U N I V E R S I T Y O F C O P E N H A G E N

F A C U L T Y O F S C I E N C E

F R E S H W A T E R B I O L O G I C A L L A B O R A T O R Y

Ecology in small aquatic ecosystems

Mikkel René Andersen

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Cover photos

Top left: The study site.

Bottom right: The study site.

Background: Dense Chara aspera bed at the study site.

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Ecology in small aquatic ecosystems

Ph.D. thesis

Author Mikkel René Andersen

Freshwater Biological Laboratory

Universitetsparken 4, 3rd floor.

2100 Copenhagen Ø

Denmark

Principal

supervisor

Kaj Sand-Jensen

Freshwater Biological Laboratory

Universitetsparken 4, 3rd floor.

2100 Copenhagen Ø

Denmark

Co-supervisor Peter A. Staehr

Department of Bioscience - Marine Diversity and Experimental

Ecology

Frederiksborgvej 399. B1.19

4000 Roskilde

Denmark

Committee Dr. Ole Petersen (Chair)

Dr. Eleanor Jennings

Dr. Torben Linding Lauridsen

University of Copenhagen

Dundalk Institute of Technology

Aarhus University

Photos All photos by Mikkel René Andersen, unless otherwise specified.

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Table of contents

ABSTRACT ......................................................................................................................... 4

DANSK RESUMÉ ................................................................................................................ 4

INTRODUCTION ................................................................................................................. 8

AIM .................................................................................................................................... 17

PAPER SYNOPSIS ........................................................................................................... 17

CONCLUSIONS AND IMPLICATIONS ............................................................................. 25

REFERENCES .................................................................................................................. 28

PAPER 1 - PROFOUND DAILY VERTICAL STRATIFICATION AND MIXING IN A SHALLOW, WIND-EXPOSED POND WITH SUBMERGED MACROPHYTES. ................ 30

PAPER 2 - RECURRING STRATIFICATION AND MIXING GENERATE EXTREME DIURNAL OXYGEN AND CARBON CYCLES IN SHALLOW VEGETATED LAKES ........ 60

PAPER 3 - DISTINCT DIURNAL PATTERNS OF ECOSYSTEM METABOLISM IN A SMALL CHAROPHYTE-LAKE ........................................................................................... 78

PAPER 4 - WHOLE-STREAM METABOLISM IN NUTRIENT-POOR CALCAREOUS STREAMS ON ÖLAND, SWEDEN .................................................................................. 110

PAPER 5 - CAUGHT BETWEEN DROUGHT AND FLOODING ON ÖLANDS GREAT ALVAR (IN SWEDISH, ENGLISH ABSTRACT) ............................................................... 139

ACKNOWLEDGEMENTS ............................................................................................... 148

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Abstract

Small ecosystems are many-fold more abundant than their larger counterparts. Both

on regional and global scale small lakes outnumber medium and large lakes and

account for a much larger surface area. Small streams are also far more common than

rivers. Despite their abundance small ecosystems are grossly understudied.

In this thesis I present new insights into the dynamic nature of small aquatic

ecosystems. I show that small lakes can stratify and that the resulting gradients are

much steeper than in larger lakes. In a 30-40 cm shallow water-column the surface

waters can be more than 200 % supersaturated in oxygen while the bottom waters

becomes anoxic. Dense charophyte stands influenced the hydrodynamics and created

favorable conditions for the apical parts in the surface waters, while the basal parts

withstood anoxia for up to 12 hours in the bottom waters. Nocturnal convective

mixing oxygenated the bottom waters and replenished the DIC pool in the surface

waters every night. Nocturnal mixing and small distances resulted in similar

metabolic signals recorded by many oxygen sensors placed across the small lake.

Respiration and gross primary production (GPP) were tightly coupled (1:1 ratio) both

in the small lakes and in the small ephemeral streams on the Great Alvar.

Downstream respiration was decoupled from GPP as respiration rates were much

higher due to agricultural impact.

Dansk resumé

Små økosystemer meget mere almindelige end deres større modstykker. Små søer og

vandløb udgør det typiske ferskvandshabitat både i Danmark og globalt, men dette til

trods er de stærkt underrepræsenteret i videnskabelige undersøgelser hvor fokus

meget oftere har været på de større ikoniske søer og floder.

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Man har ofte antaget at små søer var fuldt opblandede, mens større søer kan have

komplekse opblandings og lagdelings mønstre. Denne forsimpling af de små søer er

dog foretaget uden videnskabeligt belæg.

I kapitel 1 viser vi at helt små søer kan lagdele hvis indkommende solenergi afsættes i

toppen af vandsøjlen og den fysiske opblanding som følge af vindens friktion mod

vandoverfladen samtidigt hæmmes. Vi undersøger disse varmeflukse og den rolle tæt

bevoksning af kransnålalger har i en lille sø. Vi viser fysiske modeller som forudsiger

at søen i forhold til dens overflade areal hvorpå vinden afsættes skal være 4 gange

dybere end den er før den vil lagdele. Alligevel lagdeler den kun 30-40 cm dybe sø

næsten hver dag mellem slutningen af marts og slutningen af maj. Vi måler

temperaturforskelle på op til 15 °C mellem top og bund. Om natten opstår en meget

ustabil situation når overfladevandet køles ned til 1-5 grader under

bundvandstemperaturen, dette resulterer i kraftige konvektive strømme som

opblander vandsøjlen fuldstændigt.

Denne lagdeling muliggøres af kransnålebevoksningen, da op imod 90 % af den

indkommende solenergi afsættes i de øverste 5-20 cm af vandsøjlen som varmes op,

friktionen mellem vinden og søens overflade skaber strømhvirvler men disse svækkes

kraftigt af den tætte kransnålalgebevoksning og når ikke bundvandet der derfor

forbliver koldt. De natlige konvektive strømme påvirkes derimod kun lidt af

kransnålalgebevoksningen da de er retningsbestemte mod bunden som følge af

tyngdekraften. Dermed har kransnålalgerne en udtalt effekt på søen.

I kapitel 2 undersøger vi de gradienter som opstår som følge af daglige lagdeling af

søen. Om dagen overmættes overfladevandet med ilt til over 200 % af

atmosfæreligevægten, mens bundvandet bliver iltfrit. I overfladevandet hvor der er

lys, forbruges uorganisk kulstof i fotosyntesen og pH stiger, herved skabes de forhold

hvor kalk (CaCO3) kan fælde ud som krystaller, i denne proces frigives CO2 til

fotosyntesen uden at pH stiger yderligere.

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CaCO3 krystallerne synker mod bunden, her i det kolde bundvand hvori

respirationsprocesser har frigivet CO2 og sænket pH opløses CaCO3 krystallerne,

herved ophobes uorganisk kulstof og CO2 ved bunden om dagen. I det iltfrie

bundvand reduceres desuden ferrijern og sulfat til ferrojern og sulfid som er giftig for

mange organismer.

Den natlige opblanding bringer ilt til bunden som oxiderer de reducerede stoffer og

samtidig føres det ophobede uorganiske kulstof tilbage til overfladevandet.

Disse processer betyder at denne næringsfattige sø kan have høj produktivitet og tæt

bevoksning.

I kapitel 3 viser vi at den høje produktivitet hænger sammen med en ligeledes høj

respiration, forskellen mellem disse er tæt på nul, hvilket vil sige at

nettoproduktiviteten i systemet er meget lav. Kun i nogle få timer først på dagen er

produktiviteten høj nok til at opveje respirationen, og allerede først på eftermiddagen

er respirationen større end produktionen. Da der er masser af lys tilstede hæmmes

produktionen af mangel på CO2, i stedet bruges bikarbonat og kalk.

Vi undersøger heterogeniteten i systemet ved dels at måle på metabolismen ned

gennem vandsøjlen og på tværs af søen med mange sensorer forskellige steder i

overfladevandet. Vi finder ensartede resultater ved målinger på tværs af søen, hvilket

formentligt skyldes den effektive natlige opblanding og de korte afstande i søen. Hvis

man ikke tager højde for lagdelingen i søen får man underestimeret respirationen som

dominerer i den nederste del af vandsøjlen.

Om natten aftager respirationsraten som følge af manglende substrat.

I kapitel 4 undersøger vi metabolismen i de små vandløb på Ölands Store Alvar, hvis

øvre dele ofte tørrer ud om sommeren. Her er flere ligheder med metabolismen i de

små søer. Produktiviteten og respirationen er tæt koblet og forhold i mellem dem er

tæt på 1:1 og nettoproduktiviteten er meget lav. Længere nedstrøms løber vandet

gennem landbrugspræget opland, her stiger respirationen og afkobles dermed fra

produktiviteten. Respirationsraten aftager også om natten i vandløbene.

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Kapitel 5 (på svensk) opsummeres flere studier på Ölands Alvar, og vi viser

eksempler på de landskabsmæssige gradienter som findes her. Vandløb som tørrer ud,

rå kalkflader som oversvømmes skaber damme og udtørres igen, samt mere

permanente små søer hvor vandspejlet ændres dramatisk over året. Der gives

eksempler på biologien i disse habitater.

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Introduction

Small aquatic ecosystems are many-fold more abundant than the larger and often

iconic lakes and rivers (Downing et al. 2006). Small ecosystems have highly dynamic

physico-chemical dynamics and house species that must be adapted to such dynamic

– often extreme – environments (Wesenberg-Lund 1915, Christensen et al. 2013).

Nonetheless, small lakes and streams have been greatly understudied despite that

many new aspects of ecosystem ecology and species adaptation can apparently be

discovered and understood in this abundant, widespread but forgotten environment

(Herb & Stefan 2005). I should add, that the small aquatic ecosystem are so common

that ecosystem processes such as storage of terrestrial fixed organic carbon and CO2

emission to the atmosphere have a much greater contribution to these processes for

the entire landscape than that of medium-sized and large lakes and running waters

(Hanson et al. 2007, Sand-Jensen & Staehr 2012).

With this entrance I have very much argued for the focus and title of my

Ph.D. thesis: Ecology in small aquatic ecosystems. First of all, because small aquatic

ecosystems are greatly understudied my investigation was likely to offer new insight

and knowledge that would furthermore be relevant on the landscape level. Secondly,

being highly dynamic environments, small aquatic ecosystems are extremely

fascinating and appealing which is motivating during long field days and tiresome

data analysis. I was not let down by the results that appeared. Discovering recurring

daytime stratification and nocturnal mixing in a small charophyte-lake was a great

experience to me and my co-authors (paper 1), not to mention the documentation of

15 °C changes of water temperature and vertical gradients of the same magnitude

during a summer day. Finding anoxia in bottom waters during daytime alternating

with oxic conditions at night was a big surprise and an interesting discovery which is

obviously a huge challenge to the survival of plants, algae and immobile animals

(paper 2). Being able to present highly reproducible diurnal patterns of ecosystem

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production and respiration in a small charophyte-lake and subsequently explain them

by relationships to characteristically diurnal courses of environmental variables was

highly rewarding. It comes at a time where recent reports of extreme spatial

variability of estimates of ecosystem metabolism in medium-sized lakes by multiple

oxygen sensors placed at different locations (Van de Bogert et al. 2012) threaten to

make the approach of deploying numerous temperature, O2, pH and other sensors in

the free-water less attractive because the ecosystem estimates attained can apparently

be so noisy that useful interpretation of results are difficult (paper 3). That we, in

contrast, could demonstrate distinct diurnal courses of ecosystem metabolism (i.e.

afternoon depression of photosynthesis and declining nocturnal respiration from

sunset to sunrise, paper 3) as well as approximately balanced production and

respiration rates under highly oligotrophic conditions in small charophyte-lakes as

well as in small ephemeral streams on the open, oligotrophic limestone grasslands of

Öland, SE Sweden was also a new finding and confirmation of a hypothesis that was

originally predicted by the father of aquatic ecosystem metabolism, Howard T. Odum

more than fifty years ago (Odum 1956) (papers 4 and 5).

Abundance of small ecosystems – With my Danish background I know that very

small lakes (< 1 ha) are counted in numbers exceeding 100,000 in the country, while

medium-sized and larger lakes (> 10 ha) are 100-fold less abundant (Sand-Jensen

2001). Also small narrow streams (< 2.5 m width) stretches for 48.000 km through

the landscape compared to 1.500 km for larger Danish streams (> 8 m width, Sand-

Jensen et al. 2006). If we turn to other lowland countries such as England (Biggs et

al. 2005, Davies et al. 2008) and the American mid-West (Hanson et al. 2007) the

dominance of small lakes and streams is repeated. The same pattern is even more

pronounced on the Arctic tundra (Anderson & Stedmon 2007) and in the deltas and

backwaters of major rivers (Emmerton et al. 2007).

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On the large Swedish island of Öland, which became the region of my

investigation, there are no major streams and only one major lake (Lake Hornsjöen).

In contrast, there are many small, shallow streams, mostly ephemeral that dry out

during summer (Fig. 1) and there a many shallow lakes, often filled with charophytes

or submerged flowering plants because of the shallow, nutrient-poor and transparent

waters (paper 4 and 5).

Shallow lakes cover large surface areas of Ölands Great Alvar (Unesco

World Heritage) during winter, but are markedly reduced in size and numbers during

summer drought where many

waterbodies dry out completely. The

same changing water levels are

experienced by the many small lakes

in the abandoned limestone quarry

surrounded by the undisturbed

Räpplinge Alvar, where I made the

largest part of my Ph.D. study (Fig. 2).

This area has about twenty small lakes

and my studies could be made

undisturbed by intruders and under kind observation of the owner such that we

experienced no loss of equipment during the three year study.

I should also emphasize that we selected small lakes and streams on

Öland as study objects because they are calcareous and oligotrophic and represents a

contrast to most ecosystems in other parts of the European lowlands that have been

greatly disturbed by excessive eutrophication from urban areas and intensely

cultivated farmland (paper 4 and 5). This destructive development has not occurred to

nearly the same extent on Öland as yet. Thus, we are studying ecosystems as they

Figure 1 Ephemeral stream on Öland (Åbybäcken) that has dried

out in the summer drought.

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were throughout Europe before World War II and as they still are in unspoiled

regions and as they may once again become following reduced nutrient loadings.

Small lakes: ephemeral or permanent

and terrestrial impact – Small shallow

lakes can dry out and undergo refilling

on a regular basis, though the nature

of the processes are stochastic such

that drying-out and refilling can occur

several times a year and in different

seasons or not take place whatsoever

in wet years. The small charophyte-

lake we studied lost 73% of its water

from winter to early summer in 2014 (paper 1). According to the landowner, it dries

out completely approximately once every 15 years. Charophytes and flowering plants

can sustain complete water loss, while fish populations are more susceptible, though

they were indeed present.

