that

is about one-third of the global soil carbon pool. This is quite remarkable since peatlands occupy

only about 3% of the world’s total land area (Rydin & Jeglum, 2006). Peatlands, including Sphagnum

bogs, function as a net sink for CO2 (Clymo et al., 1998; Gorham, 1991) and as a consequence, Sphag-

num bogs play an important role in global carbon cycling (Bridgham et al., 2001a; Clymo et al., 1998;

Gorham, 1991).

Due to the extensive exploitation for fuel, agriculture and forestry over many centuries, and

the ongoing global warming, living (peat forming) peatlands have become endangered ecosystems

throughout the world (Rochefort & Price, 2003). Even nowadays (extensive) peat extraction activities

take place for commercial use in, for example, Canada, Scandinavia, Ireland and the Baltic states

(Joosten, 2009). Due to the important role of peatlands in the global carbon cycle, and because

of their unique ecological values, conservation and restoration of these ecosystems is necessary,

preventing stored CO2 being released into the atmosphere, which will lead to accelerated global

warming.

Globally much effort is dedicated to the restoration of damaged peatlands. However, the

restoration of bog remnants in particular, has proven to be fairly complicated and not always

successful (Grootjans et al., 2006; Money & Wheeler, 1999; Money et al., 2009). For the successful

conservation and restoration of Sphagnum-dominated bogs, knowledge about environmental

constrains for Sphagnum growth is necessary.


Nutrient supply in Sphagnum bogs

For their nutrient supply, Sphagnum bogs mainly depend on wet and dry atmospheric deposition.

However, it has been shown that under non-polluted conditions the annual input of nutrients

from atmospheric deposition is often insufficient to sustain the observed primary production in

these systems (Aerts et al., 1999; Aldous, 2002a, 2002b; Bowden, 1987; Bridgham, 2002; Damman,

1978, 1986; Morris, 1991; Pakarinen, 1978; Rosswall & Granhall, 1980; Urban & Eisenreich, 1988).

Therefore, other nutrients sources must be involved. Under natural conditions Sphagnum bogs

are often nitrogen-deficient (Aerts et al., 1992; Bragazza et al., 2004; Bridgham et al., 2001b;

Gunnarsson & Rydin, 2000; Li & Vitt, 1997) and therefore the availability of nitrogen is of special

interest. However, Sphagnum growth has also been shown to be limited by phosphorus (Aerts et al.,

1992; Bridgham et al., 1996), potassium (Damman, 1978; Pakarinen, 1978) and carbon dioxide (Rice

& Giles, 1996; Smolders et al., 2001).

Sources of nitrogen include N-fixation by cyanobacteria associated with Sphagnum and other

plants (Gerdol et al. 2006), the internal reallocation of nitrogen from older senescent tissues to the

metabolically active capitula (Malmer, 1988) and the mineralization of senescent Sphagnum plants

at the border of the acrotelm and catotelm. The mineralization of N has been shown to be the most

important nitrogen source for Sphagnum (Aldous, 2002a; Bowden, 1987; Bridgham, 2002; Urban &

Eisenreich, 1988). Gerdol et al. (2006) showed that direct retention of N from precipitation is less

important than recycling of mineralized N to support Sphagnum growth (Aldous, 2002b; Bowden,

1987; Bridgham, 2002; Urban & Eisenreich, 1988). The importance of re-mineralization of nitrogen

for Sphagnum growth has been demonstrated in situ by Urban & Eisenreich (1988). They calculated

the assimilation of nitrogen by plants (primarily Sphagnum) to be 66 kg ∙ ha-1 ∙ yr-1, whereas only

14.6 kg N ∙ ha-1 ∙ yr-1 was supplied by total inputs. The remainder was supplied by mineralization of

the peat.

In Sphagnum bogs the highest mineralization rates are found in the aerobic zone at the transition

zone from acrotelm to the anaerobic catotelm (Bridgham et al., 1998; Malmer, 1993; Waddington et

al., 2001). In contrast, the highest metabolic activity and nutrient uptake in Sphagna takes place in

the upper part of the plant, the capitulum (Aldous, 2002a; Johansson & Linder, 1980; Malmer, 1988;

Malmer et al., 1994; Robroek et al., 2009; Rydin & Jeglum, 2006). The spatial separation between

the actively growing photosynthesizing capitula and the mineralization of nutrients requires an

efficient nutrient transport system. Several nutrient transport mechanisms have been described

for Sphagnum bogs. Throughout the water layer nutrients are passively distributed by diffusion.

Above the water layer solutes might be transported upwards to the capitula through the extracellu-

lar capillary spaces between pendant branches and stems (Hayward & Clymo, 1982). Rydin & Clymo

(1989) demonstrated the internal acropetal transport of carbon and phosphorus. Complementary

to the abovementioned transport mechanisms, Baaijens (1982) and Rappoldt et al. (2003) reported

on a phenomenon called buoyancy-driven water flow as a possible mechanism for the external

transport of nutrients in a water-saturated Sphagnum layer.

Buoyancy-driven water flow

Buoyancy-driven water flow is the vertical convective flow of water in a water-saturated peat moss

layer, driven by the temperature difference between day and night. Due to the temperature drop

during the night the surface of the water layer will cool down, resulting in a relative cold layer on


top of a warmer layer. Because of the difference in density between these two layers, the cold water

will sink and the warm water will rise. Evidence for the occurrence of buoyancy-driven water flow

in a water-saturated Sphagnum layer, based on theoretical and experimental grounds, was provided

by Rappoldt et al. (2003).

The development of buoyancy flow in a water-saturated Sphagnum layer is determined by the

Rayleigh (Ra) number of that layer. Rappoldt et al. (2003) calculated that buoyancy flow occurs if

the system’s Ra number exceeds 25. For a typical peat moss layer, a temperature difference of 10

degrees between day and night will result in a Ra number of 80 which is suitable for the quick

development of buoyancy flow (Rappoldt et al., 2003). Adema et al. (2006) provided evidence for

the occurrence of buoyancy flow in the field; based on the hydraulic conductivity (k) of the Spha-

gnum layer and a temperature difference between day and night of 8˚C, in a small Sphagnum do-

minated peat bog in the Netherlands, the calculated Ra number was sufficiently high to induce

buoyancy flow.

The convective flow of water will result in the mixing of solutes as well. However, direct

evidence for nutrient transport is lacking. It is hypothesized that nutrients originating from

decomposition in the lower acrotelm, will be transported upwards by buoyancy flow and may

become available for the growing Sphagnum capitula, thereby contributing to the nutrient supply

of the Sphagnum plants. Moreover, oxygen produced by photosynthesis in the upper Sphagnum

layer will be transported downwards resulting in increased decomposition rates. In turn, nutrients

will become available to the growing Sphagnum when transported upwards by buoyancy flow.

Consequently, buoyancy flow might be an essential mechanism in the efficient recycling of

nutrients in Sphagnum bogs.

Aim and outline of this thesis

Part I: Buoyancy-driven water flow as a transport mechanism

The main objective of the first part of this thesis is to determine the importance of buoyancy-driv-

en water flow in the nutrient distribution in Sphagnum bogs. In Chapter 2 (The transport of solutes

by buoyancy-driven water flow in a water-saturated Sphagnum layer; laboratory and field evidence),

we asked ourselves the basic question whether nutrients are indeed transported by buoyancy flow.

To answer this question a straightforward, but effective mesocosm experiment was performed

in a temperature-regulated climate chamber. Buoyancy flow was generated in a water-saturated

Sphagnum matrix and the transport of solutes by buoyancy flow was visualized by the addition

and subsequent monitoring of a coloring dye. It became evident that buoyancy flow can act as a

fast and efficient transport mechanism (Chapter 2). In accordance with Rappoldt et al. (2003), a

reversal of the nutrient gradient due to the occurrence of buoyancy flow was possible and thereby

induce a stepwise increase in the nutrient concentration near the capitula. Consequently, the im-

portance of buoyancy flow as a transport mechanism in supplying the capitula is also determined

by the ability of Sphagnum capitula to enhance the uptake and assimilation by (and thus benefit

from) this increased nutrient availability. The amount of nutrients taken up by Sphagnum depends

on the nutrient concentration and the affinity of the uptake mechanism for the substrate. In the

case of, for example, temporary high ammonium concentrations in the upper Sphagnum layer due


to buoyancy-driven water flow, Sphagnum must have a suitable uptake mechanism to benefit op-

timally from the situation. Therefore, we determined the uptake kinetics of ammonium by the

capitula of S. cuspidatum and S. fallax. The possible role of the cation-binding sites in the uptake of

nutrients is taken into consideration as well. In Chapter 2 also the findings of a field experiment,

in which the transport of labeled nitrogen (15N) in a Sphagnum layer by buoyancy-driven water flow

and the subsequent uptake by the Sphagnum capitula are reported.

Whereas the laboratory experiments (Chapter 2; Rappoldt et al., 2003) are all conducted under

controlled conditions and with homogeneous samples, in the field temporal and spatial variation

in temperature and hydraulic conductivity might occur and influence the occurrence and size of

the buoyancy cells. Therefore, in Chapter 3 (Field characteristics of buoyancy-driven water flow and

its global occurrence), a series of vertical temperature profiles were recorded in a pristine Sphagnum

bog to validate the theoretical predictions in a natural situation. Based on the measured hydrau-

lic conductivities and ambient day and night air temperatures, the Ra numbers of the Sphagnum

sites were calculated. Based on these Ra numbers, the occurrence of buoyancy flow in the field

could be predicted using the model described by Rappoldt et al. (2003). Additionally in Chapter 3,

the possible occurrence of buoyancy-driven water flow in Sphagnum bogs throughout the world

was determined. Geographical Information System software was used to analyze worldwide daily

temperature data and model the occurrence of buoyancy flow in peatlands throughout the world.

The importance of buoyancy-driven water flow in the nutrient supply of Sphagnum and nutri-

ent cycling in bogs depends on the transport rate relative to other transport mechanism. To date,

diffusion and internal transport were the known mechanisms by which nutrients were transported

throughout a water-saturated Sphagnum layer. Note that capillary transport is often mentioned as a

nutrient transport mechanism (Hayward & Clymo, 1982), but this type of transport is only possible

above the water table and therefore not taken into account here.

Diffusion and internal transport are both slow processes. For example, the diffusion coefficients

for oxygen and ammonium are respectively 1.96 ∙ 10-5 and 1.95 ∙ 10-5 cm2 ∙ s-1 at 20°C (Boudreau,

1997). Internal transport is estimated to distribute solutes throughout the plant with a half time of

about 11 days, an estimation based on the symplasmic apical transport of 14C (Rydin & Clymo, 1989).

Moreover, in a review on internal transport in non-vascular plants (Raven, 2003) it was stated that

there is no evidence for symplastic transport in Sphagna faster than can be accounted for by dif-

fusion. As a consequence, we expect buoyancy-driven water flow to play an important role in the

nutrient distribution in Sphagnum bogs. In earlier studies (Aldous, 2002b; Bridgham, 2002) the

contribution of translocation to the nitrogen supply of the capitula was shown to be significant.

Although, the ability of Sphagnum to transport nitrogen internally was widely assumed (Bonnett et

al., 2010; Bragazza et al., 2005; Gerdol et al., 2006; Limpens & Berendse, 2003; Limpens & Heijmans,

2008; Malmer, 1988), internal transport of nitrogen had never been demonstrated. The assumption

that N is transported internally is mainly based on the observations of internally transported car-

bon and phosphorus (Rydin & Clymo, 1989). This idea was supported by the often observed higher

C:N ratios in stems than in capitula (e.g. Malmer, 1988) and are taken as an indication for the inter-

nal reallocation of N from the stem to capitula. However, experimental evidence for the internal

reallocation of N was lacking. Chapter 4 (Physiological evidence for internal acropetal transport of

nitrogen in Sphagnum cuspidatum and S. fallax) deals with the contribution of internal transport

in the upward translocation of mineralized nitrogen in Sphagnum bogs. Two Sphagnum species,


Sphagnum cuspidatum and S. fallax, were used in experiments in which diffusion and capillary

transport were excluded and the internal transport of nitrogen was monitored. For both Sphagnum

cuspidatum and S. fallax, a slow but significant acropetal transport of nitrogen through an internal

mechanism was observed. Moreover, the rate at which nitrogen was transported internally was

estimated and its importance relative to buoyancy-driven water flow, is discussed.

Part II: The importance of carbon dioxide for the growth of Sphagnum

The second part of this thesis focuses on the importance of carbon dioxide for the growth of

Sphagnum. In contrast to vascular plants, Sphagnum mosses lack a cuticle and stomates to regu-

late photosynthesis (Rydin & Jeglum, 2006), but are surrounded by an external water film through

which gas exchange for photosynthesis is taking place. The photosynthetic rate of Sphagnum

mosses has been shown to be a compromise between external water content and the availability

of CO2 (Schipperges & Rydin, 1998; Silvola, 1990; Titus et al., 1983). At low water contents, dehydra-

tion inhibits photosynthesis whereas at very high water contents Sphagnum species may suffer

from carbon limitation due to very thick boundary layers (Jauhiainen & Silvola, 1999; Rice & Giles,

1996; Silvola, 1990; Titus et al., 1983; Williams & Flanagan, 1996). Since the diffusion of CO2 is about

104 times lower in water than in air, external water films can form large barriers for gas exchange,

reducing the supply of CO2 towards the carbon assimilating cells resulting in a reduced photosyn-

thetic rate (Bowes & Salvucci, 1989; Rice & Giles, 1996; Silvola, 1990; Williams & Flanagan, 1996).

To overcome this problem many aquatic plant species make use of carbon concentrating

mechanisms (CCM), which enhances the accumulation of carbon under water (Maberly & Madsen,

2002). The most frequently used mechanism is the use of bicarbonate (HCO3

-) as a carbon source

in photosynthesis (Prins & Elzenga, 1989). Sphagnum mosses lack such a CCM. Like most aquatic

bryophytes (Raven et al., 1985), Sphagnum mosses are known to be obligate CO2 users (Bain &

Proctor, 1980) and are therefore solely dependent on the diffusive supply of CO2 to the site of carbon

fixation. In obligate CO2 users high rates of underwater photosynthesis can only be sustained when

the leaves are exposed to high concentrations of CO2 (Jauhiainen & Silvola, 1999; Silvola, 1990).

Because CO2 is continuously produced by aerobic and anaerobic decomposition processes

dissolved CO2 concentrations are normally much higher in upper peat layers than atmospheric

ones (16 μmol ∙ L-1 vs. 100-5000 μmol ∙ L-1; Bridgham & Richardson, 1992; Lamers et al., 1999; Silvola,

1990; Yavitt et al., 1997; Glatzel et al., 2004; Smolders et al., 2001; Waddington et al., 2001). High CO2

concentrations can compensate for low diffusion rates and ensure the substrate delivery for pho-

tosynthetic carbon fixation to be sufficient (Maberly & Madsen, 2002; Silvola, 1990). The refixation

of CO2 from decomposition processes has been unambiguously demonstrated by Rydin & Clymo

(1989) and Turetsky & Wieder (1999). This so-called substrate-derived CO2 has been shown to be

an important carbon source for aquatic and emergent Sphagnum mosses (Baker & Boatman, 1990;

Paffen & Roelofs, 1991; Riis & SandJensen, 1997; Roelofs, 1983; Smolders et al., 2001; Smolders et al.,

2003). As a consequence, increased ambient atmospheric CO2 concentrations (up to twice ambient)

appeared to have limited effect on the growth of Sphagnum as outlined by Smolders et al. (2001).

Chapter 5 (The importance of groundwater carbon dioxide in the restoration of Sphagnum bogs)

focuses on the importance of CO2 for Sphagnum in a field situation. The study was performed

in the “Dwingelderveld”, a nature reserve in the Netherlands characterized by numerous small,


damaged Sphagnum bogs, distributed throughout the area. Since the start of restoration measures,

the developmental success between bogs has varied significantly; some bogs developed well,

whereas others did not. Peat extraction has removed the bulk of organic material and the highly

decomposed, humified peat which is left behind, has only limited CO2 production rates (Bridgham

& Richardson, 1992; Glatzel et al., 2004; Tomassen et al., 2004; Waddington et al., 2001). Therefore,

for the successful restoration of cut-over Sphagnum bogs an additional carbon source might be

essential for the re-establishment of Sphagnum mosses. It is hypothesized that in these hydrologi-

cally degraded bog remnants the restoration of Sphagnum growth is limited by the availability of

CO2. Bog waters analysis showed that the well-developed bogs received C-rich water from outside

the bogs. It was concluded that high CO2 availability is a pre-requisite for the successful re-esta-

blishment of Sphagnum mosses and subsequent bog development. Despite the obvious importance

of a high CO2 availability for Sphagnum, the physiological background of this apparent high CO

2

requirement of Sphagnum has never been established. Therefore, the physiological background of

carbon uptake by Sphagnum was investigated as well.

In Chapter 5 the plants used in the experiments were grown under ambient CO2 conditions.

However, For Sphagnum fuscum, a hummock forming species, acclimation to CO2 levels has

been shown (Jauhiainen & Silvola, 1999). Culturing plant under high CO2 availability resulted in

lower photosynthetic rates compared to plants that were grown under CO2-limiting conditions

(Jauhiainen & Silvola, 1999). Therefore, we expected physiological adaptations in carbon dioxide

uptake in response to the CO2 concentration during the culturing period. Therefore in Chapter

6 (Photosynthesis of three Sphagnum species after acclimatization to high and low carbon dioxide

availability) the physiological characteristics of carbon uptake by three different Sphagnum species

was investigated for plants grown for a long period at high and low CO2 availability.

Chapter 7 (Summary and synthesis) is a combined summary and synthesis of this thesis. The

main focus in this chapter lies on the ecological importance of buoyancy flow in Sphagnum-dom-

inated bogs. For different Sphagnum habitats (hollows, lawns and hummocks) the contribution of

buoyancy flow in the nutrient supply of Sphagnum will be compared to other nutrient transport

mechanisms. Finally, some findings in this thesis will be discussed in relation to the restoration

and conservation of Sphagnum bogs.

Chapter 2

The transport of solutes by buoyancy-driven water flow in a water-saturated Sphagnum layer; laboratory and field evidence

Wouter Patberg

Gert Jan Baaijens

Christian Fritz

Ab Grootjans

Rodolpho Iturraspe

Alfons Smolders

Theo Elzenga


Abstract

Sphagnum bogs depend for their nutrients mainly on atmospheric deposition.

Yet, the main nutrient source for Sphagnum growth has been shown to be

the mineralization of organic material. The highest mineralization rates are

found in lower peat layers at the border between acrotelm and catotelm.

The highest metabolic activity and nutrient uptake, however, takes place in

the capitula, found at the top of the living Sphagnum layer. This separation

between the actively growing capitula and the site of nutrient mineralization

requires an efficient nutrient transport system. Several nutrient transport

mechanisms in bogs have been described; diffusion, internal transport and

capillary transport. Complementary to these mechanisms, buoyancy-driven

water flow was proposed as an external nutrient transport mechanism in

a water-saturated peat layer. Buoyancy flow is the vertical movement of

water driven by the difference in density between water layers, which is the

result of nocturnal cooling. This chapter shows, by means of a mesocosm

experiment, that solutes are rapidly and in large quantities transported

by buoyancy-driven water flow in a Sphagnum matrix. As a consequence,

nutrient concentrations are stepwise increased around the capitula.

Consequently, the importance of buoyancy flow in the nutrient supply of

Sphagnum is also determined by the nutrient uptake capacity of Sphagnum.

The uptake kinetics of ammonium by Sphagnum indicates that Sphagnum is

able to benefit efficiently from a stepwise increase in ammonium availability.

Therefore, compared to diffusion and internal transport, buoyancy flow

seems to be a quantitatively important transport mechanism for nitrogen in

a water-saturated bog. Additionally, the transport of N by buoyancy-driven

water flow and subsequent uptake by the capitula was shown in a field

situation. Other solutes like carbon dioxide and oxygen will be redistributed

as well by buoyancy flow and this presumably also plays an important role

in ecosystem functioning.

The transport of solutes – 19

Introduction

One of the main characteristics of Sphagnum bogs is the accumulation of peat (Clymo et al.,

1998). Due to the wet and acidic environment in bogs the production of Sphagnum exceeds the

decomposition of organic material, resulting in the accumulation of organic material or peat

(Clymo & Hayward, 1982). Sphagnum is functioning as an ecosystem engineer by contributing to the

creation of this environment (Van Breemen, 1995). Consequently, peatlands function as a net sink

for CO2 (Clymo et al., 1998; Gorham, 1991). Sphagnum dominated bogs receive nutrients mainly by

atmospheric deposition (e.g. Van Breemen, 1995). Sphagnum mosses are able to very efficiently in-

tercept nutrients from precipitation and recycle them (Li & Vitt, 1997; Malmer, 1988; Rudolph et al.,

1993; Woodin & Lee, 1987). Consequently, they are able to dominate these low nutrient ecosystems

(Aldous, 2002a; Li & Vitt, 1997; Malmer, 1988; Van Breemen, 1995).

However, it has been shown that under non-polluted conditions the annual input of nutri-

ents by precipitation is often insufficient to sustain the observed primary production in these

systems (Aerts et al., 1999; Aldous, 2002a, b; Bowden, 1987; Bridgham, 2002; Damman, 1978, 1986;

Morris, 1991; Pakarinen, 1978; Rosswall & Granhall, 1980; Urban & Eisenreich, 1988). The release of

minerals by decomposition of organic material has been shown to be an important nutrient source

to support Sphagnum growth (Aldous, 2002a; Bowden, 1987; Bridgham, 2002; Gerdol et al., 2006;

Urban & Eisenreich, 1988).

The highest mineralization rates are found in the aerobic zone of the acrotelm at the border of

the anaerobic catotelm (Bridgham et al., 1998; Malmer, 1993; Waddington et al., 2001). In contrast,

the highest metabolic activity and nutrient uptake in Sphagna takes place in the upper part of the

plant, the capitulum (Aldous, 2002a; Johansson & Linder, 1980; Malmer, 1988; Malmer et al., 1994;

Robroek et al., 2009; Rydin & Jeglum, 2006). Together, these processes result in a gradient in nutri-

ent concentration, from low in the upper layer of a Sphagnum bog to high in the lower parts were

decomposition takes place. In addition, the oxygen produced by the photosynthesizing capitula in

the top layer and the consumption of oxygen by decomposition processes in deeper acrotelm layer

will result in decreasing oxygen levels with increasing depths (Lloyd et al., 1998; Redinger, 1934).

The spatial separation between the actively growing, photosynthesizing capitula and the

mineralization of nutrients requires an efficient nutrient transport system. Several nutrient

transport mechanisms have been described for Sphagnum bogs. Nutrients are passively distributed

throughout the water layer by diffusion. Above the water layer solutes might be transported

upwards to the capitula through the extracellular capillary spaces between pendant branches and

stems (Hayward & Clymo, 1982). Nutrients are also transported internally by Sphagnum. Rydin &

Clymo (1989) demonstrated the internal acropetal transport of carbon and phosphorus. In Chap-

ter 4 of this thesis evidence for internal transport of nitrogen is provided. Complementary to

these mechanisms, Baaijens (1982) and Rappoldt et al. (2003) reported on a phenomenon called

buoyancy-driven water flow, as a possible external nutrient transport mechanism in a water-

saturated peat moss layer.

Buoyancy flow is the vertical redistribution of water, generated by nocturnal cooling. During

the night the upper water layer cools down, leading to relatively dense cold water on top of warmer

water. If the temperature drop is sufficiently large, the cold water sinks leading to mixing of the

water column. Evidence for the occurrence of buoyancy-driven water flow in a water-saturated


Sphagnum layer was provided, based on theoretical and experimental grounds, by Rappoldt et

al. (2003). They showed that for a typical peat moss layer a temperature difference of about 10

degrees between day and night will result in a Rayleigh number (Ra) suitable for the development

of buoyancy flow. Adema et al. (2006) provided field evidence for buoyancy-driven water flow in

a Sphagnum dominated peat bog. Hydraulic conductivity and temperature measurements in a

pristine bog in Tierra del Fuego, Argentina, indicated that buoyancy flow events occur regularly.

Moreover, daily air temperature data collected around the world indicate that buoyancy-driven

water flow is very likely to occur on a regular basis in peat lands throughout the world (Chapter 3).

Flow of water will result in the mixing of solutes and this has been proposed to be the most im-

portant ecological consequence of buoyancy-driven water flow (Adema et al., 2006; Rappoldt et al.,

2003). However, direct evidence for nutrient transport is lacking. This study focuses on buoyancy-

driven water flow as a nutrient transport mechanism in a water-saturated Sphagnum layer. It is

hypothesized that nutrients originating from decomposition will be transported upwards and may

become available for the growing capitula of Sphagnum and thereby contributing to the nutrient

supply of the Sphagnum plants.

A mesocosm experiment was set up to trace the transport of solutes during the occurrence of

buoyancy-driven water flow in a Sphagnum matrix. The contribution of buoyancy flow in the nutri-

ent supply of Sphagnum also depends on the nutrient uptake capacity of Sphagnum. Therefore, the

uptake kinetics of ammonium by the capitula of S. cuspidatum and S. fallax were determined.

Additionally, in a pristine Argentinean bog a 15N source was placed in the deeper acrotelm and the

uptake of labelled nitrogen by the capitula was measured with and without the obstruction of con-

vective flow. The importance of buoyancy-driven water flow for several nutrients and its impor-

tance with respect to other nutrient transport mechanisms in a Sphagnum bog will be discussed.

Materials and methods

Mesocosm experiment

A container (h=11.5 cm d=20 cm) was completely filled with demineralized water and Sphagnum

magellanicum mosses to create a water-saturated Sphagnum matrix. The container was insulated

with a ten centimetre thick layer of foam to prevent radial heat loss and placed on a vibration free

foundation in a climate controlled room. “Day” temperature was set at 20°C (14 hrs) and “night”

temperature at 8°C (10 hrs). Light conditions were 50 µmol ∙ m-2 ∙ s-1, continuously (Hansatech Quan-

titherm Light meter). The occurrence of buoyancy flow in the Sphagnum matrix was monitored by

continuously measuring the vertical temperature profile using an array of five chromel-alumel

thermocouples placed in the centre of the bucket at 5, 20, 40, 70 and 110 mm depth connected to

a Graphtec GL200 midi logger (Graphtec Corp., Yokohama, Japan) on which data were logged and

stored with one minute intervals. A vertical temperature profile was also monitored continuously

in a similar container filled with wet potting soil (providing a matrix in which no convective flow

was expected to occur).

A blue dye (Coomassie Brilliant Blue G, No. B0770, Sigma Aldrich) was used to mimic the transport

of dissolved compounds by buoyancy flow. A volume of 500 mL of water was extracted from the


Sphagnum matrix by a siphon followed by the resettlement of the matrix for an hour. Subsequently,

at the onset of “night” (t=0), 200 mL of Brilliant Blue solution (66 mg/L) was layered through a tube

(3*5mm*40 cm) on the bottom of the Sphagnum matrix from a 500 mL Erlenmeyer flask with a flow

rate of approximately 1.3 mL · s-1. The temperature of the Brilliant Blue solution was 4°C to assist the

positioning of the layer at the bottom of the container. The homogeneous Sphagnum matrix had

a density of approximately 4 g DW ∙ L-1, which is comparable to a natural, green and growing peat

moss layer (Clymo, 1970).

After 15 minutes (t=15) the first water samples were taken from the mesocosm at 5, 55 and

105 mm depth, using an array of three black norprene tubes (l = 400 mm, 4.8 mm outer and 1.6

mm inner diameter; Saint-Gobain Performance Plastics, Verneret, France) in combination with a

peristaltic pump (Masterflex L/S model 7519-25, Cole Parmer Instrument company). Water samples

were 1 mL each and collected in disposable polystyrene cuvettes (10 ∙ 4 ∙ 45 mm; Sarstedt, Nüm-

brecht, Germany). Water samples were taken at 15 minutes intervals during the first 90 minutes of

the experiment and at increasingly longer intervals during the remainder of the experiment. The

experiment lasted for 25 hours. Care was taken that the dead volume in the tubes was excluded

from the samples taken. After 2.5 hours additional samples (1 mL) were taken with a pipette at the

periphery of the Sphagnum matrix at 5 mm depth. Immediately after sampling the extinction of the

water samples was measured on a double-beam spectrophotometer (Uvikon 940, Kontron Instru-

ments, Germany) at 580nm with demineralized water as a reference. Potential effects of tempera-

ture on the extinction values of the water samples were determined by simultaneously sampling a

solution with a known concentration of Brilliant Blue. No temperature effects were observed.

The abovementioned experiment was repeated eight times. Data were plotted and fitted using

graphing software (Prism version 4.03, 2005; GraphPad Software, Inc., San Diego, CA, USA).

The Sphagnum magellanicum plants used in the mesocosm experiment were collected in

August 2009 in a small bog located in the “Dwingelderveld” (N52°50’, E6°26’), a nature reserve in the

north of the Netherlands. The mosses used in this experiment were solely used to create a Sphag-

num matrix with acrotelmic characteristics. To minimize the adsorption and/or uptake of Brilliant

Blue during the experiment by the Sphagnum mosses they were incubated for at least 5 days in a

Brilliant Blue solution (66 mg ∙ L-1) prior to the experiment. Before the plants were used to set up the

matrix they were rinsed twice with demineralized water. The Sphagnum matrix was set up at least

12 hours before the start of the experiment to allow equilibration, thereby avoiding possible leak-

age of Brilliant Blue from the Sphagnum plants into the matrix solution during the experiment.

Uptake kinetics

Experimental design and analysis

To determine the uptake kinetics of ammonium by the capitula of Sphagnum cuspidatum and S. fal-

lax, the upper 2 cm of the plants were incubated for two hours in solutions containing 0, 5, 10, 25,

50 and 100 µmol 15NH4Cl ∙ L-1 and 20mM MES at pH = 4. Per concentration and species, three capitula

were incubated in 85 mL solution in a square Petri-dish (120*120mm; Greiner bio-one GmbH), in

triplicate. The capitula were rinsed three times with demineralized water before and after the treat-

ment. The experiment was performed in a climate chamber (light 185 µmol ∙ m-2 ∙ s-1; 18˚C).

To distinguish between the amount of ammonium taken up internally (assimilated) and

bound to the cell wall, an additional experiment was performed. The experimental set up was


exactly the same as the experiment described above except, to rinse of the 15NH4

+ from the cell wall,

in this experiment the plants were rinsed for two minutes with 100 mL 1mM KCl + 0.5 mM CaCl2

solution (at 150 rpm) after incubation. Subsequently, the plants were dried for at least 48 hours

at 80°C. From each Petri-dish the capitula were pooled and grinded to a fine powder using a ball

miller (Retsch MM2, Haan, Germany). Per sample the %N and %15N were measured at the Univer-

sity of California Davis Stable Isotope Facility, Davis, California, USA, using a PDZ Europa ANCA-

GSL elemental analyzer interfaced to a PDZ Europa 20-20 isotope ratio mass spectrometer (Sercon

Ltd., Cheshire, UK). The data are expressed as the amount of 15N taken up (assimilated and bound to

the cell wall) by the plants, in µmol ∙ g DW-1 calculated by the formula (%15N * %N / 100)*10000/15,

where %N is the percentage of total N in the sample, %15N is the percentage of 15N of total N and 15

is the molecular weight of the stable N isotope.

Plant material

Sphagnum cuspidatum Ehrh. Ex Hoffm. plants were collected in a pool in a small bog in the forestry

“Gasselterveld”, the Netherlands (N52°57.420’, E6°43.857’). Sphagnum fallax (klinggr.) Klinggr. plants

were collected in a small bog in the “Dwingelderveld” (N52°49.135’, E06°29.491’). Before being used

in the experiment, the plants acclimated for two weeks in a greenhouse. In the greenhouse, natural

light was supplemented with high pressure sodium lamps to obtain a 14 hour photoperiod. During

this period the plants were kept wet with demineralized water. No nutrients were supplied to the

plants.

Field experiment Rancho Hambre, Tierra del Fuego, Argentina

In February 2009 the transport of labeled nitrogen (15N) by buoyancy-driven water flow in a field

situation was examined. The location was a pristine Sphagnum magellanicum bog complex, named

“Rancho Hambre” (54°45’S 67°49’W), located in the Argentinean province of Tierra del Fuego and

characterized by the presence of numerous water hollows of different size (Mataloni & Tell, 1996).

See Grootjans et al. (2010) for a more detailed site description.

The experiment was performed in two contrasting Sphagnum habitats; a pool dominated by the

aquatic moss S. fimbriatum, growing with their capitula at the level of the water table, and a lawn

completely dominated by S. magellanicum with the capitula growing approximately 20 cm above

the water table. See figure 2 for the experimental design. Per site, two treatments were applied and

compared to a control situation. In both treatments a 15N source was introduced by placing an agar

cube (50*50*50 mm) containing 10 mg 15NH4Cl ∙ g-1 agar at 10 cm depth below the capitula. In the

first treatment, convective transport in the Sphagnum layer was excluded by the placement of a two

centimeter thick agar disc (d=30 cm) just below the capitula. Before positioning the agar disc, the

upper two centimeter of the Sphagnum plants were removed and placed back on top of the disc. In

the second treatment convective flow was not blocked. The upper two cm of the Sphagnum mosses

were cut off and immediately placed back, to rule out any effect of cutting. Cutting of the top 2 cm

also will block possible internal transport of 15N. Control values were obtained from a third treat-

ment in which the Sphagnum plants were kept intact and no 15N source was applied. All treatments

were performed in triplicate. The distance between the experimental plots was at least one meter.

All plots were sampled twice: just before and 10 days after application of the labeled nitrogen (10

February 2009). Per sample ten capitula were collected and transported in polyethylene bags to the


laboratory where they were dried for at least 24 hours at 80°C. The samples were ground to a fine

powder using a ball miller (Retsch MM2, Haan, Germany). The stable nitrogen isotope composi-

tion was measured for each sample with a Carlo Erba NA 1500 elemental analyzer (Thermo Fisher

Scientific Inc.) coupled online via a Finnigan Conflo III interface with a Thermo-Finnigan Delta-

Plus mass-spectrometer. Values of 15N are expresses as percentage of total nitrogen concentration.

Statistical differences in 15N concentrations between treatments and species were tested using

a two-way ANOVA with species and treatment as fixed factors (SPSS for Windows version 16.0.1,

2007; SPSS Inc., Chicago, IL, USA). The assumption of homogeneity of variance was not met, not

even after transformation of the data. According to Heath (1995), the analysis of variance appears

not to be greatly affected by heterogeneity in variance if sample sizes are more or less equal. There-

fore, we decided to continue our analysis using non-transformed data.

Air temperature data of the months January and February were obtained from a climate station

located adjacent to the Rancho Hambre bog complex.

Results

Mesocosm experiment

A clear difference between the vertical temperature profiles in the Sphagnum matrix and the

container with potting soil was observed (figure 1a and b). Due to absence of convective flow in the

potting soil, heat transfer takes place solely by conduction, resulting in a stratified temperature

pattern and gradually decreasing temperatures during the nocturnal cooling period. The cooling

of the Sphagnum matrix proceeds differently. Most remarkable is the small temperature increase

during the nocturnal cooling in the upper Sphagnum layer after approximately 95 minutes.

According to Rappoldt et al. (2003) and Adema et al. (2006) a telltale sign of the upwards transport

of warmer water by buoyancy flow. Moreover, in the presence of convective flow the layers will

mix and the appearance of stratification will be less, which is clearly shown by the smaller

temperature differences between the layers in the Sphagnum matrix when compared to the potting

soil temperature profile.

Buoyancy flow has also been shown to result in a more efficient nocturnal cooling than the

diurnal heating, indicated by a decreasing average temperature with increasing depth (cf. Rappoldt

et al., 2003; Adema et al., 2006). This effect was also observed in the present study (figure 1d). In

the Sphagnum matrix, the average temperature at 110 mm depth is about 0.75˚C lower than at the

surface of the matrix. In contrast, the average daily temperature in the container filled with potting

soil is slightly increasing with depth.

The addition of the Brilliant Blue solution at the beginning of the night, has led to a steep Brilliant

Blue gradient in the Sphagnum matrix, with extinction values of 0.948, 0.056 and 0.063 at 105, 55

and 5 mm depth, respectively (figure 1c). The concentration of Brilliant Blue is reaching equili-

brium at the end of the experiment (t=1500). The course to equilibrium, without the occurrence

of buoyancy flow, can be fitted by a single exponential function, as plotted in figure 1c using the

data from the first 95 minutes (before the occurrence of buoyancy flow) and the equilibrium value

at t=1500.


However, between 90 and 120 minutes the Brilliant Blue concentration at 105 mm depth suddenly

drops from where it slowly increases to the equilibrium after 25 hours. The sudden drop at t=120

follows the small temperature increase in the upper Sphagnum matrix indicative for the occur-

rence of buoyancy flow. Up to that moment the levels of Brilliant Blue at 5 and 55 mm depth were

at a constant low level but reach equilibrium relatively fast after that moment. Striking is the high

concentration of Brilliant Blue at the edge of the upper matrix layer following the occurrence of

buoyancy flow, reaching a concentration much higher than the fitted line based on exponential

decay. If no mass flow mixing occurs and Brilliant Blue is solely redistributed by diffusion, the

equilibrium value is the maximum concentration that will be reached in the upper Sphagnum layer

and the minimum concentration for the layer at 105 mm. However, in the Sphagnum matrix, after

the occurrence of buoyancy flow, the Brilliant Blue gradient is reversed, indicating the transport of

Brilliant Blue by mixing of the different layers.

Figure 1. Temperature profiles and Brilliant Blue course during the nocturnal period of the mesocosm experiment. (a) and (b)

show the course of the vertical temperature profile in the container with wet potting soil (control) and in the Sphagnum matrix,

respectively. The numbers in the temperature courses in (a) indicate the depths at which the temperature was measured. In (b)

the occurrence of buoyancy flow is clearly visible by the small temperature increase after approximately 95 minutes (indicated

by the vertical grey line). (c) shows the course of the Brilliant Blue solution throughout the Sphagnum matrix. The solution was

added at the bottom of the Sphagnum matrix at t=0. The amount of Brilliant Blue at depths of 5, 55 and 110 mm is shown by re-

spectively closed, grey and open circles. Additional samples taken with a pipette in the upper layer of the Sphagnum matrix are

indicated by an open square. The initial concentration of Brilliant Blue at 110 mm depth is indicated by an open diamond. The


black curve indicates the mixing of the Brilliant Blue throughout the Sphagnum matrix following a single exponential decay

using the data from the first 95 minutes of the experiment (before buoyancy flow starts) and the equilibrium value reached at

t=1500 (r2=0.9970). At the moment of buoyancy flow development (vertical grey line), a sudden decrease of Brilliant Blue at 110

mm depth was observed. (d) shows the average 24-hrs temperature with depth relative to the temperature at 5 mm depth for

the Sphagnum matrix (closed circles) and the potting soils container (open circles) The points refer to the measurements in (a)

and (b). Buoyancy flow results in a more efficient nocturnal cooling relative to the diurnal heating, indicated by a decreasing

average temperature with depth. This is not the case in the container filled with potting soil.

Uptake kinetics of NH4

+

The uptake of 15N as function of the concentration 15NH4Cl, for Sphagnum cuspidatum and S. fallax,

are shown in figure 2. A distinction is made between the total amount of ammonium taken up

(the fraction bound to the cell wall and the fraction taken up internally), the fraction 15N taken up

internally only (which are the 15N concentrations after rinsing) and the fraction bound to the cell

wall only (adsorption) which is derived from the difference between the total amount and adsorp-

tion. Hyperbolic curves according to the formula A=Vmax

*[15N]/(Km

+[15N]) + c were fitted to the data.

Except for the internal assimilated 15N which shows a linear response to the 15N concentration. The

uptake kinetic parameters for total uptake and adsorption are shown in table 1.

Table 1. Kinetic uptake parameters of ammonium (±SD) for Sphagnum cuspidatum and S. fallax

S. cuspidatum S. fallax

Total uptake Adsorption Total uptake Adsorption

Vmax

(µmol 15N ∙ g DW-1 ∙ hr-1) 31.3 (±4.1) 13.7 (±1.8) 20.3 (±2.4) 8.1 (±0.6)

Km

(µmol 15N ∙ L-1) 125.6 (±28.8) 60.0 (±18.4) 102.1 (±22.8) 49.5 (±10.3)

c 0.02 (±0.3) -0.15 (±0.3) -0.1 (±0.2) -0.2 (±0.1)

Figure 2. The amount of 15N (µmol ∙ g DW-1) taken up by Sphagnum cuspidatum and S. fallax when incubated for two hours at

different concentrations of 15NH4Cl at 18˚C. A distinction is made between overall uptake of 15N (assimilated and adsorption –

solid circles) and assimilated only (open circles) and 15N bound to the cell wall (adsorption (grey circles). Shown are means and

standard deviations of three replicates. Data of overall uptake and adsorption are fitted to the hyperbolic curve A=Vmax

*[15N]/

(Km

+[15N]) + c and assimilation only data to the curve y=ax+b.


Figure 3. The Rancho Hambre experiment. (a) Temperature difference between day and night from January 1st to February

28 2009, measured adjacent to the Rancho Hambre bog complex. The experimental period is indicated by the grey box.

A temperature difference of 8˚C is indicated by the dotted line. During the experimental period at least four times a difference

of 8˚C between day and night was measured, which has been shown to be sufficient for the formation of buoyancy-driven

water flow in Sphagnum fimbriatum sites. (b) The relative increase in 15N concentration in capitula of Sphagnum magellanicum

(open symbols) and S. fimbriatum (closed symbols) 10 days after placing the 15N source in the acrotelm. The solid line gives the

average value of all non-buoyancy flow treatments (all treatments except the 15N – BF treatment at the S. fimbriatum site) and the

dashed lines indicate the 95% interval of these samples. (c) A cross section of the upper part of a Sphagnum bog showing the

schematic view of the experimental set-up. The experiment consisted of three treatments; control, 15N-no BF and 15N – BF. Each

treatment was performed in two different habitats; a site dominated by S. magellanicum and a site dominated by S. fimbriatum.

The capitula (asterisks) of these mosses growing respectively 10 and 0 cm above the water level as indicated by the grey area.

In the control treatment the plants were left untreated. In the other treatments a 15N source was placed in the acrotelm at 10 cm

below the capitula as indicated by the small grey boxes. In the second treatment convective flow was obstructed by placing an

agar disc below the capitula (thick black horizontal line in the 15N – no BF treatment). In the third treatment, convective flow

was not obstructed (15N – BF).


15N experiment Rancho Hambre

The temperature difference between day and night adjacent to the Rancho Hambre bog complex in

February 2009 are presented in figure 3a. During the experimental period a difference between day

and night temperature of at least 8˚C occurred several times in the Rancho Hambre bog complex.

In Chapter 3 it is demonstrated that buoyancy flow develops in the Sphagnum fimbriatum sites in

the Rancho Hambre bog complex at temperature differences of ≥ 8˚C between day and night. Figure

3b shows the relative increase of 15N in the capitula of Sphagnum magellanicum and S. fimbriatum

capitula in all treatments after 10 days. Individual values per plot are shown. Main effects of spe-

cies and treatment on 15N concentration in the capitula were non-significant, (F(1,10)= 0.635, F(2,10)

= 1.246, respectively; p>0.05). Also no significant interaction effect of species * treatment was

found (F(2,10) = 0.177, p>0.05). However, in two of the non-obstructed Sphagnum fimbriatum plots

(15N - BF) a much higher increase of 15N in the capitula was observed (2.28 and 2.72%,) compared

to the average increase (0.97%) in the other treatments in which transport by buoyancy flow was

not to be expected due to either too low water tables (all S. magellanicum sites) or obstruction of

transport of 15N by convective flow (S. fimbriatum, no BF).

Discussion

The mesocosm experiment clearly demonstrates that solutes are transported by buoyancy-driven

water flow in a Sphagnum matrix. These findings indicate that buoyancy-driven water flow acts as

an external nutrient transport mechanism in water-saturated Sphagnum habitats and thereby con-

tributes to the supply of nutrients to the Sphagnum capitula in the upper bog layer and thereby to

the recycling of nutrients. To date, diffusion and internal transport were the known mechanisms

by which nutrients were transported throughout a water-saturated Sphagnum layer. Note that cap-

illary transport is often mentioned as an (important) nutrient transport mechanism (Hayward &

Clymo, 1982), but this type of transport is only possible above the water table and therefore not

taken into account here. The mesocosm experiment presented here clearly shows buoyancy flow

to distribute solutes more rapidly and in larger amounts than is possible by diffusion (figure 1)

with the reversal of the Brilliant Blue gradient indicates mixing of the layers.

It was assumed that diffusion was the only mechanism by which the Brilliant Blue was redis-

tributed throughout the Sphagnum matrix before buoyancy flow started to occur. However, based

on the concentration Brilliant Blue at t=0 at a depth of 105 mm, and the diffusion coefficient for

Brilliant Blue, the decaying values in the first 95 minutes of the experiment and the equilibrium

values at t=1500 are too high to be explained by diffusion alone. Other mechanisms play a role in

the redistribution of Brilliant Blue in this phase of the experiment. This could involve small scale

convective flow, adsorption by the Sphagnum plants and/or diffusion into the hyaline cells.

However, the importance of buoyancy flow as a transport mechanism in supplying the capitula is

co-determined by the nutrient uptake capacity of the capitula. Due to buoyancy flow, nitrogen gra-

dients can be reversed in a water-saturated Sphagnum layer and thereby induce a stepwise increase

in the nitrogen concentration near the capitula. In case of a full reversal of layers the maximum

ammonium concentration in the surroundings of the capitula reflects the ammonium concentra-

tion in the deeper acrotelm. Literature values for bog water ammonium concentration are about


3 µmol ∙ L-1 for a pristine bog in Ireland and 105 µmol ∙ L-1 for a Dutch bog suffering high nitrogen

loads (Lamers et al., 2000).

In the observed uptake kinetics for ammonium by Sphagnum cuspidatum and S. fallax, the Vmax

of ammonium uptake when exposed to ammonium concentrations up to 100 µmol ∙ L-1 does not

seem to be reached. In a separate experiment in which the time dependence of uptake was deter-

mined for 100 µmol NH4

+ ∙ L-1 equilibration was reached after about 24 hours for both S. cuspidatum

and S. fallax (see Chapter 7, figure 1). These uptake characteristics allow Sphagnum to make full

use of the stepwise increase in ammonium supplied by buoyancy flow, considering that in every

24-hour period only one buoyancy flow event can be expected.

Interestingly, the observed uptake kinetics of ammonium by Sphagnum cuspidatum and S. fal-

lax indicate the existence of two different processes: a high affinity adsorption and a low affinity

internal uptake mechanism. The high affinity of the adsorption of NH4

+ to the cell wall implies that

the fixation of ammonium at low concentrations is mainly realized by adsorption (table 1). With

increasing concentrations the relative importance of adsorption to total uptake decreases; the cell

wall will saturate and increased uptake will take place by intracellular uptake. These observations

support the general assumption of the cell wall functioning as a temporal extension of nutrient

availability for intracellular uptake (e.g. Buscher et al., 1990; Clymo, 1963; Hajek & Adamec, 2009;

Jauhiainen et al., 1998). Sphagnum fallax shows a higher affinity for NH4

+ uptake than S. cuspidatum,

a difference that could be reflected by the different habitat of these two species. Sphagnum species

growing higher above the water level (hummocks) have higher cation exchange capacities (CEC)

than species growing in wetter habitats like lawns and hollows (Jauhiainen et al., 1998; Hajek,

2009; Clymo, 1963).

Earlier studies report various values for NH4

+ uptake rates by Sphagnum ranging from about

20 to 130 µmol NH4

+ ∙ g DW-1 ∙ hr-1(Jauhiainen et al., 1998; Rudolph et al., 1993; Twenhoven, 1992;

Wiedermann et al., 2009). However, these studies are difficult to compare since the amount of

NH4

+ applied differed.

As in our study, Jauhiainen et al. (1998) applied 50 µM NH4

+ to the capitula of seven Sphagnum

species in order to measure NH4

+ uptake rates. For S. cuspidatum and S. fallax they found uptake

rates of 67 and 78 µmol ∙ g DW-1 ∙ hr-1, respectively, which are high compared to the values found

in this study; 8.7 and 6.3 µmol ∙ g DW-1 ∙ hr-1 for S. cuspidatum and S. fallax, respectively. Nitrogen

uptake rates are suggested to be negatively correlated with nitrogen deposition rates and internal N

concentration (Limpens & Berendse, 2003). If so, the relatively low uptake rates might be explained

by the high N deposition at the sites were the plants were collected (28 kg ∙ ha-1 ∙ yr-1; RIVM, 2009),

which was reflected by the internal nitrogen concentration of 11.1 ± 0.6 and 10.1 ± 2.0 mg ∙ g DW-1

for S. cuspidatum and S. fallax, respectively. Another explanation for these low uptake rakes might

be the difference in pH between our experimental solution (4.0) and the one used by Jauhiainen et

al. (1998): between 5.0 and 5.5. Since our data show the importance of the cation exchange capac-

ity (CEC) of the cell wall in the ammonium uptake, a low pH, correlating with a low CEC (Richter &

Dainty, 1989; Rudolph et al., 1993), might have resulted in lower uptake rates.

Internal transport is estimated to distribute solutes throughout the plant with a half time of

about 11 days, an estimation based on the symplasmic apical transport of 14C (in NaHCO3; Rydin &

Clymo, 1989). Internal, apical transport of N showed even lower rates, a half time of 17 days (see

Chapter 4). According to a review by Raven (2003) on long distance transport in non-vascular


plants, there seems to be no evidence for symplasmic transport in Sphagna at speeds faster than

can be accounted for by diffusion. Thus, in comparison with diffusion and internal transport,

buoyancy flow seems to be a quantitative important nutrient transport mechanism in a water-

saturated Sphagnum bog.

Nitrogen

Since Sphagnum growth in peatlands is often limited by nitrogen availability (Aerts et al., 1992;

Bridgham et al., 1996; Gunnarsson & Rydin, 2000) the supply of nitrogen to the capitula is of spe-

cial interest. The main N source for Sphagnum has been shown to be re-mineralized N (Aerts et al.,

1999; Aldous, 2002b; Bridgham, 2002; Gerdol et al., 2006; Morris, 1991; Urban & Eisenreich, 1988).

The importance of re-mineralization of nitrogen for Sphagnum growth has been demonstrated in

situ by Urban & Eisenreich (1988). They calculated the assimilation of nitrogen by plants (primarily

Sphagnum) to be 66 kg ∙ ha-1 ∙ yr-1, whereas only 14.6 kg N ∙ ha-1 ∙ yr-1 was supplied by total inputs.

The remainder was supplied by mineralization of the peat. These findings are contradictory to the

idea that Sphagnum and vascular plants utilize spatially distinct nutrient pools, with Sphagnum

relying on N from precipitation and vascular plants on mineralization of senescing organic mat-

ter in the deeper acrotelm (Malmer et al., 1994; Pastor et al., 2002). Partly due to buoyancy flow,

Sphagnum can also rely on mineralized nitrogen as a major N source. Consequently, Sphagnum

mosses may outcompete vascular plants more easily and thereby enhance their ability to engineer

the ecosystem (Van Breemen, 1995).

Gerdol et al. (2006) stated that the cycling through mineralization of senescing tissues

by heterotrophic bacteria is essential for the supply of N to the growing tissues. An interesting

observation was the positive effect of high water tables on N cycling, a result that in the original

paper was left unexplained (Gerdol et al., 2006). Significantly lower upward translocation of N to

the growing capitula at low water tables was also observed by Aldous (2002b). A likely explanation

for this phenomenon is that high water tables allow the occurrence of buoyancy flow, increasing

recycling rates of nitrogen.

The experiment performed in the Rancho Hambre bog demonstrates the transport of N by

buoyancy-driven water flow in the Sphagnum fimbriatum sites and the subsequent uptake by the

capitula in the upper Sphagnum layer. The increase in 15N concentration in the treatment with

unobstructed convective flow indicates the transport of 15N by buoyancy flow. In two of the three

replicates this increase was observed. Possibly in one case buoyancy flow did not occur, or did not

result in the upward transport of 15N. Multiple factors determine the variety in the occurrence of

buoyancy flow. Although during the experiment the temperature difference between day and night

were sufficient to induce buoyancy flow, this is not a guarantee for buoyancy flow cells to occur.

Other factors co-determine the development of these cells. For example, a heterogeneity in the

vertical hydraulic conductivity might locally hamper the development of buoyancy flow. Addition-

ally, buoyancy flow occurs as adjacent cells with warm water going up and cold water going down

(Adema et al., 2006). Sphagnum capitula residing in a downward flow do not receive nutrients from

below despite the occurrence of buoyancy flow.

Ecological implications

CO2 gradients. Lloyd et al. (1998) showed an increase with depth of the CO

2 concentration in a


water-saturated Sphagnum core due to the uptake of CO2 by photosynthetic activity of the capitula in

the upper layer and the release of CO2 by decomposition of organic material in the lower acrotelm.

Since submerged Sphagnum species that inhabit peat hollows have been shown to be limited by

CO2 (Rice & Giles, 1996; Rice & Schuepp, 1995), buoyancy flow might be an important mechanism

replenishing CO2 around the capitula and enhancing photosynthesis by vertical transport of CO

2

from the deeper catotelm layer.

Oxygen gradients. The photosynthetic activity results in high oxygen levels in the top Sphagnum lay-

er and decreasing levels with depth (Adema et al., 2006; Lloyd et al., 1998). Lloyd et al. (1998) measu-

red a steep oxygen gradient in the upper four centimeters of a water-saturated Sphagnum layer,

decreasing from 300 to 0 µM. Mixing of water layers by buoyancy flow will result in a downward

transport of oxygen. Adema et al. (2006) attributed a conspicuous change in oxygen concentration

at 5 cm depth in a Sphagnum layer to the occurrence of buoyancy flow. Since the aerobic decom-

position of organic material is significantly higher than the anaerobic decomposition (Bridgham

et al., 1998; Waddington et al., 2001) the transport of oxygen to the lower parts of the acrotelm will

increase decomposition rates thereby increasing the concentrations of CO2 and nutrients like N. In

turn the nutrients will become available to the growing Sphagnum when transported upwards by

buoyancy flow. Moreover, the downward transport of O2 will increase the CO

2:O

2 ratio in the upper

Sphagnum layer and will enhance photosynthetic performance even more since photosynthesis is

inhibited by oxygen (see Chapter 6; Bowes & Salvucci, 1989; Raven, 2011; Raven et al., 2008; Skre &

Oechel, 1981).

Methane. Methane is anaerobically produced in large quantities in bogs (e.g. Gorham, 1991).

Nevertheless, emissions of methane to the atmosphere are very low (Larmola et al., 2010) due to

the oxidation of methane by methanotropic bacteria (Kip et al., 2010; Raghoebarsing et al., 2005).

The mixing of methane and photosynthetically produced oxygen by buoyancy flow might results in

lower methane emission rates, and affect global carbon cycling. In addition, the released CO2 in this

oxidation process has been shown to be a significant carbon source for Sphagnum (Raghoebarsing

et al., 2005).

Overall conclusion

Net transport by buoyancy flow occurs when a vertical gradient exists. These gradients have been

shown explicitly for CO2, CH

4 and O

2 in a water-saturated Sphagnum layer (Lloyd et al., 1998) with

large concentration differences in the upper 12 cm of the Sphagnum layer for CO2 and CH

4, and O

2

decreasing to undetectable values at 2 cm depth. According to the models described by Rappoldt

et al. (2003), the size of the buoyancy cells (dependent on the Rayleigh number of the Sphagnum

layer) can be as large as 24 cm. Based on in situ temperature measurements in a pristine Sphag-

num bog in Tierra del Fuego, Argentina, cell sizes of 3 to 26 cm were predicted. Therefore, due to

buoyancy flow these solutes can be transported and gradients can even be reversed (figure 1). The

maximal transport of nutrients per buoyancy event depends on the concentration in the lower

layer. Rappoldt et al. (2003) showed in a model that the Sphagnum matrix is well mixed after 4

consecutive buoyancy events. The mesocosm experiment shows that the Sphagnum matrix was

already mixed after one occurrence of buoyancy flow.


The recycling of nutrients is of great importance in the functioning of Sphagnum bogs and has been

suggested to explain the high carbon burial rates despite the low primary productivity, also known

as the “Paradox of Peatlands” (Raghoebarsing et al., 2005). This chapter shows that buoyancy flow

might be essential for the efficient recycling of nutrient by Sphagnum at least under waterlogged

conditions.

Chapter 3

Field characteristics of buoyancy-driven water flow and its global occurrence

Wouter Patberg

Erwin Adema

Myra Boers

Gert Jan Baaijens

Christian Fritz

Ab Grootjans

Rodolfo Iturraspe

Alfons Smolders

Theo Elzenga


Abstract

Nutrients enter Sphagnum bogs mainly by atmospheric deposition.

However, to sustain their annual production, this must be supplemented

with nutrients released by decomposition processes in the peat layer. The

capitula are the actively growing parts of the Sphagnum plants and since

they are spatially distinct from the decomposition processes, transport of

the nutrients is essential. For Sphagnum dominated bogs, several nutrient

transport mechanisms have been described; internal transport, external

transport by diffusion, capillary action and buoyancy-driven water flow. In

Chapter 2 it was shown that nutrients are transported more rapidly and in

larger quantities by buoyancy flow than would be possible by diffusion or

internal transport. Therefore, buoyancy-driven water flow is considered to be

an important nutrient transport mechanism in water-saturated Sphagnum

layers. However, this conclusion is based on laboratory experiments,

which were conducted under controlled conditions, whereas in the field

temporal and spatial variation in temperature and hydraulic conductivity

might occur and affect the occurrence and size of the buoyancy cells. This

chapter focuses on the occurrence of buoyancy flow in the field. Vertical

temperature profiles and vertical hydraulic conductivities were measured in

a pristine Sphagnum bog to validate the theoretical predictions in a natural

situation. Subsequently, Ra numbers of the Sphagnum sites were calculated

and the possible occurrence of buoyancy flow in the field was predicted

using the model described by Rappoldt et al. (2003). The calculated Ra

numbers indicate the Sphagnum fimbriatum layer to be suitable for the

development of buoyancy flow, which is supported by the course of the

vertical temperature profiles. Moreover, the predicted starting time of

buoyancy flow development very well correlates to the observed starting

time of buoyancy flow.