There are important implicit scaling functions concerning water retention

time, external loading of organic material and nutrients as well as incident irradiance

and wind exposure to surface area and water volume of lakes (Sand-Jensen & Staehr

2007, Staehr et al. 2012). Small lakes have a longer coastline relative to surface area

than large lakes; i.e. the contact to the surrounding terrestrial landscape is higher. In

fact for the same shape, the length of the coastline relative to surface area declines in

proportion to the linear dimensions of the lake. Therefore, small shallow lakes tend to

have shorter water retention time, greater external loading of organic material and

nutrients to surface area and volume than large deeper lakes, and are also likely to be

more shaded by riparian vegetation and less exposed to wind (Sand-Jensen & Staehr

Figure 2 Arial photograph of an abandoned limestone quarry,

dominated by ponds and small lakes, seen as predominantly as

dark brown patches © Google.

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2007, Staehr et al. 2012). Small forest lakes can be so extensively shaded that

autochthonous production is negligible and the metabolism is entirely based on input

of terrestrial material. In contrast, small lakes located in open landscapes host very

productive plant communities whose primary production by far outweighs the input

of allochthonous material that may come from the low plants in the surroundings.

Commonly, temperate lakes on open agricultural landscapes receive heavy external

nutrient loads resulting in blooms of microalgae, floating duckweeds or submerged

plants and anoxia following die-back and decomposition of the produced plant

material.

The small lakes on Öland, in contrast, are surrounded by nutrient-poor

open grassland with thin soils on the very slowly weathering Ordovician limestone.

Thus, external input of nutrients and organic material is low and the lake water is

crystal clear (Sand-Jensen et al. 2010, paper 5). Ecosystem processes should,

therefore, mostly be of autochthonous character and gross primary production and

community respiration pretty close to each other.

Warming, cooling and hydrodynamics – Small shallow lakes can respond much faster

to meteorological drivers than large, deep lakes because the smaller water column

requires less energy to heat up or cool down. It has mostly been over-looked and

forgotten, but already 40 years ago, Martin (1972) showed that very small lakes can

undergo daily thermal stratification. The fast response to meteorological conditions

and the fact that small shallow lakes can indeed stratify open up the possibility of

very dynamic and complex thermal regimes. Nonetheless, small shallow lakes are

usually assumed to be fully mixed with little justification (Branco & Torgersen

2009).

In contrast, it is realized that large lakes have complex thermodynamics

(Staehr et al. 2009), probably because scientific studies and knowledge of these lakes

are much more comprehensive. Since the early 1900, lakes have been classified based

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on their stratification pattern (Hutchinson & Löffler 1956), and refined classifications

have been introduced more recently (Lewis (1983). A deep lake located at our

latitude stratifies during summer, mix in autumn, can have reverse temperature

stratification under winter-ice and become fully mixed following ice-out in spring.

We were very interested in analyzing how stratification and mixing

patterns are in small shallow lakes with dense submerged vegetation because

vegetation should greatly impede wind-induced mixing by offering great resistance to

water movements (Losee & Wetzel 1993, Sand-Jensen & Pedersen 1999). Also,

dense submerged vegetation generates extremely steep light attenuation and the

possibility of particularly strong surface warming. Both strong dissipation of

turbulent energy and strong warming of surface waters should facilitate formation of

vertical density gradients. Indeed it turns out that the temporal and spatial thermal

pattern of a deep temperate lake from spring, through summer to autumn is repeated

in the shallow charophyte-lake during every 24-hours day-night cycle in summer

(paper 1) with great consequences for water chemistry (paper 2).

Stratification and water chemistry – There are several reasons why temperature and

coupled stratification-mixing patterns are crucial for understanding physico-chemical

and biological processes in lakes. The thermocline acts like a boundary between

surface waters and bottom waters such that different processes result in diverging

chemistry. The density gradient between the two water layers marks the lower limit

to where turbulent mixing can effectively penetrate such that dissolved ions and gases

are predominantly transported by slow molecular diffusion, while dense particles

influenced by gravity can sink into the bottom waters (Boehrer & Schultze 2008).

In the surface mixed layer organic production by photosynthesis will

typically surpass respiration during daytime leading to accumulation of oxygen,

increase of pH and depletion of dissolved inorganic carbon (DIC). In the bottom

waters, being physically separated from surface waters by the density gradient, the

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opposite processes (i.e. depletion of oxygen, decline of pH and DIC accumulation)

take place leading to strong vertical gradients.

The crucial question is now how these processes behave and influence

chemical gradients in small lakes that develop dense stands of macrophytes and

possibly form vertical density gradients despite shallow water. In dense stands of

charophytes and flowering plants with steep vertical light attenuation, the chemical

gradients are enhanced by the contrast between well-illuminated surface waters

warming up during daytime and shaded bottom waters. When vertical stratification

develops on a daily basis within macrophyte stands we anticipate the development of

much more profound temporal and vertical gradients of substrates and products

involved in photosynthesis and plant respiration as well as oxygenic and anoxic

bacterial processes than usually encountered in large lakes. pH changes coupled to

photosynthesis and respiration can also induce precipitation of calcium carbonate in

surface waters and dissolution in bottom waters. The rate of these processes and the

resulting vertical gradients should be particularly strong because all metabolic

activity is packed in a shallow water column. Perhaps the dynamics could become

intermediate between that experienced in the upper few millimeters of well-

illuminated surface sediments where diffusion processes and extremely high

metabolic rates normalized to volume prevail (Jørgensen & Revsbech 1985) and the

conditions in meter-thick well-mixed water columns of lakes. If stratification lasts

long enough, surfaces waters may become depleted in plant nutrients and bottom

waters enriched in the same nutrients.

In the case of development of strong vertical oxygen gradients bacterial

processes and animals will be greatly influenced. Anoxia induces sulphate, nitrate,

manganese and iron reduction and animals may try to escape from the risk of anoxia

and accumulation of sulphide and reduced iron. The stratification-mixing regime sets

the scene for all the biological and chemical processes and their direct and indirect

ecological consequences. This scene should be quite different in small shallow lakes

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with dense macrophyte stands than the well-known scene in the open water column

and even within the littoral vegetation of large lakes where vertical and horizontal

turbulence and water flow should be much stronger and weaken the gradients.

Ecosystem metabolism – Technological improvements of O2 sensors as well as other

sensors for free-water measurements of temperature, light, pH, conductivity, etc. have

made it possible to estimate ecosystem processes of gross primary production (GPP),

net ecosystem production (NEP) and respiration and the environmental conditions

regulating them without enclosing the organisms in bottles and chambers under

unnatural environmental conditions. After years of progress and optimism, we have

now been warned that perhaps we have been over-optimistic concerning the

potentials of free-water measurements to determine the processes and demonstrate

their regulation because of surprisingly high differences in metabolism estimates

derived from multiple sensors at different locations in medium-sized lakes (Van de

Bogert et al. 2007, Van de Bogert et al. 2012). Experience suggested that we ought to

maintain optimism regarding the ability of single and multiple sensors to yield

reproducible estimates of ecosystem metabolism in small lakes (Christensen et al.

2013) because shorter vertical and horizontal distances may reduce delays between

processes actually occurring and being registered and at the same time homogenizing

oxygen (and other chemical signals) signals from different sections of the small lakes.

This motivated us to perform the whole-lake studies in the small Charophyte-lake

(papers 2 and 3).

Studies of ecosystem studies of streams are the classical ones which were

initiated already in the 1920-1930ies. Because of the unidirectional flow, oxygen

balances can be established by the two-station method of H. T. Odum and the later

refinements. One challenge is to ensure accurate determination of air-water gas

exchange. We made direct measurements of gas exchange using flow chambers in the

small alvar streams on Öland and were able to obtain highly accurate and

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reproducible results. We were particularly interested in these streams because they

are oligotrophic and collect virtually no sediment because most sediment is washed

out during winter stormflow following the low-flow spring and summer period and

what remains of organic particles is metabolized when the streams dry out in

summer. We hypothesized that autochthonous production and community respiration

should be very low compared to most other, more nutrient-rich and flow-stable

temperate streams and production and respiration should be in approximate balance.

The small shallow lakes, being filled with water for most of the year and indeed

collecting sediment, develop a dense cover of charophytes and should, thus, have

much higher rates of ecosystem production and respiration. This was an interesting

contrast that we wanted to evaluate (paper 5).

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Aim

The aim of this thesis was to:

i. Investigate surface heat fluxes and stratification-mixing in a small charophyte

dominated lake.

ii. Investigate if macrophytes themselves can influence the hydrodynamics of the

lake sufficiently to cause it to stratify.

iii. Investigate the physico-chemical gradients that may develop in a stratified

charophyte-lake.

iv. Investigate ecosystem metabolism of small oligotrophic lakes and streams.

v. Investigate if the horizontal and vertical heterogeneity of metabolic signals in

such systems differ from larger lakes and rivers.

Paper synopsis

Paper 1 - Profound daily vertical stratification and mixing in a small, shallow, wind-

exposed lake with submerged macrophytes.

We studied a small (< 1000 m2), shallow (< 0.6 m) lake with dense submerged

macrophytes located in an open landscape on Öland, SE Sweden, between March and

May to investigate thermal regimes, surface heat fluxes and effects of macrophytes

on stratification and mixing processes.

0

5

10

15

20

25

30

35

March April May

Wate

r te

mp

era

ture

(C

)

Figure 3 Surface water temperature (dashed line) and bottom water temperature (bold line) in the shallow lake

during the investigation.

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The small lake heated up from March to May. Profound daytime temperature

differences developed between surface and bottom-waters ranging from 3 °C in

March to 15 °C in May (Fig 3).

Maximum relative thermal resistance to mixing (RTRM) exceeded 50 (a literature-

derived value for the certain onset of stratification) on 11 days in April and 25 days in

May while the mixed depth dropped from 100 % of the water column to just 25 %

(calculations of mixed depth showed, however, that the small lake stratified

moderately even in late March). Nocturnal cooling of surface waters to 1-5 °C below

bottom water temperature led to full convective mixing of the water column every

night. Nocturnal surface cooling and convective mixing were enhanced by the

extraordinary daytime warming of surface waters above air temperatures. Convective

mixing was only weakly affected by the charophytes.

The daytime focal depth of the thermocline was 25 cm below the water surface in

early May and just 15 cm in late May following a parallel shallowing of the lake

bringing the charophyte canopy closer to the water surface. The strength of

stratification peaked in the early afternoon although diel wind speeds were highest at

this time (Fig. 4).

The coinciding peaks in wind speed and strength of stratification was possible

because the dense macrophyte cover rapidly attenuated depth penetration of the

-50

100

150

200

250

03 06 09 12 15 18 21 2400

0

50

Time of Day

RT

RM

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03 06 09 12 15 18 21 2400

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dep

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%)

a b

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peed

(m

s-1

)

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ol m

-2 s

-1)

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tem

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( C

)

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idit

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%)

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25

03 06 09 12 15 18 21 2400

Time of Day

Su

rf.

wate

r te

mp

. (°

C)

a b c

d e

a b

Figure 4 Diel averages of RTRM (dimensionless) (panel a) and diel averages of windspeed (m s-1)

( panel b). Blue line is March, green line is April and red line is May.

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radiative fluxes, while also greatly attenuating the depth penetration of wind-induced

turbulent mixing. Thus, by facilitating build-up of temperature, chemical and density

gradients the macrophytes profoundly influenced their own environment.

Paper 2 - Recurring stratification and mixing generate extreme diurnal oxygen and

carbon cycles in shallow vegetated lakes.

Vertical stratification-mixing patterns are main determinants of biogeochemistry.

Here we show that a small, wind-exposed, shallow (ca 0.4 m) lake with submerged

macrophytes underwent recurring daytime stratification and nocturnal mixing during

summer accompanied by extreme variations in temperature, oxygen, pH and

dissolved inorganic carbon (DIC) with time and depth. During daytime stratification,

surface waters attained 230 % oxygen saturation and strong CO2 depletion (< 10 %

air saturation, Fig. 5).

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Figure 5 Time series of temperature, O2, pH, ANC, calcite saturation index, DIC, individual carbon species with depth in a

shallow charophyte-lake during six days.

a, Temperature isopleths calculated from measurements at 5-cm depth intervals.

b, Oxygen measured at 0.08 m (dark blue), 0.24 m (green) and 0.34 m (red) below the water surface.

c, pH (blue) and ANC (green) in surface waters (0.08 m).

d, DIC (red), CO32- (orange) and CO2 (blue) in surface waters (0.08 m).

Where b-d background color show day/night cycle (white = day, grey = night).

Deeper waters were colder and became anoxic. In the cold anoxic bottom waters,

reduced compounds such as ferrous iron and sulphide accumulated during the day

and CO2 built up to more than 1500 % super-saturation (Fig. 6).

0.08 m

0.24 m

0.34 m

100

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500

O2 (

M)

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1.5

2.0

8.0

9.0

10.0

AN

C (

meq

L-1

)p

H

2

4

6

8

10

12

14

Calc

ite s

atu

ratio

n in

dex (g

reen

)

0.0

0.5

1.0

1.5

0.0

0.1

0.2

0.3

0.4

0 12

12 August

0 12

13 August

0 12

14 August

0 12

15 August

0 12

16 August

0

CO32-

CO2

DIC

DIC

(m

M)

CO

32- / C

O2 (m

M)

0.2

0

Wate

r d

ep

th (

m)

5 C

10 C

15 C

20 C

25 C

30 C

35 C

0.1

a

b

c

d

0.3

0.4

a

21

Figure 6 Depth profiles of Fe2+, sulphide and DIC in a shallow charophyte lake during a diurnal cycle.

At 6:00 (red), 11:00 (blue), 16:00 (green) and 22:00 (orange) o’clock. The water column was vertically mixed at 6.00 o’clock

and stratified below 0.20 m at 11:00 - 16.00 and below 0.25 m at 22:00 o’clock.

High daytime pH in surface waters induced CaCO3 precipitation while releasing CO2

for ongoing photosynthesis without further pH rise. The majority of the precipitated

CaCO3 was re-dissolved in bottom waters leading to a buildup of DIC (Fig. 6).

Vertical gradients disappeared during nocturnal convective mixing which oxygenated

the bottom waters and regenerated the DIC pool in the surface waters.

These processes add new dimensions to our understanding of the regulation of

ecosystem photosynthesis and respiration and the adaptation of sessile plants and

mobile animals to the extreme variability of environmental stressors.

Paper 3 - Distinct diurnal patterns of ecosystem metabolism in a small charophyte-

lake.

We wanted to characterize the temporal and spatial variability of metabolic

parameters: gross primary production (GPP), respiration (R) and net ecosystem

production (NEP) in a small, shallow lake with dense charophyte stands. To do so we

collected data from many O2 sensors placed along a vertical mid-lake profile and

across the lake surface in late May and early June. Similar diurnal patterns derived

both from individual surface sensors and multiple sensors. Maximum NEP-rates

0 10 20 30 40

0.0

0.1

0.2

0.3

Fe2+

(M)

Wate

r d

ep

th (

m)

0 0.1 0.2 0.3 0.4

Sulphide (M)

1 2 3

DIC (mM)

22

occurred between 8:00 and 11:00 am and was followed by strong afternoon

depression with rates close to zero (Fig. 7).

While NEP rates dropped along with DIC and

CO2 concentrations, O2 concentration, pH and

temperature all rose profoundly in the surface

waters from morning to late afternoon.