Additionally, worldwide daily temperature data were analyzed to model

the possible occurrence of buoyancy flow in peatlands throughout the

world. The results from this GIS analysis indicate that many peatlands are

subjected to temperature differences between day and night of 8˚C or more,

which is sufficient for the development of buoyancy flow. In the month July

about 70% of the peatlands have at least 5 days with temperature differences

between day and night suitable for the development of a buoyancy flow

event. From this analysis it is apparent that buoyancy flow is a worldwide

occurring phenomenon in peatlands.

Field characteristics of buoyancy-driven water flow – 35

Introduction

Sphagnum bogs receive nutrients mainly by precipitation (Van Breemen, 1995). However, to support

their annual production, Sphagnum mosses are supported in their nutrient supply by nutrients

released by decomposition processes in the peat (Aldous, 2002a; Bowden, 1987; Bridgham, 2002;

Gerdol et al., 2006; Urban & Eisenreich, 1988). The capitula are actively growing parts of the Sphag-

num plants (Johansson & Linder, 1980; Malmer, 1988; Malmer et al., 1994; Robroek et al., 2009;

Rydin & Jeglum, 2006) and since they are spatially distinct from the decomposition processes,

transport of the nutrients is essential. For Sphagnum dominated bogs several nutrient transport

mechanisms have been described. Internal transport (Rydin & Clymo, 1989; Chapter 4), external

transport by diffusion, by capillary action (Hayward & Clymo, 1982) or by buoyancy-driven water

flow (Rappoldt et al., 2003; Adema et al., 2006; Chapter 2).

Buoyancy-driven water flow is the vertical convective flow of water in a water-saturated

Sphagnum layer, driven by the temperature difference between day and night (Baaijens, 1982;

Rappoldt et al., 2003) . During the night the upper water layer cools down more rapidly than

the layers below, leading to relatively dense cold water on top of warmer water. Due to density

differences between these two layers the colder and denser water will sink and the warmer water

will rise. Consequently, buoyancy flow occurs as “cells” consisting of adjacent regions with up-

ward and downward flow (Adema et al., 2006; Rappoldt et al., 2003). Evidence for the occurrence of

buoyancy-driven water flow in a water-saturated Sphagnum layer was provided on theoretical and

experimental grounds by Rappoldt et al. (2003).

Whether an inversion in the vertical temperature profile leads to instability and the genera-

tion of buoyancy flow depends on the system’s Rayleigh number, a dimensionless number that

depends on several parameters, including vertical hydraulic conductivity and the temperature dif-

ference between day and night. Rappoldt et al. (2003) showed that buoyancy flow occurs if the

system’s Rayleigh (Ra) number exceeds a value of 25. For a typical peat moss layer, a temperature

difference of 10 degrees between day and night will result in a Ra number of 80 which is suitable

for the quick development of buoyancy flow (Rappoldt et al., 2003).

Adema et al. (2006) provided evidence for the occurrence of buoyancy flow in a small Sphagnum

dominated peat bog in the Netherlands. Based on the hydraulic conductivity (k) of the Sphagnum

layer and a temperature difference between day and night of 8˚C, , the calculated Ra number was

sufficiently high to induce buoyancy flow. In Chapter 2, the convective transport of solutes by

buoyancy-driven water flow is demonstrated in both a mesocosm experiment and a field situation.

In the mesocosm experiment it was shown that buoyancy flow transports nutrients more rapidly

and in larger quantities than would be possible by diffusion (Chapter 2) or by internal transport

(Chapter 4). Therefore, buoyancy-driven water flow is considered to be an important nutrient

transport mechanism in water-saturated Sphagnum layers.

Whereas the laboratory experiments are all conducted under controlled conditions and with

homogeneous samples, in the field temporal and spatial variation in temperature and hydraulic

conductivity might occur and affect the occurrence and size of the buoyancy cells. Therefore, a

series of vertical temperature profiles were measured in a pristine Sphagnum bog to validate the

theoretical predictions in a natural situation. Based on the measured hydraulic conductivities and

ambient day and night air temperatures, the Ra numbers of the Sphagnum sites were calculated.


Based on these Ra numbers, the possible occurrence of buoyancy flow in the field could be predicted

using the model described by Rappoldt et al. (2003).

Additionally, Geographical Information System software was used to analyze worldwide daily

temperature data and to model the possible occurrence of buoyancy flow in peatlands throughout

the world. Our findings will be discussed with respect to the supply and cycling of nutrients in

Sphagnum dominated bogs.


Rancho Hambre, Tierra del Fuego, Argentina

Field measurements were performed in February 2005 in Rancho Hambre (54°45’S 67°49’W), a

Sphagnum magellanicum bog complex in the Argentinean province of Tierra del Fuego, extensively

covered by pools of different size (Mataloni & Tell, 1996). A more detailed site description is given

in Grootjans et al. (2010).

In a Sphagnum fimbriatum pool site and a S. magellanicum lawn site, respectively three and

ten intact peat monoliths were collected in 25 cm long PVC tubes with an internal diameter of 108

mm. The tubes were sealed by two PVC caps, stored at 4°C and analyzed the next day. For different

lengths of the cores the vertical hydraulic conductivity (k) was measured (in situ) using the con-

stant head method as described by Adema et al. (2006). Different lengths of the core were obtained

by removing parts of the monolith from the bottom. This process was repeated until the hydrau-

lic conductivity became too large to measure. For each length the hydraulic conductivity was

measured in triplicate. From these measurements the hydraulic conductivity of each segment was

calculated. In each core the depth of the transition from acrotelm to catotelm was determined.

For a period of thirteen days temperature profiles were measured in a Sphagnum fimbriatum pool

using eight copper-constantan-copper thermocouples connected to a Campbell Scientific AM25T

solid state multiplexer with an internal reference RTD (Resistance Temperature Detector). The

thermocouples were placed on 1, 6, 14, 25, 39, 56, 76, and 99 mm depth. The data were collected us-

ing a Campbell Scientific CR10x multi-channel data logger which measured every second of which

the average was stored every minute.

By using the temperature and the vertical hydraulic conductivity data, Ra numbers for the

upper 12 cm of the cores were calculated for each day during this 13 day period (see Rappoldt et al.,

2003). The following formula was used

Ra = k α ΔT √(t0/D

eff).

In which Ra is the dimensionless Rayleigh number, k is the vertical hydraulic conductivity (m ∙ s-1);

α is the thermal expansion coefficient (K-1); ΔT is the difference between maximum day and mini-

mum temperature of the subsequent night (˚C); t0 is the duration of the low temperature phase in

seconds (the value for t0 was 43200s (12h; Rappoldt et al., 2003) and D

eff is the thermal diffusivity

(m2 ∙ s-1). Both the thermal expansion coefficient (α) and the thermal diffusivity (Deff

) of water are

temperature dependent. For each day the values of α and Deff

were determined using the minimum,

maximum and average temperature, resulting in three calculated Ra values per day. The time of


onset of buoyancy flow development was derived from the telltale deviation from the monotonic

decrease in temperature in the vertical temperature profile and was compared with the predicted

time, based on the Ra numbers calculated according to Rappoldt et al. (2003).

The global occurrence of buoyancy-driven water flow

To quantify the potential global occurrence of buoyancy flow in peat bogs, air temperature data of

peatlands around the world were analyzed by using a Geographical Information System (ArcGis

software, ESRI, Redlands, CA, USA). The following digital maps were used: a map with the distri-

bution of global peatland distribution (Yu et al., 2010). Daily day and night temperature data were

obtained from NASA (https://lpdaac.usgs.gov; dataset MOD11C1 – Version 005, format: HDF-EOS;

Year: 2009; layers used: Daytime LST & Nighttime LST). Average monthly temperature data were

obtained from the same website (dataset: MOD11C3 – Version 005, format: HDF-EOS, year: 2009,

layers used: Daytime LST). Nitrogen-deposition data were derived from the Oak Ridge National

Laboratory Distributed Active Center (ORNL DAAC) site (https://daac.ornl.gov).

For all peatlands we calculated the daily temperature difference between the maximum day

temperature and the minimum temperature of the subsequent night. The minimum temperature

difference for buoyancy flow to occur is 8˚C or more (Adema et al., 2006; Rappoldt et al., 2003).

For each month we calculated the area of peatlands were ≥2, ≥5, ≥10 or ≥20 buoyancy flow events

occurred. Only peatlands with possible Sphagnum growth were included in the analysis. We

assumed that when the average monthly daytime temperature was lower than 8oC, ice formation

would prevent the development of buoyancy flow and/or plant growth would be restricted.

Additionally, global nitrogen deposition data were combined with the distribution of buoyancy

flow events. Based on the response of Sphagnum to increased nitrogen deposition as proposed by

Lamers et al. (2000), three categories were distinguished: <12, 12-18 and > 18 kg N ∙ ha-1 ∙ yr-1 (Lamers

et al., 2000).

Results

Local characteristics of buoyancy flow, Rancho Hambre, Tierra del Fuego

The hydraulic conductivity (k) values for cores of S. magellanicum and S. fimbriatum at different

depths are shown in the figures 1a and b, respectively. The hydraulic conductivity in the upper layer

of the S. fimbriatum cores is the highest (ranging from 0.3 to 0.5 m ∙ s-1) with k values decreasing with

increasing core depth. In Sphagnum fimbriatum cores the depths at which the transition between

acrotelm and acrotelm was found varied between 10 and 25 cm. In S. magellanicum the hydraulic

conductivity is close to zero and does hardly vary with depth. In two of the S. magellanicum cores

(4 and 9) the k values are higher in the upper layer (figure 1b). For all S. magellanicum cores the

transition from acro- to catotelm was found at 25 cm depth.

Temperature measurements Rancho Hambre

The temperature measurements in a Sphagnum fimbriatum pool in the Rancho Hambre bog complex

show a daily temperature cycle for several depths (figure 2a). The difference between day and night

temperature decreases with depth and results in a reversal of the temperature gradient during the


night. The small temperature increases in the upper Sphagnum layer during nocturnal cooling

(arrow in figure 2c) are clear indications that buoyancy-driven water flow has occurred (figure 2a).

The small temperature increases in the upper water layer are the result of upward movement of

warmer water and the sinking of cooler water (Adema et al., 2006; Rappoldt et al., 2003; Chapter 2).

Figure 1. Vertical hydraulic conductivity (in m ∙ s-1) at different depths of S. fimbriatum (a) and S. magellanicum (b) cores.

Symbols represent the average hydraulic conductivity (± SD) and are placed in the centre of the vertical line which represents

the length and depth of the associating core segment.

Figure 2b shows the calculated daily Ra numbers, ranging from 25 to 153, for each day for S.

fimbriatum core 2. The Rayleigh numbers in figure 2b describe a state of the Sphagnum matrix

calculated for a 24 hour period. However, it is very likely that the state of the Sphagnum matrix

during a particular 24 h period is also influenced by the conditions during previous day or days.

Therefore, the average Ra number for the complete period of 13 days was calculated as well. The Ra

number for the Sphagnum matrix for the complete experimental period was calculated to be 81.

Based on the calculated Rayleigh numbers, the earliest possible times of onset of buoyancy

flow were determined according to the theoretical model described by Rappoldt et al. (2003). This

model describes the relation between the Rayleigh number and the time of onset based on a 12

day/12 night temperature regime. Day/night temperature changes are either assumed to follow a

block wave or a sine wave, resulting in slightly different times of onset between these approaches.


Based on the sinus wave model, the times of onset were determined for the development of

buoyancy flow in the S. fimbriatum matrix during the experimental period. These are indicated by

the vertical lines in figure 2a. In general, at all days, the calculated time of onset is preceding the

temperature peaks which are indicative for the occurrence of buoyancy flow. Figure 2c is a detailed

view of the temperature profile at day 2 showing the typical temperature increase during nocturnal

cooling due to buoyancy flow (arrow) and the calculated time of onset (vertical black line).

Figure 2. a) Vertical temperature profile in a Sphagnum fimbriatum pool in the Rancho Hambre bog complex, Tierra del Fuego,

Argentina. The measuring period lasted for 13 full days. Temperature was measured at 1 (red), 6 (light blue), 14 (orange), 25 (light

green), 39 (dark blue) , 56 (dark green), 76 (brown) and 99 mm (black) depth. The dashed vertical lines indicate the times of onset

for the development of buoyancy flow determined by the theoretical model of Rappoldt et al. (2003). B) the calculated Ra numbers

for the S. fimbriatum layer during the measuring period. For each day three Ra numbers were calculated using the thermal expan-

sion coefficient (α) and the thermal diffusivity (Deff

) according to the minimum, maximum and average temperature of that day.

C) Vertical temperature profile at day 2. The vertical line indicates the calculated time of onset which is preceding the temperature

peak, pointed out by the arrow, which is indicative for the occurrence of buoyancy flow. Colors of the lines are as in figure a.


The global occurrence of buoyancy flow

Figure 3 shows per month the relative amount of peatlands with a) the occurrence of ≥2 and ≥5

buoyancy flow events, b) the occurrence of < 2 events, c) without sufficient temperature data and

d) without Sphagnum growth.

In the months June to September in about 80% of the peatlands a buoyancy flow event occurs at

least twice. In 40 to 70% of the peatlands it occurs at least 5 times and in 10 to 20% of the peatlands

at least 10 times. Peatlands with occurrences of 20 times or more are scarce (in general ≤ 1%; 4% in

April), however to establish that for a particular pixel ≥20 buoyancy flow events could have taken

place, the number of days with missing data must be very low. Therefore it is possible that the

number pixels with ≥20 buoyancy flow event is actually higher.

Figure 3. The relative amount of peatlands per month where buoyancy flow events occurred <2, ≥2, ≥5 or where it was assumed

that no Sphagnum growth could occur. Dark grey indicates the percentage of pixels with peatlands for which insufficient data

were available to calculate the number of occurrences of a buoyancy flow event for that particular month.

In figure 4 the distribution of peatlands throughout the world are shown. Indicated in green are the

peatlands with the occurrence of at least 5 buoyancy flow events in July 2009, in red the peatlands

with less then 5 buoyancy flow events, in orange peatlands without sufficient data for the analysis

and in grey peatlands with an average growth temperature <8˚C. In 70% of the peatlands a tem-

perature difference of at least 8˚C occurs at least 5 times (see also figure 3). In 28% of the peatlands

insufficient temperature data are available. In only 0.5% of the peatlands buoyancy flow occurs

less then 5 times. In southern Argentina and Chile the average monthly temperature is lower than

8˚C and therefore not taken into account in the analysis.

To obtain an indication whether Sphagnum growth indeed depends on the occurrence of buoy-

ancy flow for transport and cannot sustain growth on atmospheric N-deposition, we also plot-

ted the atmospheric nitrogen deposition (in kg ∙ ha-1 ∙ yr-1) in the year 1993. Based on the growth

response of Sphagnum to increasing N input (Lamers et al., 2000) a distinction is made between

areas with annual N loads of <12, 12-18 and >18 kg ∙ ha-1 ∙ yr-1. The areas with the highest nitrogen

load are North-Western Europe, East-Asia and small parts of North and South America. Remark-

ably, most peatlands do not coincide with these high N load areas and are thus likely to depend on

buoyancy flow transported nitrogen.


Figure 4. The occurrence of buoyancy flow in peatlands throughout the world for the month July 2009. Peatlands with the

occurrence of at least 5 buoyancy flow events are green, peatlands with less than 5 buoyancy flow events are red, in grey

peatlands the temperature was too low to allow growth and peatlands without sufficient temperature data for the analysis are

in orange. The squares indicate an atmospheric nitrogen deposition ≥ 12 (green) or ≥ 18 kg ∙ ha-1 ∙ yr-1 (grey).

Discussion

Local characteristics of buoyancy-driven water flow

Based on the vertical hydraulic conductivity of the Sphagnum fimbriatum cores and the temperature

differences between day and night, the calculated daily Ra numbers varied from 25 to 153 and

according to the model of Rappoldt et al. (2003) the S. fimbriatum layer is thus suitable for the

development of buoyancy-driven water flow. Indeed, the typical small temperature increases

during the nocturnal cooling in figure 2 very clearly indicates the regular occurrence of buoyancy-

driven water flow in the Sphagnum fimbriatum pool in Rancho Hambre.

The calculated starting time of buoyancy flow development based on the Ra number, correlates,

with exception of days 11 and 13 both with a Ra of 25, very well with time of the small tempera-

ture increases indicative of buoyancy flow. It should be noted that the calculated Rayleigh number

describes a state of the Sphagnum matrix for a single day/night period, ignoring the temperature

history during the previous days of the site. This could have been of influence at days 11 and 13,

on which the buoyancy flow event occurs much faster than the modelled time of onset. These

two days are characterized by a relatively low day temperature. As a consequence, during the cool-

ing period at night the temperature of the surface layer drops fast below the still relatively high

temperatures of the lower layers. Possibly, this explains the discrepancy with the model, which,

based solely on the relatively small diurnal temperature difference and ignoring the previous days,

predicts a very late time of onset of the buoyancy flow event.

In agreement with the model is the observation that at day 8, which has a calculated Ra number

of 153, no buoyancy flow event is apparent. At very high Ra numbers the model of Rappoldt et al.

(2003) predicts that the size of the cells become very small and as a consequence are not detectable


anymore with the used sensor array.

The vertical hydraulic conductivity values in the upper 12 cm of the S. magellanicum cores were in

general (with the exception of two cores) too low to result in Ra numbers suitable for the develop-

ment of buoyancy flow (data not shown). Furthermore, in the observed S. magellanicum lawns the

water table is located about 20 cm below the top of the Sphagnum plants which form an insulating

layer, preventing the development of a cool water surface layer and instability in the water column

(Van der Molen & Wijmstra, 1994). If buoyancy flow would nevertheless occur, solutes transported

from deeper layers to the upper water layer would still have to be transported to the capitula by

capillary transport. In this case, buoyancy flow only acts as an auxiliary transport mechanism and

its relative importance in the nutrient supply to the capitula is determined by the height of the

capitula above the water level. Such a situation can be found in Sphagnum lawns and the transition

zones between pools and hummocks.

Global occurrence of buoyancy-driven water flow

The results from the GIS analysis indicates that many peatlands are subjected to temperature

differences between day and night of 8˚C or more, several days each month during the growing

season. At these sites, buoyancy flow events could occur, resulting in sufficient mixing of the water

column and efficient recycling of nutrients.

In the month July about 70% of the peatlands have at least 5 days and about 90% at least 2 days,

with temperature differences between day and night suitable for the development of a buoyancy

flow event. From this analysis it is apparent that buoyancy flow is occurring in peatlands on a

global scale.

For weather stations at latitudes above 50˚, the fraction of days with Ra numbers >100 during

the months June and July was calculated by Rappoldt et al. (2003). From this analysis they con-

cluded that buoyancy flow occurs on a large scale , but is more likely in continental areas than in

coastal areas. This distinction between coastal and continental sites was not found in the present

study. This is possibly due to the fact that in the current study a temperature difference of 8 oC

was used as the sole criterion, while Rappoldt et al. (2003) calculated the Ra, taking into account

the effect of temperature on thermal expansion coefficient and thermal diffusivity. This approach

leads to different results at very low temperatures and does not affect our conclusions, since we

have included only pixels with and average monthly temperature higher than 8oC. Furthermore

our study focused on Sphagnum dominated peatlands, while Rappoldt et al. (2003) included all

weather station data available.

The mesocosm experiment in Chapter 2 has shown buoyancy flow to be an efficient and rapid

nutrient transport mechanism when compared to diffusion and internal transport. Uptake charac-

teristics for ammonium of Sphagnum (Chapter 2) imply that Sphagnum can profit from stepwise in-

creases in nitrogen availability. Therefore, even very infrequent occurrences of buoyancy flow can

make a significant contribution to the nutrient availability in a water-saturated Sphagnum layer.

The relative importance of transport of nutrients from deeper water layers to the top layer

with the capitulum, is higher when atmospheric input is low. This is especially the case for

nitrogen availability which is often considered growth limiting in Sphagnum bogs (Aerts et al.,

1992; Bridgham et al., 1996; Gunnarsson & Rydin, 2000). Since areas with a high nitrogen depo-

sition load only slightly overlap with the area covered with peatlands (figure 4), the importance


of buoyancy flow in the transport and recycling of nitrogen in Sphagnum bogs, is hardly dimin-

ished by increased, anthropogenic N deposition. Therefore, we conclude that buoyancy flow is

a worldwide occurring phenomenon which significantly contributes to the nutrient supply and

nutrient recycling in Sphagnum bogs.

The importance of buoyancy flow

Buoyancy-driven water flow has been shown to act as an external nutrient transport mechanism

in water-saturated Sphagnum habitats (Chapter 2), thereby contributing to the supply of nutrients

to the Sphagnum capitula in the upper bog layer and thus to the efficient recycling of nutrients.

Moreover, in comparison with diffusion and internal transport, buoyancy flow seems to be a quan-

titatively important nutrient transport mechanism (Chapter 2 and 4). The present study shows that

buoyancy-driven water flow is a worldwide occurring phenomenon in peatlands.

A Sphagnum bog can consist of hollows, lawns and hummocks. As buoyancy flow is restricted

to the water layer in a Sphagnum bog, direct supply of nutrients from deeper layers to the capitulum

by buoyancy flow only takes place in hollows. In lawns buoyancy flow can assist capillary driven

nutrient transport and in hummocks buoyancy flow probably is relatively unimportant or does

not occur. As the initial successional stage of a Sphagnum bog is the colonization of aquatic Sphag-

num species of water bodies followed by the invasion of hummock forming species, buoyancy flow

seems to be particularly important in the early stages of bog development. Efficient nutrient use

might be of great importance in creating dominance over vascular plants by keeping the nutrient

concentrations low in the lower acrotelm and thereby facilitating conditions beneficial to Sphag-

num growth.

With the regular occurrence of buoyancy flow, the nitrogen concentration in the acrotelmic

water will be determined by the decomposition rate in catotelm, the depth of the buoyancy flow

cells, the depletion in the top water layer by uptake and assimilation and dilution by rain water.

One can easily imagine how such a complicated process, that prevents the build up of high nutri-

ent levels in the deeper layers and thereby the establishment of vascular plants, can be disrupted

by high nitrogen loads.

Chapter 4

Physiological evidence for internal acropetal transport of nitrogen in Sphagnum cuspidatum and S. fallax

Wouter Patberg

Bikila Warkineh Dullo

Alfons Smolders

Ab Grootjans

Theo Elzenga


Abstract

Several mechanisms for acropetal or upward transport of nutrients have

been described for Sphagnum layers; diffusion, buoyancy-driven water

flow, capillary transport and internal transport. This chapter focuses on the

contribution of internal transport in the translocation of nitrogen from the

catotelm, where nutrients are released by decomposition of organic mate-

rial, to the acrotelm where growth of the Sphagnum plants takes place. The

internal transport of nitrogen has often been assumed to be present, but has

never been explicitly demonstrated before.

The ability of Sphagnum to transport nitrogen internally was investigated

by monitoring the transport of labeled nitrogen in two Sphagnum species,

S. cuspidatum and S. fallax, in experiments in which diffusion and external

transport were prevented. For both species a slow, but significant acropetal

transport of nitrogen through an internal mechanism was observed. Within

the time frame of the experiments, the internal transport of nitrogen was

only observed when nitrogen was supplied as NH4

+ and not as NO3

-. The

speed at which the internal transport took place was estimated at 5 mm ∙

day-1 and the equilibration between donor and acceptor parts of the plants

was characterized by half time value of 17 days. Relative to other transport

mechanisms, like buoyancy flow and capillary transport, internal transport

represents only a minor fraction of the upward transport of externally sup-

plied nitrogen to the capitula. The main importance of internal transport

is therefore considered to be the reallocation of internally catabolized

nitrogen compounds.

Physiological evidence for internal acropetal transport – 47

Introduction

Sphagnum mosses are the most dominant plant species in bogs. For their nutrient supply they de-

pend mainly on atmospheric deposition. Bogs are, therefore ombrotrophic ecosystems and under

natural conditions they are usually nitrogen-deficient (Bragazza et al., 2004; Bridgham et al., 2001b;

Gunnarsson & Rydin, 2000; Li & Vitt, 1997). Sphagnum mosses are able to survive due to their very

efficient nitrogen utilization (Bridgham, 2002; Li & Vitt, 1997). Sphagnum utilizes nitrogen from

atmospheric deposition with an efficiency that ranges from 50 to 90% (Aldous, 2002a; Li & Vitt,

1997). Woodin & Lee (1987) even measured a retention of 100% of inorganic nitrogen at an unpol-

luted site, whereas chloride and sulphate were passing freely through the moss mat. The efficiency

of nitrogen retention by Sphagnum clearly results in a competitive advantage of Sphagnum over

vascular plants. Aldous (2002a), for instance, showed that vascular plants could take up less than

1% of N recently added by wet deposition.

Nonetheless, under non-polluted conditions, the annual input of nitrogen by atmospheric

deposition is not sufficient to support the observed primary production in Sphagnum dominated

ecosystems (Aerts et al., 1999; Aldous, 2002a, b; Bowden, 1987; Bridgham, 2002; Damman, 1978,

1986; Morris, 1991; Pakarinen, 1978; Rosswall & Granhall, 1980; Urban & Eisenreich, 1988). Mass

balance calculations have shown that direct retention of N from precipitation is less important

than recycling of mineralized N to support Sphagnum growth (Aldous, 2002b; Bowden, 1987;

Bridgham, 2002; Urban & Eisenreich, 1988).

The highest mineralization rates have been found in the aerobic zone of the acrotelm at the border

of the anaerobic catotelm (Bridgham et al., 1998; Malmer, 1993; Waddington et al., 2001). On the

other hand, the highest metabolic activity and nutrient uptake in Sphagnum mosses was measured

in the upper part of the plant, the capitulum (Aldous, 2002a; Johansson & Linder, 1980; Malmer,

1988; Malmer et al., 1994; Robroek et al., 2009; Rydin & Jeglum, 2006). Since the capitula are spa-

tially separated from of the layer where mineralization occurs, transport of nitrogen to the capitula

is necessary for the required recycling of nitrogen in a Sphagnum bog.

Several nutrient transport mechanisms have been described for Sphagnum bogs; diffusion,

buoyancy-driven water flow (Adema et al., 2006; Rappoldt et al., 2003; Chapter 3), capillary transport

(Clymo & Hayward, 1982) and internal transport (Rydin & Clymo, 1989). Our study focuses on the

contribution of internal transport in the upward translocation of nitrogen in a Sphagnum bog.

Sphagnum plants are lacking both roots and a vascular transport system. Rydin & Clymo (1989),

however, demonstrated the internal acropetal transport of carbon and phosphorus in Sphagnum

fallax. They showed the presence of numerous plasmodesmata linking stem cells which create a

possible symplastic (cytoplasm to cytoplasm flow, contrasting apoplastic transport which indi-

cates the flow of solutes through the cell walls) transport pathway. Furthermore, Ligrone & Duckett

(1998b) found cytological evidence for nutrient translocation in Sphagnum. In a light- and electron

microscope study they revealed that the cells in the central region of Sphagnum stems have a

highly specialized cytoplasmic organization which has only been described for assumed solute-

conducting cells in mosses (Ligrone & Duckett, 1994). The presence of these cells, referred to as

“conducting parenchyma cells”, strongly suggests a cellular specialization in symplastic transport

(Ligrone & Duckett, 1998a). Internal transport of nitrogen has often been assumed (Aldous, 2002b;

Bonnett et al., 2010; Bragazza et al., 2005; Gerdol et al., 2006; Limpens & Berendse, 2003; Limpens


& Heijmans, 2008; Malmer, 1988) but, to our knowledge, has never been demonstrated before.

Here, the ability of Sphagnum to transport nitrogen internally is investigated. Sphagnum

cuspidatum and S. fallax are used in experiments in which diffusion and capillary transport are

excluded and the internal transport of labeled nitrogen is monitored. An attempt is made to eluci-

date the mechanism of transport, apo- or symplastic. By killing a small part of the stem cells sym-

plastic transport is excluded while the transport of solutes through the apoplast is still possible.

The contribution of internal transport in the nutrient supply to Sphagnum and to the nutrient

cycling in a Sphagnum bog with respect to other nutrient transport mechanisms in a Sphagnum bog

will be discussed.


Plant material

Sphagnum cuspidatum Ehrh. Ex Hoffm. plants were collected in a pool at the edge of a small bog

in the nature reserve “Dwingelderveld” (N52°49.777’, E6°25.994’) in the north of the Netherlands.

Sphagnum fallax (klinggr.) Klinggr. plants were collected in a small bog adjacent to the

“Dwingelderveld” (N52°49.135’, E06°29.491’). The mean annual nitrogen deposition in the north of

the Netherlands is 28 kg ∙ ha-1 ∙ yr-1 (RIVM, 2009).