Inorganic carbon limitation of photosynthesis

and temperature enhancement of respiration

could account for the profound afternoon

depression of NEP. Nocturnal respiration declined systematically from sunset to

sunrise due to falling temperature and presumably depletion of respiratory substrates.

Mean temperature-corrected respiration rates at sunrise were 63% of that at sunset.

The dense charophyte canopy accounted for 90% of ecosystem respiration and the

entire primary production. Mean daily estimates of GPP and R varied only 2-fold and

small, negative NEP-rates varied less between surface sensors at different locations

across the lake (Table 1).

Figure 7 Mean volume-weighted GPP (red) and NEP

(blue) for the entire lake, based on the seven oxygen

sensors

23

Table 1 . Mean daily rates of GPP, NEP and R derived from continuous oxygen

measurements at seven different positions (A to G) and the overall mean of all measurements

in a small lake during a week in early June. Daily minimum and maximum daily rates are in

parenthesis.

In the small oligotrophic lake rates of GPP and R were tight coupled and both about

20-fold higher than NEP (Table 1). Multiple oxygen sensors representing the main

depths and sections of the small lake could provide reliable and accurate

measurements of diurnal course and daily rates of metabolism, probably because a

relatively uniform oxygen signal was ensured by small distances and very efficient

nocturnal mixing.

Paper 4 - Whole-stream metabolism in nutrient-poor calcareous streams on Öland,

Sweden.

We studied whole-stream metabolism in three headwater non-forested stream reaches

on the island of Öland, Sweden in order to characterize the metabolism of this

unusual ecosystem and to compare it with other stream ecosystems in NW Europe.

Gross primary production (GPP) was low (< 4 g O2 m-2 d-1) with the lowest GPP

recorded in the most upstream, shallow reach draining the thin soils of the limestone

Alvar plains. Here, completely flooded terrestrial plants could account for the whole

primary production. Respiration (R) increased several-fold downstream with

increasing agricultural impact, resulting in heterotrophic stream conditions and higher

24

light requirements for GPP to outweigh the higher respiration.

Some similarities between the small oligotrophic lakes (paper 3) and the most

nutrient-poor reaches of the stream were observed. GPP and R were tightly coupled

and temperature-corrected respiration rates were highest in the beginning of the night

and decreased towards the end of the night (Fig. 8), indicating that nocturnal

respiration depleted photosynthetic products and became limited by organic

substrates in the streams too.

Broad-scale comparison of open NW

European streams showed a 1:1

relationship, indicating a tight link

between daily GPP and R during summer

(April-August) but not during winter.

We extended the range of GPP and R

measurements to include nutrient-poor

NW European streams, thereby increasing the knowledge on stream metabolism in

this region, otherwise, highly impacted by agriculture. We documented a strong

relationship between GPP and R in streams, ranging from extremely nutrient-poor to

moderately nutrient-rich conditions during spring and summer.

Paper 5 - Caught between drought and flooding on Ölands Great Alvar

The Great Alvar plain on the Swedish island of Öland is characterized by thin soils

covering the hard limestone pavements. This gives rise to widely fluctuating water

levels between winter flooding and summer drought and strong hydrological

gradients across small changes in elevation. In the semi-natural grassland, the

intermittent streams and the ponds are all strongly influenced by the fluctuating water

levels and the extremely low phosphorus availability. These factors have selected for

Figure 8 Respiration rates declining during the night in a

small nutrient-poor Alvar stream.

25

phototrophs with low metabolic rates and growth, and communities of low

photosynthesis and respiration.

Plant species were distinctively distributed according to their characteristic plant

traits along a moisture gradient from ponds to dry alvar. High root porosity to ensure

efficient oxygen transport was strongly selected for among species in wet soils, while

small, thick leaves were strongly selected for on thin, dry soils. Overall, six plant

traits could predict 66% of the variation in abundance of plant species in the

communities along the gradient.

The alvar streams had only modest biomasses during maximum development of

benthic algae in May, and community photosynthesis was 5–10 times lower than

corresponding levels in nutrient-rich streams in cultivated lowlands of Scandinavia.

During June–September streams dried out and the re-establishment of flow in winter

and spring led to an export of nutrients. Shallow ponds also dried out during summer

and had low metabolic rates just like the streams, while permanent ponds developed

dense stands of charophytes, despite undetectable levels of N and P in the water.

Photosynthesis and community respiration were in approximate balance in permanent

ponds. The maximum rates were comparable to those in eutrophic, phytoplankton-

rich lakes.

Conclusions and implications

Several concrete results have emerged from this thesis. Small lakes can stratify and

exhibit temperature differences between the surface mixed layer and the hypolimnion

that rival that of much larger lakes. Because the stratification takes place in a very

shallow water column, the resulting gradients are extremely steep. It was a new

insight that a water column of just 0.3-0.4 meter could be more than 200 %

supersaturated in the surface waters and anoxic in the bottom waters.

26

The carbon pool in the surface waters in such a lake is regenerated in DIC and CO2

which is dissolved in the anoxic bottom waters during the day and distributed in the

water column by the efficient convective nocturnal mixing. On particularly cold days

when the water column does not stratify photosynthesis in the surface water becomes

carbon-limited much earlier during the day.

While the stratification presents plants and animals with challenges for survival, it

also provides the basis on which they depend and have adapted. Physical models of

mixing resulting from surface wind shear show that the lake would have to be

roughly 4 times as deep as it was in order to stratify, and the only explanation is that

the attenuation of both radiative heat fluxes and wind-induced turbulent mixing due

to the presence of macrophytes is the reason why the lake stratifies. The macrophytes

then create the physical and chemical environment on which they themselves depend.

Potential harmful effects of elevated pH levels in the surface waters were limited by

the fact that as the day progressed productivity depended more and more on CaCO3

precipitation which release CO2 and is pH neutral.

In this highly dynamic system we observed the same metabolic patterns with

individual oxygen sensors as we did with a volume-weighted average of many

sensors placed across the lake. Respiration was underestimated when calculations of

metabolic parameters did not account for stratification, but the results were still of the

same order of magnitude. We credit the small distances and the recurring nocturnal

mixing for integrating possible local differences in metabolic rates to a common

oxygen signal.

Investigating the metabolism in detail revealed some interesting patterns. Positive

NEP rates were restricted to mornings and early afternoons. Later in the day

respiration speeded up due to elevated temperatures and productivity dwindled as the

surface water became depleted on metabolic substrates. Nocturnal respiration slowed

down during the night, even when corrected for the drop of temperature. This finding

suggests that respiration too is limited by availability of substrate. Both productivity

27

and respiration rates were high on areal basis in this oligotrophic system, while NEP

was close to 0. This validates our metabolism model as the very shallow lake would

simply be filled up if respiration did not closely follow production, and in the ~30

years in which the small lake has existed, only a few (4-10) centimeters of sediment

have accumulated.

Some of the same patterns were observed in the small intermittent streams we

investigated on the Great Alvar. Productivity and respiration was tightly coupled with

a 1:1 ratio, and respiration rates dropped markedly as the night progressed. Both

productivity and respiration was several fold lower than in other low land streams,

owing to the extremely low nutrient- and DIC-concentrations in the water and the

winter wash-out of sediments. As we moved downstream into agricultural areas,

respiration was decoupled from production as it became much larger and the

differences between the Öland streams and other lowland streams was less marked,

although productivity remained reasonably low.

This thesis has implications for conservation and ecology on a broad scale. My co-

authors and I show that stratification can occur in very small lakes. Two drivers are

necessary for a small lake to stratify:

The incoming heat flux must be attenuated unevenly through the water column.

The turbulent mixing must be insufficient to ensure full mixing of the water

column.

In this study the charophytes attenuated almost 90 % of the incoming short-wave

radiation in the top 5-20 cm of the water column. In other small lakes light could be

attenuated effectively in the surface water by dense microalgae or high densities of

humic substances.

The charophytes also effectively attenuated mixing, in other systems mixing could be

limited by the surroundings, for instance by forests or crevasses between fault lines in

mountains.

28

The implication is that many small lakes may have complex thermal regimes and may

behave vastly different than assumed in global estimates of temperature response,

carbon sinks etc. It also means that the palette of ecological niches may be larger on

local and global scales than previously assumed.

For system ecologists this thesis provides the basis for investigating physical,

metabolic and ecological patterns for a wider range of small lakes and streams.

It remains unanswered how sessile organisms survive the harsh conditions of the

bottom waters in the small charophyte-lakes.

It is my hope that this thesis may be an inspiration to physiological studies of

adaptations to extreme environmental conditions for algae, plants and animals in

small lakes.

References

Anderson, N., and Stedmon, C. A. 2007. The effect of evapoconcentration on dissolved organic carbon concentration and quality in lakes of SW Greenland. Freshwater Biology 52:280-289. Biggs, J., Williams, P., Whitfield, M., Nicolet, P., and Weatherby, A. 2005. 15 years of pond assessment in Britain: results and lessons learned from the work of Pond Conservation. Aquatic Conservation: Marine and Freshwater Ecosystems 15:693-714. Boehrer, B., and Schultze, M. 2008. Stratification of lakes. Reviews of Geophysics 46. Branco, B. F., and Torgersen, T. 2009. Predicting the onset of thermal stratification in shallow inland waterbodies. Aquatic Sciences-Research Across Boundaries 71:65-79. Christensen, J., Sand‐Jensen, K., and Staehr, P. A. 2013. Fluctuating water levels control water chemistry and metabolism of a charophyte‐dominated pond. Freshwater Biology 58:1353-1365. Davies, B., Biggs, J., Williams, P., Lee, J., and Thompson, S. 2008. A comparison of the catchment sizes of rivers, streams, ponds, ditches and lakes: implications for protecting aquatic biodiversity in an agricultural landscape. Hydrobiologia 597:7-17. Downing, J., Prairie, Y., Cole, J., Duarte, C., Tranvik, L., Striegl, R., McDowell, W., Kortelainen, P., Caraco, N., and Melack, J. 2006. The global abundance and size distribution of lakes, ponds, and impoundments. Limnology and Oceanography 51:2388-2397. Emmerton, C. A., Lesack, L. F., and Marsh, P. 2007. Lake abundance, potential water storage, and habitat distribution in the Mackenzie River Delta, western Canadian Arctic. Water Resources Research 43. Hanson, P. C., Carpenter, S. R., Cardille, J. A., Coe, M. T., and Winslow, L. A. 2007. Small lakes dominate a random sample of regional lake characteristics. Freshwater Biology 52:814-822. Herb, W. R., and Stefan, H. G. 2005. Model for wind-driven vertical mixing in a shallow lake with submersed macrophytes. Journal of Hydraulic Engineering 131:488-496.

29

Hutchinson, G., and Löffler, H. 1956. The thermal classification of lakes. Proceedings of the National Academy of Sciences of the United States of America 42:84. Jørgensen, B. B., and Revsbech, N. P. 1985. Diffusive boundary layers and the oxygen uptake of sediments and detritus. Limnology and Oceanography 30:111-122. Lewis, W. M. 1983. A revised classification of lakes based on mixing. Canadian Journal of Fisheries and Aquatic Sciences 40:1779-1787. Losee, R. F., and Wetzel, R. C. 1993. Littoral flow rates within and around submersed macrophyte communities. Freshwater Biology 29:7-17. Martin, N. 1972. Temperature fluctuations within English lowland ponds. Hydrobiologia 40:455-470. Odum, H. 1956. Primary production in flowing waters. Limnology and Oceanography 1:102-117. Sand-Jensen, K. 2001. Lakes - A Protected Nature Type (Danish). Gad Publishing. Sand-Jensen, K., Baastrup-Spohr, L., Winkel, A., Moller, C. L., Borum, J., Brodersen, K. P., Lindell, T., and Staehr, P. A. 2010. Plant distribution patterns and adaptations in a limestone quarry on Oland. Svensk Botanisk Tidskrift 104:23-31. Sand-Jensen, K., Friberg, N., and Murphy, J. 2006. Running Waters: Historical development and restoration of lowland Danish streams. Aarhus Universitetsforlag. Sand-Jensen, K., and Pedersen, O. 1999. Velocity gradients and turbulence around macrophyte stands in streams. Freshwater Biology 42:315-328. Sand-Jensen, K., and Staehr, P. A. 2007. Scaling of pelagic metabolism to size, trophy and forest cover in small Danish lakes. Ecosystems 10:128-142. Sand-Jensen, K., and Staehr, P. A. 2012. CO2 dynamics along Danish lowland streams: water-air gradients, piston velocities and evasion rates. Biogeochemistry 111:615-628. Staehr, P., Bade, D., Bogert, M., Koch, G., Williamson, C., Hanson, P., Cole, J., and Kratz, T. 2009. Lake metabolism and the diel oxygen technique: State of the science. Staehr, P. A., Baastrup-Spohr, L., Sand-Jensen, K., and Stedmon, C. 2012. Lake metabolism scales with lake morphometry and catchment conditions. Aquatic Sciences 74:155-169. Van de Bogert, M. C., Bade, D. L., Carpenter, S. R., Cole, J. J., Pace, M. L., Hanson, P. C., and Langman, O. C. 2012. Spatial heterogeneity strongly affects estimates of ecosystem metabolism in two north temperate lakes. Limnology and Oceanography 57:1689. Van de Bogert, M. C., Carpenter, S. R., Cole, J. J., and Pace, M. L. 2007. Assessing pelagic and benthic metabolism using free water measurements. Limnology and Oceanography: Methods 5:145-155. Wesenberg-Lund, C. 1915. Insektlivet i ferske vande. Gyldendal, Nordisk forlag.

30

Paper 1 - Profound daily vertical stratification and mixing in a shallow, wind-exposed pond with

submerged macrophytes.

31

Profound daily vertical stratification and mixing in a shallow, wind-exposed

pond with submerged macrophytes.

Mikkel René Andersen1, Kaj Sand-Jensen1, R. Iestyn Woolway2 and Ian D.

Jones3.

1 Biological Institute, Freshwater Biological Laboratory, University of

Copenhagen, Universitetsparken 4, 2100 Copenhagen, Denmark.

2 Department of Meteorology, Reading University, Reading, RG6 6BB, United

Kingdom.

3 Centre for Ecology & Hydrology, Lancaster Environment Centre, Library

Avenue,

Bailrigg, Lancaster, LA1 4AP, United Kingdom.

Submitted to Freshwater Biology

Keywords: temperature stratification, vertical mixing, pond, macrophytes,

charophytes

32

Summary

1) Ecology of small shallow lakes and ponds have been grossly understudied in

freshwater ecology although they are 100-fold more abundant than large, deep

lakes and cover a much larger area globally. Mixing patterns are essential

because they regulate distribution of gases, solutes and organisms. Here, we

studied a small (< 1000 m2), shallow (< 0.6 m) pond with dense submerged

macrophytes located in an open landscape on Öland SE Sweden between

March and May to investigate thermal regimes, surface heat fluxes and effects

of macrophytes on stratification and mixing processes.

2) The pond heated up from March to May as surface heat fluxes were positive.

Profound daytime temperature differences developed between surface and

bottom waters ranging from 3 °C in March to 15 °C in May. Maximum relative

thermal resistance to mixing (RTRM) exceeded a threshold of 50 on 11 days in

April and 25 days in May while the mixed depth dropped from 100 % of the

water column to just 25 %. Nocturnal cooling of surface waters to 1-5 °C below

bottom waters temperature led to full convective mixing of the water column

every night. Nocturnal surface cooling and convective mixing were enhanced

by the extraordinary daytime warming of surface waters above air

temperatures.

3) The daytime focal depth of the thermocline was 25 cm below the water surface

in early May and just 15 cm in late May following a parallel shallowing of the

pond bringing the charophyte canopy closer to the water surface. The strength

of stratification peaked in the early afternoon although diel wind speeds were

highest at this time. The dense macrophyte cover rapidly attenuated depth

penetration of radiative fluxes and wind-induced mixing.

4) Dense macrophyte stands can influence their own environment by facilitating

build-up of temperature, chemical and density gradients while lack of

macrophytes permits continuous mixing and uniform conditions.

33

Introduction

Ponds are far more abundant than lakes, and ponds have a combined surface area

which far exceed that of lakes (Downing et al. 2006). While it is well known that

lakes have complex thermodynamics (Boehrer & Schultze 2008), studies of surface

forcing, mixing and stratification in ponds are rare. Ponds are typically treated as

fully mixed systems with little or no justification (Branco & Torgersen 2009). This is

problematic as stratification and mixing dynamics in the water column are major

determinants of environmental conditions (Branco et al. 2005), distribution,

metabolism and survival of organisms (Vad et al. 2013). Ecosystem properties such

as carbon metabolism (Staehr et al. 2010), the flux of gases between water and air

(Boehrer & Schultze 2008, Coloso et al. 2011) and production rates of the

greenhouse gas, methane are also highly influenced by thermodynamics (Bastviken et

al. 2011).

Accurate characterization of heat exchange between lakes and atmosphere

is important for analysis of lake hydrodynamics (Lofgren & Zhu 2000). Positive net

heat input results in a positive buoyancy flux, stabilizing the warmer surface layer,

while a net surface heat loss cools the surface waters and promotes vertical mixing

(Imberger 1985). Wind-induced vertical mixing produced by wind shear on the

surface acts as a destabilizing force (Imberger & Hamblin 1982). This mechanical

vertical mixing deepens the mixed layer and reduces the likelihood of temperature

stratification in wind-exposed shallow lakes (Imberger 1985, Boehrer & Schultze

2008, Branco & Torgersen 2009). Thus, vertical temperature stratification in lentic

ecosystems takes place when the stabilizing influence of surface heating from solar

radiation and infrared radiation from the sky exceeds the destabilizing influence of

turbulent mixing generated by the wind and cooling of the surface waters (Gorham &

Boyce 1989, Imboden & Wüest 1995). In general, the shallower the lake, and the

34

more wind-exposed, the less likely is it to stratify (Imberger 1985, Gorham & Boyce

1989).

It was, therefore, a surprise that a very shallow (< 0.6 m) and small (<

1000 m2) pond exposed to the wind on the open calcareous Alvar plains on the Island

of Öland, Sweden, apparently underwent profound vertical stratification during most

days and full mixing every night according to vertical oxygen dynamics (Andersen et

al. 2015). Formation of anoxia in bottom waters during the day was probably made

possible by strong vertical density stratification, because surface waters were always

oxygenated and strongly supersaturated during the day. The pond, though, had a

dense macrophyte cover that can result in strong vertical light attenuation. High light

attenuation in humic waters can increase surface water temperatures due to absorbed

radiation (Persson & Jones 2008, Read & Rose 2013) but increase in surface water

temperature also causes a lake to lose more heat from sensible, latent and long-wave

fluxes than one with a cooler surface (Persson & Jones 2008) and, when this heat loss

exceeds incoming heat, will promote mixing. Thus, vegetation cover can potentially

affect the hydrodynamics in lakes if the macrophyte canopy is dense enough to

significantly enhance light attenuation. This effect will be stronger if the macrophytes

are located close to the surface as the macrophytes will absorb more energy relative

to the water column, and this energy will subsequently be dissipated in a smaller

volume of water. In transparent oligotrophic waters the strong light attenuation effect

of macrophytes will dominate as light attenuation directly in the water is small. Thus,

the effect of macrophyte light attenuation on hydrodynamics should be stronger in

clear than unclear waters. Similarly, if cover is sufficiently dense, macrophytes can

inhibit mechanical mixing by dissipating turbulent kinetic energy (Sand-Jensen &

Mebus 1996, Folkard et al. 2007).

We therefore initiated continuous measurements of temperature structure

at high spatial and temporal resolution in a calcareous pond with dense charophyte

beds, in order to investigate if the charophytes could influence the stratifying and

35

mixing dynamics sufficiently to cause diel stratification. To calculate heat fluxes a

meteorological measuring station was established next to the pond.

Our specific objectives in this study were to determine: (i) the heat fluxes

in a small pond with dense macrophyte cover, (ii) when and where stratification and

mixing take place during daily and monthly periods, and (iii) the impact of

macrophytes on stratification and mixing dynamics.

Materials and methods

Site description

The investigation was conducted in a small permanent pond in an abandoned

limestone quarry on Räpplinge Alvar on Öland, SE Sweden (56.81168°N, 16.6094°E;

Sand-Jensen & Jespersen 2012). The quarry supports about 20 temporary and

permanent small ponds (Sand-Jensen et al. 2010, Christensen et al. 2013). The

substratum in the quarry consists of exposed solid limestone pavements, which are

almost devoid of vegetation over large areas. The quarry which is surrounded by the

natural Alvar was abandoned about 30 years ago. The area is kept open by grazing

horses.

The local climate is quite dry (mean annual precipitation 510 mm; 1960-

1990), with moderately cold winters (January mean -1.2o C) and mild summers (July

mean 16.2o C) (SMHI 2013). The precipitation is evenly distributed throughout the

year (monthly mean range 32-54 mm), but temperature variations lead to large

seasonal differences in evapotranspiration and water availability (SMHI 2013).

Between April and August in 2010 local surface temperature on the exposed

limestone pavements exceeded 40o C on 37 days (Sand-Jensen & Jespersen 2012).

The drainage water from the limestone soils filling the shallow ponds has a high acid-

neutralizing capacity and a pH of 8.0 at air saturation (Sand-Jensen et al. 2010,

Christensen et al. 2013). The ponds have extremely low concentrations of soluble

36

inorganic nitrogen and phosphorus close to the limit of detection (Christensen et al.

2013).

A meteorological station was established next to the study pond 2.0 m

above the water surface. The station was equipped with sensors for incident

irradiance (HOBO PAR sensor (400-700 nm): S-LIA-M003, Onset Computers,

Bourne, MA, USA), wind speed and direction (HOBO anemometer and direction, S-

WSET-A, Onset Computers), air temperature and relative humidity (HOBO U23 Pro

v2, Onset Computers). Measurements were stored on a data logger (HOBO micro

station, H21-002, Onset Computers). A Swedish meteorological station is located at

Kalmar Airport 25 km away. This national station offered measurements of daily

precipitation, wind speed and direction when data collection failed for a short period

at our station. We used the regression equation established between the two sets of

measurements when both stations operated to convert Kalmar Airport measurements

to the pond setting. Wind speed was closely correlated (r2: 0.68, P < 0.0001) between

our station and the Kalmar station nearby.

Maximum water level, surface area and water volume in the pond are set

by overflow across its rim onto the adjacent Alvar plains. Water depths were

measured in a grid of 258 measurements across the pond surface. Water level in the

pond was measured at 10 minute intervals with an accuracy of 3 mm by recording

pressure differences between a submerged water level data logger (HOBO U-20-001-

04, Onset Computers) and a similar logger in air allowing continuous calculations of

water depth, surface area and water volume of the pond, while correcting for

atmospheric changes in barometric pressure.

From early March through May of 2014, maximum water depth dropped

from 0.59 to 0.30 m and surface area declined from 972 to 661 m2 (Fig. S1). The

maximum fetch across the pond in the main West-East wind direction ranged from 10

to 30 m. Water volume was reduced from early March through May from 343 to 99

m3 as a result of higher evaporation than precipitation (Fig. S1). Evaporation and

37

precipitation were the main components in the water balance during the period (data

not shown).

Sediments were dominated by fine mineral particles with a high content

of calcium carbonate (47% of dry mass) and a medium level of organic matter (10%)

(M. Andersen and K. Sand-Jensen, pers. comm. 2013). Sediments were deposited

directly on top of the hard limestone and varied in thickness from 4.0 to 9.5 cm

(mean 5.8 cm). The pond was covered by dense vegetation of charophytes. The

dominant species was Chara aspera and additional charophyte species included C.

vulgaris, C. virgata and C. globularis. Phanerogams comprised small populations of

Potamogeton crispus, Potamogeton natans and the emergent plants Phragmites

australis, Typha latifolia, Alisma plantago-aquatica and Alisma lanceolata.

Pond measurements

Vertical temperature and light profiles were measured in the middle of the pond at 8-

10 positions and about 5-cm depth intervals from the water surface to the sediment

surface using small temperature-light sensors (HOBO UA-002-64, Onset Computers)

logging the signals every 10 minutes. Measurements were conducted from March to

late May 2014. Sensors were calibrated relative to each other before and after use by

setting them up in shallow water in the pond in natural daylight (10-cm depth and no

vegetation) for 24 hours and subsequently correcting the response of the individual

sensors relative to the mean value of all sensors. Temperature readings were in full

agreement with measurements by a high-precision thermometer. Because the HOBO-

loggers work in steps of 0.14 °C, we judge this as the absolute accuracy. Irradiance

data through the water column were recorded in May and used in relative mode to

calculate light attenuation between sensors positioned at different depths.

Temperature-light sensors were mounted on a vertical steel peg rising

from a heavy steel plate buried in the sediment. Individual sensors were fastened to

thin 5-cm long plastic brackets keeping the sensors horizontal and pointing in

38

different directions to avoid internal shading and minimize influence on the natural

temperature, light and flow regimes.

Temperature profiles were used to construct isopleths of temperature with

depth and time and to calculate the relative thermal resistance to mixing (RTRM,

Wetzel (2001)). RTRM is the non-dimensional ratio between the difference in density

of bottom and surface water normalized to the difference in density between waters at

temperatures of 4.0 and 5.0 °C:

RTRM=(ρ

z2-ρ

z1)

(ρ4-ρ

5)

,

where ρ is specific mass density of water (kg m-3), ρz1 is for the surface water, ρz2 is ρ

at the bottom water and ρ4-ρ5 is the difference in specific mass density of water at 4

°C and 5 °C respectively. Specific mass density of water was calculated from water

temperature according to Bigg (1967).

Temperature profiles through the water column at the time of maximum

Wetzel stability were used to determine the depth interval in which the maximum

change in water temperature occurred. The mid-point of this depth interval is an

analogue to the focal depth of the thermocline (zThCline) used for lakes.

Light data within the water column were not recorded for March and

April, but for May light data were collected and profiles of daily irradiance with

depth (z, m) below the water surface (Ez) were integrated over the day and used to

determine the mean daily light attenuation coefficients (η, m-1) with depth below the

water surface (z) by linear regression analysis according to:

Ln (Ez) = -η*z.

The attenuation coefficient was used to calculate the depth (z10%) at which the

subsurface irradiance was reduced to 10 % according to:

z10% = 2.3*η-1.

Biomass samples were collected in the charophyte bed on three occasions. Six to ten

randomly located cores (inner diameter 10 cm) were placed over the vegetation and

39

gently pushed into the sediment. All above-ground charophyte material within the

cores was removed by hand, carefully rinsed and dry weight (DW) determined after

48 h at 105 °C. Mean biomass density was calculated both per unit surface area and

per unit water volume.

Air temperature, relative humidity, PAR-light and wind speed were

measured every minute and stored every 10 minutes. The first HOBO temperature-

light sensor which was fully submerged was used for surface water temperature. The

MatLab© version of Lake Heat Flux Analyzer (Woolway et al. 2015) was used to

calculate surface heat fluxes, net incoming short-wave radiation (Qsin), the reflected

component of short-wave radiation (Qsr), sensible heat flux (Qh), latent heat flux (Qe),

incoming long-wave radiation (Qlin), outgoing long-wave radiation (Qlout), net long-

wave heat flux (Qlnet; Qlin - Qlout) and total surface heat flux (Qtot), as well as the

dimensionless drag coefficient, CD, and the transfer coefficient for latent heat, CE.

The transfer coefficient for sensible heat was assumed equal to that for latent heat

(Zeng et al. 1998). Lake Heat Flux Analyzer calculates fluxes and transfer

coefficients from standard, established equations in the air-water literature, including

calculating the transfer coefficients, CD10 and CE10, at the standard reference height of

10 m. Turbulent flux equations are based on Zeng et al. (1998), incoming long-wave

radiation is modelled after Crawford and Duchon (1999) and Fresnel’s equation is

used to calculate the reflected solar radiation (Woolway et al. 2015). The wind

energy flux, referenced to 10 m, P10, was calculated following (Wüest et al. 2000) as

P10 = ρa*CD10*U103, where ρa is the density of air calculated as Verburg and

Antenucci (2010) and U10 is the wind speed at 10 m, calculated from the measured

wind speed and atmospheric stability using Lake Heat Flux Analyzer.

Results

Meteorological variables

Surface irradiance reached daily peaks at noon above 1000 µmol m-2 s-1 on most of

the investigated days from March to May (Fig. 1). Days of lower irradiance were

40

scattered throughout the period. Wind speed was moderate (< 4 m s-1) during 94% of

the time and only on 12 % of the days was the maximum wind speed above 6 m s-1.

Wind speed peaked in the afternoon to 2-2.5 times the nocturnal wind

speed, and there was no difference between months (Fig. 2). PAR light had slightly

lower midday peaks in March than May. Surface water temperatures followed air

temperatures but were consistently higher; both peaked in the afternoon and both

increased from March to May. The relative humidity dropped in the afternoon at

increasing air temperature and declined from March to April, but did not differ

between April and May.

Light attenuation and charophyte density

Irradiance was rapidly attenuated with depth in the pond because of the high biomass

density of charophytes. The mean daily light attenuation coefficient from just below

the water surface to 8 cm above the sediment surface was 10-25 m-1, resulting in

absorption of 90 % of subsurface irradiance in the upper 9-23 cm of the water column

(Fig. 3). The depth at which subsurface irradiance was reduced to 10 % (z10%)

shallowed significantly during May along with falling maximum water depth (z10% =

0.75 * zmax - 0.12; r2: 0.39, P < 0.001) because falling water depth led to the canopy of

the charophyte vegetation being closer to the pond surface. The pond water itself is

highly transparent with light attenuation coefficients of only about 0.5 m-1. The steep

reduction of irradiance with water depth is due to charophytes having high areal

biomasses of 642-773 g DW m-2 in March-May. Biomass density per unit volume

was 2275-2569 g DW m-3 in March-May corresponding to biomass specific light

attenuation coefficients of 6-10 m-1 (kg DW m-3)-1 after correcting for background

light attenuation in the water.