The plants were collected and stored overnight (dark, 4°C) in plastic containers. Before being

used in the experiment the plants were cut to a length of 10 cm and rinsed three times with demin-

eralized water. Only visually non-damaged and fully green plants were used in the experiments.

Experimental design

To measure the ability of Sphagnum to transport nitrogen internally, three experiments were per-

formed in which the transport of solutes by diffusion and capillary transport was excluded. The ex-

perimental set up for these experiments consisted of two compartments divided by a barrier created

by placing two square Petri-dishes (120*120mm; Greiner bio-one GmbH) against each other. Both

compartments were filled with 85 mL of diluted artificial rainwater (100 times) containing 20 mM

MES buffer (pH = 4.0). To one of the two compartments, the donor compartment, labeled nitrogen 25

µmol ∙ L-1 15NH4Cl (98 atom %; Sigma Aldrich Inc., product number 299251) or 10 µmol ∙ L-1 K15NO

3 (98

atom %; Sigma Aldrich Inc., 335134) was added. The other compartment was called the acceptor dish.

Both solutions were not in contact with each other. Three plants with a length of 10 cm were placed

over the rim dividing the two compartments. The 10 cm long plants had green stems and were there-

fore considered to be alive and healthy. Two Sphagnum species were used in this experiment, Sphag-

num cuspidatum and S. fallax. The compartments were covered with a lid to reduce evapotranspira-

tion. All experiments took place in a climate controlled room at 18±1°C and a 16L:8D photoperiod.

Experiment 1: acropetal transport of nitrogen

In the first experiment the internal transport of ammonium and nitrate from the stem to the capitula

(acropetal transport) was measured. The upper 2 cm of the plants (including the capitulum) were

placed in the acceptor dish. The 8 cm long stem was placed in the donor dish. Plants were harvested

after 1, 2, 4 and 8 days.


Experiment 2: Basipetal transport of nitrogen

The potential transport of nitrogen from the capitula to the stem (basipetal transport) was

determined by placing the upper 2 cm of the Sphagnum plants in the donor dish and the stem in

the acceptor dish. Nitrogen was applied as ammonium (15NH4Cl) or nitrate (K15NO

3). Plants were

harvested at day eight.

Experiment 3: Apoplastic transport of nitrogen

To distinguish between apoplastic and symplastic transport of ammonium the plants were again

placed with the stem in the donor compartment, but a small segment of the stem was killed by

steam using a modification of the method described by Rydin & Clymo (1989). Two centimeter

below the capitula a stem section of ca. 1 cm was treated with steam for 60 seconds. The adjoining

sections of the stem were protected from the steam by temporarily covering them with cork. Vital-

ity of the steam-treated and untreated stem sections was determined by FDA staining (Elzenga et

al., 1991; Heslop-Harrison & Heslop-Harrison, 1970). Vital cells were distinguished from non-vital

cells based on their fluorescein green appearances using a Zeiss fluorescence microscope. During

the experiment, the steamed part of the stem became white whereas the untreated parts remained

green. Plants were harvested at day four.

In all three experiments leakage of 15N from the donor to the acceptor compartment by capil-

lary flow along the stem was excluded by covering the stem at the rim dividing the two compart-

ments with white Vaseline (Lamers & Indemans, ‘s Hertogenbosch). The efficiency of this waxy

barrier was tested visually by using the dye Brilliant Blue (Coomassie Brilliant Blue G, No. B0770,

Sigma Aldrich) as a marker. In the acceptor compartment no Brilliant Blue was observed when wax

was used.

A second test for leakage and contamination of the acceptor dishes with 15N, three capitula

(so called “contamination control capitula”) were placed in the acceptor compartment during the

experiment and were harvested together with the other capitula and stems. These capitula indicate

a possible change in 15N concentration in the capitula during the experiment when not exposed to a

source of labeled nitrogen. All experiments were performed in triplicate resulting in 3*3 plants per

sampling day.

Analyses

Upon harvesting the Sphagnum plants were separated into the upper first centimeter (the capitu-

lum) and the undermost seven centimeters of the stem. A 2 cm segment bridging the rim was not

included in the analysis to allow the grinding of the tissue without interference of possible wax

remains. After harvesting the plants were three times thoroughly rinsed with demineralized water

and dried for at least 48 hours at 80°C. Subsequently, the plants were grinded individually to a fine

powder using a ball miller (Retsch MM2, Haan, Germany). The contamination capitula were pooled

to reduce the number of samples. For each sample the %N and %15N (stable nitrogen isotope com-

position) was measured with a Carlo Erba NA 1500 elemental analyzer (Thermo Fisher Scientific

Inc.) coupled online via a Finnigan Conflo III interface with a Thermo-Finnigan DeltaPlus mass-

spectrometer. Additionally, the natural occurring 15N concentration in the capitula and stems of

the Sphagnum plants was determined (day=0 samples).

The data are expressed as the amount of 15N assimilated in the plants (in µmol ∙ g DW-1 and was


calculated by the formula (%15N * %N / 100)*10000/15, where %N is the percentage of total N in

the sample, %15N is the percentage of 15N of total N and 15 is the molecular weight of the stable N

isotope.

Statistical analyses

In the acropetal experiment the 15N concentrations of the capitula and “contamination control

capitula” in the acceptor dishes at day 1, 2, 4 and 8 were compared to the values in the capitula

and stems at day zero by using a one way ANOVA, with day as independent variable. A Dunnett’s

post-hoc test was performed in case of differences within factors. In both the “basipetal” and

the “apoplastic” experiment, a t-test was used to determine differences in 15N concentrations in

capitula or stems between day 0 and the end of the incubation period.

In general for each factor we had three replicates and three plants per replicate.

Prior to analysis, all data were transformed (1/x) when necessary to meet the assumption of

homogeneous variance. All statistical analysis were performed by using SPSS for Windows (version

16.0.1, 2007; SPSS Inc., Chicago, IL, USA).

Hyperbolic curves were fitted to the ammonium uptake data using graphing software (Prism

version 4.03, 2005; GraphPad Software, Inc., San Diego, CA, USA).

Results

For both Sphagnum cuspidatum and S. fallax a slow but significant acropetal transport of ammoni-

um through an internal mechanism was observed (figure 1). A one way ANOVA showed a significant

difference in 15N concentration between days in the capitula of both S. cuspidatum (F(4,40)=12.389,

p<.01) and S. fallax (F(4,39)=8.522). In the capitula of S. cuspidatum a significant increase of 15N was

observed after four (p=0.12) and eight days (p<.01); 4.8 ± 0.4 and 7.1 ± 0.9 µmol ∙ g DW-1, respectively,

compared to 3.7 ± 0.3 µmol 15N ∙ g DW-1 at the beginning of the experiment (day=0). For S. fallax a

significant increase of 15N in the capitula was observed at day eight (p=0.001); 4.0 ± 0.3 µmol ∙ g

DW-1 compared to 2.8 ± 0.2 at day=0. The concentration of 15N in the contamination control capitula

remained very low throughout the experiment (average value; p>0.05), from which we concluded

that no contamination occurred and that the concentration of 15N in the Sphagnum mosses was

not affected by the experimental procedure itself. Most obvious is the rapid uptake of 15NH4

+ by the

stems of both Sphagnum cuspidatum and S. fallax for which within one day respectively 64 and 84%

of the maximum concentration is reached (figure 1).

For nitrate the uptake by the stems of S. cuspidatum shows a similar pattern as ammonium;

74% is taken up within the first day (figure 1). On the other hand, hardly any NO3

- was taken up by

the stems of Sphagnum fallax.

A one way ANOVA showed no significant difference in 15N concentration between days in

the capitula of S. cuspidatum (F(4,40)=1.940, p>.05) and S. fallax (F(4,40)=1.225, p>.05). Also no

significant difference between 15N concentration in the contamination control capitula between

days was observed from which we assumed that no contamination occurred.


Figure 1. Mean 15N concentration (± SE) in the stems (grey circles), capitula (black circles) and contamination capitula (open

circles; ± SD) in Sphagnum cuspidatum (a,c) and Sphagnum fallax (b,d) measured after 0, 1, 2, 4 and 8 days of incubation of the

stems in 15NH4Cl (a and b) or K15NO

3 (c and d). Because of large differences in 15N concentration between the capitula and stems

these values are plotted distinctively on respectively the left and the right y-axes. Notice that the concentration of NO3

- in the

donor compartment was lower than ammonium (10 vs. 25 µmol ∙ L-1). Significant increase of 15N in the capitula compared to

the concentration at the start of the experiment (day = 0) are indicated by an asterisk (p<0.05). When no error bar is visible the

standard error is lower than the size of the circles. For clarity the untransformed data are shown.

In the experiment designed to determine possible basipetal transport of nitrogen, the 15NO3

- and 15NH

4+ was readily taken up from the experimental solution by the capitula in both Sphagnum

cuspidatum and S. fallax within eight days (figure 2). However, no significant increase of 15N in the

stems was observed after 8 days in S. cuspidatum with either ammonium or nitrate (t(16)=-2.048

and t(16)=0.163, resp., p>0.05) and also S. fallax showed no significant increase with NH4

+ and NO3

-

(t(16)= -1.523 and t(16)=0.584, resp., p>0.05; figure 2).

When a 1 cm segment of the stem was killed by steam no significant differences in 15N con-

centration between the capitula at day 0 and day 4 were observed for both Sphagnum cuspidatum

(t(15)= -0.76, p>0.05) and S. fallax (t(15)= -0.600, p>0.05; figure 3) and the acropetal transport as

demonstrated in figure 1 is inhibited.


Figure 2. The average concentration of 15N (± SE) per gram dry weight in the stems (black circles), capitula (white circles) and

contamination capitula (± SD; grey circles) in Sphagnum cuspidatum and Sphagnum fallax measured after the incubation of the

capitula in 15NH4Cl or K15NO

3 for 0 and 8 days. No significant differences in 15N concentration between days in the stems of both

S. cuspidatum and S. fallax were observed. When no error bar is visible the standard error is lower than the size of the circles.

For clarity the untransformed data are shown.

Discussion

Internal transport of nitrogen in Sphagnum

For both Sphagnum cuspidatum and S. fallax a slow but significant acropetal transport of nitrogen

through an internal mechanism was observed (figure 1). In this chapter we describe that live stems

of S. cuspidatum and S. fallax do take up both, NH4

+ and NO3

- , although with different efficiencies,

and that nitrogen supplied as NH4

+ is subsequently transported acropetally to the capitulum. The

difference between NH4

+ and NO3

- uptake efficiency will also have been determined by the concen-

trations in which both N species were applied, 25 and 10 µmol ∙ L-1 for NH4

+ and NO3

-, respectively.

Yet, these values were chosen since they represent natural bog water concentrations under natural

conditions.

After uptake, both NO3

- and NH4

+ are assimilated into the amino acid glutamine (Gln) and

subsequently converted into other amino acids (Kahl et al., 1997; Rudolph et al., 1993). Therefore, it

is assumed that transport of N takes place in the form of amino acids.

When a segment of the stem is killed by steam, Sphagnum plants no longer show a significant


Figure 3. The average concentration (± SE) of 15N in the stems (black circles), capitula (open circles) and contamination capitula

(± SD; grey circles) in Sphagnum cuspidatum and S. fallax measured after the incubation of the stems in 15NH4Cl for 0 and 4 days.

A one centimeter segment of the stem was killed by steam. No significant differences in 15N concentration between days in the

capitula of both S. cuspidatum and S. fallax were observed. When no error bar is visible the standard error is lower than the size

of the circles. For clarity the untransformed data are shown.

transport of 15N to the capitula within four days. Killing a segment of the stem by steam, blocks

symplastic transport of compounds, but should still allow apoplastic transport (Rydin & Clymo,

1989). Therefore, these results are indicative for the symplastic nature of acropetal transport of

nitrogen, and are in full agreement with the findings of Ligrone and Duckett (1998b) and Rydin

& Clymo (1989) who demonstrated cellular specializations of Sphagnum for symplastic transport.

Nevertheless, the duration of four days might be too short to totally exclude a small contribution by

apoplastic transport. An alternative hypothesis is the diffusive transport of NH4

+ and NO3

- through

the cell wall and the hyaline cells present in the epidermis of the stem of Sphagnum. However, dif-

fusive extracellular transport would result in similar transport rates in both acropetal and basipetal

direction. In contrast, 15N is not being transported into the stem due to basipetal transport of 15N in

experiment 2. In our experiment we only observed net transport of nitrogen from stem to capitula.

A possible explanation for this unidirect transport might be the higher sink strength for N of the

capitulum, relative to the stem, which can be expected because growth is taking place exclusively

in the capitulum.

Since mineralized nitrogen is an important nitrogen source (Aerts et al., 1999; Aldous, 2002b;

Bridgham, 2002; Gerdol et al., 2006; Morris, 1991; Urban & Eisenreich, 1988), that becomes available

in the deeper layers, distinct from the growing capitulum in the top layer, the supply of nitrogen

depends on upward-directed transport mechanisms (Aldous, 2002b; Bridgham, 2002). Uptake by

the stem and subsequent internal transport to the growing parts is thus a possible pathway for

mineralized nitrogen.

Internal, stem-mediated transport can be involve in re-allocation of nitrogen from older,

senescing tissue, a nutrient retention mechanism common in vascular plants (Aerts, 1990, 1995;

Chapin, 1980; Vitousek, 1982) and in transport of nitrogen taken up from the external medium

by the stems. This distinction must be taken into consideration when reviewing the importance

of different processes in the transport of externally mineralized nutrients. Gerdol et al. (2006)

stated that the mineralization of N is more important than the enzymatic reallocation of N.


Our experimental procedure only involves the translocation of the externally mineralized nitro-

gen. Higher C:N ratios in stems than in capitula are often observed in Sphagnum (e.g. Malmer, 1988)

and are taken as an indication for the internal reallocation of N from the stem to capitula. This

assumption, in combination with the internal transport of C and P (Rydin & Clymo, 1989), were

reasons for the general acceptance of internal transport in Sphagnum (Bonnett et al., 2010; Bragazza

et al., 2005; Gerdol et al., 2006; Limpens & Berendse, 2003; Limpens & Heijmans, 2008; Malmer,

1988). However, experimental evidence for the internal reallocation of N was lacking. Our findings

provide physiological evidence for internal transport of N taken up from the external medium.

There is, however, no reason to assume that the same mechanism does not transport N internally

released from senescing tissue.

NH4

+ and NO3

-

Within the time frame of the experiments the internal transport of nitrogen was only observed

when nitrogen was supplied as NH4

+ and not as NO3

-. According to a review by Rudolph et al. (1993)

this difference might have a metabolic origin. After uptake NO3

- is reduced to NH4

+, which takes

place in the chloroplast. The subsequent assimilation into amino acids also takes place in the

chloroplast. On the other hand, the assimilation of NH4

+ can take place in the cytosol (Rudolph

et al., 1993). Taking symplastic transport into account (see below), the N present in the cytosol is

available for transport whereas the amino acids in the chloroplast are more or less ‘fixed’.

The uptake of NO3

- by the stems differed between the two species. The stems of S. fallax hardly

took up NO3

- within the eight days of the experiment. Wiedermann et al. (2009) showed NO3

- to

be taken up in small amounts by capitula of Sphagnum balticum and S. fuscum from a solution

containing in total four N forms (NH4

+, alanine and glutamine and NO3

-). According to Woodin &

Lee (1987), most absorption of nitrate takes place in the capitulum and decreases down the stem.

With the stems hardly taking up NO3

- the transport to the capitula can not be expected.

The rate of internal transport

In earlier studies (Aldous, 2002b; Bridgham, 2002) the contribution of translocation to the nitrogen

supply of the capitula was shown to be significant. However, these studies did not distinguish be-

tween different types of transport. Based on the uptake and internal transport of ammonium data by

S. cuspidatum (figure 1) the rate by which nitrogen is transported internally can be estimated. Since

already after one day the maximum concentration of 15N in the stem is nearly reached, we can as-

sume that the concentration in the stem is almost constant for the duration of the experiment. Since

there is a lag period of almost 4 days before labeled N appears in the capitulum and we know the

length of the stem segment between the donor compartment and the receiving capitulum (which is

2 cm), we can calculate the speed of the acropetal, symplastic transport process: 5 mm/d. From the

increase in the capitulum in the four days following the lag, 2.3 µmol ∙ g DW-1 or 12% of the total N

taken up by the stems, we can calculate a half time value of equilibration between donor and recep-

tor sections of the plant: 17 days. Notice that this calculation is based on the assumption that the

amount of 15N in the capitula is a function of the amount of 15N taken up by the stems in the preced-

ing four days. It should be noted that the delay and apparent transport speed taken the assimilation

of NH4

+ into amino acids is also included. This calculated half time value for N is higher than the

estimated half time value of 11 days for the internal transport of C and P (Rydin & Clymo, 1989).


Since the internal transport of nitrogen is a mechanism for efficient nitrogen use, the transport

rate is expected to be reduced under high N content in the capitula (Bragazza et al., 2004). The

plants used in these experiments were collected from an area with a high load of atmospheric

nitrogen deposition. It has been demonstrated that Sphagnum mosses subjected to a high N sup-

ply accumulate elevated amounts of N (Nordin & Gunnarsson, 2000; Van der Heijden et al., 2000;

Limpens & Berendse, 2003; Limpens et al. 2011). Moreover, The capitula of the Sphagnum cuspida-

tum plants used in our experiments also showed a relatively high N content: 15.3 ± 2.2 mg ∙ g-1. If

indeed internal transport rates are negatively affected by nitrogen supply, our estimates are likely

to be an underestimation for Sphagnum residing in non-polluted areas and internal transport

might be more important.

The importance of internal transport

The uptake of ammonium by the living stems and the subsequent transport of N to the capitula

shows that internal transport very likely functions as a mechanism in supplying the capitula with

N. Moreover, it implies that Sphagnum mosses are able to compete with microbes and vascular

plant roots for available soil nitrogen (see Bridgham, 2002) and thereby might function as a N

retention step contributing to the efficient use of N by Sphagnum in ombrotrophic bogs.

However, the importance of internal transport in the nutrient supply of Sphagnum and nutrient

cycling in bogs depends on the transport rate relative to other transport mechanism. In a review of

the internal transport in non-vascular plants (Raven, 2003) it is claimed that there is no evidence for

symplastic transport in Sphagna faster than can be accounted for by diffusion. Next, the rate at which

nitrogen is transported internally is very slow compared to the transport rates by buoyancy-driven

water flow (Rappoldt et al., 2003). Chapter 2 shows buoyancy flow to be a fast and effective nutrient

transport mechanism in bog water. The uptake kinetics of Sphagnum cuspidatum and S. fallax show

the ability of Sphagnum to take up large amounts of ammonium relatively fast, indicating buoyancy

flow to be a very effective nutrient transport mechanism in supplying the capitula with nitrogen.

Therefore, with the regular occurrence of buoyancy flow, the supply of nitrogen by internal

transport might be insignificant, compared to the supply of nitrogen by buoyancy flow. Indeed,

buoyancy flow is restricted to water-saturated Sphagnum habitats and, because of its dependence

on varying physical parameters like the difference in temperature between day and night, an

irregularly occurring phenomenon. The importance of internal transport might therefore reside in

its continuous character, continuously supplying Sphagnum with N which contrasts with the pulse

wise supply of N by buoyancy flow (and of course precipitation). For Sphagnum species that form

hummocks that extend above the water surface and do not benefit from buoyancy flow, internal

transport is, next to capillary transport, a possible acropetal pathway for nutrients. Clymo (1973)

estimated the average velocity by capillary flow to be 0.4 mm ∙ min-1. However, this rate, and the

concomitant nutrient transport, is dependent of several factors, like evaporation, plant density

and pore water nutrient concentrations (Clymo & Hayward, 1982). Moreover, during extracellular

transport nutrients may be lost to microorganism or vascular plant roots. Compared to the external

transport mechanisms (buoyancy flow and capillary transport) internal transport very likely rep-

resents a minor contribution in the upward transport of externally supplied N to the capitula. The

main importance of internal transport is therefore considered to be the reallocation of internally

catabolized nitrogen compounds.

Chapter 5

The importance of groundwater carbon dioxide in the restoration of Sphagnum bogs

Wouter Patberg

Gert Jan Baaijens

Alfons Smolders

Ab Grootjans

Theo Elzenga


Abstract

Essential for successful bog restoration is the re-establishment of Sphagnum

mosses. High CO2 availability has been shown to be of great importance for

the growth of Sphagnum mosses. In well-developed Sphagnum bogs large

amounts of CO2 are produced by decomposition processes in the peat layer.

In cut-over Sphagnum bogs this carbon source is often absent or strongly re-

duced. Therefore, for the successful restoration of cut-over Sphagnum bogs

an alternative, additional carbon source might be essential for the re-estab-

lishment of Sphagnum mosses. This chapter focuses on the role of CO2 in the

development of Sphagnum bogs in a field situation.

Study area is one of the largest wet heathland reserves in Western Europe

and is characterized by many small damaged Sphagnum bogs. Rewetting

measures resulted in large developmental differences between bogs; some

bogs developed markedly well, whereas others did not. Of ten small bogs the

developmental success was quantified using aerial photographs and surface

water and groundwater samples were collected. In addition, the physiologi-

cal characteristics of carbon dioxide uptake of two Sphagnum species were

determined.

Water chemistry analysis revealed that the total inorganic carbon concen-

tration (TIC) in the nearby groundwater of the well-developed bogs, is sig-

nificantly higher than that of poorly developed bogs. The CO2 availability in

the surface water of the investigated bogs was positively correlated to the in-

organic carbon in the groundwater. It is concluded that the well-developing

bogs are fed by a carbon-rich groundwater inflow from outside the bog.

The CO2 uptake kinetics of both Sphagnum species are characterized by a

high compensation point and a low affinity, both indicating an adaptation

to a high CO2 availability.

The present findings indicate that high carbon dioxide availability is a pre-

requisite for the successful re-establishment of Sphagnum mosses in peat

bog restoration projects and that carbon-rich groundwater can apparently

substitute for the decomposing peat layer as a source of CO2. Therefore, the

availability of CO2 should be included in bog restoration feasibility studies.

The importance of groundwater – 59

Introduction

Due to the extensive exploitation for fuel, agriculture and forestry over many centuries, living (peat

forming) mires have become endangered in most of North-Western Europe (Rochefort & Price,

2003). Even nowadays (extensive) peat extraction activities take place for commercial use in, for

example, Canada, Scandinavia, Ireland and the Baltic states (Joosten, 2009). Due to the important

role of peatlands in the global carbon cycle, and because of their unique ecological values, globally

much effort is dedicated to the restoration of damaged mires. However, the restoration of large bog

remnants in particular, has proven to be fairly complicated and not always successful (Grootjans et

al., 2006; Money & Wheeler, 1999; Money et al., 2009).

Essential for successful bog restoration is the re-establishment of Sphagnum mosses followed

by the re-development of a functional acrotelm, leading to a self-sustaining system (Money &

Wheeler, 1999; Money et al., 2009; Smolders et al., 2003). Since wet conditions are essential for

Sphagnum growth (Robroek et al., 2009), the creation of suitable wet conditions is a prerequisite in

restoring peatlands (e.g. Money et al. 2009). Often rewetting is realized by inundating large areas to

ensure wet conditions throughout the year (Money & Wheeler, 1999; Smolders et al., 2003). The wa-

ter layer can be colonized by aquatic Sphagnum species, especially Sphagnum cuspidatum, to form

dense mats on which peat forming species like S. magellanicum and S. papillosum might establish

(Money & Wheeler, 1999; Wheeler & Shaw, 1995). However, this method often results in large water

bodies in which Sphagnum growth is severely hampered. The lack of success in re-colonization of

aquatic Sphagnum species in rewetted bog remnants has been ascribed to the limited availability

of light and/or CO2 (Money & Wheeler, 1999; Smolders et al., 2001; Smolders et al., 2003; Wheeler &

Shaw, 1995).

Like most aquatic bryophytes (Raven et al., 1985) Sphagnum mosses are known to be obligate

CO2 users (Bain & Proctor, 1980) and are solely dependent on the diffusive supply of CO

2 to the site

of carbon fixation (Rubisco - ribulose-1,5-bisphosphate carboxylase-oxygenase). In very wet condi-

tions the Sphagnum mosses are surrounded by a thick water layer which lowers CO2 conductivity

resulting in a reduced photosynthetic rate (Silvola, 1990; Williams & Flanagan, 1996). Consequent-

ly, high rates of underwater photosynthesis can only be sustained when the leaves are exposed to

high levels of CO2 (Jauhiainen & Silvola, 1999; Paffen & Roelofs, 1991; Silvola, 1990; Smolders et al.,

2003).

In well developed bogs CO2 is produced in large quantities by decomposition processes in the

peat layer (Bridgham & Richardson, 1992; Glatzel et al., 2004; Smolders et al., 2001; Waddington

et al., 2001). Carbon dioxide concentrations in the pore water can reach up to several millimo-

lar (Smolders et al., 2001; Smolders et al., 2003), compensating low diffusion rates and ensuring

sufficient substrate delivery for photosynthetic carbon fixation (Maberly & Madsen, 2002; Silvola,

1990). This so-called substrate-derived CO2 has been shown to be an important carbon source for

aquatic and emergent Sphagnum mosses (Baker & Boatman, 1990; Paffen & Roelofs, 1991; Riis &

SandJensen, 1997; Roelofs, 1983; Smolders et al., 2001; Smolders et al., 2003). Under more reductive

conditions the methane production in the catotelm may become higher than the production of

CO2. This methane can be oxidized by methanotrophic bacteria to CO

2, which then can be used as a

carbon source by Sphagnum mosses (Kip et al., 2010).

Cut-over peat bogs often lack this source of additional inorganic carbon. This type of damage


can often be found in North-Western Europe (Joosten, 2009). Peat extraction has removed the bulk

of organic material. The highly decomposed, humified peat which is left behind, has only limited

CO2 (and methane) production rates (Bridgham & Richardson, 1992; Glatzel et al., 2004; Tomassen

et al., 2004; Waddington et al., 2001). Therefore, for the successful restoration of cut-over Sphag-

num bogs an additional carbon source might be essential for the re-establishment of Sphagnum

mosses.

This study focuses on the role of CO2 in the development of Sphagnum bogs in a field situation.

The study area is one of the largest wet heathland reserves in Western Europe and is characterized

by numerous small Sphagnum bogs. They have been damaged by drainage and small scale peat ex-

cavations in the past. From 1988 onwards, rewetting measures have been carried out, but the devel-

opmental success has varied significantly between bogs; some bogs developed well, whereas oth-

ers did not. It is hypothesized that in these hydrologically degraded bog remnants the restoration

of Sphagnum growth is limited by the availability of CO2. We expect that the well-developing bogs

are being fed by lateral, carbon-rich, groundwater inflow. Groundwater with high inorganic carbon

concentrations entering a bog, will release high amounts of CO2 when it comes into contact with

the more acidic water around the Sphagnum mass. The higher availability of CO2 will stimulate the

growth of aquatic Sphagnum mosses and subsequent bog development. We also determined the

CO2 requirement for two Sphagnum species (S. cuspidatum and S. fallax), which are abundant in

well-developing bogs.

Materials & Methods

Study area

Study area is the “Dwingelderveld”, one of Europe’s largest wet heathland areas (about 3500 hectares),

situated in the north of the Netherlands (52°49’6.71”N 6°27’28.48”E). The landscape consists of pine

forest, wet and dry heathland and many small peat bogs scattered throughout the area (figure 1).

The presence of boulder clay underneath the reserve is responsible for the generally wet character

of the area. Wind erosion resulted in differences of up to 5 meters in height of the Pleistocene

sand cover. During the second half of the last century most of the wet heathland and bogs became

desiccated by drainage activities both in the reserve (for the benefit of pine plantations) and also in

the surrounding brook valleys (for the benefit of agriculture). Many small bogs were also subjected

to small scale peat cutting by farmers. These activities have ended around 1950.

In 1988 large scale rewetting measures, i.e. closing of drainage ditches and cutting of trees, were

initiated with variable results in developmental success of the different bogs. Some bogs developed

well and are characterized by a luxurious growth of Sphagnum spp., whereas others did not and

mainly consist of open water with marginal occurrence of Sphagnum mosses (Grootjans et al.,

2003).

An interesting observation by Grootjans et al. (2003) was that bogs with abundant Sphagnum

growth were located in old erosion gullies, while bogs without Sphagnum-dominated succession

were found outside or at the edge of these gullies. In these erosion gullies impermeable podsolic

layers stretch out beyond the border of the bogs itself. The groundwater levels in the sandy hills are

generally higher than in the gullies. Since the vertical conductance of these podsolic layers is very


low, a horizontal sub-surface flow of groundwater towards the bogs is facilitated, prolonging the

residence time of water in the soil and allowing the groundwater to become possibly enriched with

inorganic carbon (Grootjans et al., 2003; figure 2).