Surface fluxes

The pond heated up throughout the period with consistently positive daily

accumulated Qtot values (Fig. 4). Net incoming solar radiation followed the expected

diurnal cycle. Reflected short-wave radiation was greatest during mornings and

41

evenings, even though the incoming radiation was low at this time, as the low angle

of the incident light resulted in a large fraction being reflected (Fig. 4). The net long-

wave radiation (with positive values indicating heating i.e. Qlin - Qlout), being

dependent on both outgoing and incoming long-wave radiation and therefore

dependent on air temperature, water temperature, relative humidity and cloud cover

showed a relatively complex diel cycle, but one that did not change greatly between

day and night. Both latent and sensible heat fluxes (where positive values indicate

cooling), however, had dramatic diel cycles with much greater cooling during the

day, driven by the large increases in wind speed and water temperature and the

marked day-time reduction in relative humidity (Figs. 4 and S2). As the wind energy

flux is proportional to the cube of the wind speed it was far greater during the day

than the night (Fig. 4).

Transfer coefficients

The transfer coefficients for latent heat, referenced to 10 m, CE10, ranged between

0.7x10-3 and 4.4x10-3, with an average of 2.13xI0-3, while the drag coefficients,

referenced to 10 m, CD10, generally had lower maximum and average values but the

same minimum, ranging between 0.7x10-3 and 3.2x10-3, with an average of 1.76x10-3.

Throughout the period CD10 was 17.6 % lower than CE10 (Fig. S3). Both transfer

functions dropped at midday to 45-66 % of the maximum nocturnal values and both

were slightly higher in May than in April. A few exceptional measuring points lead to

March standing out as very different from the other two months because a hail and

snow storm passed the site in two of the measuring days greatly affecting the

atmospheric stability (Fig. 5).

Stratification

The RTRM did not exceed the threshold value for stratification of 50 in March, and

RTRM was only above 50 once in the first half of April (Fig. 6). For the last half of

April and all of May the RTRM exceeded the threshold every day, except for six

particularly cold and windy days. The RTRM dropped to negative values almost

42

every night. In May the RTRM exceeded 50 around 9 am, peaked early in the

afternoon at 200 and stayed above 50 until 8 pm. While the RTRM exceeded 50 in

May, the mixed depth was reduced to 25 % of the water column during the day but

the pond was fully mixed every night (Fig. 7). Although the RTRM did not exceed 50

in March, day-time surface temperatures were regularly a few degrees higher than

bottom temperatures (Figs 8 & 9) and calculations showed that the mixed depth did

not remain at 100 % of the water column throughout the day, but dropped to 87 % of

the water column around midday. The temperature differences between surface and

bottom waters exceeded 15 °C on some warm days in late May (Fig. 8). Surface

waters were consistently colder than bottom waters at night. Isopleth plots of

temperature clearly showed daily stratification and nocturnal cooling (Fig. 9), weak

or no stratification in late March, stratification on most days in late April and strong

stratification on most days in late May. Regardless of the strength of stratification the

water column was fully mixed every night.

Discussion

Daily stratification

Daytime stratification and nocturnal mixing were recurring phenomena in this small

and densely vegetated pond on the open alvar. The mixed depth during daytime was

below 100 % of the water column for all three months, but at 100 % every night and

there was a distinct temperature decline from surface to bottom waters during the day

with a reversal during the night. From mid-April to the end of May the cycle was

profound; relative thermal resistance to mixing (RTRM) was above the threshold

value of 50 during most of these days and the daytime temperature gradients rose to

over 15 °C in less than half a meter of water. The strength of the diel stratification

typically peaked around midday despite the wind speed also peaking then, clearly

demonstrating that wind-induced mixing was not sufficient to fully mix the pond.

This consistent pattern developed despite the shallow water and the wind-exposed

location. Calculations of reduced mixed depth during the day throughout the period,

43

even on days when RTRM was below 50, suggest that the suggested threshold value

for stratification at RTRM > 50 is too large, at least in shallow systems.

Gorham and Boyce (1989) derived an empirical equation for determining which lakes

will typically stratify based on their length (L), approximated as the square root of the

surface area, and their depth (H): H = 0.34L0.5; a lake would not be expected to

stratify unless its actual depth was greater than H predicted by this equation.

Although both surface area and depth varied in the pond in this study, by this

estimate the pond would always be significantly shallower than the expected depth, at

least 1.7 m, necessary for stratification. Indeed, the implication of this empirical fit is

that any pond less than a meter deep would have to have a smaller surface area than

75 m2 in order to be likely to stratify. While this equation is only an approximation,

nevertheless it is indicative that, a priori, ponds of the size studied here might not be

expected to stratify, particularly if situated in an exposed location. This is reiterated

by Branco and Torgersen (2009) who also found that it was unlikely for a small

wind-exposed pond to stratify. Gorham and Boyce (1989) and Branco and Torgersen

(2009) worked with lakes with no macrophyte cover, and indeed in the pelagic of

larger lakes macrophytes are of little or no influence. However, in the littoral zone or

in small ponds macrophytes can enhance light attenuation, increase surface warming

and reduce vertical mixing to a large extent. It has been well documented that an

increase in light attenuation, by affecting the depths at which incoming solar radiation

is absorbed, can substantially reduce mixed depths and increase stratification (e.g.

Kling 1988, Persson & Jones 2008, Gaiser et al. 2009). Lake studies have

documented that submerged macrophyte beds can increase temperature stratification

by strong light absorption (Dale & Gillespie 1977). In the present study high density

of charophytes through most of the water column was, no doubt, a key to the

development of strong density gradients and restriction of the mixed layer to the

uppermost part of the water column during the day.

44

Incident irradiance only penetrated a few centimeters into the charophyte

canopy. The depth at which 10 % of surface irradiance is left then makes a proper

estimation of the depth of the upper canopy. During May, when continuous irradiance

data were available, the thermocline depth was just 0-9 cm (average 5 cm) into the

charophyte canopy (Fig. 3). Dense charophyte vegetation resulted in a very uneven

distribution of incoming short-wave energy throughout the water column as almost

all the energy was absorbed in the upper 3-cm of the vegetation. These findings

accord with model predictions and empirical comparisons of temperature dynamics

between macrophyte beds and open water locations by Herb and Stefan (2005a, b).

Falling water depth throughout May brought the upper part of the charophyte canopy

and the main attenuation of short-wave energy closer to the surface and, thereby,

further inhibited wind-induced mixing and reduced the mixed layer depth. As the

pond stratified all of the incoming heat would be confined to the mixed layer and

additionally stabilize the water column.

Strong reduction of local flow velocities within dense macrophyte stands

and restriction of strong turbulence to the upper few cm of the canopy have been

documented in shallow streams (Sand-Jensen & Mebus 1996, Sand‐ Jensen &

Pedersen 1999). Effective dampening of local flow and turbulence by macrophyte

beds can also account for their ability to reduce mixing and thus stimulate particle

sedimentation and reduce resuspension (Barko & James 1998, Sand‐ Jensen 1998,

Vermaat et al. 2000). It is therefore likely that the macrophytes influenced the mixing

processes both through light attenuation and by inhibition of mechanical mixing.

Nocturnal mixing

The regular pattern of daytime stratification and nighttime mixing was driven by the

pronounced diel cycle in the heat fluxes and wind mixing. Solar and atmospheric

long-wave radiation heated the pond during the day, but the shallowness of the mixed

layer enabled surface temperatures to climb rapidly, promoting significant cooling

through outward long-wave and turbulent heat loss. Although winds calmed

45

markedly during the night, the temperature differential between water and air was

still sufficiently high to allow significant cooling to take place each night. The heat

loss cooled surface waters down to 1-5 °C below bottom waters temperatures and

produced an unstable water density profile resulting in penetrative convective mixing

(Imberger 1985) to the sediment surface. Whilst the wind energy flux would also

have some impact, it was an order of magnitude lower at night than during the day

(Fig. 4g), emphasizing the importance of the nocturnal cooling. The dominance of the

nocturnal cooling was in line with Read et al. (2012) who showed that for small lakes

penetrative cooling frequently generates more surface turbulence than wind shear.

High densities of submerged macrophytes have relatively little influence on mixing

processes by natural convection caused by surface cooling (Herb & Stefan 2005b).

Natural convection carries potential energy down from the surface via plunging

thermals and although submerged macrophyte surfaces may reduce the kinetic energy

generated by the plunging downward flow, the mixing depth is not greatly influenced

(Herb & Stefan 2005b). The sinking plumes imply the existence of coherent rising

plumes because of the continuity of mass, and the surface layer is subject to intense

stirring (Imboden & Wüest 1995). According to Deardorff et al. (1969) and Wüest

(1987), sinking water parcels still have part of their kinetic energy left (e.g. 30%)

when they reach the bottom of the mixed layer and can, therefore, penetrate the

density gradient and push heavier water from below into the mixed layer leading to

its deepening. This situation is very different from the influence of macrophytes on

turbulence induced by wind shear on the water surface which must penetrate the

canopy from above via undirected isotropic eddies which are effectively dissipated by

contact with macrophyte surfaces (Sand‐ Jensen & Pedersen 1999, Herb & Stefan

2005b). Because the energy of sinking plumes is more directed than that of wind-

induced mixing, plumes generated by surface cooling generally have greater mixing

efficiency (Imboden & Wüest 1995).

46

A further impact of the temperature increases associated with heating being

concentrated in a shallow surface layer was for there to be a strongly unstable

atmosphere above the pond. This resulted in transfer coefficients being higher than

often reported (see Verburg & Antenucci 2010), which, in turn, contributed to

increased wind mixing and turbulent heat loss. The transfer coefficients, CD10 and

CE10, were also around 25 % higher during the night than the day (Fig.5), owing to

the low nocturnal wind speeds, unstable atmospheric conditions and smooth flow

conditions (see Verburg & Antenucci, 2010). This nighttime increase in transfer

coefficients was a further factor driving the nocturnal mixing processes.

Implications of stratification-mixing patterns

The dynamics of vertical mixing is crucial because of its overriding impact on

physical, chemical and biological conditions (Branco et al. 2005). The recurring

daytime stratification and nighttime mixing is not unique for the shallow open ponds

with dense charophyte vegetation studied here. It can also develop in the littoral zone

with submerged vegetation of large lakes (Herb & Stefan 2005a, Herb & Stefan

2005b, Coates & Folkard 2009) as well as in small, wind-protected lakes devoid of

submerged macrophytes where dense growth of phytoplankton or humic water lead to

strong vertical light attenuation and surface warming (Gu et al. 1996, Ford et al.

2002, Song et al. 2013).

Temperature itself is a key variable for the distribution and metabolic

activity of all organisms. In our study pond, sessile organisms and macrophytes were

exposed to diel temperature amplitudes of 1.9°C-18.9°C in the surface waters and

0.5°C-6.1°C in the bottom waters between late March and the end of May. This

highly variable environment is both a challenge to survival and a trigger of highly

variable metabolic rates with time. Assuming that only the temperature range of 17.0

°C influences metabolism, then for typical Q10-values of 1.5-3 metabolic rates can be

expected to change between 2 and 8-fold.

47

The diurnal stratification and mixing of the shallow lakes and ponds are

accompanied by profound vertical dynamics of pH, oxygen, nutrients and redox

potentials (Gu et al. 1996, Ford et al. 2002, Branco et al. 2005, Song et al. 2013). In

our study pond, for example, daytime stratification is accompanied by anoxia and

accumulation of CO2, sulphide and reduced ferrous-Fe in the lower 10 cm of the

water column, while oxygen reappears and sulphide and ferrous-Fe disappear in the

bottom waters during nocturnal mixing (Andersen et al. 2015). Macrophytes and the

sessile fauna in the sediment and in the lower part of the water column do not only

have to withstand profound diel temperature excursions but also alternating oxic-

anoxic and oxidized-reduced conditions.

Small water bodies are far more common world-wide than the iconic, charismatic

lakes often studied (Downing et al. 2006). Their temperature, mixing, and

stratification dynamics, which drive the lake ecology, are therefore of great interest; a

huge number of them will have complex thermal patterns resulting in very dynamic

stratification and mixing mechanisms as documented here.

A priori, it is not obvious that a pond, such as the one studied here, should stratify at

all. Almost certainly this regular diel stratification is promoted by the macrophyte

presence both influencing the vertical absorption of solar radiation and the

mechanical mixing within the pond. The macrophytes are thereby profoundly

influencing their own environment.

References Andersen, M. R., Kragh, T., and Sand-Jensen, K. 2015. Recurring stratification and mixing generate extreme diurnal oxygen and carbon cycles in shallow vegetated lakes. in prep. Barko, J. W., and James, W. F. 1998. Effects of submerged aquatic macrophytes on nutrient dynamics, sedimentation, and resuspension. In: E. Jeppesen, M. Sondergaard, M. Sondergaard and K. Christoffersen (eds.). The structuring role of submerged macrophytes in lakes. Springer, New York. 423 p. : 197-214. Bastviken, D., Tranvik, L. J., Downing, J. A., Crill, P. M., and Enrich-Prast, A. 2011. Freshwater methane emissions offset the continental carbon sink. Science 331:50-50. Bigg, P. 1967. Density of water in SI units over the range 0-40 C. British Journal of Applied Physics 18:521. Boehrer, B., and Schultze, M. 2008. Stratification of lakes. Reviews of Geophysics 46.