Figure 1. Aerial photograph of the research area in the “Dwingelderveld”. All bogs in this part of the nature reserve are outlined

by a black line. The bogs used in this field study are indicated by a number corresponding to the numbers used in table 1. The

numbers of the poorly developed bogs are underlined. The locations of the sampling sites are indicates by black circles. The

white spot in the Diepveen (6) is open water as proven by field observations.

Classifying peat bogs

In 10 bogs 20 sampling sites were selected (figure 1; table 1). Aerial photographs of 1982 and 2006

were used to determine the developmental success of each of the sampling sites. The increase in

surface area covered by Sphagnum mosses was used as a measure for the success of bog develop-

ment. With the use of image analysis software (ImageJ, version 1.41o, National Institute of Health,

USA) the area of open water per sampling site in both 1982 and 2006 was calculated based on grey

scale differences between vegetation and open water. The developmental success was determined

by calculating the relative decrease of open water surface between 1982 and 2006. The following

formula was used: (%OW1982

- %OW2006

) / %OW1982

, where %OW is the surface percentage of the

bog occupied by open water in 1982 or 2006. In this aerial photograph analysis the vegetation was

assumed to be bog vegetation dominated by Sphagnum mosses. This was validated by field observa-

tions. In some cases, Cootjes Veen, Groote Veen East and Veerles Veen, the aerial photograph analy-

sis had to be adjusted. Here the vegetation was dominated by vascular plants (e.g. Molinia caerulea)

instead of Sphagnum mosses. Consequently these bogs were classified as poorly developed bogs.


Table 1. Names, coordinates, the percentage open water in 1982 and 2006, the decrease of open water and the developmental

success of the investigated bogs in the “Dwingelderveld”. The developmental success of the bogs was determined by calculating

the relative decrease of open water surface in 2006 compared to the surface open water in 1982. A “+” indicates a well developed

bog and a “-“ indicates a poorly developed bog. For three bogs the developmental success was not determined by aerial photo-

graphs as indicated by an “*”. See the materials and methods section for a more detailed explanation. The numbers in the first

column correspond with the numbers in figure 1

# Name Coordinates

Surface

open water %

Decrease

open water

(%)

Develop mental

success

1982 2006

1 Barkmans Veen N52°49.423’ E6°26.283’ 29 6 78 +

2 Groote Veen N52°49.178’ E6°25.991’ 51 0 100 +

3 Reigersplas N52°50.019’ E6°26.946’ 62 0 100 +

4 Adderveen N52°49.885’ E6°27.058’ 74 43 42 -

5 Cootjes Veen N52°49.000’ E6°26.244’ 0 0 * -

6 Diepveen N52°49.140’ E6°26.404’ 52 56 -8 -

2 Groote Veen East N52°49.171’ E6°26.243’ 71 0 * -

7 Kliploo N52°50.082’ E6°26.380’ 100 98 2 -

8 Schurenberg N52°49.551’ E6°25.951’ 100 83 17 -

9 Veerles Veen N52°49.003’ E6°25.992’ 61 0 * -

10 Zandveen N52°49.694’ E6°26.471’ 85 69 19 -

Water sampling and analysis

Groundwater and surface water samples were taken at the sampling sites in February and April

2007, August and October 2008 and September 2009. Groundwater samples were collected using

piezometers (Ø32 mm PVC tubes with nylon filters) and a peristaltic pump. The piezometers

were placed just outside the bogs on the slopes of the gully and always with the filters above the

impervious layer. The exact position of the piezometers was chosen such that they intercepted the

groundwater flowing into the bogs, based on the results of a previous hydrological study of the area

(Verschoor et al., 2003). One day before sampling the water present in the piezometers was discard-

ed to allow refilling with fresh groundwater. Surface water samples were taken in the bog close to

the piezometers. 30 mL airtight bottles were filled by gently submersing them in the surface water.

Water samples were transported in a cool box to the laboratory where pH and TIC measurements

were performed immediately. The remaining samples were stored frozen until further analyses.

Precipitation data of the “Dwingelderveld” were obtained from the Royal Netherlands Meteorologi-

cal Institute (www.knmi.nl, station number 327 “Dwingeloo”).

The concentration of Total Inorganic Carbon (TIC) in the water samples was determined by

measuring the CO2, released after acidifying the samples to a pH <3, using an Infra-Red Gas Analyzer

(IRGA, ABB Advance Optima). The pH of the water samples was determined using a combined pH

electrode with an Ag⁄AgCl internal reference (Cole Parmer Instrument Company, Illinois, USA) and

a PHM 64 pH meter (Radiometer, Copenhagen). The concentrations of CO2 and bicarbonate in the

water samples were calculated based on the pH and the TIC concentration (Prins & Elzenga, 1989).


Concentrations of nitrate (NO3), ammonium (NH

4) and chloride (Cl) were measured colourimetri-

cally according to Geurts et al. (2008) and potassium (K) by flame photometry by using an Auto

Analyzer 3 system (Bran+Luebbe, Norderstedt, Germany). Aluminium (Al), calcium (Ca), iron (Fe),

magnesium (Mg), manganese (Mn), sodium (Na), total phosphorus (P), sulphur (S), silicon (Si) and

zinc (Zn) were measured using an ICP Spectrometer (IRIS Intrepid II, Thermo Electron Corporation,

Franklin, MA).

Figure 2. A cross section of a part of the study area in the “Dwingelderveld”. The small bogs are situated in the gullies which

are surrounded by the higher Pleistocene sand cover. A layer of boulder clay is present underneath the whole area, resulting

in wet conditions throughout the area. The bogs are characterized by the presence of podsolic layers, responsible for the wet

conditions and bog development. Note the lack of peat development in the bog in the forefront which lies outside the gully.

Groundwater samples were taken adjacent to the border of the bogs, as indicated by the piezometer.

CO2 uptake characteristics

Carbon dioxide uptake characteristics of Sphagnum cuspidatum Ehrh. Ex Hoffm. and S. fallax

(klinggr.) Klinggr., two pioneer Sphagnum species important in the initial stage of bog formation

(Money, 1995; Smolders et al., 2003), were determined by measuring the photosynthetic activity

(A) at different CO2 concentrations at saturating light conditions (1500 µmol ∙ m-2 ∙ s-1; Hansatech

Quantitherm Light meter). Plants were collected in April 2008 in two small Sphagnum bogs in the

Dwingelderveld area.

A capitulum was placed in a closed thermostatic cuvette containing 1 mL of measuring

solution (see below) which was stirred continuously. The photosynthetic evolution of oxygen was

measured by a Clark electrode located at the bottom of the cuvette in combination with a millivolt

recorder. The temperature of the measuring solution was 21°C.

Any inorganic carbon present in the hyaline cells or adhering water was removed by illuminat-

ing the capitula with 1000 µmol ∙ m-2 ∙ s-1 for at least 60 minutes while keeping them in a CO2 free

medium. When the capitula showed a steady, low rate of oxygen uptake it was assumed that no sig-

nificant CO2 stores were left in the hyaline cells. The solution was then removed from the cuvette

by using a syringe and replaced by a solution with the desired CO2 concentration. Different CO

2

concentrations ranging from 0 to 800 µmol ∙ L-1 in 10 times diluted artificial rainwater (Smolders et

al., 2001) containing 20 mM MES pH = 5.5 were created of which 1 mL was used in the cuvette and


1 mL was used to exactly determine the Total Inorganic Carbon concentration, by using an infrared

gas analyzer (CO2 analyzer model no. S151, QUBIT Systems Inc., Kingston, ON, Canada). The pH of

the measuring solution was determined using a pH microelectrode (type MI-406, Microelectrodes

Inc., Bedford, NH, USA) in combination with an Ag⁄AgCl micro-reference electrode (type MI-401,

Microelectrodes Inc., Bedford, NH, USA) and a millivolt meter. The concentrations of CO2 and bi-

carbonate in the water samples were calculated based on the pH and the TIC concentration (Prins

& Elzenga, 1989).

Per measurement one capitulum was used and each capitulum was used for a maximum of

three measurements. Large branches were trimmed to fit in the cuvette. After usage capitula were

frozen at -80°C, ground to a powder and the chlorophyll concentration was determined according

to Lichtenthaler (1987).

Statistical analysis

Data were tested for normality using a Kolmogorov-Smirnov test and equality of variance using

Levene’s test. The assumption of homogeneity of variance was not always met, not even after

transformation of the data. According to Heath (1995), the analysis of variance appears not to be

greatly affected by heterogeneity in variance if sample sizes are more or less equal. Therefore, we

decided to continue our analysis using non-transformed data. Differences in groundwater TIC

concentration and surface water CO2 concentration between well and poorly developed bogs, were

tested using a mixed model, with bog development (well and poor) as fixed factor and sampling

date as repeated measure. Differences between sampling dates were determined by using Bonfer-

roni’s post-hoc test. The Pearson correlation coefficient was determined for the relation between

groundwater TIC and surface water CO2 concentrations (both log transformed). The mixed model

and the Pearson correlation test were performed using SPSS for Windows (version 16.0.1, 2007;

SPSS Inc., Chicago, IL, USA).

Additionally, the data were analyzed by applying principal component analyses (PCA) by using

Aabel (version 3.0.3; Gigawiz Ltd. Co., Tulsa, OK, USA). All data were normalized. A hyperbolic

curve was fitted to the CO2 uptake data using graphing software (Prism version 4.03, 2005; Graph-

Pad Software, Inc., San Diego, CA, USA).

Results

Classification of peat bogs

Based on the aerial photographs and field observations the developmental success of the selected

bogs was classified into two categories; well developed bogs, with an open water decrease of at

least 78% and poorly developed bogs with a decrease of open water less than 20% (table 1). The

“Adderveen”, however, is an exception with an open water decrease of 42%. For most sites the field

observations confirmed the classification based on the aerial photograph analysis: well developed

bogs showed luxurious and dominant growth of Sphagnum spp. without or with little open water,

whereas in poorly developed bogs marginal Sphagnum spp. growth was accompanied by the pres-

ence of vascular plants and open water. Open water surface decreased in all sites between 1982 and

2006, except for Diepveen, where the open water surface slightly increased with 8%.


Table 2. Water analysis of the groundwater and surface water of well (+) and poorly (-) developed bogs; average values for all

sampling dates are given with standard deviation (SD). Ion concentrations are given in µmol ∙ L-1

Groundwater Surface water

+ - + -

mean SD mean SD mean SD mean SD

TIC 4983.7 802.4 2765.6 1426.6 1230.3 736.8 768.1 638.4

HCO3

- 152.7 102.9 64.2 72.0 15.2 17.9 25.0 50.9

CO2

4831.0 756.2 2701.4 1389.7 1215.1 730.3 743.1 625.5

pH 4.8 0.3 4.5 0.5 4.3 0.3 4.7 0.5

NH4

94.6 54.7 84.4 78.5 76.5 70.3 71.6 80.0

NO3

7.4 14.6 8.3 12.5 8.3 13.9 9.4 14.8

K 14.1 10.6 25.5 24.2 26.4 19.2 34.2 27.7

P 2.6 4.1 1.9 1.9 4.7 7.8 3.9 7.6

Al 42.2 31.1 36.4 33.0 10.1 7.5 7.5 9.3

Ca 61.9 59.6 51.9 32.1 30.5 34.5 34.2 32.0

Cl 246.4 58.1 341.2 181.7 256.3 80.3 289.5 152.3

Fe 61.1 24.0 24.5 18.2 42.4 107.2 12.1 13.9

Mg 67.0 21.4 42.4 26.7 26.3 14.0 30.3 13.0

Mn 0.5 0.2 1.0 1.2 0.6 0.3 1.2 1.5

Na 231.4 38.2 284.8 130.2 192.3 48.0 227.0 94.6

S 39.2 18.1 53.5 55.9 22.0 15.8 27.9 21.8

Si 263.2 105.8 121.0 83.9 35.8 22.1 19.3 21.9

Zn 8.7 13.7 5.4 5.3 0.9 0.9 0.8 1.0

Water chemistry

Bog water chemistry data are shown in table 2. The difference in groundwater composition between

the well developed and poorly developed bogs is illustrated by a principal component analysis (figure

3). The groundwater samples from well and poorly developed bogs appear as clusters in the PCA

diagram along the first principal component axis that is dominated by high TIC, iron (Fe) and silicium

(Si), indicating that good Sphagnum development is associated with groundwater rich in inorganic

carbon. The groundwater is relatively acid and CO2 is the main inorganic carbon species (table 2).

The chemical composition of groundwater and surface water is clearly different, with concentrations

of most multivalent minerals and inorganic carbon being higher in the groundwater (table 2).

Groundwater TIC concentrations are shown per location in figure 4. The average ground-

water TIC concentration near the well developed bogs was 4983 ± 802 µmol ∙ L-1 and ranged from

2620 to 6215 µmol ∙ L-1. The poorly developed bogs, with an average TIC concentration of 2765 ±

1426 µmol ∙ L-1, showed a much wider range in TIC concentration both between measurements at


individual locations and between locations. A significant main effect of category on groundwater

TIC concentrations was found (F(1,12) = 20.246, p=0.001). In the well developed bogs the average

CO2 concentration in the surface water was 1215 ± 730 µmol ∙ L-1 and for the poorly developed bogs

743 ± 625 µmol ∙ L-1 (table 2). For surface water CO2 concentration also a significant main effect of

category was found, F(1,13) = 6.063, p=0.029.

Figure 3. Principal Component Analysis (PCA) biplot of all groundwater samples and selected environmental variables. Each

symbol represents a sampling location at one of the sampling dates. Black circles are the well developed bogs and the open

circles represent the poorly developed bogs. The first axis explains 27% of the variation and second principal component

accounts for 20 % of the variation.

Figure 4. Box plot showing the total inorganic carbon (TIC) concentration in the groundwater in µmol ∙ L-1 per location of

both well (gray boxes) and poorly (white boxes) developed bogs. Box plots are composed of minimum, maximum, 25%, 75%

quartiles and the median. Where used, north, south, east and west, indicate sampling sites at one bog.


Figure 5. Groundwater total inorganic carbon concentrations (TIC, solid lines) and surface water CO2 concentrations (dashed

lines) for the well developed bogs (filled symbols) and the poorly developed bogs (open symbols) shown per sampling date.

Symbols represent mean values in µmol ∙ L-1. Bars represent standard deviations and are one sided for readability of the graph.

Different letters mean significant differences between sampling dates, tested for groundwater TIC and surface water CO2

concentrations separately.

Figure 5 shows the changes over time in the total inorganic carbon and CO2 concentration of both

groundwater and surface water of well and poorly developed bogs. A significant main effect of

date on both groundwater TIC and surface water CO2 concentrations was found (F(4,48) = 5.734 and

F(4,52) = 6.126, respectively, p<0.01); the groundwater TIC concentration was significantly higher

on August 15 2008 than in February 8 2007 (p<0.05). For the surface water CO2 concentrations

significant higher values were found in April and September (figure 5). However, no significant

interaction effect of category * date was found for both groundwater TIC (F(4,48) = 0.566, p>0.05)

and surface water CO2 concentrations (F(4,52) = 1.317, p>0.05). In other words, groundwater TIC

and surface water CO2 concentrations in well and poorly developed bogs, respectively, were not

affected differently by date.

The logarithm of the groundwater TIC concentration was positively and significantly correlated

to the logarithm of the CO2 concentrations in the surface water. However, this correlation was the

strongest in the well developed bogs; Pearson’s r=0.521 (p<0.01) compared to a value of r= 0.286

(p<0.05) in the poorly developed bogs (figure 6).


Figure 6. The relation between the total inorganic carbon (TIC) concentration in the groundwater and the CO2 concentration in

the surface water for well (filled circles) and poorly (open circles) developed bogs. Water samples collected in April and August

are in black, others in gray. Lines are regression lines for well (solid line, r2=0.272) and poorly (dashed line, r2=0.082) developed

bogs. For clarity of presentation, the non-transformed data are shown.

Figure 7. Dose-response curves of photosynthetic activity as a function of the carbon dioxide concentration for Sphagnum

cuspidatum (filled symbols; r2=0.935) and S. fallax (open symbols; r2= 0.974).

CO2 uptake characteristics

The photosynthetic rate of Sphagnum cuspidatum and S. fallax as a function of CO2 concentration

are shown in figure 7. A hyperbolic curve according to the formula A=Vmax

*[CO2]/(K

m+[CO

2]) + c was

fitted to the data. From the curve the compensation point (Γ) for CO2 was calculated (table 3).

Table 3. Carbon dioxide uptake characteristic (±SD) for Sphagnum cuspidatum and S. fallax. See text for explanation of the parameters

S. cuspidatum S. fallax

Vmax

(nmol O2 ∙ s-1 ∙ mg chl-1) 19.8 ± 1.5 33.3 ± 2.6

Km

(µmol CO2 ∙ L-1) 133.2 ± 31.1 231.4 ± 52.5

Γ (µmol CO2 ∙ L-1) 10.2 7.2

c -1.4 ± 0.4 -1.1 ± 0.6


Discussion

Restoration success

The situation in the “Dwingelderveld” is characterized by a number of small Sphagnum bogs that

were subjected to identical restoration measures, but differing in groundwater influence and

developmental success. This offered us the opportunity to study the importance of CO2 in the

development of Sphagnum bogs in a field situation.

The aerial photograph analysis revealed distinct differences in restoration success. The results

were based on a quantitative approach; the occurrence of a high cover of Sphagnum species in

general. The success of the restoration was also evaluated by Everts et al. (2002) using total species

composition. Their results were in agreement with the results from our approach using aerial

photographs.

Evidence for influence of local groundwater flows

The PCA analysis using all main groundwater chemical data showed that well developed and poorly

developed bogs separate well, and that total inorganic carbon (TIC) differences are largely respon-

sible for that; the groundwater nearby the well developed bogs contains a significantly higher TIC

concentration than groundwater nearby the poorly developed bogs (figure 4).

We also found that CO2 concentrations in the surface water of the well developed bogs were

significantly higher than in the poorly developed bogs. Moreover, the CO2 availability in the surface

water of the investigated bogs is positively correlated to the inorganic carbon concentration in the

groundwater (figure 6). Carbon dioxide is very likely released from the carbon-rich groundwater

upon entering the acidic bog environment resulting in an increased CO2 availability stimulating

Sphagnum growth. Higher groundwater levels in the surrounding sandy areas will result in a flow

of the local groundwater towards the bogs. The concave shaped impermeable layer, essential for

bog formation, will result in the uni-directional flow of the local groundwater towards the bogs.

An alternative hypothesis is that the high inorganic carbon concentrations found in the

groundwater nearby the well developed bogs has its origin in the decomposition of organic material

in the bog. However, the chemical signature of the groundwater indicates an origin from outside

the bog. This was particularly clear in the silicium values, which are generally much higher in

water that has been in contact with mineral sediments for a longer period (Engelen & Jones, 1986).

Moreover, the groundwater near the Diepveen and the Zandveen which are poorly developed bogs,

contained high TIC concentrations despite the absence of accumulated organic material (figure 4).

Additionally, the placement of the piezometers on the concave shaped impermeable layers ensures

the sampling of inflowing water. Therefore, the results presented are in agreement with the hy-

pothesis that the well-developing bogs are fed by a lateral, carbon-rich, groundwater inflow.

To investigate differences between well and poorly developed bogs, a principal component

analysis was performed based on the chemical composition of the groundwater. Since the deve-

loping Sphagnum capitula are in close contact with surface water and only indirectly influenced by

groundwater this seems counter-intuitive. However, surface water composition is more influenced

by short term changes compared to the more stable composition of groundwater. Next, fluxes of

CO2 are more important than the resulting concentrations. The growth of Sphagnum mosses resul-

ting from the release of carbon rich groundwater will generate some positive feedbacks which will


result in an enhancement of the submerged Sphagnum growth (figure 8). The resulting accumula-

tion of organic matter will enhance the internal generation of CO2 from decomposition processes.

Furthermore, it will result in a decrease of the water depth which will increase light availability.

Figure 8. Schematic view of the positive feedbacks concerning CO2 availability in a Sphagnum dominated bog resulting from

the input of carbon rich groundwater. In a well developed bog the decomposition of accumulated organic matter results in a

high availability of CO2 stimulating Sphagnum growth, which in turn increases the accumulation of organic matter. During the

initial stages of bog development organic matter is absent and the inflow of carbon rich groundwater can substitute for the or-

ganic matter as a source of CO2, stimulating Sphagnum growth and thereby inducing the internal positive feedback mechanism

concerning CO2 availability. Additionally, the accumulation of organic matter decreases water depths, increasing light and CO

2

availability stimulating Sphagnum growth as well.

Seasonality of groundwater input

Some seasonality in groundwater input appears to be visible. In April and August we observed

increased CO2 concentrations in the well developed bogs compared to the poorly developed bogs

(figure 5). In autumn and winter we did not observe differences in CO2 in the surface water of the

bogs.

In spring and (early) summer lowered surface water levels (due to evaporation) will result in

an increase of the hydrological gradient resulting in an increased inflow of groundwater into the

bogs. In autumn and winter, this gradient will be decreased by higher surface water levels due to

an increase in precipitation. Additionally, strong rainfalls will lead to an increased run off towards

the bogs of very superficial groundwater which is relatively poor in CO2. Interestingly, this period

from April to August represent the growing season of the Sphagnum mosses (Clymo, 1970). High

CO2 consumption in the well developed and growing Sphagnum bogs, could even have caused a

lower than expected CO2 concentrations difference between well and poorly developed bogs.

Why do some Sphagnum species require high CO2 concentrations?

Smolders et al. (2003) showed very poor growth of five Sphagnum species under inundated

conditions on strongly humified, ‘black’ peat. Carbon dioxide concentrations in the water layer

remained very low in this situation (<20 µmol ∙ L-1). The same species, however, developed very


well on weakly humified, ‘white’ peat. In short, low carbon availability in combination with low

diffusion rates of CO2 in water severely reduces CO

2 availability and limitation of Sphagnum growth

is very likely to occur.

The requirement for high CO2 availability by Sphagnum can partly be explained by the

mechanisms of CO2 uptake that are determined by the physiological characteristics of both

Sphagnum cuspidatum and S. fallax (table 3; figure 7). Both Sphagnum species are characterized by

high CO2 compensation values, the CO

2 concentration at which CO

2 fixation by photosynthesis bal-

ances CO2 loss by respiration. Air-equilibrated water contains a CO

2 concentration of 10 - 20 µmol

∙ L-1 between 25 and 10°C. The high compensation values of S. cuspidatum and S. fallax imply that

under air-saturated conditions no, or extremely limited, net carbon accumulation can occur. In

the acidic bog environment, where no reservoir of bicarbonate is present to replace the CO2 that

is taken up, this is especially relevant and Sphagnum growth will not occur when CO2 is provided

exclusively through equilibration with air. The high Km

values of 231.4 and 133.2 µM CO2 for S. cuspi-

datum and S. fallax, respectively, further indicate that even when CO2 is present at a concentration

that is higher than air-saturated, carbon utilization is not optimal. In the investigated Sphagnum

species CO2 concentrations up to 400 - 500 µM are still not saturating (figure 7). Like most aquatic

plants lacking a carbon concentrating mechanism the kinetic properties of CO2 uptake of the in-

vestigated Sphagnum species indicate an adaptation to a high CO2 availability (Raven et al., 1998).

Under natural conditions the stagnant bog water will result in thick boundary layers and long

diffusion path lengths when compared to the well stirred conditions during the measurements,

lowering the apparent affinity for CO2 concentration even more. Additionally, photosynthetically

produced oxygen will accumulate in the boundary layer and because of the competition between

CO2 and O

2 at the site of Rubisco, photosynthetic rate is negatively affected by this increase in O

2

(Bowes & Salvucci, 1989). The importance of diffusion rates on CO2 availability for Sphagnum has

been shown by Baker and Boatman (1985). They showed the ability of Sphagnum cuspidatum to form

smaller and thinner leafs under low CO2 availability. This will reduce the boundary layer resistance

and facilitate CO2 uptake. Paffen & Roelofs (1991) concluded that a dissolved CO

2 concentration of

at least 750 µmol ∙ L-1 is necessary for the optimal growth of Sphagnum cuspidatum and the subse-

quent formation of floating vegetation. This was shown by the remarkable low average CO2 concen-

trations in the surface water of the poorly developing sites Schurenberg North, Cootjes Veen South

and Groote Veen East; 233, 287 and 122 µmol ∙ L-1, respectively. At those sites Sphagnum growth was

severely hampered.

For the successful re-establishment of aquatic Sphagnum species an additional carbon source

seems to be crucial. This field study shows that the inflow of carbon-rich groundwater can substi-

tute for the peat layer as a source of CO2 during the initial stages of bog development.

Other explanations for restricted Sphagnum growth in peat ponds

Of course, many factors affect the re-establishment of Sphagnum and subsequent bog develop-

ment (Money et al., 2009). In the majority of the studied bogs, Sphagnum growth appears to be

limited by the availability of CO2. However, there are several interesting exceptions. This is illus-

trated by the high CO2 availability in some of the poorly developed bogs (figure 4). Nutrient and

light limitation might be responsible for failing Sphagnum re-colonization (e.g. Money & Wheeler,

1999; Smolders et al., 2003). A shortage in nutrients would probably prevent an increase in produc-


tion at enhanced CO2 concentrations in natural ecosystems (Kramer, 1981). The Diepveen and the

Zandveen are two bogs lacking the re-colonization of Sphagnum under conditions of apparently

sufficient inorganic carbon (figure 4). However, nutrient limitation seems not to be responsible

for this since no clear difference in groundwater or surface water chemistry compared to the other

bogs was found (data not shown). Smolders et al. (2003) concluded that the availability of both

light and CO2 have to be sufficient to enable submerged Sphagnum to reach high photosynthetic

and growth rates. These factors might indeed affect the Sphagnum development in both Diepveen

and Zandveen South in which the water depth is generally several meters in the centre of the bog

and on average >50 cm at the edges, very probably hampering Sphagnum growth due to the reduced

light availability. Additionally, physical constraints like wind and wave action possibly severely

hamper Sphagnum growth in open water bodies (Money et al., 2009). The lack of re-colonization of

Sphagnum mosses and hampered growth of already established Sphagnum mosses has often been

ascribed to high levels of atmospheric nitrogen deposition (Lamers et al., 2000; Money & Wheeler,

1999; Twenhoven, 1992). The ammonium availability in the surface water at all sampling sites (ta-

ble 2) reflects the high nitrogen loads as present in the north of the Netherlands, on average 28 kg ∙

ha-1 ∙ yr-1 (Limpens et al., 2003; RIVM, 2009). However, since no significant differences in nitrogen

availability are found between well and poorly developed bogs (data not shown) the current ni-

trogen deposition is not the determining factor for bog developmental success. Moreover, Tomas-

sen (2004), suggested that bog vitality is much less affected by high nitrogen deposition if other

environmental factors, such as water table and the availability of other nutrients (such as CO2), are

optimal.

Thresholds in the restoration of bogs

The present study demonstrates that Sphagnum bogs in the “Dwingelderveld” are part of the

total landscape hydrology instead of being hydrologically distinct entities. This might be the

case for many damaged Sphagnum bogs and it implies a landscape approach for successful bog

restoration. The current findings clearly show that high CO2 availability is a pre-requisite for the

successful re-establishment of Sphagnum mosses and subsequent bog development. Therefore,

CO2 availability should be included in bog restoration feasibility studies.

Chapter 6

Photosynthesis of three Sphagnum species after acclimatization to high and low carbon dioxide availability

Wouter Patberg

Jan Erik van der Heide

Theo Elzenga


Abstract

A high CO2 availability stimulates the growth of both aquatic and emergent

Sphagnum species. As shown in the previous chapter, the physiological

parameters of CO2 uptake by Sphagnum also show an adaptation to a high

CO2 availability; a high CO

2 compensation point and a low affinity for CO

2.

However, from literature it is known that Sphagnum is able to acclimatize to

different CO2 levels. For example, culturing plants under high CO

2 availabil-

ity, results in lower photosynthetic rates compared to plants that are grown

under CO2-limiting conditions. In this chapter, the physiology of carbon

uptake by Sphagnum (substrate specificity, affinity and plasticity of carbon

assimilation) was determined for three Sphagnum species grown for long

periods at high and low CO2 availability.

The CO2 compensation point and the K

m values of the high and low CO

2

grown S. cuspidatum plants indicate that primary production is limited un-

der air-equilibrated conditions. Remarkably, in S. cuspidatum the low CO2

treated plants were capable of higher photosynthetic rates compared to the

high CO2 treated plants at similar, high CO

2 concentrations. This difference

was not found for S. fallax and S. magellanicum. Possibly, this reflects the

difference in habitat: S. cuspidatum is a submerged aquatic species, while S.

fallax and S. magellanicum are both emergent species.

Considering the high CO2 compensation point, the low affinity for CO

2, the

absence of a carbon concentrating mechanism and the limited morphologi-

cal and physiological plasticity of the plants when exposed to a low external

CO2 concentration, primary production by Sphagnum is expected to be ex-

tremely low when solely supplied with atmospheric CO2. This agrees with

our findings in Chapter 5 that when an organic layer is lacking, i.e. during

the initial stages of bog development, an alternative external CO2 source

seems to be essential for the successful (re-)establishment of Sphagnum.

Photosynthesis of three Sphagnum species – 77

Introduction

Bogs ecosystems are very wet and acidic and are often dominated by mosses of the genus Sphagnum

(Clymo & Hayward, 1982). The decomposition of organic material in bogs is slower than the photo-

synthetic fixation of CO2, resulting in the accumulation of peat (Clymo et al., 1998). Since Sphagnum

bogs function as carbon sink they play an important role in global carbon cycling (Bridgham et al.,

2001a; Gorham, 1991).

In contrast to vascular plants Sphagnum mosses lack a cuticle and stomates to regulate

photosynthesis (Proctor, 2008). Sphagnum mosses are surrounded by an external water film

through which gas exchange for photosynthesis is taking place. Since the diffusion of CO2 is about

104 times lower in water than in air, the diffusional barrier formed by the external water films

reduces the supply of CO2 to the carbon assimilating cells and, consequently, the photosynthetic

rate (Bowes & Salvucci, 1989; Rice & Giles, 1996; Silvola, 1990; Williams & Flanagan, 1996).

The photosynthetic rate of Sphagnum mosses has been shown to be a compromise between

external water content and the availability of CO2 (Schipperges & Rydin, 1998; Silvola, 1990; Titus

et al., 1983). At low water contents, dehydration inhibits photosynthesis whereas at very high

water contents Sphagnum species may suffer from carbon limitation due to very thick boundary

layers (Jauhiainen & Silvola, 1999; Rice & Giles, 1996; Silvola, 1990; Titus et al., 1983; Williams

& Flanagan, 1996). For Sphagna, a morphological difference between aquatic and non-aquatic

species was demonstrated by Rice and Schuepp (1995); aquatic Sphagnum species have, compared

to non-aquatic taxa, longer and thinner leaves and consequently a thinner boundary layer.

The growth of submersed aquatic macrophytes is often limited by CO2 (Raven et al., 1985; Rice

& Schuepp, 1995). To overcome the diffusion barrier many aquatic plant species make use of a

carbon concentrating mechanism (CCM), which enhances the accumulation of carbon (Maberly &

Madsen, 2002). The mechanism most often found is the utilization of bicarbonate (HCO3

-) as a car-

bon source in photosynthesis (Prins & Elzenga, 1989). Most aquatic bryophytes, however, lack such

a CCM and are known to be pure CO2 users (Bain & Proctor, 1980; Raven et al., 1998; Raven et al.,

1985). By performing pH drift experiments, Bain and Proctor (1980) demonstrated that Sphagnum

cuspidatum is a pure CO2 user and exclusively depends on diffusion of CO

2 to the site of carbon

fixation. Due to the diffusional barrier presented by the water layer surrounding the plants high

rates of photosynthesis can only be sustained when the leaves are exposed to high levels of CO2

(Jauhiainen & Silvola, 1999; Raven et al., 1985; Silvola, 1990).

A high CO2 availability has been shown to stimulate the growth of both aquatic and emergent

Sphagnum species (Baker & Boatman, 1990; Jauhiainen & Silvola, 1999; Paffen & Roelofs, 1991; Riis

& SandJensen, 1997; Roelofs, 1983; Smolders et al., 2001; Smolders et al., 2003). Chapter 5 describes

a field study, which demonstrates the importance of a high CO2 availability for the successful re-

establishment of Sphagnum and subsequent bog development. Despite the obvious importance

of a high CO2 availability for Sphagnum, the physiological background of this apparent high CO

2

requirement of Sphagnum has never been established.

The physiological characteristics of aquatic plants lacking a CCM indicate an adaptation to

high CO2 availability: a high CO

2 compensation point concentration and a low affinity for CO

2.

With these characteristics it is likely that Sphagnum will be limited by the diffusion of CO2 under

air equilibrated conditions (Raven et al., 1985). Chapter 5 shows the need for high CO2 availability


based on the physiological background of carbon uptake by Sphagnum cuspidatum and S. recurvum.

However, the Sphagnum plants used in that experiment were grown under ambient CO2 conditions.

For Sphagnum fuscum, a hummock forming species, acclimation to high CO2 levels has been shown.

Culturing plants under high CO2 availability results in low photosynthetic rates compared to plants

that are grown under CO2-limiting conditions (Jauhiainen & Silvola, 1999). In the present study,

the physiological background of carbon uptake by Sphagnum (substrate specificity, affinity and

plasticity of carbon assimilation) was determined for plants grown for a long period at high and

low CO2 availability. Three Sphagnum species (Sphagnum cuspidatum, S. fallax and S. magellanicum)

were grown for four months under high or low CO2 availability. The Sphagnum species used in this

study occupy different ecological niches. Sphagnum fallax and S. magellanicum both are emergent

and grow above the water surface, while S. cuspidatum is growing completely submerged. From an

evolutionary perspective, emergent species might be adapted to water holding capacity and less to

low CO2 levels (Rice & Schuepp, 1995).

The high and low CO2 S. cuspidatum plants were used to measure the photosynthetic response

at different CO2 concentrations. At CO

2 concentrations close to the saturation level, low CO

2

grown Sphagnum cuspidatum plants exhibited a higher photosynthetic rates compared to the

high CO2 grown plants. At this CO

2 level, the photosynthetic rate of S. fallax and S. magellanicum

was measured as well. However, differences in photosynthetic rate between treatments were not

observed. In addition, supplemental to the pH drift experiments performed on S. cuspidatum by

Bain and Proctor (1980) similar pH drift experiments were carried out to test carbon utilization by

S. fallax and S. magellanicum; both species were shown to be pure CO2 users as well.


pH drift experiment

For the pH drift experiment Sphagnum fallax (klinggr.) Klinggr. and S. magellanicum Brid. were collected

in a small bog in the “Dwingelderveld”, a nature reserve in the north of the Netherlands (N52°49.178’,

E6°25.991’). The upper two cm of ten plants were incubated in a closed 250 mL Erlenmeyer flask com-

pletely filled with ten times diluted artificial rainwater (Smolders et al., 2001) supplemented with

1 mM NaHCO3. The flasks were kept at 20˚C by placing them in a water bath. The solution was con-

tinuously and slowly stirred. The flasks were illuminated by a halogen lamp (FL 103, Walz, Effeltrich

Germany) with a light intensity of approximately 350 µmol · m-2 · s-1. The pH of the solution was

measured continuously for at least 6 hours using a combined pH electrode with an Ag⁄AgCl internal

reference electrode (Cole Parmer Instrument Company, Illinois, USA) in combination with a home

made amplifier (input impedance 1011 Ohm) and a Campbell Scientific CR10X data logger. The experi-

ment was terminated when no further pH increase could be observed. At the end of the experiment,

three 1 mL samples were taken from each Erlenmeyer flask for total inorganic carbon (TIC) measure-

ments. TIC concentration was measured using an infrared gas analyzer (CO2 analyzer model no. S151,

Qubit Systems Inc., Kingston, ON, Canada). The ratio between HCO3

- and CO2 in the TIC samples was

calculated based on the pH (Prins & Elzenga, 1989). For each species the pH drift experiment was per-

formed in triplicate. Carbon uptake, CO2 compensation point and half-saturation constant (K

m) for

CO2 uptake by S. fallax and S. magellanicum were calculated according to Maberly and Spence (1983).


CO2 uptake experiment

Sphagnum cores were collected in April 2010 from two small bogs in the “Dwingelderveld”. Three

species of Sphagnum were collected, Sphagnum cuspidatum Ehrh. Ex Hoffm., S. fallax (klinggr.)

Klinggr. and S. magellanicum Brid.. Per species four homogeneous 10 cm thick sections of 18 cm

by 25 cm were cut out of the Sphagnum carpet and gently placed in a glass container (18*25 cm,

height 10 cm) and transported to a greenhouse. During transport the water table in the containers

was kept at approximately 2 cm below the capitula. In the greenhouse the cores were cut to a depth

of 5 cm and placed in plastic nets which, in turn, were mounted in the same glass container. The

sides of the containers were covered with black plastic sheets to keep out the light. The containers

were filled with artificial rainwater (according to Smolders et al. (2001) except for NH4NO

3 of which

100 µmol ∙ L-1 was used). The S. cuspidatum cores were placed in the containers with the capitula at

the water level, S. fallax with their capitula ~1 cm and S. magellanicum ~3 cm above the water table.

Growth was compensated for by lowering the nets with the Sphagnum cores in order to keep the

water level equal with respect to the top of the capitula. During the experiment the containers were

continuously fed with artificial rainwater (~20 mL ∙ hr-1) from 40 liter containers by using black

norprene tubes (l = 400 mm, 4.8 mm outer and 1.6 mm inner diameter; Saint-Gobain Performance

Plastics, Verneret, France) in combination with a peristaltic pump (Masterflex L/S model 7519-25,

Cole Parmer Instrument company). The solution left the container through an overflow located 3

cm below the rim. For each species, two containers were fed by artificial rainwater bubbled with

carbon dioxide (Carbon Dioxide 4.0, Linde AG, Munich, Germany) and two containers with air-

equilibrated artificial rainwater, resulting in a ‘high’ and ‘low’ CO2 treatment, respectively. Vascular

plants were removed from the Sphagnum cores on a regular basis. In the greenhouse, natural light

was supplemented with high pressure sodium lamps to induce a 14 hour photoperiod. The plants

acclimated for two weeks before the treatments started. After four months the photosynthetic rate

of the Sphagnum species at different CO2 concentrations was measured (see below).

During the culture period the CO2 concentration and pH of the pore water in the containers

were measured every two months. Water samples were collected using 10 cm long Teflon Rhizons

(Eijkelkamp, Agrisearch, Giesbeek, the Netherlands) which were placed diagonally in the middle of

the Sphagnum cores. The total inorganic carbon (TIC) concentrations were measured using an Infra

Red Gas Analyzer (IRGA; Li-7000 CO2 / H

2O analyzer, Li-Cor, Inc., USA); The pH was measured using a

Metrohm 780 pH meter (Metrohm, Herisau, Switzerland) together with a combined Metrohm glass

electrode (Metrohm 6.0258.010). The CO2 concentration in the water samples was subsequently

calculated based on the pH and the TIC concentration (Prins & Elzenga, 1989). During the growth

period the pigment content of the mosses was determined three times with regular intervals. Per

container five capitula were randomly collected, pooled and pigments were determined according

to Lichtenthaler (1987).

CO2 uptake characteristics were determined by measuring photosynthetic activity (A) at differ-

ent CO2 concentrations. Photosynthetic activity was determined by the photosynthetic evolution

of oxygen by a capitulum placed in a closed thermostatic cuvette containing 1 mL of measuring

buffer (see below) at 18 ± 0.2˚C and saturating light conditions (1500 µmol ∙ m-2 ∙ s-1; Hansatech

Quantitherm Light meter). The solution in the cuvette was stirred continuously. Per measurement

one capitulum was used and each capitulum was used for only one measurements. Large branch-

es were trimmed to fit in the cuvette. Oxygen was measured by a Clark electrode located at the


bottom of the cuvette in combination with a millivolt recorder (Kipp and Zonen BD40; Delft, the

Netherlands) connected to a Graphtec GL200 midi logger (Graphtec Corp., Yokohama, Japan) on

which data were logged every second. The measuring buffer consisted of 10 times diluted artificial

rainwater (Smolders et al., 2001) and 20 mM MES set at pH = 4.0 using NaOH. During the initial

phase of the experiment, the oxygen concentration in the measuring buffer was in equilibrium

with air (21%) which seemed to reduce photosynthetic rate considerably. Therefore, the oxygen

concentration was reduced by flushing the measuring buffer with N2 which resulted in an average

(±SD) oxygen concentration in the buffer of 9% (±3).

Different CO2 concentrations were obtained by diluting 20 to 600 µL of 0.1 M NaHCO

3 solution

with 35 mL of buffer of which 1.5 mL was injected (Hamilton 2.5 mL syringe) into the cuvette whereof

0.5 mL was instantly retaken for the immediate determination of the CO2 concentration. Total inor-

ganic carbon (TIC) concentrations were measured using an Infra Red Gas Analyzer (IRGA; Li-7000

Co2/H2O analyzer, Li-Cor, Inc., USA). After usage the fresh weight of the capitula was determined

and the plant material was subsequently frozen at -80°C for until determination of the chloro-

phyll concentration according to Lichtenthaler (1987). Photosynthetic activity is expressed as the

amount of oxygen produced (nmol O2 ∙ s-1) on a fresh weight (g FW) and chlorophyll (mg) basis.

Prior to each measurement residual inorganic carbon from the culture medium, present in the

hyaline cells or adhering water was reduced as much as possible by illuminating the capitula with

1000 µmol ∙ m-2 ∙ s-1 for at least 60 minutes while keeping them in a CO2 free medium.

Statistical analysis

Hyperbolic curves were fitted to the carbon dioxide uptake data, using graphing software (Prism

version 4.03, 2005; GraphPad Software, Inc., San Diego, CA, USA).

Per species t-tests were performed to indicate differences between high and low CO2 avail-

ability on photosynthetic performance by using SPSS for Windows (version 16.0.1, 2007; SPSS Inc.,

Chicago, IL, USA).

Results

pH Drift experiment

If only free CO2 is taken up, the final pH value of a pH drift experiment will be between 8 and 10,

whereas in case of HCO3

- uptake the pH can reach a value between 11 and 12 (Bain & Proctor, 1980;

Prins & Elzenga, 1989). So, a distinction between a CO2 and a HCO

3- user can be made, based on this

difference in maximum pH.

In the pH drift experiment maximum pH values of 8.1 ± 0.03 and 8.4 ± 0.18 were measured for S.

fallax and S. magellanicum, respectively, indicating that these Sphagnum species are not able to utilize

HCO3

- as a carbon source, and are therefore defined as strictly CO2 users. Figure 1 shows the photo-

synthetic activity (A) (expressed as relative carbon uptake compared to the maximum carbon up-

take at the beginning of the experiment) of Sphagnum fallax and S. magellanicum as a function of CO2

availability during the pH drift experiment. Hyperbolic curves according to the formula A=Vmax

*[CO2]/

(Km

+[CO2]) + c were fitted to the data (see figure 1). From the curves the CO

2 compensation point con-

centrations (Γ), the affinity for CO2 (K

m(CO2)) and photorespiration (c) were determined, see table 1.


Figure 1. Photosynthetic activity as a function of CO2 concentration of Sphagnum fallax (a) and S. magellanicum (b) during

the pH drift experiment. Photosynthetic activity is expressed as the CO2 uptake relative to the maximum CO

2 uptake at the

beginning of the pH drift experiment (in %).

Table 1. The affinity for CO2 (K

m), CO

2 compensation point (Γ) and photorespiration (c) (±SD)for Sphagnum fallax and S. magel-

lanicum determined from the relation between CO2 availability and photosynthetic activity as shown in figure 1

S. fallax S. magellanicum

Km

(µmol CO2 ∙ L-1) 14.4 ± 5.0 24.5 ± 2.9

Γ (µmol CO2 ∙ L-1) 7.2 ± 2.1 8.5 ± 0.9

c 60% ± 8.3 47% ± 3.9

CO2 uptake experiment

CO2 concentration, pH and chlorophyll content during growth phase

Per treatment there were two containers per species. However, per species and treatment plants

from only one container were used in the photosynthesis measurements. For these containers the

average, minimum and maximum CO2 concentrations and average pH values measured in the pore

water during the growth phase of the experiment are shown in table 2.


Table 2. Average, minimal and maximum carbon dioxide concentrations (n=3; in µmol ∙ L-1) and average pH (n between

brackets) in the pore water of the containers during the growth phase of the experiment. + = high and - = low CO2 treatment

[CO2] (µmol ∙ L-1)

pHmean min. max.

S. cuspidatum+ 8539 4930 13243 3.7 (3)

- 337 227 507 4.6 (2)

S. fallax+ 7829 4033 14126 4.2 (3)

- 184 104 276 4.4 (2)

S. magellanicum+ 5392 4562 6599 4.3 (2)

- 183 85 248 4.3 (2)

The pigment content of the three Sphagnum species after growth for four months at the two

different CO2 treatments are given in figure 2. Per species and treatment the pigment concentration

of five pooled capitula was determined. For all three species the plants grown at the low CO2 regime

contained higher pigment concentrations (chl a, b and carotene) compared to plants grown at high

CO2. This higher pigment content in the low CO

2 treated plants might be indicative of an increased

investment in the photosynthetic apparatus under more severe CO2 limitation.

Figure 2. Pigment content of Sphagnum cuspidatum, S. fallax and S. magellanicum grown at high and low CO2 availability for a

period of four months. The white bars represent chlorophyll a, light grey bars chlorophyll b and the dark grey bars carotene,

all in mg ∙ g FW-1. Per species and pigment type, the left bar represents the high (+) and the right bar the low (-) carbon dioxide

treatment. Each bar represents one sample of five pooled capitula.

Photosynthetic response to CO2

The photosynthetic rate of Sphagnum cuspidatum grown at high and low CO2 availability as a

function of CO2 concentration is shown in figure 3. Hyperbolic curves according to the formula

A=Vmax

*[CO2]/(K

m+[CO

2]) + c were fitted to the data. From the curves the CO

2 compensation point

concentrations (Γ) were calculated. The maximum photosynthetic rate (Vmax

), the affinity for CO2

(Km

) and CO2 compensation point are presented in Table 3. Based on visual inspection and to reduce

the degrees of freedom in the fitting procedure, one Km

value was fitted for both treatments.


Table 3. CO2 uptake characteristics (±SE) for Sphagnum cuspidatum grown for three months at high carbon dioxide availability

(+) and at low carbon dioxide availability (-). See text for explanation of the parameters

+ -

Vmax

(nmol O2 ∙ s-1 ∙ FW-1) 2.71 ± 0.29 2.77 ± 0.27

Km

(µmol CO2 ∙ L-1) 11.60 ± 3.52 11.60 ± 3.52

Γ (µmol CO2 ∙ L-1) 15.26 11.35

c -1.54 ± 0.19 -1.37 ± 0.16

For pure CO2 users the CO

2 compensation point is the lower limit for net C fixation. The CO

2

compensation point concentrations for the high and low CO2 grown S. cuspidatum plants are 15.26

and 11.35 µmol CO2 ∙ L-1, respectively (table 3). Considering that air-equilibrated water contains a CO

2

concentration of 10 - 20 µmol ∙ L-1 between 25 and 10°C, the CO2 compensation point found for S.

cuspidatum (both treatments) indicates that primary production is limited by CO2 diffusion under

air-equilibrated conditions. Under air-equilibrated conditions no net growth can be expected,

since the minimal carbon gain during the light period is expected to be more than offset by the

carbon loss due to respiration during the dark period.

The Km

value for S. cuspidatum grown at high and low CO2 availability is 11.60 µmol ∙ L-1 (table

3), indicative for the fixation of CO2 by Rubisco (lacking a CCM). Consequently, Rubisco is far from

being saturated in air-equilibrated conditions and high CO2 concentrations are needed for optimal

photosynthesis.

More remarkably is the difference in photosynthetic rate between high and low grown S.

cuspidatum at higher CO2 concentrations (figure 3 and 4). Based on fresh weight (figure 4a) the

photosynthetic rate of S. cuspidatum is on average significantly higher in the low CO2 treatment

(1.24 ± 0.23 nmol O2 ∙ s-1 ∙ g FW-1), than in the high CO

2 treatment (0.75 ± 0,18; t(6)=-3.411, p<0.05).

The exact CO2 concentrations at which photosynthetic rate were measured are mentioned in the

accompanying text.

The average photosynthetic rates of S. fallax in the high and low CO2 treatments were 1.47 ± 0.53

and 3.08 ± 1.45, respectively. For S. magellanicum the average photosynthetic rate was 1.66 ± 1.04 for

the high and 2.33 ± 1.29 for the low CO2 treatment. For both S. fallax and S. magellanicum the average

rates of photosynthesis were highest in the low CO2 treatment but no significant differences be-

tween treatments were observed; t(6)=-2.343, p=0.058 and t(9)=-0.934, p=0.375, respectively.


Figure 3. Net photosynthetic rate of Sphagnum cuspidatum grown at high and low CO2 availability, measured at different CO

2

concentrations at 18±0.2˚C. Photosynthetic rate is expressed as the amount of oxygen released based on fresh weight. The data

are fitted to hyperbolic curves of the form A=Vmax

* [CO2] / (K

m + [CO

2]) + c, where V

max is the maximum photosynthetic rate, K

m

the CO2 concentration at which half of the maximum rate of photosynthesis is reached and c is a constant.

Figure 4. Box plots showing the photosynthetic rate (in nmol O2 ∙ s-1 ∙ g FW-1) of S. cuspidatum, S. fallax and S. magellanicum

based on fresh weight (a) and based on chlorophyll content (b). Plants were grown for four months at a high (+) or low (-) carbon

dioxide availability. The average (± SD) carbon dioxide concentrations at which photosynthetic rates were measured were 177 ±

9 µmol ∙ L-1 (+) and 150 ± 10 (-) for S. cuspidatum; 151 ± 4 (+) and 153 ± 9 (-) for S. fallax and 158 ± 6 (+) and 159 ± 11 (-) for S. magellani-

cum. The box plots are composed of minimum, maximum, 25%, 75% quartiles and the median. Significant differences between

treatments are indicated by an asterisk (p<0.05). The amount of used capitula is noted between brackets in figure a.


Based on chlorophyll content (figure 4b) the photosynthetic rates also show a significant difference

between treatments for S. cuspidatum, t(6)=-2.858, p<0.05) with the low CO2 grown plants giving a

higher rate (26.57 ± 4.16 nmol O2 ∙ s-1 ∙ mg Chl-1) than the high CO

2 plants (16.77 ± 5.46). For S. fal-

lax and S. magellanicum the differences in photosynthetic rate between treatments differed not

significantly, t(6)=-0.516, p=0.624 and t(9)=1.467, p=0.177, respectively. For S. fallax the photosyn-

thetic rates based on chlorophyll content were on average lower in the high CO2 treatment than the

average rate measured in the low CO2 treated plants; 56.55 ± 22.37 and 66.54 ± 33.25, respectively.

On the other hand, the average photosynthetic rates of S. magellanicum based on chlorophyll con-

tent were highest in the high CO2 treatment, 92.98 ± 55.19 compared to 49.51 ± 43.30 in the low CO

2

treatment.

Discussion

Kinetic properties of CO2 uptake by Sphagnum

Like most aquatic bryophytes, Sphagnum has been shown to be a pure CO2 user. For S. cuspidatum

this was shown by Bain and Proctor (1980) and the results presented here show it for S. fallax

and S. magellanicum. CO2 uptake by S. cuspidatum, as shown here, is characterized by a high CO

2

compensation point and a Km

value consistent with Rubisco being the primary CO2 fixing enzyme

and with the absence of a carbon concentrating mechanism (table 1 and 2) (Raven et al., 1985). For

submerged aquatic macrophytes a low affinity for CO2 is common and K

m values are usually in the

range of 30 – 70 µM CO2 (Bowes & Salvucci, 1989; Raven et al., 1985). The K

m values for S. cuspidatum,

S. fallax and S. magellanicum found in this study, are lower (11.6, 14.4 and 24.5 µmol ∙ L-1, resp.) and

very likely a consequence of the low oxygen levels used and the vigorous stirring during the photo-

synthesis measurements. Due to the competition of CO2 and O

2 at the site of Rubisco, K

m values and

CO2 compensation points are higher with increasing O

2 concentrations. Therefore, the presented

Km

values are very likely lower compared to Km

values at ambient oxygen levels. This is supported

by the higher calculated Km

values (133.2 and 231.4 µmol ∙ L-1 for S. cuspidatum and S. fallax, resp.)

obtained from the CO2 response curve in Chapter 5, which were determined at ambient O

2 levels.

The difference in Km

values with differing O2 concentration stresses the influence of O

2 availability

on CO2 uptake. This is especially of interest when regarding the high O

2 concentrations in the up-

per Sphagnum layer due to photosynthetic activity (Lloyd et al., 1998). In the stagnant bog water,

the boundary layers will be substantially thicker, compared to the well-stirred conditions in the

presented experiments, reducing the affinity for CO2 even more. The K

m values determined from

the pH drift experiments are likely to be an underestimation, compared to the values in the natural

situation, due to the reservoir of HCO3

-, present at high pH, that can replenish the CO2 that is taken

up and which is not present in the acidic bog situation.

Despite the variability in Km

and compensation point due to O2 availability (and other

environmental conditions as light and temperature (e.g. Maberly & Spence, 1983)), these kinetic

properties are suggestive of optimization for operation at high CO2 concentrations at the active site

of Rubisco, a condition that is not being met under air-equilibrated conditions. The CO2 acquisition

characteristics are typical for an aquatic plant lacking a CCM (Raven et al., 1985).


Plasticity of CO2 assimilation characteristics

Main focus of this chapter, however, is the difference in CO2 uptake characteristics between

Sphagnum plants subjected to different CO2 concentrations. In principle, for a submerged plant that

can only utilize CO2 there are three possible strategies to increase carbon fixation rates at external

concentrations of CO2 that are slightly higher than the compensation concentration: 1. Increase

in the affinity of the primary CO2 fixing enzyme (lower K

m(CO2)); 2. Increase in the photosynthetic

capacity (higher Vmax

) and 3. Decrease in dark respiration (less negative c). In figure 5 the effects of

these three strategies on net photosynthesis at low external CO2 concentration are illustrated.

Figure 5. Visualization of the photosynthetic response as a function of CO2 concentration for the three possible strategies that

can be applied by a submerged Sphagnum plant, in order to increase carbon fixation rates at low external CO2 concentrations.

The strategies are: (i) increasing the affinity of the primary CO2 fixing enzyme (lower K

m); (ii) increasing the photosynthetic

capacity (higher Vmax

) and (iii) decreasing dark respiration (less photorespiration). The strategies curves are plotted relative to

a control curve, using arbitrary units on both axes. At air-equilibrated conditions, water contains a CO2 concentration of 10 - 20

µmol ∙ L-1 between 25 and 10 °C which is indicated by the grey box.

Between treatments, no (significant) difference in CO2 affinity (K

m) and compensation point (Γ) by

S. cuspidatum were observed. Increased affinity and lowering the CO2 compensation point are strat-

egies shown by micro-organisms and submersed angiosperms to gain more carbon in response to

a decreased DIC availability (Bowes, 1996). Despite the fact that Sphagnum lacks a biophysical and

biochemical mechanism to increase the concentration of C at the site of fixation by Rubisco, S.

cuspidatum shows some adaptation to CO2 availability. However, our results indicate that S. cuspida-

tum does increase its photosynthetic capacity under CO2-limiting conditions, possibly to increase

carbon fixation at low external CO2 availability. This is shown by the difference in V

max between S.

cuspidatum grown in the high and the low CO2 treatments; the low CO

2 treated plants were capable

of higher photosynthetic rates compared to the high CO2 plants at similar CO

2 concentrations. This

might be caused by an increase in the chlorophyll concentration under CO2 limiting conditions


compared to high CO2 availability. This was shown for all three Sphagnum species (figure 2). The

higher concentrations of chlorophyll probably allows the mosses to gain more CO2 (Rice, 1995) and

thus lead to an increase in photosynthetic performance. For S. magellanicum this inverse correlation

between chlorophyll content and CO2 availability was already shown by Smolders et al. (2001).

Jauhiainen and Silvola (1999) showed a reduced photosynthetic efficiency for Sphagnum fuscum

when grown under high CO2 availability, compared to plants grown under low CO

2 conditions. Our

findings are in line with the general pattern to down-regulate photosynthetic performance at high

carbon availability (e.g. Maberly & Madsen, 2002). On the other hand, increased dry weight by the

production of non-structural carbohydrates under high CO2 levels (Van Der Heijden et al., 2000)

might cause the lower photosynthetic rate based on weight (figure 4). However, these increases are

small on a dry mass base and therefore not taken into account in this study.

Possible morphological adaptation to increased CO2 uptake

Neither S. fallax, nor S. magellanicum were able to enhance photosynthetic performance in comparison

with the high CO2 grown plants, despite CO

2 limitation (indicated by an increased chlorophyll con-

tent in the low CO2 treatment). An explanation could be found in the morphological differences be-

tween the Sphagnum species. A mechanism to increase photosynthetic performance under low CO2

conditions is to enhance the supply of CO2 by reducing the boundary layer thickness. Aquatic plant

species commonly change morphology in order to reduce boundary layer resistance under CO2-lim-

ited conditions (Bowes & Salvucci, 1989; Rice & Schuepp, 1995). Sphagnum cuspidatum was shown to

be able to form thinner leaves when grown submerged at low CO2 availability (Baker & Boatman, 1985;

Rydin & McDonald, 1985). In the present study, leaf morphology was not determined. However, since

S. cuspidatum was grown submerged, a morphological adaptation of S. cuspidatum to low CO2 levels

very likely contributes to the significant higher photosynthetic rate of plants grown under low CO2

levels compared to the high CO2 plants. When growing emergent, like S. fallax and S. magellanicum in

this experiment, the formation of thinner leaves is not to be expected, since this will not results in a

reduced boundary layer thickness when growing under low CO2 availability. Moreover, for emergent

growing Sphagnum plants, plant morphology must compromise the possible conflicting require-

ments of water holding capacity (and conduction) and free gas exchange for photosynthesis (Proctor,

2008). Since the formation of thinner leaves will result in a decreased water holding capacity, this

morphological adaptation is not to be expected in S. fallax and S. magellanicum.