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Branco, B., Torgersen, T., Bean, J. R., Grenier, G., and Arbige, D. 2005. A new water column profiler for shallow aquatic systems. Limnology and Oceanography: Methods 3:190-202. Branco, B. F., and Torgersen, T. 2009. Predicting the onset of thermal stratification in shallow inland waterbodies. Aquatic Sciences-Research Across Boundaries 71:65-79. Christensen, J. P. A., Sand-Jensen, K., and Staehr, P. A. 2013. Fluctuating water levels control water chemistry and metabolism of a charophyte-dominated pond. Freshwater Biology 58:1353-1365. Coates, M. J., and Folkard, A. M. 2009. The effects of littoral zone vegetation on turbulent mixing in lakes. Ecological Modelling 220:2714-2726. Coloso, J. J., Cole, J. J., and Pace, M. L. 2011. Short-term variation in thermal stratification complicates estimation of lake metabolism. Aquatic Sciences 73:305-315. Crawford, T. M., and Duchon, C. E. 1999. An improved parameterization for estimating effective atmospheric emissivity for use in calculating daytime downwelling longwave radiation. Journal of Applied Meteorology 38:474-480. Dale, H., and Gillespie, T. 1977. The influence of submersed aquatic plants on temperature gradients in shallow water bodies. Canadian Journal of Botany 55:2216-2225. Deardorff, J. W., Willis, G. E., and Lilly, D. K. 1969. Laboratory investigation of non-steady penetrative convection. Journal of Fluid Mechanics 35:7-31. Downing, J., Prairie, Y., Cole, J., Duarte, C., Tranvik, L., Striegl, R., McDowell, W., Kortelainen, P., Caraco, N., and Melack, J. 2006. The global abundance and size distribution of lakes, ponds, and impoundments. Limnology and Oceanography 51:2388-2397. Folkard, A. M., Sherborne, A. J., and Coates, M. J. 2007. Turbulence and stratification in Priest Pot, a productive pond in a sheltered environment. Limnology 8:113-120. Ford, P. W., Boon, P. I., and Lee, K. 2002. Methane and oxygen dynamics in a shallow floodplain lake: the significance of periodic stratification. Hydrobiologia 485:97-110. Gaiser, E. E., Deyrup, N. D., Bachmann, R. W., Battoe, L. E., and Swain, H. M. 2009. Effects of climate variability on transparency and thermal structure in subtropical, monomictic Lake Annie, Florida. Fundamental and Applied Limnology/Archiv für Hydrobiologie 175:217-230. Gorham, E., and Boyce, F. M. 1989. Influence of lake surface area and depth upon thermal stratification and the depth of the summer thermocline. Journal of Great Lakes Research 15:233-245. Gu, R., Luck, F. N., and Stefan, H. G. 1996. Water Quality Stratification In Shallow Wastewater Stabilization Ponds. Wiley Online Library. Herb, W. R., and Stefan, H. G. 2005a. Dynamics of vertical mixing in a shallow lake with submersed macrophytes. Water Resources Research 41. Herb, W. R., and Stefan, H. G. 2005b. Model for wind-driven vertical mixing in a shallow lake with submersed macrophytes. Journal of Hydraulic Engineering 131:488-496. Imberger, J. 1985. The diurnal mixed layer. Limnology Oceanography 30:737-770. Imberger, J., and Hamblin, P. 1982. Dynamics of lakes, reservoirs, and cooling ponds. Annual Review of Fluid Mechanics 14:153-187. Imboden, D. M., and Wüest, A. 1995. Mixing mechanisms in lakes. Pages 83-138. Physics and Chemistry of Lakes. Springer. Kling, G. W. 1988. Comparative transparency, depth of mixing, and stability of stratification in lakes of Cameroon, West Africa. Limnology and Oceanography 33:27-40. Lofgren, B. M., and Zhu, Y. 2000. Surface energy fluxes on the Great Lakes based on satellite-observed surface temperatures 1992 to 1995. Journal of Great Lakes Research 26:305-314. Persson, I., and Jones, I. D. 2008. The effect of water colour on lake hydrodynamics: A modelling study. Freshwater Biology 53:2345-2355. Read, J. S., Hamilton, D. P., Desai, A. R., Rose, K. C., MacIntyre, S., Lenters, J. D., Smyth, R. L., Hanson, P. C., Cole, J. J., and Staehr, P. A. 2012. Lake‐size dependency of wind shear and convection as controls on gas exchange. Geophysical Research Letters 39.

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Read, J. S., and Rose, K. C. 2013. Physical responses of small temperate lakes to variation in dissolved organic carbon concentrations. Limnology and Oceanography 58:921-931. Sand-Jensen, K., Baastrup-Spohr, L., Winkel, A., Møller, C. L., Borum, J., and Brodersen, K. P. 2010. Ett kalkbrott på Ölands alvar. Svensk Botanisk Tidskrift 104:23-30. Sand-Jensen, K., and Jespersen, T. S. 2012. Tolerance of the widespread cyanobacterium Nostoc commune to extreme temperature variations (-269 to 105 degrees C), pH and salt stress. Oecologia 169:331-339. Sand-Jensen, K., and Mebus, J. R. 1996. Fine-scale patterns of water velocity within macrophyte patches in streams. Oikos:169-180. Sand‐Jensen, K. 1998. Influence of submerged macrophytes on sediment composition and near‐bed flow in lowland streams. Freshwater Biology 39:663-679. Sand‐Jensen, K., and Pedersen, O. 1999. Velocity gradients and turbulence around macrophyte stands in streams. Freshwater Biology 42:315-328. SMHI 2013. Swedish Meteorological and Hydrological Institute. http://www.smhi.se/klimatdata/meteorologi/temperatur/dataserier-med-normalvarden-1.7354. Song, K., Xenopoulos, M. A., Buttle, J. M., Marsalek, J., Wagner, N. D., Pick, F. R., and Frost, P. C. 2013. Thermal stratification patterns in urban ponds and their relationships with vertical nutrient gradients. Journal of Environmental Management 127:317-323. Staehr, P. A., Bade, D., Van de Bogert, M. C., Koch, G. R., Williamson, C., Hanson, P., Cole, J. J., and Kratz, T. 2010. Lake metabolism and the diel oxygen technique: State of the science. Limnology and Oceanography: Methods 8:628-644. Vad, C. F., Horvath, Z., Kiss, K. T., Toth, B., Pentek, A. L., and Acs, E. 2013. Vertical distribution of zooplankton in a shallow peatland pond: the limiting role of dissolved oxygen. Annales de Limnologie-International Journal of Limnology 49:275-285. Verburg, P., and Antenucci, J. P. 2010. Persistent unstable atmospheric boundary layer enhances sensible and latent heat loss in a tropical great lake: Lake Tanganyika. Journal of Geophysical Research: Atmospheres (1984–2012) 115. Vermaat, J. E., Santamaria, L., and Roos, P. J. 2000. Water flow across and sediment trapping in submerged macrophyte beds of contrasting growth form. Archiv für Hydrobiologie 148:549-562. Wetzel, R. 2001. Limnology, lake and river ecosystems. Academic. San Diego. Woolway, R. I., Jones, I. D., Hamilton, D. P., Maberly, S. C., Muraoka, K., Read, J. S., Smyth, R. L., and Winslow, L. A. 2015. Automated calculation of surface energy fluxes with high-frequency lake buoy data. Environmental Modelling & Software 70:191-198. Wüest, A. 1987. Ursprung und Grösse von Mischungsprozessen im Hypolimnion natürlicher Seen. Diss. Naturwiss. ETH Zürich, Nr. 8350, 1987. Wüest, A., Piepke, G., and Van Senden, D. C. 2000. Turbulent kinetic energy balance as a tool for estimating vertical diffusivity in wind‐forced stratified waters. Limnology and Oceanography 45:1388-1400. Zeng, X., Zhao, M., and Dickinson, R. E. 1998. Intercomparison of bulk aerodynamic algorithms for the computation of sea surface fluxes using TOGA COARE and TAO data. Journal of Climate 11:2628-2644.

Figure legends.

Figure 1.

Surface irradiance (a), air temperature (b), relative humidity (c) and wind speed (d)

measured next to the pond at 2.0 m above ground level during the investigation.

50

Figure 2.

Diel averages of wind speed (a), surface irradiance (b), air temperature (c), relative

humidity (d) and surface water temperature (e) for March (blue line), April (green

line) and May (red line).

Figure 3.

Daily mean light attenuation coefficients in the pond from immediately below the

surface to 8 cm above the sediment surface (dotted line), daily mean depth to which

10 % of subsurface light remains (white surface) and daily mean focal depth of the

thermocline (full line). The sediment surface is shown in black.

Figure 4.

Diel averages of incoming short-wave radiation (a), reflected short-wave radiation

(b), net long-wave radiation (c), latent heat flux (d), sensible heat flux (e),total heat

flux (f) and wind energy flux (g) for March (blue line), April (green line) and May

(red line).

Figure 5.

Diel averages of transfer coefficients (dimensionless) for March (blue line), April

(green line) and May (red line). CD10 (a) and CE10 (b).

Figure 6.

Relative thermal resistance to mixing (RTRM) calculated during the investigation.

Threshold value for onset of stratification (RTRM = 50) and no relative thermal

resistance to mixing (RTRM = 0) shown as dotted lines.

Figure 7.

Diel averages of relative thermal resistance to mixing (RTRM) (a) and mixed depth

(percentage of the water column) (b) for March (blue line), April (green line) and

May (red line).

Figure 8

Surface water temperature (dashed line) and bottom water temperature (bold line) in

the pond during the investigation.

51

Figure 9

Daily time course of water temperature with depth in the pond during 5 days in late

March, April and May based on measurements at 5-cm depth intervals every 10

minutes. The white area (25th May) marks a period when sensors were retrieved and

redeployed.

Supplementary figure legends

Figure S1.

Temporal changes in surface area (dashed line) and water volume of the pond (full

line; upper panel) and maximum water depth (dotted line) and precipitation (columns;

lower panel).

Figure S2.

Heat fluxes calculated during the investigation. Incoming short-wave radiation (a),

reflected short-wave radiation (b), net long-wave radiation (c), latent heat flux (d),

sensible heat flux (e) and total heat flux (f).

Figure S3.

Transfer coefficients (dimensionless) calculated during the investigation. CD10 (top

panel) and CE10 (bottom panel).

52

Figures

Figure 1

0

1000

2000

3000

PA

R (

µm

ol m

-2 s

-1)

-5

0

5

10

15

20

25

Air

tem

pera

ture

(C

)

March0

2

4

6

April May

Win

d s

peed

(m

s-1

)

20

40

60

80

0

Rela

tive h

um

idit

y (

%)

a

b

c

d

53

Figure 2

Figure 3

0

1

2

3

03 06 09 12 15 18 21 2400

Time of Day

Win

d s

peed

(m

s-1

)

0

500

1000

1500

03 06 09 12 15 18 21 2400

Time of Day

PA

R (

µm

ol m

-2 s

-1)

0

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10

15

20

03 06 09 12 15 18 21 2400

Time of Day

Air

tem

p.

( C

)

50

60

70

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90

100

03 06 09 12 15 18 21 2400

Time of Day

Rela

tive h

um

idit

y (

%)

0

5

10

15

20

25

03 06 09 12 15 18 21 2400

Time of Day

Su

rf.

wate

r te

mp

. (°

C)

a b c

d e

54

Figure 4

Figure 5

0

200

400

600

800

03 06 09 12 15 18 21 2400

Time of Day

Qsin

(W

m-2

)

0

20

40

60

03 06 09 12 15 18 21 2400

Time of Day

Qsr

(W m

-2)

-150

-100

-50

0

03 06 09 12 15 18 21 2400

Time of Day

Qln

et (W

m-2

)

0

50

100

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200

03 06 09 12 15 18 21 2400

Time of Day

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Figure 6

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Figure 9

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57

Supplementary figures

Figure S1

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Figure S2

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60

Paper 2 - Recurring stratification and mixing generate extreme diurnal oxygen and carbon cycles in shallow

vegetated lakes

61

Recurring stratification and mixing generate extreme diurnal oxygen and

carbon cycles in shallow vegetated lakes

Mikkel Rene Andersen, Theis Kragh and Kaj Sand-Jensen

Freshwater Biological Laboratory, Biological Institute, University of Copenhagen,

Universitetsparken 4, DK-2100 Copenhagen Denmark

Target Journal: Nature Communications

Environmental conditions in small lakes (< 1 ha) have been grossly understudied

although they globally exist in millions and are several-fold more abundant than

larger lakes1,2. Vertical stratification-mixing patterns are main determinants of

biogeochemistry and metabolism of organisms3, but small, shallow lakes have

usually been assumed to be homogeneously mixed, though with little

justification4. Here we show that a small, wind-exposed, shallow (ca 0.4 m) lake

with submerged macrophytes underwent recurring daytime stratification and

nocturnal mixing during summer accompanied by extreme variations in

temperature, oxygen, pH and inorganic carbon with time and depth. During

daytime stratification, surface waters attained 230 % oxygen saturation and strong

CO2 depletion (<10 % air saturation), while 6-14 oC colder bottom waters

developed anoxia and accumulated reduced iron, sulphide and >1500 % CO2

saturation. High daytime pH in surface waters induced carbonate precipitation

releasing CO2 for ongoing photosynthesis without further pH rise, while most

precipitated CaCO3 was re-dissolved in bottom waters. Vertical gradients

disappeared during nocturnal mixing injecting oxygen into bottom waters for

aerobic respiration and regenerated inorganic carbon into surface waters for

62

photosynthesis. These processes add new dimensions to our understanding of the

regulation of ecosystem photosynthesis and respiration and the adaptation of

sessile plants and mobile animals to the extreme variability of environmental

stressors.

Turbulent mixing of the water column in shallow lakes is expected to prevent

formation of vertical gradients of temperature, oxygen and solutes. Hydraulic models

predict that small, wind-exposed temperate lakes (1000 m2) should be deeper than 3

m to become vertically stratified5. While no vertical gradients are expected to form,

intense daytime photosynthesis and nocturnal respiration may generate profound

diurnal cycles of temperature and solutes6,7. It was, therefore, unexpected that a 0.4

m-deep lake underwent strong temperature stratification during daytime and

nocturnal mixing. Here we show that recurring stratification and mixing, because of

the presence of submerged macrophytes, generate extreme vertical and diurnal cycles

of pH, inorganic carbon and oxygen - including anoxia and reducing conditions in

bottom waters during daytime and oxic conditions at night. This overlooked

dynamics should be common in small vegetated lakes and within macrophyte beds in

large lakes and adds new dimensions to our understanding of biogeochemical cycles,

macrophyte adaptation and animal behavior.

We recently discovered an unexpected behavior of the great pond snail

(Lymnaea stagnalis) in shallow lakes dominated by characean macroalgae during

summer in open south-Swedish habitats8. The snails concentrated in surface waters

before noon to ventilate the vascularized lung with atmospheric air as if they rapidly

needed to recover from a threatening oxygen debt. A similar behavior has been

described for dragonfly larvae actively ventilating the gills in the rectum with

63

atmospheric air9. Oxygen concentrations were high in the surface waters before noon8

and it was unclear where the snails had developed the suggested oxygen debt that led

to their awkward behavior and risky exposure to predators. The environmental

conditions should be highly variable considering the high macrophyte density in the

shallow water. To document the conditions, we measured light, temperature, oxygen,

pH and conductivity at high vertical resolution in one of the shallow lakes during

weeks in May and August along with meteorological parameters. During a diurnal

cycle, we also determined the vertical distribution of dissolved inorganic nutrients,

sulphide, ferrous iron, calcium, total (DIC) and individual carbon species (i.e., DIC =

CO2 + HCO3- + CO3

2-) closely linked to photosynthesis and respiration. Acid

neutralizing capacity (ANC = HCO3- + 2 CO3

2- + OH- - H+) was measured directly

during the diurnal cycle and was calculated from conductivity during longer periods

because HCO3- is the main determinant of ANC and the dominant anion is closely

linearly related to conductivity (Suppl. Fig. S1).

The shallow lake developed strong daytime vertical stratification of

temperature and specific density on four days during the week in May (Fig. 1) and all

examined days in August (Fig. 2). No vertical stratification formed on two cold

windy days in May (Fig. 1). At night, the water column was always fully mixed.