However, when based on chlorophyll content, the photosynthetic rate of S. fallax and

S. magellanicum grown at low CO 2 availability were on average lower than the rate of the high

CO2 plants, which is opposite to the differences between treatments when based on fresh weight

(figure 3), indicative for an increased photosynthetic performance at low CO2 availability probably

due to the increased investment in the photosynthetic apparatus.

There is a substantial difference between species, and to a lesser extent between treatments, in

the variation of the photosynthetic rate (figure 4); S. fallax and S. magellanicum show a variation of

at least 300% whereas S. cuspidatum exhibits a more narrow range. Sphagnum fallax and S. magel-

lanicum grown under low CO2 levels show a greater variation in photosynthetic rate than the plants

grown under high CO2 (figure 4). Due to their emergent growth S. fallax and S. magellanicum were

not directly in contact with the CO2 concentrations in the water, possibly leading to varying CO

2

availability throughout the containers and consequently less uniform adaptations by the plants.


Ecological consequences of low affinity CO2 uptake

Because of the competition between CO2 and O

2 at the site of Rubisco, photosynthetic rate, and

consequently Km

and the compensation point, is affected by the ratio between CO2 and O

2 (Bowes

& Salvucci, 1989; Maberly & Spence, 1983). For Sphagnum this was shown by Skre & Oechel (1981)

and here it became evident during the set up of this experiment; in an air-equilibrated measuring

buffer containing 21% O2 the photosynthetic rate of Sphagnum cuspidatum was severely inhibited at

a CO2 concentration of 20 µmol ∙ L-1. Reduced O

2 levels resulted in an increased photosynthetic per-

formance. Due to the production of O2 and the uptake of CO

2 as a result of photosynthetic activity in

combination with the low gas diffusion rates in water, the CO2/O

2 ratios are very likely to decrease

rapidly having negative consequences for photosynthetic performance. Methanotrophic bacteria,

globally occurring in symbiosis with Sphagnum mosses (Kip et al., 2010; Larmola et al., 2010), have

been shown to be of great importance by providing Sphagnum with substantial amounts of CO2 by

oxidizing CH4 using oxygen derived from photosynthesis (Raghoebarsing et al., 2005). We believe

that the consumption of oxygen by these methanotrophs, which reduces the O2 concentration in

the close vicinity of the photosynthesizing cells, is at least as important for the successful growth

of aquatic Sphagnum species.

Considering the low affinity for CO2, the absence of a carbon concentrating mechanism and the

limited morphological and physiological reactions of the plants to low external CO2 concentration,

primary production by Sphagnum is expected to be extremely low when solely supplied with

atmospheric CO2. Growth of Sphagnum therefore requires an additional CO

2 source. Due to aerobic

decomposition processes in the peat layer, additional CO2 will be produced in Sphagnum bogs

(Bridgham & Richardson, 1992; Glatzel et al., 2004; Lamers et al., 1999; Smolders et al., 2001; Wad-

dington et al., 2001) and as a consequence pore water CO2 concentrations can reach up to several

millimoles (Smolders et al., 2001; Chapter 5), thereby ameliorating the effects of low diffusion rates

(Maberly & Madsen, 2002; Silvola, 1990). This agrees with our findings in Chapter 5 that when an

organic layer is lacking, i.e. during the initial stages of bog development, an alternative external

CO2 source seems to be essential for the successful (re-)establishment of Sphagnum.

Chapter 7

Summary and synthesis

Summary and synthesis – 93

The importance of buoyancy-driven water flow in Sphagnum dominated bogs

The requirement of nutrient transport

The motivation for this thesis is based on the findings by Baaijens (1982) and Rappoldt et al. (2003).

They report on a phenomenon called buoyancy-driven water flow, which is the occurrence of

convective flow in water-saturated Sphagnum layers when the temperature difference between day

and night is sufficiently large. During the night, the surface of the peat moss layer cools and results

in a relatively denser and colder water layer on top of warm water. When the density difference

become large enough the cold water in the top layer sinks and warm water rises. It was hypothesized

that in this flow of water, solutes will be transported as well. Therefore, buoyancy-driven water flow

was proposed as a newly discovered mechanism for the translocation of nutrients in a Sphagnum

dominated peat bog.

In bogs, the mineralization of organic matter has been shown to be the most important

nutrient source for Sphagnum (Aerts et al., 1999; Aldous, 2002a, b; Bowden, 1987; Bridgham, 2002;

Damman, 1978, 1986; Morris, 1991; Pakarinen, 1978; Rosswall & Granhall, 1980; Urban & Eisenreich,

1988). In contrast, the highest metabolic activity and nutrient uptake takes place in the capitula

(Aldous, 2002a; Johansson & Linder, 1980; Malmer, 1988; Malmer et al., 1994; Robroek et al., 2009;

Rydin & Jeglum, 2006). The spatial distinction between mineralization and capitula requires an

efficient nutrient transport system.

Diffusion, internal transport and capillary transport were the known nutrient transport

mechanisms in Sphagnum bogs. Complementary to these mechanisms, buoyancy-driven water

flow was hypothesized to be a possible external nutrient transport mechanism, redistributing

nutrients from lower Sphagnum layers to the capitula, and vice versa. Evidence for the development

of buoyancy-driven water flow in a water-saturated Sphagnum layer was provided, based on

theoretical and experimental grounds, by Rappoldt et al. (2003). However, direct evidence for the

transport of solutes was lacking and the importance of buoyancy flow in nutrient transport in

Sphagnum bogs remained unclear. This thesis provides direct evidence for the transport of solutes

by buoyancy flow. Moreover, it is demonstrated that buoyancy flow transports nutrients in such

quantities that it, relative to other transport mechanisms, plays an important role in the redistribu-

tion of nutrients in a water-saturated Sphagnum layer.

The transport of solutes by buoyancy flow

The mesocosm experiment in Chapter 2 unequivocally demonstrates the transport of solutes by

buoyancy-driven water flow in a water-saturated Sphagnum matrix. Moreover, the experiment

shows that due to buoyancy flow a reversal of the gradient can take place in a relatively short period

of time. The findings of this mesocosm experiment indicate that buoyancy-driven water flow acts

as an efficient external nutrient transport mechanism in water-saturated Sphagnum habitats and

thereby can contribute to the supply of nutrients to the Sphagnum capitula in the upper bog layer

and the recycling of nutrients.

The uptake capacity of ammonium by the capitula

In a water-saturated Sphagnum layer, a stepwise increase of solutes near the capitula can be


induced due to the reversal of the gradient by buoyancy flow (Chapter 2, figure 1). The importance

of buoyancy flow as a nutrient transport mechanism in supplying the capitula is also determined

by its ability to absorb and take up pulses of high nutrient concentrations.

Sphagnum has been shown to be opportunistic in its N uptake (Twenhoven, 1992; Woodin et

al., 1985). The strong cation exchange capacity of the cell wall of Sphagnum is often regarded as

an efficient mechanism to retain cations when supplied by rain water (Bates, 1992; Buscher et al.,

1990). Ammonium, the dominant form of nitrogen in bog water, has been shown to be retained

very efficiently by Sphagnum (Li & Vitt, 1997; Wiedermann et al., 2009; Williams et al., 1999;

Jauhiainen et al., 1998; Twenhoven, 1992). Additionally, the very short lag phase of the substrate

inducible enzyme nitrate reductase enables Sphagnum assimilating even short pulses of nitrogen

(Woodin et al., 1985). Moreover, Sphagnum is very well able to deal with pulse-wise supply of N by

the accumulation of a surplus of N in N-rich amino acids like arginine and asparagine (Baxter et al.,

1992; Karsisto, 1996; Limpens & Berendse, 2003; Nordin & Gunnarsson, 2000).

The observed uptake kinetics for ammonium by Sphagnum cuspidatum and S. fallax (Chapter

2) very well fit the opportunistic nitrogen uptake characteristics. Ammonium uptake is not satu-

rated by concentrations up to 100 µmol ∙ L-1 (Chapter 2, figure 2). The time over which the uptake

rates can be maintained also determines the ability of Sphagnum to benefit from the high nutrient

availability caused by buoyancy flow. In a separate experiment the time dependence of uptake was

determined for NH4

+ (figure 1). It was shown that, when exposed to NH4

+, for 190 hours, the capitula

of S. cuspidatum and S. fallax take up 17 and 13% of the final value within one hour and 76 and

64% within 24 hours, respectively. Together, the uptake characteristics enable Sphagnum to benefit

from a stepwise increase in ammonium (or cation) availability which is the case after precipitation

and buoyancy flow events.

Figure 1. The increase of 15N in capitula of S. cuspidatum (open symbols) and S. fallax (filled symbols; in µmol ∙ g DW-1) over

time when incubated in 100 µmol 15NH4Cl ∙ L-1. Each symbol represents the average of three capitula, which were incubated

in a square Petri-dish filled with 50 mL of experimental solution (see materials and methods section in Chapter 4) containing

the labeled nitrogen, 20 mM MES (pH=4.0) and 100 times diluted artificial rainwater. Error bars represent standard deviations.

The experiment took place in a climate controlled room at 18±1 °C and a 16L:8D photoperiod and a light intensity of 185 µmol

∙ m-2 ∙ s-1).


In Chapter 2 the role of the cation exchange sites in ammonium uptake was demonstrated as

well. The adsorption of ammonium by the cell wall is most important at lower concentrations.

With increasing concentrations the relative importance of adsorption to total uptake decreases;

the cell wall will saturate and increased uptake will take place by intracellular uptake. Compared

to the cation exchange, the active intracellular uptake of ammonium is a slow process. These

observations support the general assumption of the cell wall functioning as a temporal extension

of nutrient availability for intracellular uptake (Buscher et al., 1990; Clymo, 1963; Hajek & Adamec,

2009; Jauhiainen et al., 1998).

The internal transport of nitrogen

The relative importance of buoyancy flow should be weighed against the contribution of the other

transport mechanisms occurring in a water-saturated Sphagnum layer: diffusion and internal

transport. The mesocosm experiment demonstrated that the transport of solutes by buoyancy flow

can be much faster than is possible by diffusion alone. Moreover, buoyancy flow can transport

more solutes upwards than is possible by diffusion. In Chapter 4 the internal transport rate of

nitrogen in Sphagnum was determined. The experiments were performed using two Sphagnum

species, S. cuspidatum and S. fallax, occupying respectively hollows and pools, both wet habitats

where buoyancy flow is likely to occur.

Until this present study it was generally assumed that nitrogen was translocated by an

internal transport mechanism, but direct evidence for such a mechanism was lacking. In Chapter

4 physiological evidence for the internal acropetal transport of nitrogen in Sphagnum is provided.

The findings in Chapter 4 are indicative for symplastic transport of nitrogen, which is in line with

the findings of Ligrone and Duckett (1998b) and Rydin & Clymo (1989) who demonstrated cellular

specializations in Sphagnum for symplasmic transport. No basipetal transport of nitrogen was

observed. Therefore, it seems to be a mechanism that supplies the capitulum with nitrogen and

thereby contributes to the efficient use of N.

The amount of N transported internally to the capitula is low compared to the amounts

potentially transported upwards by buoyancy flow and, subsequently, taken up by the capitula.

The uptake kinetics of the capitula show a much faster uptake rate for ammonium than can be

supplied by internal transport (Chapter 2, figure 2). When exposed to 25 µmol 15NH4

+ ∙ L-1 the uptake

by the capitula of S. cuspidatum is 5.6 ± 2.1 µmol ∙ g DW-1 ∙ hr-1, whereas the transport of this amount

by internal transport takes at least four days. Therefore, with the regular occurrence of buoyancy

flow, the supply of nitrogen by internal transport will be insignificant, compared to the supply of

nitrogen by buoyancy flow. Thus, in comparison with diffusion and internal transport, buoyancy

flow seems to be a quantitatively important nutrient transport mechanism in a water-saturated

Sphagnum habitats.

Buoyancy flow is restricted to water-saturated Sphagnum habitats and, because of its

dependence on varying physical parameters (i.e. the difference in temperature between day

and night) an irregularly occurring phenomenon. The importance of internal transport might

therefore reside in its continuous character, supplying the capitulum slowly, but steadily, with

N and contrasting with the pulsed supply of N by buoyancy flow and atmospheric deposition.

Moreover, during extracellular transport nutrients may be lost to microorganism or vascular plant

roots. For Sphagnum species that form hummocks that extend above the water surface and do not


benefit from buoyancy flow, internal transport is, next to capillary transport, a possible acropetal

pathway for nutrients. Clymo (1973) estimated the average velocity by capillary flow to be

0.4 mm ∙ min-1. However, this rate, and the concomitant nutrient transport, is dependent on several

external factors, like evaporation, plant density and pore water nutrient concentrations (Clymo &

Hayward, 1982).

Based on the uptake by the stems and internal transport to the capitula of ammonium by S.

cuspidatum, the rate by which nitrogen is transported internally was estimated at 5 mm ∙ day-1,

which is in accordance with a half time value of equilibration between the stem and capitula of 17

days. Compared to external transport mechanism (buoyancy flow and capillary transport), internal

transport is very likely of limited importance for the upward transport of externally supplied N to

the capitula. The main function of internal transport is therefore assumed to be the reallocation of

internally broken down N.

Since the internal transport of nitrogen is a mechanism for efficient nitrogen use, the trans-

port rate is expected to be reduced under high N availability (Bragazza et al., 2004). The transport

rate of solutes by buoyancy-driven water flow is independent from the internal concentration,

thus also supplying the capitula with nitrogen (and other nutrients) when there is a low demand.

The assimilation of N in amino acids by Sphagnum enables the Sphagnum plants to take up the N

and store it for later use. With the regular occurrence of buoyancy flow, and thereby the supply of N

to the capitula, the sink strength for N of the capitula will be reduced and consequently the rate of

internal transport as well. Thus, the relatively low contribution of internal transport in the trans-

port of N in a water-saturated Sphagnum layer will very likely be reduced even more in the presence

of buoyancy flow.

Ecological importance of buoyancy flow

Chapter 3 clearly shows the regular occurrence of buoyancy-driven water flow in a Sphagnum pool

in a field situation. Moreover, the theoretical models of Rappoldt et al. (2003) on the develop-

ment of buoyancy flow can be applied to the field situation. Based on the Ra numbers, calculated

from the vertical hydraulic conductivity of the Sphagnum cores and the difference in temperature

between day and night, the occurrence and starting times of buoyancy flow development was very

well predictable. The results from the GIS study indicate that many peatlands throughout the world

are subjected, several days each month during the growth season, to temperature difference be-

tween day and night, which are suitable for the development of buoyancy flow.

Sphagnum bogs can consist of a patchwork of hollows, lawns and hummocks. As buoyancy

flow is restricted to the water layer in a Sphagnum bog, direct supply of nutrients from deeper layers

to the capitulum by buoyancy flow only takes place in hollows. The transport of nitrogen by buoy-

ancy-driven water flow and the subsequent uptake by the capitula in the upper Sphagnum layer in

a field situation was demonstrated by the field experiment performed in the Rancho Hambre bog

complex, Argentina (Chapter 2; figure 2). The increase in 15N concentration in the S. fimbriatum

capitula in the treatment with unobstructed convective flow, indicates the upward transport of 15N by buoyancy flow. As expected, in the S. magellanicum sites, however, no increase in 15N in the

capitula was observed.

Moreover, in the observed S. magellanicum lawns (Chapter 3) the water table is located about 20

cm below the top of the Sphagnum plants which form an insulating layer (Van der Molen & Wijm-


stra, 1994), preventing the development of a cool water surface layer and instability in the water col-

umn. If buoyancy flow would nevertheless occur, solutes transported from deeper layers to the upper

water layer would still have to be transported to the capitula by capillary transport (or by diffusion in

case of, for example, CO2). In this case buoyancy flow only acts as an auxiliary transport mechanism

and its relative importance in the nutrient supply to the capitula is determined by the height of the

capitula above the water level. Such a situation could be found in Sphagnum lawns and the transition

zones between pools and hummocks. As the initial successional stage of a Sphagnum bog is the colo-

nization of aquatic Sphagnum species of water bodies followed by the invasion of hummock forming

species, buoyancy flow seems to be particularly important in the early stages of bog development.

An overview of the (expected) relative importance of buoyancy flow, diffusion, internal

transport and capillary transport in three different Sphagnum habitats (hollow, lawn and hummock)

are presented in figure 2.

Figure 2. A schematic cross section of three different Sphagnum habitats (hollows, lawns and hummocks) indicating the

relative importance (in %) of buoyancy flow (light grey), diffusion (dotted grey), internal transport (dark grey) and capillary

transport (medium grey) in the redistribution and cycling of nutrients. The three habitats differ in the height of the capitula

(indicated by the asterisks) relative to the water table level (indicated by the thick dashed horizontal lines). In hollows, buoy-

ancy flow is the major mechanism by which nutrients are transported. Since the capitula are at the water level in hollows,

buoyancy flow can directly contribute to the supply of nutrients to the capitula. Diffusion and internal transport take place as

well in hollows but has been shown to be less rapid and effective than buoyancy flow. Even though buoyancy flow might occur

irregularly, the opportunistic nutrient uptake capacity of Sphagnum will result in a significant contribution of buoyancy flow

even at a few occurrences of buoyancy flow. Since buoyancy flow is restricted to the water layer its contribution will be less in

lawns and very likely totally absent in hummocks. In contrast, capillary transport only takes place above the water table and

therefore only contributes to nutrient transport in lawns and hummocks. Since the plant density is higher in hummocks than

in lawns, capillary transport will be faster and consequently more important in hummocks than in lawn. Moreover, in lawns

buoyancy flow might occur, reducing the relative contribution of capillary transport in lawns compared to hummocks. With

increasing heights of the capitula above the water table the relative importance of diffusion will be less and completely reduced

to zero in hummocks. Internal transport has been shown to be a slow mechanism. As a consequence, internal transport very

likely plays a minor role in the distribution of nutrients in especially hollows and hummocks because of the presence of the

faster mechanisms buoyancy flow and capillary transport, respectively. In lawns the relative importance of internal transport

is expected to be greatest since buoyancy flow is less effective and capillary transport will be less fast due to the low plant

density in these habitats.


Nitrogen

The main N source for Sphagnum has been shown to be re-mineralized N (Aerts et al., 1999; Aldous,

2002b; Bridgham, 2002; Gerdol et al., 2006; Morris, 1991; Urban & Eisenreich, 1988). The importance

of re-mineralization of nitrogen for Sphagnum growth has been demonstrated in situ by Urban &

Eisenreich (1988). They calculated the assimilation of nitrogen by plants (primarily Sphagnum) to

be 66 kg ∙ ha-1 ∙ yr-1, whereas only 14.6 kg N ∙ ha-1 ∙ yr-1 was supplied by total inputs. The remainder

was supplied by mineralization of the peat.

Maybe one would expect that vascular plants may be better competitors for this source of

nitrogen by scavenging the peat for mineralized N with their fine roots (Backeus, 1990; Jackson

et al., 1990) and as a consequence vascular plants will outcompete Sphagnum mosses. This is,

however, not the case. Instead, Sphagnum is capable of very efficient (re-)use of mineralized

nitrogen, creating a low nutrient environment which consequently contributes to their domi-

nance over vascular plants.

As already mentioned by Gerdol et al. (2006), these findings are contradictory to the general idea

that Sphagnum and vascular plants utilize spatially distinct nutrient pools, with Sphagnum relying

on N from precipitation and vascular plants on mineralization of senescing organic matter in the

deeper acrotelm (Malmer et al., 1994; Pastor et al., 2002).

The importance of nutrient transport for efficient nutrient recycling is generally accepted.

For example, Aldous (2002b) mentions the translocation of nitrogen as a key process in bog nu-

trient cycling. Blodeau et al. (2006) states that Sphagnum mosses are the dominant species in

northern peatlands, in part because they have the capability to conserve nitrogen by transferring

it from lower, inactive parts of their stem to apices where new biomass is formed (Aldous, 2002a,

b; Malmer, 1988). However, the contribution of the different nutrient transport mechanisms in

nutrient recycling was never determined. In this thesis we demonstrate that buoyancy-driven

water flow is an important mechanism contributing to the recycling of mineralized nutrients in

Sphagnum bogs. Consequently, Sphagnum mosses may outcompete vascular plants more easily

and thereby enhance their ability to engineer the ecosystem (Van Breemen, 1995).

Carbon dioxide

In waterlogged Sphagnum habitats, the stagnant bog water will result in thick boundary layers and

long diffusion path, lengths which reduces the supply of CO2 to the carbon assimilating cells and,

consequently, the photosynthetic rate (Bowes & Salvucci, 1989; Rice & Giles, 1996; Silvola, 1990;

Williams & Flanagan, 1996). Consequently, high rates of underwater photosynthesis can only be

sustained when the leaves are exposed to high levels of CO2

(Jauhiainen & Silvola, 1999; Paffen &

Roelofs, 1991; Silvola, 1990; Smolders et al., 2003). Submerged Sphagnum species that inhabit peat

hollows have been shown to be limited by CO2 (Chapter 5; Rice & Giles, 1996; Rice & Schuepp, 1995).

On the other hand, high carbon dioxide concentrations haven been shown to stimulate Sphagnum

growth (e.g. Smolders et al., 2001; Chapter 5). However, doubling atmospheric CO2 concentrations

appear to have rather limited effects on the growth of Sphagnum (Heijmans et al., 2001; Hoosbeek

et al., 2001; Jauhiainen et al., 1994; Toet et al., 2006).

The high CO2 requirements for Sphagnum is determined by physiological characteristics of the

CO2 uptake mechanism of Sphagnum, as shown in Chapters 5 and 6. Sphagnum mosses have been

shown to be pure CO2 users (Bain & Proctor, 1980; Chapter 6) and therefore exclusively depend on


diffusion of CO2 to the site of carbon fixation. The kinetic properties of CO

2 uptake indicate an ad-

aptation to a high CO2 availability. The investigated Sphagnum species are characterized by a high

CO2 compensation value, the CO

2 concentration at which CO

2 fixation by photosynthesis balances

CO2 loss by respiration. Air-equilibrated water contains a CO

2 concentration of 10 - 20 µmol ∙ L-1

between 25 and 10°C. The high compensation value of Sphagnum implies that under air-saturated

conditions no, or extremely limited, net carbon accumulation can occur. In the acidic bog envi-

ronment, where no reservoir of bicarbonate is present to replace the CO2 that is taken up, this is

especially relevant and Sphagnum growth will not occur when CO2 is provided exclusively through

equilibration with air. Furthermore, The high Km

values of 231.4 and 133.2 µM CO2 for S. cuspidatum

and S. fallax, respectively, further indicate that even when CO2 is present at a concentration that is

higher than air-saturated, carbon utilization is not optimal.

Remarkably, Sphagnum cuspidatum was shown to be able to form thinner leaves when grown

submerged at low CO2 availability (Baker & Boatman, 1985; Rydin & McDonald, 1985), which

will reduce the boundary layer resistance and facilitate CO2 uptake. Moreover, in Chapter 6 it is

demonstrated that S. cuspidatum is able to increase its photosynthetic capacity under CO2-lim-

iting conditions, possibly to increase carbon fixation at low external CO2 availability. Very likely

due to an increased Rubisco concentration under CO2 limiting conditions compared to high CO

2

availability.

Buoyancy flow will result in net transport when a vertical gradient exists, as for example is the

case for nutrients. Uptake and assimilation of CO2 will result in depletion in the zone of the capitula

where most photosynthetic activity takes place. In contrast, in the lower acrotelm CO2 is released

as a consequence of the decomposition of organic material. An increasing CO2 concentration with

depth has been shown in a water-saturated Sphagnum layer (Lloyd et al., 1998). Therefore, buoyancy

flow is very likely an important mechanism also in replenishing CO2 in the upper Sphagnum layer

and enhancing photosynthesis.

Oxygen

Photosynthetic activity in the top layer of the Sphagnum matrix will also result in the production of

oxygen. Due to the thick boundary layers and the low diffusion rates of oxygen in water the oxygen

will accumulate in the upper Sphagnum layer during the day, resulting in a decreasing gradient with

depth (Adema et al., 2006; Lloyd et al., 1998). Lloyd et al. (1998) measured a steep oxygen gradient

in the upper four centimeters of a water-saturated Sphagnum layer, decreasing from 300 to 0 µM.

The mixing of the water layers by buoyancy flow will result in a net downward transport of oxygen.

Adema et al. (2006) attributed a conspicuous change in oxygen concentration at 5 cm depth in a

Sphagnum layer to the occurrence of buoyancy flow.

Because of the competition between CO2 and O

2 at the site of Rubisco, photosynthetic rate is

negatively affected by the accumulation of O2 in the surroundings of the capitula (Chapter 6; Bowes

& Salvucci, 1989; Raven, 2011; Raven et al., 2008; Skre & Oechel, 1981). Together with the upwards

transport of carbon dioxide by buoyancy flow, the CO2:O

2 ratio in the upper Sphagnum layer will

increase and thereby enabling the mosses to photosynthesize. However, during the day the CO2:O

2

ratio decreases again due to photosynthetic activity. Therefore, photosynthetic rates of aquatic

Sphagnum species might be highest in the beginning of the day.

The downward transport of oxygen very likely will increase decomposition rates since


the aerobic decomposition of organic material is significantly higher than the anaerobic

decomposition (Bridgham et al., 1998; Waddington et al., 2001). Consequently, this might result

in a positive feedback mechanism in which the concentrations of CO2 and nutrients like N and

P will be increased by the downward supply of oxygen. These nutrients will become available to

the growing Sphagnum when transported upwards by buoyancy flow which in turn will stimulate

photosynthesis. Consequently, under N-limiting conditions, the transport of oxygen by buoyancy

flow might even determine primary production.

Moreover, the negative effect of oxygen on photosynthesis might give rise to another impor-

tant feature of the symbiosis between Sphagnum and methanotrophic bacteria. The consumption

of oxygen by the methanotrophs, which reduces the O2 concentration in the close vicinity of the

photosynthesizing cells and thereby enhance photosynthesis. This might at least be as important

for the successful growth of aquatic Sphagnum species as the concomitant CO2 release.

Methane

Inseparable from the availability and redistribution of O2 and CO

2 in Sphagnum bogs is the presence

of methane. Under more reductive conditions the methane production in the catotelm may become

higher than the production of CO2. This methane can be oxidized by methanotrophic bacteria to

CO2, which then can be used as a carbon source by Sphagnum mosses (Kip et al., 2010; Raghoebars-

ing et al., 2005). Methane is anaerobically produced in large quantities in bogs (e.g. Gorham, 1991).

Nevertheless, emissions of methane to the atmosphere are relatively low (Larmola et al., 2010) due

to the activity of methanotropic bacteria. The mixing of methane and photosynthetically produced

oxygen by buoyancy flow might have an important role in 1) the CO2 supply to the capitula and 2)

the low methane emission rates, thereby playing a significant role in the global carbon cycling.

Other determinants for the importance of buoyancy flow

Basically, the nutrient concentration in acrotelmic water will be determined by the decomposition

rate in the catotelm and the depletion in the top water layer by uptake and assimilation and

dilution by rain water. As discussed above, buoyancy flow has its effect on the distribution of

multiple nutrients which mutually interact. Not mentioned are phosphorus and potassium, which

have been shown to limit Sphagnum growth (Aerts et al., 1992; Bridgham et al., 1996; Hoosbeek et

al., 2002) and the organic peat layer being an important source for these nutrients (Bates, 1992;

Damman, 1978, 1986).

The effect of buoyancy flow on the nutrient concentration and distribution will be determined

by two other factors. First, the depth of the buoyancy cells: the deeper the cells the more mixing

takes place which very likely results in a higher nutrient availability. Second, the frequency of

buoyancy flow events. Because of its dependence on the difference in temperature between day

and night, buoyancy flow is an irregularly occurring mechanism. The more buoyancy flow events

occur the more the water layer will be mixed, which results in higher decomposition rates, the

amount of transported nutrients and photosynthetic activity (see above).

At low frequencies, gradients are allowed to be build up and relatively large amounts of solutes

might be transported at once. Consequently, the transport and nutrient availability might not be in

synchrony with the requirements of Sphagnum for primary production. For nitrogen this might not

pose a problem, since the pulse-wise availability of N is buffered by the opportunistic uptake and


assimilation characteristics of Sphagnum (Chapter 2). In contrast, photosynthesis will very likely

benefit more from a frequent occurrence of buoyancy flow. Photosynthetic activity will result in

lowered CO2 and increased O

2 levels in the surroundings of the capitula, which in turn will inhibit

photosynthesis. Since this inhibition can occur within the period of one day, overall photosyn-

thetic rates will be enhanced optimally with a diurnal occurrence of buoyancy flow.

The relative importance of transport of nutrients from deeper water layers to the top layer with

the capitula, is higher when atmospheric input is low. This is especially relevant for nitrogen since

atmospheric N deposition has increased significantly during recent decades (Vitousek, 1982). An

increased N deposition results in higher ammonium concentrations in the bog water (Lamers et al.,

2000). Under such conditions the decreasing nitrogen gradient with depth will be reduced or even

be completely diminished, thereby reducing the net upward transport of nitrogen by buoyancy

flow to zero. Under high N availability, Sphagnum growth has been shown to shift from a nitrogen

to a phosphorus limitation (Aerts et al., 1992). Consequently, the importance of buoyancy-driven

water flow as a nutrient transport mechanism will also shift from nitrogen to phosphorus. More-

over, the availability of CO2 might become important under high N loads as well. However, since ar-

eas with a high nitrogen deposition load only slightly overlap with the area covered with peatlands

(Chapter 3, figure 4), the importance of buoyancy flow in the transport and recycling of nitrogen in

Sphagnum bogs, is hardly diminished by increased, anthropogenic N deposition.

Restoration and conservation of Sphagnum bogs

Throughout the world, Sphagnum bogs have become greatly endangered and consequently much

effort is dedicated to the restoration of damaged bogs and the conservation of bog remnants.

Therefore, studying the functioning of Sphagnum bogs is inseparable to the restoration and

conservation of these ecosystems. Chapter 5 focuses on the restoration of Sphagnum bogs. As

mentioned in the previous paragraph, several studies have reported on the stimulation of Sphag-

num growth by high CO2 concentrations (Baker & Boatman, 1990; Jauhiainen & Silvola, 1999; Paffen

& Roelofs, 1991; Riis & SandJensen, 1997; Roelofs, 1983; Smolders et al., 2001; Smolders et al., 2003).

Chapter 5 and 6 show the physiological background of the high CO2 needs of Sphagnum. In addition

to these studies, Chapter 5 reports on a field study in which the significance of CO2 availability for

the successful re-establishment of Sphagnum and subsequent bog development is demonstrated.

Study area was the “Dwingelderveld”, a nature reserve in the north of the Netherlands

characterized by several small damaged Sphagnum bogs throughout the area. After rewetting

measures were taken, the developmental success between these bogs varied significantly; some

bogs developed well, whereas others did not. It was shown that the poorly developed bogs were

limited in CO2, whereas the successful re-establishment of Sphagnum in the well developed bogs

was correlated with high CO2 availability.

Groundwater essential for bog development; a paradox

The findings in Chapter 5 clearly show that high CO2 availability is a pre-requisite for the successful

re-establishment of Sphagnum mosses and subsequent bog development. In well developed

bogs CO2 is sufficiently available due to the decomposition of organic material in the peat layer


(Bridgham & Richardson, 1992; Glatzel et al., 2004; Smolders et al., 2001; Waddington et al., 2001).

However, damaged bogs subjected to peat cuttings or drainage often lack such a carbon source

and an additional CO2 source is needed for Sphagnum growth. Therefore, one of the conclusions of

Chapter 5 is that CO2 availability should be included in bog restoration feasibility studies.

Remarkably, water chemistry analysis revealed that the well developed bogs in the “Dwingelderveld”

(Chapter 5) received carbon rich groundwater from outside the bogs, increasing CO2 concentrations

in the bog stimulating Sphagnum growth and thereby bog development. Interestingly, this

dependence of Sphagnum growth on groundwater input hides a paradox; bogs exists due to being

isolated from the groundwater. In such an ombrotrophic, low nutrient environment Sphagnum

mosses have a competitive advantage over vascular plants. Moreover, groundwater is often

characterized by a high pH and a high Ca2+ concentration and has been shown to be toxic for most

Sphagnum species (Skene, 1915; Clymo & Hayward, 1982).

The need for carbon under high N loads

A large number of studies have focussed on the negative effects of an increased atmospheric

nitrogen deposition on the growth of Sphagnum and the functioning of Sphagnum bogs (Limpens

et al., 2011). The lack of re-colonization of Sphagnum mosses and hampered growth of already

established Sphagnum mosses has often been ascribed to high levels of atmospheric nitrogen

deposition (Lamers et al., 2000; Li & Vitt, 1994; Money & Wheeler, 1999; Twenhoven, 1992). Chapter

5 demonstrates that the successful restoration of Sphagnum bogs is possible under high nitrogen

loads. It is hypothesized that CO2 can compensate the negative effects of a high nitrogen deposition

on an ecological as a physiological level.

Sphagnum mosses lack a regulatory mechanism for nitrogen uptake. As a consequence,

internal nitrogen concentration increases with increasing nitrogen deposition rates (Bragazza et

al., 2005; Lamers et al., 2000; Limpens et al., 2011). However, under high levels of N deposition

levels, Sphagnum is not able to filter out all the N from precipitation and nitrogen leaches to the

rhizosphere were it becomes available for vascular plants, thereby making the bog vulnerable

to invasions by competitive vascular plants that require a high N supply. The reduced growth of

Sphagnum is often attributed to the shading by vascular plants (Berendse et al., 2001; Heijmans et

al., 2001; Lamers et al., 2000; Limpens et al., 2011).

After uptake nitrate is reduced to NH4

+ prior to assimilation, while ammonium is directly

assimilated into glutamine (Rudolph et al., 1993). Subsequently, glutamine is converted into other

amino acids (Kahl et al., 1997; Rudolph et al., 1993). With increasing N deposition, plants are no

longer N-limited, but will still take up N (Lamers et al., 2000). Continued assimilation of N leads to

accumulation of free amino acids (Baxter et al., 1992; Karsisto, 1996; Nordin & Gunnarsson, 2000).

Under these conditions a decrease in Sphagnum growth was observed (Baxter et al., 1992; Nordin

& Gunnarsson, 2000), possibly the result of the accumulation of amino acids requiring carbon

and energy (Baxter et al., 1992; Nordin & Gunnarsson, 2000). Hence, under high N availability

an additional need of carbon is very likely. Paffen & Roelofs (1991) showed that high ammonium

concentrations in the water layer had no major effects on the growth of submerged growing S.

cuspidatum when simultaneously a high concentration of 1000 µmol CO2 ∙ L-1 was applied.

High CO2 levels might enable Sphagnum to increase their competitive strength over vascular

plants by enabling continued growth and N assimilation, keeping the nitrogen concentrations low


in the pore water and thereby gain competitive strength over vascular plants.

The importance of carbon under high N availability is supported by the following findings.

In Sphagnum, under normal conditions, NH4

+ is stored in amino acids having relatively high C:N

ratios like glutamine (5:2) (Kahl et al., 1997; Nordin & Gunnarsson, 2000; Rudolph et al., 1993).

However, under increased nitrogen loads, N is accumulated in amino acids with lower C:N ratios,

mostly arginine (Nordin & Gunnarsson, 2000), which has a C:N ratio of 3:2, the lowest all amino

acids. This shift in amino acid accumulation suggests an economical use of carbon under high N

loads. Thus, the high carbon dioxide needs of Sphagnum are very likely enhanced under high N

availability.

Moreover, the interaction between CO2 availability and high levels of atmospheric N deposi-

tion might have an effect on nutrient uptake as well. The accumulation of N in N-rich rich amino

acids has been shown to be at the expense of C rich amino acid like phenylalanine (Smolders et al.,

2001), which is a precursor of cell wall compounds like polymeric uronic acids and phenolic com-

pounds. A study performed by Richter and Dainty (1989) on the cell wall ion exchange capacity of

Sphagnum russowii suggests that polymeric uronic acids account for over half the cation exchange

capacity (CEC) and phenolic compounds for about 25%. With its high CEC, the cell wall also plays

an important role in nutrient uptake (see Chapter 2). As mentioned above, high N loads reduces the

amounts of amino acid important in cell wall assimilation and composition. This might result in

changes in CEC and consequently in nutrient uptake processes. Additionally, high concentrations

of NH4

+ in bog water (as a consequence of high N loads) directly affect cation composition at the

cell wall and possibly thereby the nutrient balance in Sphagnum.

Concluding remarks

Sphagnum mosses are known for their ability to engineer their environment (Van Breemen, 1995).

One of these engineering abilities is to maintain low nutrient concentrations in the pore water,

preventing increased vascular plant cover and keeping the competitive advantage. Depending

on Sphagnum habitat, different transport mechanism play a role in this efficient scavenging for

nutrients. In this thesis we demonstrated that buoyancy-driven water flow plays an important role

in the distribution of nutrients in Sphagnum bogs. Buoyancy flow might also have an important

influence on primary production and decomposition rates by preventing the building up of

gradients that have a negative feedback on these processes (N, CO2 and O

2). In this context, the

disruption of the nutrient balance in these ecosystems by high N atmospheric deposition loads are

easily understood.

Buoyancy flow, a worldwide occurring, but poorly studied phenomenon in Sphagnum bogs

will have to be taken account when we want to understand bog functioning.

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Samenvatting

Samenvatting – 121

De aanleiding van dit proefschrift was een fenomeen dat door Baaijens (1982) en Rappoldt et

al. (2003) is beschreven. Dit fenomeen, ‘buoyancy-driven water flow’ genaamd, is een convectie-

stroming in de bovenste laag van een met water verzadigde veenmoslaag, die optreedt als gevolg

van het temperatuurverschil tussen dag en nacht. ’s Nachts koelt het oppervlak van de veenmos-

laag af, wat resulteert in een relatief koude, zware laag bovenop warmer, lichter water. Als het

dichtheidsverschil tussen deze twee lagen groot genoeg is dan zinkt de bovenste, zwaardere laag

en komt de warme, ondergelegen laag naar boven.

Hoogvenen zijn voor de aanvoer van nutriënten voornamelijk aangewezen op atmosferische

depositie. Het blijkt echter dat onder niet vervuilde omstandigheden de jaarlijkse aanvoer van

nutriënten middels depositie niet voldoende is voor de waargenomen primaire productie in deze

systemen. Er moeten derhalve andere nutriëntenbronnen bij betrokken zijn. Mineralisatie van or-

ganisch materiaal is de belangrijkste bron van nutriënten voor veenmossen in deze ecosystemen.

De grootste metabolische activiteit en de grootste opname van nutriënten vindt echter plaats in

het capitulum, het bovenste deel van een individuele veenmosplant. De ruimtelijke scheiding tus-

sen mineralisatie en de capitula vereist een efficiënt systeem voor het transport van nutriënten.

Diffusie, intern transport en capillair transport waren de bekende wegen waarlangs nutriën-

ten worden getransporteerd in een hoogveen. Aanvullend op deze mechanismen werd buoyancy

flow beschreven als mogelijk mechanisme waarlangs nutriënten van lagere veenlagen naar boven

en vice versa, kunnen worden getransporteerd. Bewijs voor het optreden van buoyancy flow werd

geleverd door Rappoldt et al. (2003) op theoretische en experimentele gronden. Echter, direct

bewijs voor het transport van opgeloste stoffen ontbrak en het belang van buoyancy flow in het

transport van nutriënten in een hoogveen bleef onduidelijk. Dit proefschrift levert direct bewijs

voor het transport van opgeloste stoffen middels buoyancy flow. Bovendien laat dit proefschrift

zien dat buoyancy flow nutriënten transporteert in zulke hoeveelheden dat het, ten opzichte van

andere transportmechanismen, een belangrijke rol speelt in de nutriëntenvoorziening van een

veenmoslaag wanneer deze met water is verzadigd.

Transport van nutriënten middels buoyancy flow

Het mesocosm experiment in hoofdstuk 2 laat zien dat opgeloste stoffen snel én in grote

hoeveelheden getransporteerd kunnen worden door buoyancy flow in een Sphagnum-matrix.

Hieruit kan worden geconcludeerd dat buoyancy flow een efficiënt extern transportmechanisme is

in een waterverzadigde Sphagnum-laag en kan bijdragen aan de voorziening van nutriënten in de

bovenste laag van een hoogveen en het hergebruik van nutriënten.

De opnamecapaciteit van ammonium door veenmossen

Daarnaast laat het mesocosm experiment zien dat als gevolg van buoyancy flow er een sterke

plotselinge, of stapsgewijze, toename van nutriënten in de bovenste Sphagnum-laag kan op-

treden. Door buoyancy flow getransporteerde nutrienten zijn alleen van ecologisch belang als

Sphagnum planten het vermogen hebben om die in korte tijd toegenomen hoeveelheid nutriënten

daad werkelijk te benutten. In hoofdstuk 2 is de opnamekinetiek van Sphagnum voor ammonium

bepaald. De resultaten laten zien dat Sphagnum heel goed kan profiteren van de stapsgewijze be-

schikbaarheid van ammonium. In hoofdstuk 2 is ook de rol van de celwand als ionenwisselaar

in de opname van nutriënten gedemonstreerd. Adsorptie van ammonium aan de celwand vindt


vooral plaats bij lagere concentraties. Met toenemende concentraties neemt het belang van

adsorptie in verhouding tot de totale opname af; de celwand raakt dan verzadigd en een toename

in de opname zal plaatsvinden via interne opname. Vergeleken met adsorptie aan de celwand is de

actieve interne opname een langzaam proces. Deze waarnemingen steunen het algemene idee dat

de celwand functioneert als een tijdelijke bufferopslag voor nutriënten, voordat ze intern worden

opgenomen.

Het interne transport van stikstof

Het relatieve belang van buoyancy flow moet afgewogen worden tegen de bijdrage van de andere

transportmechanismen, diffusie en intern transport, die op kunnen treden in een waterver-

zadigde veenmoslaag. Uit het mesocosm experiment in hoofdstuk 2 blijkt dat buoyancy flow een

sneller transportproces is dan diffusie. Daarnaast is gebleken dat er meer voedingsstoffen middels

buoyancy flow getransporteerd kunnen worden dan via diffusie. In hoofdstuk 4 is de interne

transportsnelheid van stikstof in Sphagnum bepaald. Tot deze studie werd algemeen aangenomen

dat stikstof intern door Sphagnum wordt getransporteerd, maar direct bewijs daarvoor ontbrak.

Hoofdstuk 4 levert fysiologisch bewijs voor het opwaartse transport van stikstof in Sphagnum.

Echter, ten opzichte van de hoeveelheid stikstof die mogelijk door buoyancy flow wordt getrans-

porteerd (en vervolgens wordt opgenomen), is de hoeveelheid die intern naar de capitula wordt

getransporteerd klein). Onder omstandigheden waarin buoyancy flow regelmatig optreedt, is de

bijdrage van intern transport aan de voorziening van stikstof bijna verwaarloosbaar klein. Dus

in vergelijking met diffusie en intern transport is buoyancy flow kwantitatief het belangrijkste

mechanisme voor het transport van nutriënten.

Het ecologische belang van buoyancy flow

Hoofdstuk 3 laat zien dat buoyancy flow onder veldomstandigheden ook voorkomt. Middels een

GIS-studie is gebleken dat in vele hoogvenen over de gehele wereld, tijdens het groeiseizoen,

meerdere dagen per maand, temperatuurverschillen tussen dag en nacht optreden die geschikt

zijn voor de ontwikkeling van buoyancy flow.

De belangrijkste bron voor stikstof voor Sphagnum is de mineralisatie in de diepere lagen in

een hoogveen. Het is gebleken dat Sphagnum deze stikstof efficiënt hergebruikt. Het transport

van stikstof naar de capitula is hierbij van groot belang. In hoofdstuk 2 wordt het transport van

stikstof middels buoyancy flow en de daaropvolgende opname door de capitula aangetoond in een

veldsituatie.

Ook voor de beschikbaarheid van koolstofdioxide in een Sphagnum-veen kan buoyancy

flow van groot belang zijn. In waterverzadigde Sphagnum-habitats kan de stilstaande waterlaag

resulteren in dikke grenslagen en grote diffusieweerstanden. Hierdoor wordt de toevoer van CO2

naar de planten vertraagd, resulterend in lagere fotosynthesesnelheden en vervolgens in een ver-

minderde groei. Hoge fotosynthetische activiteit onder water kan alleen worden bereikt onder

hoge CO2- beschikbaarheid. Het is gebleken dat ondergedoken Sphagnum-soorten in hun groei

worden gelimiteerd door de CO2-beschikbaarheid. De behoefte aan hoge CO

2 beschikbaarheid

wordt bepaald door fysiologische karakteristieken van Sphagnum (Hoofdstuk 5 en 6). Veenmos-

sen kunnen alleen CO2 gebruiken als koolstofbron en zijn volledig afhankelijk van diffusie voor

hun koolstofvoorziening. De opnamekinetiek voor CO2 laat een aanpassing zien aan een hoge

Samenvatting – 123

CO2 beschikbaarheid (Hoofdstuk 5 en 6). In hoofdstuk 6 wordt bovendien gedemonstreerd dat

Sphagnum cuspidatum in staat is de fotosynthetische activiteit te verhogen onder CO2 limiterende

omstandigheden, waarschijnlijk door de koolstoffixatiecapaciteit te verhogen.

Opname en assimilatie van CO2 in de laag waarin de capitula zich bevinden resulteert in

verlaging van de CO2 concentratie. In de diepere lagen daarentegen, komt veel CO

2 vrij bij de afbraak

van organisch materiaal. Door buoyancy flow wordt CO2 in de bovenste lagen aangevuld, wat tot

een hogere fotosyntheseactiviteit kan leiden. Ook voor het transport van zuurstof kan buoyancy

flow van groot belang zijn. Bij fotosynthese wordt zuurstof geproduceerd. Aangezien fotosynthese

voornamelijk plaatsvindt in de capitula in de bovenste Sphagnum laag, zal zuurstof zich daar opho-

pen. Vanwege de competitie tussen CO2 en O

2 voor binding aan RuBisCo, zal de fotosyntese worden

geremd. Door het optreden van buoyancy flow zal de zuurstofconcentratie in de bovenste laag afne-

men. In combinatie met het opwaartse transport van CO2 zal de fotosynthese gestimuleerd worden.

Bovendien zal waarschijnlijk de afbraak van organisch materiaal in diepere lagen gestimuleerd

worden door het neerwaartse transport van zuurstof. Dit kan resulteren in een positieve terugkop-

peling waarbij door het neerwaartse transport van zuurstof de concentraties CO2, N en P onderin

toenemen, die vervolgens opwaarts worden getransporteerd middels buoyancy flow en beschikbaar

komen voor de veenmossen, leidend tot hogere fotosynthese, meer zuurstof, etc. Daarnaast zal

door de beschikbaarheid van zuurstof in diepere lagen de methaanemissie reduceren als gevolg

van oxidatie tot CO2. Op deze manier draagt buoyancy flow bij aan de Global Carbon Cycle.

Factoren die de effecten van buoyancy flow bepalen

In principe wordt de concentratie van nutriënten in het veenwater bepaald door afbraaksnelheid

van organisch materiaal en de benutting ervan in de bovenste laag door veenmossen en de verdun-

ning door regenwater. Zoals hierboven genoemd heeft buoyancy flow invloed op de verdeling van

alle opgeloste stoffen die onderling op elkaar inwerken.

De invloed van buoyancy flow op de nutriëntenconcentratie en -verdeling wordt mede bepaald

door twee andere factoren. Ten eerste de diepte van de buoyancy cells: hoe dieper de buoyancy cells des

te meer menging zal plaatsvinden, wat waarschijnlijk resulteert in een hogere beschikbaarheid van

nutriënten. Ten tweede is de frequentie van optreden van belang. Hoe vaker buoyancy flow optreedt,

des te meer zal de waterlaag gemengd zijn, resulterend in hogere decompositiesnelheden, hogere

hoeveelheden nutriënten die getransporteerd worden en hogere fotosynthetische activiteit.

Het relatieve belang van het transport van nutriënten uit diepere lagen naar de bovenste laag

waar de capitula zich bevinden, is groter naarmate de inbreng vanuit de atmosfeer laag is. Dit

geldt met name voor stikstof, waarvan de afgelopen decennia de atmosferische depositie sterk is

toegenomen. Een hoge stikstofdepositie resulteert in hoge ammoniumconcentraties in het veen-

water en vlakt de gradiënt in de stikstofconcentratie met de diepte af. Zonder concentratiegradiënt

is het netto transport van stikstof middels buoyancy flow nul. Echter, aangezien de gebieden met

een hoge stikstofdepositie slechts voor een klein deel overlappen met de gebieden waar buoyancy

flow op kan treden (Hoofdstuk 3, figuur 4), wordt het belang van buoyancy flow voor het transport

van nutriënten nauwelijks beïnvloedt door een toename in stikstofdepositie.

Herstel en behoud van hoogvenen

Over de hele wereld worden hoogvenen sterk bedreigd. Als gevolg daarvan wordt er veel moeite


gedaan om deze ecosystemen te restaureren en overblijfselen ervan te behouden. Hoofdstuk

5 handelt over het herstel van hoogvenen. Hierin wordt een veldstudie beschreven waarin het

belang van de beschikbaarheid van CO2 voor de groei van veenmossen en de daaropvolgende

ontwikkeling van hoogveen wordt gedemonstreerd. Studiegebied was het Dwingelderveld, een

groot natuur gebied in het noorden van Nederland. Dit gebied wordt gekenmerkt door meerdere

kleine hoogveentjes die, wat ontwikkeling betreft sterk van elkaar verschillen; sommigen zijn goed

ontwikkeld, andere niet. Het bleek dat de slecht ontwikkelde veentjes gelimiteerd werden door CO2.

Dit in tegenstelling tot de goed ontwikkelde veentjes, waarin de succesvolle groei van veenmossen

was gecorreleerd aan een hoge CO2 beschikbaarheid. De bevindingen in laten daarom zien dat een

hoge CO2 beschikbaarheid een vereiste is voor een succesvolle vestiging van veenmossen en het

daaropvolgende herstel van een hoogveen. In goed ontwikkelde, onaangetaste hoogvenen is altijd

voldoende CO2 aanwezig door de afbraak van organisch materiaal in een dik veenpakket. In aan getaste

hoogvenen daarentegen, ontbreekt deze organische laag regelmatig als gevolg van vervening en/

of drainage. Een aanvullende CO2-bron is dan nodig voor de succesvolle groei van Sphagnum. Eén

van de conclusies van hoofdstuk 5 is, dat de CO2-beschikbaarheid meegenomen moet worden in

studies naar de haalbaarheid van herstel van hoogvenen.

Opmerkelijk was dat de goed ontwikkelde veentjes in het Dwingelderveld koolstofrijk

grondwater ontvingen van buiten de veentjes, waardoor de CO2 concentratie in de veentjes stijgt en

de groei van de veenmossen en daarmee de ontwikkeling van het hoogveen, wordt gestimuleerd.

In de afhankelijkheid van de groei van de veenmossen van grondwater schuilt een interessante

paradox: veenmossen hebben een competitief voordeel ten opzichte van hogere planten in een

ombrotroof, nutriëntenarm milieu. Grondwater wordt vaak gekarakteriseerd door een hoge pH,

hoge calciumconcentraties en hoge nutriëntenconcentratie. Deze omstandigheden zijn zeer

ongunstig voor veenmossen waardoor ze doorgaans hun bestaansrecht ontlenen aan het feit dat

ze afgesloten zijn van grondwaterinvloeden.

Dankwoord

Dankwoord – 129

De afgelopen vijf jaar heb ik met veel plezier aan dit proefschrift gewerkt. Ik heb mogen werken

aan zeer interessante en intrigerende planten en ecosystemen. De combinatie van veldwerk en

laboratoriumexperimenten en het combineren van de disciplines ecologie en fysiologie maak-

ten het een afwisselend en fascinerend onderzoek. Ik heb ontzettend veel geleerd en ik heb hele

mooie en bijzondere plekken van de wereld gezien. Uiteindelijk heeft het allemaal geleid tot dit

proefschrift. Zeker niet in de laatste plaats hebben de mensen met wie ik de afgelopen jaren heb

samengewerkt daaraan bijgedragen. Sterker nog, zonder al die mensen was dit proefschrift nooit

tot stand gekomen. Iedereen ontzettend bedankt! Een aantal wil ik in het bijzonder noemen.

Allereerst mijn promotoren Theo Elzenga en Ab Grootjans. Jullie hebben mij de mogelijkheid

geboden om promotieonderzoek te gaan doen. Dank jullie wel voor het vertrouwen dat jullie in

mij gesteld hebben. Theo, dank je voor je begeleiding de afgelopen jaren. Op wetenschappelijk

gebied heb ik veel van je geleerd. Ik heb ontzettend veel prijs gesteld op de vrijheid die je mij hebt

gegeven waardoor ik mijn eigen ideeën kon ontwikkelen en uitvoeren. Bij naderende onzekerheid

wist je me altijd wel weer te overtuigen, op te peppen en te motiveren om vervolgens weer met

frisse moed aan de slag te gaan en het overzicht te bewaren. Maar bovenal wil ik je bedanken voor

de prettige samenwerking in de afgelopen jaren. Bovendien ben je de eerste die ik ken die “Fillmore

East - June 1971” van The Mothers ook een geweldige plaat vindt. Ab, ik heb grote bewondering voor

jouw positieve en kritische houding ten opzichte van de wetenschap. Onze samenwerking heeft

mijn kennis vergroot en mijn blik op de wetenschap verruimd. Dank daarvoor.

Ook mijn co-promotor Fons Smolders heeft een belangrijke bijdrage geleverd aan dit proef-

schrift. Ondanks je rol van begeleider op afstand was je altijd zeer betrokken en bereid om mee te

denken over alle facetten van het onderzoek. Ook heb je me altijd voorzien van nuttig commentaar

op mijn manuscripten.

Gert Jan Baaijens, brein achter het fenomeen buoyancy flow, bedankt voor je enthousiasme,

inspirerende ideeën, mooie verhalen en gastvrijheid.

Christian Fritz, mede dankzij jou heb ik een onvergetelijke tijd gehad in Ushuaia en omstreken.

Jacob Hogendorf, ik dank je voor je bewaarzucht en je goede zorgen voor mijn mossen. Ze

staan aan de basis van al mijn experimenten!

Robert, met de afronding van dit proefschrift komt er ook een einde aan het koffiedrinken op

de dinsdagochtend. Ondanks dat deze traditie nog niet zo lang bestaat, ga ik het zeker missen.

De afgelopen jaren heb ik mij op het laboratorium van Plantenfysiologie bijzonder thuis gevoeld.

Zonder iedereen bij naam te noemen wil ik alle labgenoten daarvoor bedanken. Een aantal in het

bijzonder. Marten, bedankt voor al je hulp en expertise tijdens het ontwikkelen en uitvoeren van

mijn experimenten, de oneliners, de overheerlijke koffie, de FC Groningen-kranten en je luis-

terend oor, maar bovenal voor je goeie gezelschap. Jan Henk, ook bij jou heb ik me altijd in goed

gezelschap bevonden. Jouw kritische kijk op de wetenschap (en de verrichtingen van de FC) en je

enthousiasme voor de biologie heb ik altijd zeer gewaardeerd en hebben ongetwijfeld hun weer-

slag gevonden in dit proefschrift.

Ook wil ik mijn mede-aio’s Fatma en Ika hier speciaal noemen. Het was mij een waar genoegen

om samen met jullie in hetzelfde schuitje te zitten. Fatma, ik heb grote bewondering voor je gedre-

venheid en vastberadenheid. En dat in combinatie met een immer goed humeur! Ika, jij was mijn

klankbord, steun en toeverlaat op het lab. En daarbuiten, gezellig biertjes drinken en concerten


bezoeken met Germaine en Chris. Ik ga er van uit dat we elkaar in de toekomst nog regelmatig gaan

zien!

Tijdens mijn onderzoek heb ik het voorrecht gehad om een aantal studenten te mogen begeleiden

tijdens hun bachelor of masteronderzoek. Arne, Arrie, Bikila, Charlotte, Jan Erik en Myra; jullie

hebben fantastisch werk geleverd en dat heeft dan ook geleid tot een grote bijdrage aan dit proef-

schrift. Ik ben jullie daar zeer dankbaar voor. Daarnaast hoop ik dat ik mijn enthousiasme voor het

onderzoek een beetje aan jullie heb kunnen overdragen. Wat ik wel weet, is dat ik veel plezier heb

gehad aan de samenwerking met jullie.

Steven, jouw rol in dit proefschrift is groter dan je denkt. Ik vind het geweldig dat je straks als

paranimf aan mijn zijde staat.

Mijn zus, Mirjam, bedankt voor je hulp bij de layout van dit proefschrift. Zonder jou was het

me niet gelukt!

Mijn ouders, Harry en Janny, jullie hebben altijd met mij meegeleefd en onvoorwaardelijk

gesteund op alle fronten en daar ben ik jullie ontzettend dankbaar voor.

Lieve Silke en lieve Veerle, geweldig dat jullie er zijn! Jullie hebben mij de afgelopen jaren met

beide benen op de grond gehouden; het schrijven van een proefschrift is slechts bijzaak.

Lieve Germaine, de afgelopen jaren hebben voor een groot deel in het teken gestaan van dit

proefschrift. Nu is het af. Tijd voor iets nieuws! Dank je voor je nimmer aflatende vertrouwen en je

liefdevolle steun.

Wouter

Wou

ter Patberg Solute tran

sport in

Sphagnum

dom

inated

bogs T

he ecop

hysiological eff

ects of mixin

g by convective fl

ow

Wouter Patberg



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Sphagnum Solute transport in Sphagnum

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