Maximum daytime differences between temperatures in surface and bottom waters on

days with vertical stratification were 11.2-14.4oC in May and 6.2-8.9oC in August

(Suppl. table S1). The steepest vertical temperature gradient (0.3-1.9oC cm-1) formed

at 14.5-18.5-cm depth in May and 15.9-22.5-cm in August. This exceptionally strong

daytime vertical stratification can be explained by the dense charophyte vegetation

(5685-11340 g fresh weight m-3) reaching up to from the bottom to a few cm below

the water surface and effectively attenuating both the radiative heat flux and the

turbulent wind-driven mixing with depth. Thus, 90% of the daytime radiative heat

flux was absorbed within the upper 11-cm of water column in May (Suppl. table. S2).

The lake water was transparent and the vegetation was responsible for most (98%) of

64

the vertical light attenuation. Water turbulence generated by wind shear on the lake

surface must penetrate the macrophyte canopy from above via undirected isotropic

eddies which are effectively dissipated by contact with macrophyte surfaces10,11,

thereby, preventing full mixing. On two cold and windy days in May turbulence was

sufficiently strong to disrupt the formation of vertical temperature stratification. At

night, cooling of surface waters to 0.1-4.1 oC below bottom temperatures produced

inverse unstable water density profiles resulting in penetrative convective mixing of

the entire shallow water column. Submerged macrophytes, even of high density, have

relative little influence on convective mixing caused by surface cooling12. Natural

convection carries potential energy down from the surface via directed plunging

thermals and although submerged macrophyte surfaces may reduce the kinetic energy

generated by the plunging downward flow, the mixing depth is not greatly

influenced10,11. The sinking plumes induce coherent rising plumes because of the

continuity of mass resulting in intense mixing13. Because the daytime heat flux is

absorbed within a very thin surface layer it becomes markedly warmer than the air

(Suppl. Fig. S2) and is cooled that more strongly during the evening and night

inducing effective convective mixing.

Alternating daytime stratification and nocturnal mixing and steep vertical

attenuation of photosynthetic irradiance can account for the astonishing vertical and

temporal dynamics of oxygen, pH, ANC and inorganic carbon species (Figs 1 and 2).

Oxygen and pH in surface waters (2, 9 and 16 cm) followed the same diurnal course

on six days in May according to the balance between photosynthesis and respiration.

Maximum values (205-235 % O2 saturation, pH 9.1-9.6) were recorded in the early

afternoon and minimum values (12-25 % O2 saturation, pH 7.1-8.0) shortly after

sunrise. The lowest oxygen concentrations at sunrise were observed after warm

nights because nocturnal respiration increased with temperature14. During vertical

mixing, oxygen and pH at 23 cm below the water surface followed the same pattern

as at the surface. During stratification, in contrast, oxygen dropped to zero and pH to

65

7.0-7.5 at 23 cm water depth because of respiratory oxygen consumption and CO2

release. Intermittent vertical stratification (at noon, May 28) led to immediate decline

of oxygen and pH showing that respiration was much higher than photosynthesis at

this depth.

Continued photosynthesis and rising pH in the surface waters from

morning to afternoon were accompanied by falling DIC, ANC and free CO2 and

increasing CO32- as a result of coupled photosynthesis and calcification (i.e., Ca2+ + 2

HCO3-→ CaCO3 (precipitated) + CO2 (assimilated)15. The decline of ANC in surface

waters could either be due to CaCO3 precipitating directly on charophyte surfaces

coupled to photosynthesis and/or to formation and sinking of minute calcite crystals

in the water because of high pH and CO32- (i.e. HCO3

- + OH-→ CO32-)16. The calcite

saturation index, representing the ionic molar product of Ca2+ and CO32- relative to

the solubility product at ambient temperature3,16, was high in the afternoon on all days

(i.e. >10) and should promote calcite formation (Fig. 2). Daytime loss of ANC in

surface waters was apparently due to precipitation and sinking of calcite crystals

which were re-dissolved in bottom waters because of high CO2 concentrations

resulting in accumulation of DIC and ANC until vertical mixing in the evening and at

night. This interpretation was supported by measurements of diurnal cycles and

vertical chemical profiles (Fig. 3).

Diurnal cycles resembled each other very closely during all examined

days in August (Fig. 2). Following minimum oxygen concentrations at all depths

shortly after sunrise, oxygen concentration rapidly increased in surface waters as

temperature stratification was established, while oxygen simultaneously declined in

bottom waters turning fully anoxic around midday and staying anoxic for the next 12

hours until midnight before effective convective mixing generated homogeneous

physico-chemical conditions again. Surface water pH was also lowest (about 8)

shortly after sunrise and highest (9.4-9.6) in the afternoon after many hours of

photosynthesis and was accompanied by DIC and ANC decline, drop of CO2 to

66

almost zero and increase of CO32-. Daytime loss of ANC in surface waters caused by

CaCO3 precipitation averaged 0.57 meq L-1 on five consecutive days, but 0.51 meq L-

(90%) had returned to surface waters on the next day following vertical mixing

because most CaCO3 precipitated from surface waters during daytime photosynthesis

was solubilized in bottom waters with low pH and excess CO2.

This interpretation of carbon dynamics was confirmed by direct analyses

of the vertical distribution of ANC and Ca2+ during a full diurnal cycle (Fig. 3).

Daytime decline of ANC and Ca2+ in surface waters by calcification was

accompanied by increasing concentrations in bottom waters by carbonate dissolution.

The latter process continued throughout the night giving rise to increasing

concentrations of ANC, Ca2+ and DIC in the water column until photosynthesis

picked up after sunrise. These tightly coupled vertical and diurnal cycles of

precipitation and dissolution of carbonates have not been disclosed before. The

implications are very important because daily calcification delivers protons and

ensures conversion of HCO3- to free CO2 for continued photosynthesis preventing pH

to rise to strongly inhibiting levels (i.e., Ca2+ + 2 HCO3-→ CaCO3 + CO2)

8,15, while

direct HCO3- use without calcification leads to release of OH- and continued pH rise

(HCO3- → CO2 + OH-)17. The daytime net decline of DIC in surface waters averaged

0.77 mM, and 0.48 mM of this amount represented direct uptake and 0.29 mM

represented CO2 use coupled with CaCO3 precipitation of the same magnitude. Most

of the daytime decline of the DIC pool (91%) was restored by respiration and

carbonate dissolution before the next morning. Sediment incubations confirmed this

pattern showing the release of two moles of DIC for every mole of O2 consumed in

the process, because of concomitant respiration and CaCO3 dissolution (org. C + O2 +

CaCO3 → Ca2+ + H2O + 2HCO3-, Sand-Jensen, Petersen, Kragh & Andersen pers.

comm.).

The contrast to deeper lakes is stunning. In deep lakes temperature

stratification is permanent during several summer months with surface waters

67

gradually being depleted and bottom waters gradually enriched in DIC, ANC and

Ca2+ 16,18. The injection of these soluble substances are delayed until autumn overturn

after light has become limiting to photosynthesis thereby preventing the stimulation

of photosynthesis that takes place daily by nocturnal mixing in the shallow lake.

Anoxic conditions in bottom waters after midday on days of vertical

temperature stratification had further consequences (Fig. 3). During anoxia, sulphide

and ferrous iron accumulated because of reduction of sulphate and ferric iron served

as electron acceptors during anaerobic respiration19. Sulphide accumulation in

bottoms waters was small, while accumulation of ferrous iron was larger and

continued throughout the stratification period showing that ferric iron was a more

important electron acceptor than sulphate during anoxia. Sulphide and ferrous iron

were re-oxidized following vertical mixing when oxygen was reintroduced to the

bottom waters. Ammonium accumulated to a small extent in the bottom waters

during temperature stratification, while concentrations of nitrate and phosphate

remained close to the limits of detection (< 0.3 µM, data not shown) stressing the

oligotrophic nature of the calcareous lake.

This study adds new dimensions to the understanding of environmental

conditions and biogeochemistry in shallow vegetated lakes, in that recurring vertical

stratification during daytime and nocturnal mixing generated unexpected and

profound diurnal cycles of oxygen, pH and inorganic carbon species. Shallow lakes

in open habitats have hitherto been considered to be permanently mixed and if

profound oxygen depletion developed it was confined to heavily organically polluted

lakes and to nocturnal periods during degradation of algal blooms in hypereutrophic

lakes2,3. Our study showed that temperature stratification and mixing were recurring

diurnal phenomena during summer in a shallow vegetated lake because dense

charophyte stands strongly attenuated depth penetration of light and wind-driven

turbulence during the day, while surface cooling at night induced penetrative

convective mixing. Daytime decoupling between photosynthesis at high irradiance in

68

the upper canopy and respiration in darkness in the lower canopy generated oxygen

accumulation and DIC depletion in surface waters and anoxia and DIC accumulation

in bottom waters, while nocturnal mixing injected regenerated DIC for

photosynthesis in surface waters and oxygen for oxygenic respiration in bottom

waters. If regenerated DIC had been trapped in stagnant bottom water, charophyte

photosynthesis would become severely constrained. Despite the oligotrophic

environment and low growth rates typical of charophytes20, profound diurnal cycles

of oxygen, pH and DIC, nonetheless, developed in the lake because of gradual

development of a substantial charophyte-biomass attaining high metabolic rates of

the community8.

Concerning adaptations to the extreme environmental variability, basal

parts of charophyte tissues exposed to bottom waters would have to withstand up to

12 hours of anoxia and accumulation of potentially toxic sulphide and ferrous iron

every day, while apical tissues experienced wider diurnal amplitudes in temperature

and oxygen, though no anoxia. Charophytes, in contrast to flowering plants, lack air

lacunae for longitudinal oxygen transport from apical to basal tissues located in

anoxic water and rhizoids in anoxic sediments. How they cope with this metabolic

challenge is not known, though anoxic fermentation is most likely. Preliminary

experiments show that basal parts are indeed alive and have retained their

photosynthetic and respiratory activity (data not shown). Mobile animals can move

over short distances in the shallow lake to escape from the worst environmental

stress21. For example, vertical upward movement from 23 to 16 cm below the surface

in the lake during daytime stratification in May would be sufficient to escape anoxia.

In order to unravel whether the large facultative air-breathing Lymnaea snails had

suffered from earlier exposure to anoxia and toxic reduced ions in the bottom waters

or low oxygen concentrations in surface waters at sunrise, we would need to track

their position over time. This requires new technology not yet available.

69

More generally, our findings of extreme vertical and diurnal variability of

temperature, oxygen, pH and solutes should be widespread in shallow vegetated lakes

that are found in millions throughout the world. Small lentic water bodies (< 1 ha)

have been grossly understudied, although they are 100-fold more abundant than

larger (> 10 ha) intensely studied lakes1,2. The preference for studying large lakes

have given us a wrong perception of environmental conditions, species adaptations

and ecosystem processes for the natural range of lentic water bodies22,23. Our findings

suggest that the evolutionary processes and the geographical dispersal of organisms

in freshwaters could be particularly important in small lakes because they are highly

abundant and variable24 and the environmental conditions can be extremely

challenging and drive adaptation to high and globally rising temperatures and oxygen

stress25.

Methods summary

The study was conducted in one of several small, shallow lakes located in the

sparsely vegetated calcareous grassland at Greby on Öland, SE Sweden (56.81168°N,

16.6094°E)8. The examined lake varied in surface area from 630-745 m2 in late-May

to 825-859 m2 in mid-August and maximum depth from 0.30 to 0.46 m. According to

methods described previously26, meteorological parameters (incident light, wind

speed and temperature) were measured next to the lake at 2.0 m above the ground and

vertical profiles of light and temperature were measured at the deepest site at 5 cm

depth intervals at 10 minutes intervals. Depth profiles of dissolved oxygen and pH

were measured at 7cm depth intervals at 1 minute time intervals logging the mean

signal every 10 minutes. Conductivity was also measured at 10-minutes intervals and

corrected to 20 °C.

During a diurnal cycle, water samples were collected at 7 cm depth

intervals and analyzed for DIC27, ANC, calcium, ferrous iron, sulphide, ortho-

phosphate, nitrate and ammonium by standard methods28. Weekly surface samples for

70

measurements of ANC (meq. L-1), pH and specific conductivity (Cond, µ Siemens

cm-1) were used to construct closely linear relationships of conductivity to total ANC

(= HCO3- + 2 CO3

2- + OH- + H+): total ANC = 0.009399 * Cond - 0.1410 (r2 = 0.72).

This relationship enabled continuous estimates of DIC and proportions of individual

carbon species from measurements of temperature, pH and specific conductivity29.

71

28

0.02 m

0.09 m

0.16 m

0.23 m

100

200

300

400

500

600

O2 (

M)

7

8

9

10

pH

0.5

1.0

1.5

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0.3

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2.5

0.5

0.6

0.7

0.8

DIC

CO2 CO32-

DIC

(m

M)

CO

32- / C

O2 (m

M)

0.0

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1.0

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2.0

2.5

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0.2

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25 May

0 12

26 May

0 12

27 May

0 12

28 May

0 12

29 May

0

DIC

(m

M)

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32- / C

O2 (m

M)

DIC

CO2

CO32-

0.2

0W

ate

r d

ep

th (

m)

5 C

10 C

15 C

20 C

25 C

30 C

35 C

0.1

a

b

c

d

e

0.02 m

0.23 m

72

Fig. 1.Time series of temperature, O2, pH, ANC, DIC and individual carbon

species with depth in a shallow charophyte lake during six days in May.

a, Temperature isopleths calculated from measurements at 5-cm depth intervals.

b, c, Oxygen and pH measured at 0.02 m (dark blue),0.09 m (light blue), 0.16 m

(green) and 0.23 m (red) below the water surface.

d, DIC (red), CO32- (orange) and CO2 (blue) in surface waters (0.02 m).

e, DIC (red), CO32- (orange) and CO2 (blue) in deeper waters (0.23 m).

Where b-e background color show day/night cycle (white = day, grey = night).

73

Fig. 2. Time series of temperature, O2, pH, ANC, calcite saturation index, DIC,

individual carbon species with depth in a shallow charophyte-lake during six

days in August.

0.08 m

0.24 m

0.34 m

100

200

300

400

500

O2 (

M)

0.5

1.0

1.5

2.0

8.0

9.0

10.0

AN

C (

meq

L-1

)p

H

2

4

6

8

10

12

14

Calc

ite s

atu

ratio

n in

dex (g

reen

)

0.0

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0.4

0 12

12 August

0 12

13 August

0 12

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0 12

15 August

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16 August

0

CO32-

CO2

DIC

DIC

(m

M)

CO

32- / C

O2 (m

M)

0.2

0

Wate

r d

ep

th (

m)

5 C

10 C

15 C

20 C

25 C

30 C

35 C

0.1

a

b

c

d

0.3

0.4

a

74

a, Temperature isopleths calculated from measurements at 5-cm depth intervals.

b, Oxygen measured at 0.08 m (dark blue), 0.24 m (green) and 0.34 m (red) below

the water surface.

c, pH (blue) and ANC (green) in surface waters (0.08 m).

d, DIC (red), CO32- (orange) and CO2 (blue) in surface waters (0.08 m).

Where b-d background color show day/night cycle (white = day, grey = night).

Fig. 3. Depth profiles of DIC, ANC, Ca2+, Fe2+, ∑H2S and NH4+ in a shallow

charophyte lake during a diurnal cycle. Measurements on May 26 at 6.00 (red),

11.00 (blue), 16.00 (green) and 22.00 o’clock (orange). The water column was

vertically mixed at 6.00 o’clock and stratified below 0.20 m at 16.00 and below 0.25

m at 22.00 o’clock.

1 2 3

0.0

0.1

0.2

0.3

DIC (mM)

Wate

r d

ep

th (

m)

0 1 2 3 4

06:00

11:00

16:00

22:00

ANC (meq L-1

)

0 0.5 1 1.5 2

Ca2+

(mM)

0 10 20 30 40

0.0

0.1

0.2

0.3

Fe2+

(M)

Wate

r d

ep

th (

m)

0 0.1 0.2 0.3 0.4

Sulphide (M)

0 5 10 15 20 25 30

NH4+ (M)

75

Supplementary

Fig S1. ANC in lake water as a function of specific conductivity. ANC =

0.009399*Sp.cond.-0.1410. R2=0.72. The line is forced through coordinates (15,0).

The specific conductivity of rainwater in the region is 15 μS cm-1.

Fig S2. Surface water temperature (full line) and air temperature (dashed line)

for 5 days in May and 5 days in August.

0 100 200 300 4000

1

2

3

4

Sp. conductivity (S cm-1

)

AN

C (

meq

L-1

)

5

10

15

20

25

30

35

0 0 0 012 12 12 12 12 12 0 12 0 12 0 12 0 120 00 0

May August25 26 27 28 29 12 13 14 15 16

Tem

pera

ture

(C

)

76

Table S1. Diel minimum and maximum differences between surface and bottom

water temperature in May and August. Negative values denote surface water

colder than bottom water.

Date Minimum Maximum

25-05-2014 -0.10 12.86

26-05-2014 -1.43 14.36

27-05-2014 -3.98 0.57

28-05-2014 -4.11 3.93

29-05-2014 -3.63 11.18

12-08-2013 -1.62 6.22

13-08-2013 -1.52 8.63

14-08-2013 -1.24 6.20

15-08-2013 -1.43 8.91

16-08-2013 -0.96 7.94

Table S2. Mean daily vertical attenuation coefficients and depths at which 10 %

of surface light remains.

Date

Light attenuation

coefficient (m-1)

Depth at which 10 % of

surface light remains (m)

25-05-2014 22.7 0.10

26-05-2014 17.5 0.13

27-05-2014 21.6 0.11

28-05-2014 25.3 0.09

29-05-2014 22.7 0.10

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Aquatic Botany 41, 5-40 (1991). 18 McConnaughey, T. A. et al. Carbon budget for a groundwater-fed lake: Calcification supports

summer photosynthesis. Limnology and Oceanography, 1319-1332 (1994). 19 Jørgensen, B. B. in Marine Geochemistry (eds H.D. Schulz & M. Zabel) 173-207 (Springer, 2000). 20 Kautsky, L. Life strategies of aquatic soft bottom macrophytes. Oikos, 126-135 (1988). 21 Iversen, T. M. The ecology of a mosquito population (Aedes communis) in a temporary pool in a

Danish beech wood. Archiv für Hydrobiologie 69, 309-332 (1971). 22 Hanson, P. C., Carpenter, S. R., Cardille, J. A., Coe, M. T. & Winslow, L. A. Small lakes dominate a

random sample of regional lake characteristics. Freshwater Biology 52, 814-822 (2007). 23 Sand-Jensen, K. & Staehr, P. A. Scaling of pelagic metabolism to size, trophy and forest cover in

small Danish lakes. Ecosystems 10, 128-142 (2007). 24 Williams, P. et al. Comparative biodiversity of rivers, streams, ditches and ponds in an agricultural

landscape in Southern England. Biological Conservation 115, 329-341 (2004). 25 Verberk, W. C. & Bilton, D. T. Respiratory control in aquatic insects dictates their vulnerability to

global warming. Biology letters 9, 20130473 (2013). 26 Andersen, M. R., Jones, I. D., Woolway, R. I. & Sand-Jensen, K. Profound daily stratification and

vertical mixing in a shallow, wind-exposed pond with submerged macrophytes (in prep). (2015). 27 Vermaat, J. E. & Sand-Jensen, K. Survival, metabolism and growth of Ulva lactuca under winter

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Paper 3 - Distinct diurnal patterns of ecosystem metabolism in a small charophyte-lake

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Distinct diurnal patterns of ecosystem metabolism in a small charophyte-lake

Theis Kragh, Mikkel René Andersen and Kaj Sand-Jensen, Freshwater Biological

Section, Biological Institute, University of Copenhagen, Universitetsparken 4, 2100

Copenhagen

Target journal: Limnology and Oceanography

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Abstract

To characterize the temporal and spatial variability of metabolism (gross primary

production (GPP), respiration (R) and net ecosystem production (NEP)) in a small,

shallow Swedish lake with dense charophyte stands, we collected data from many O2

sensors placed along a vertical mid-lake profile and across the lake surface in late

May and early June. Similar diurnal patterns derived from single surface sensors and

multiple sensors showed maximum NEP-rates between 8 and 11 am and strong

afternoon depression with rates close to zero accompanying profound rise of O2, pH

and temperature and depletion of inorganic carbon and CO2 from morning to late

afternoon. Inorganic carbon limitation of photosynthesis and temperature

enhancement of respiration could account for profound afternoon depression of NEP.

Nocturnal respiration declined from sunset to sunrise due to falling temperature and

presumably depletion of respiratory substrates. Mean temperature-corrected

respiration rates at sunrise were 63% of that at sunset. The dense charophyte canopy

accounted for 90% of ecosystem respiration and the entire primary production. Mean

daily estimates of GPP and R varied only 2-fold and small, negative NEP-rates

varied less between surface sensors at different locations across the lake. In

conclusion, multiple oxygen sensors representing the main depths and sections of the

lake can provide reliable and accurate measurements of diurnal course and daily

rates of metabolism in small lakes probably because a relatively uniform oxygen

signal is ensured by small distances and nocturnal mixing. During colder periods of

continuous mixing a single mid-station sensor should provide reliable metabolism

estimates.

Introduction

Ecosystem metabolism is essential to the understanding of carbon cycling and

functional properties of gross primary production (GPP), respiration (R) and net

ecosystem production (NEP; Odum 1957, Kelly et al. 1983, Staehr et al. 2012c).

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During recent years there has been renewed interest in ecosystem metabolism of

freshwaters because of technical improvements and the recognition that freshwaters

are hot spots for storage of terrestrially fixed carbon and CO2 emission to the

atmosphere (Cole et al. 2007, Sand-Jensen & Staehr 2012).

Most determinations of ecosystem characteristics and metabolism are from large to

medium-sized lakes (> 10 ha), while small lakes and ponds (< 1 ha) which are

particularly abundant in the landscape are grossly underrepresented in the studies

(Downing et al. 2006, Hanson et al. 2007, Staehr et al. 2012a). In Denmark, for

example, there are about 1000 lakes larger than 10 ha but more than 100,000 lakes

smaller than 1 ha, whose combined contribution to CO2 emission by far exceeds that

of the larger lakes (Sand-Jensen & Staehr 2007, Staehr et al. 2012a). Ecosystem

metabolism changes markedly from small to large lakes because of the reduced input

of water, organic carbon and nutrients relative to surface area and volume in

gradually larger and deeper lakes and the increase of incident irradiance, wind

exposure and turbulence (Sand-Jensen & Staehr 2007, Staehr et al. 2012a). Thus,

most small lakes exhibit strongly negative rates of NEP and high atmospheric CO2

evasion relative to surface area (Staehr et al. 2012a), though nutrient-poor lakes in

terrestrial landscapes with thin soils of low organic carbon export may show positive

NEP and release O2 to the atmosphere during summer (Christensen et al. 2013). This

situation calls for stronger future emphasis on rates and regulations of ecosystem

metabolism in small lakes.

Technological improvements of O2 sensors have made it easier to use free-water

measurements to obtain continuous estimates of ecosystem metabolism in many

freshwater localities and reach broad-scale overviews of their role in carbon balances

of the landscape. Estimates of whole-lake metabolism have usually been based on a

single O2 sensor located in surface waters at the deepest point of the lake with the

implicit assumption that it is representative of the pelagic waters, if not the

metabolism of the entire lake. However, spatial heterogeneity of estimates of

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ecosystem metabolism may exists both within and between pelagic and littoral

habitats depending on underlying differences in ecosystem structure and extent of

physical isolation of water masses (Vadeboncoeur et al. 2002, Vadeboncoeur et al.

2006, Van de Bogert et al. 2007, Langman et al. 2010, Staehr et al. 2012b, Van de

Bogert et al. 2012). Within-lake heterogeneity may be due to measurements of strong

O2 signals from “hot spots” of intensive daytime photosynthesis and nocturnal

respiration in dense periphyton or macrophyte communities alternating with weak O2

signals from “cold spots” of low metabolism on naked sediments and clear pelagic

waters (Van de Bogert et al. 2012). Detected O2 signals are, in addition to the

underlying metabolic activity, influenced by physical processes of transport, mixing

and atmospheric gas exchange causing sensors to measure on water parcels of

different metabolic and physical time history (Mackay et al. 2011, Van de Bogert et

al. 2012). Under conditions of low wind and water mixing, distinct differences in

recorded metabolism may exist between sensors located in littoral and pelagic

habitats (Van de Bogert et al. 2007, Sadro et al. 2011a). In contrast, homogenization

of water masses due to strong wind forcing and mixing and/or short horizontal and

vertical distances may generate a uniform integrated O2 signal in the water across the

lake despite variation of metabolism among sites and over time.

Here we focused on ecosystem metabolism of a small lake (ca. 1000 m2) and tested

the reproducibility of estimates of multiple sensors covering the horizontal and

vertical gradients in the lake. We measured directly gas exchange velocity as a

function of wind velocity and fetch allowing us to construct a model to account for

atmospheric gas exchange for every 10-minute estimate of metabolism among sites.

Because the small, shallow lake is covered by charophytes across the entire bottom

and undergoes recurring mixing during summer nights, we expect that the variability

of daily estimates of metabolism derived from O2 sensors located in surface waters

across the lake should be relatively small compared with the surprisingly high

variability published for larger lakes (Van de Bogert et al. 2012).

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Regulation of metabolic rates takes place at temporal scales of seconds, minutes and

hours, while ecosystem metabolism based on free-water measurements is usually

expressed on a daily basis due to lack of sensitivity at shorter time scales. Many

important metabolic regulations (i.e. afternoon depression of productivity by lower

CO2 and higher O2, and falling respiration during the night by declining temperature

and lower availability of respiratory substrates; Markager & Sand-Jensen 1989,

Alnoee et al. 2015) are missed at the integrated daily scale, but may turn up on

shorter time scales and may also reveal greater short time variability among sensors

with different spatial location. The temporal variability of temperature, O2, CO2 and

pH can be very profound in small lakes like the one we studied (e.g. Christensen et

al. 2013).

Our overall goal was to determine rates of GPP and NEP at 10-minute intervals to

test whether the mean metabolic signal for all sensors and the signal for individual

sensors resembled each other and showed the same diurnal course. The two specific

goals was to test the possibility of afternoon depression of GPP and NEP and the

decline of R as the night progresses. We tested directly the possibility of afternoon

depression of NEP because of inorganic carbon limitation by comparing

photosynthetic rates of apical charophyte shoots during in situ incubations in the

afternoon in naturally DIC- and CO2-depleted surface water versus enriched bottom

waters. We also tested whether respiration rates declined as the night progresses and

evaluated to what extent falling temperature and depletion of respiratory substrates,

following temperature correction, could account for the predicted decline. Finally,

direct measurements of sediment respiration allowed us to evaluate its contribution to

ecosystem respiration relative to that of the dense charophyte canopy as respiration in

the clear water was negligible.

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Materials and methods

Study site and lake characteristics - The study was conducted in a small lake located

in a limestone quarry abandoned 30 years ago on Räpplinge Alvar on Öland, SE

Sweden (56.81168°N, 16.6094°E). The area is very sparsely vegetated grassland

(alvar) with thin soils covering the hard Ordovician limestone. The lake is fed by

rainwater and runoff from the almost naked limestone surfaces and overflow from

nearby lakes during periods of heavy rain (Christensen et al. 2013). The lake was 847

m2 large and had a maximum depth of 46 cm and a mean depth of 21 cm during the

first intensive study period in June 8th -15th , 2013, while it was 700 m2 large and had

maximum and minimum depths of 34 and 29 cm during the second study period in

May 24th – 30th, 2014. A georeferenced bathymetric chart was created from 258

measurements of depth and position in a grid allowing extrapolation limits between

measurements of less than 1 m across the lake surface. Changes in water level were

followed continuously with an accuracy of 3 mm by recording pressure differences

between a submerged water level data logger (HOBO U 20 – 001-04, Onset

Computers, Bourne, USA) and a similar logger in air. The lake had transparent water

and very low summer concentrations of ammonium (about 2 µM), nitrate

(undetectable), ortho-phosphate (about 0.06 µM) and phytoplankton chlorophyll

(about 1 µg L-1; Sand-Jensen et al. 2010). The lake sediment was 4-10 cm thick

calcareous gyttja of low organic content (10% of dry mass) deposited on top of the

solid limestone surface (Sand-Jensen et al. 2010, Andersen and Sand-Jensen 2013,

pers. comm). The lake bottom was covered by dense charophyte vegetation (mainly

Chara aspera and some C. contraria, C. virgata and C. vulgaris) with less than 10%

representation of submerged angiosperms (Myriophyllum spicatum, Potamogeton

crispus and Zannichellia palustris; Sand-Jensen et al. 2010). The lake was free of

shading bank vegetation and mostly of emergent vegetation except for scattered

Typha angustifolia and Phragmites australis at location F (see map in Fig. 3).

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A meteorological station was established next to the lake at 2.0 m above the ground.

The station was equipped with sensors for incident irradiance (HOBO PAR sensor

(400-700 nm) S-LIA-M003, Bourne Ma, USA), wind speed and direction (Hobo

anemometer and direction, S-WSET-A), air temperature and relative humidity

(HOBO U23 Pro v2) that took measurements every 1 minute and averaged readings

for every 10 minutes on a HOBO micro station data logger (H21-002).

Temperature, oxygen and air-water gas exchange - The horizontal variability of

water temperature and dissolved O2 was determined across the lake in June 2013 by

deploying seven MiniDOT sensors (PME, Vista, Ca, USA) at different positions

immediately above the charophyte canopy and 8-12 cm below the lake surface

(positions in Fig. 3). In May 2014, the vertical variation of temperature and dissolved

O2 was recorded at five depths through the water column and the charophyte canopy

at a 31-cm deep site by deploying one MiniDOT sensor at 2 cm and four Firesting

Pyroscience fiber optic sensors (Aachen, Germany) at 9, 16, 23 and 30 cm depth

below the surface. Before each deployment MiniDOT sensors and Firesting optodes

were calibrated in air-saturated and anoxic water. After each deployment, all sensors

were tested in air-saturated water for several hours to make sure that no sensor drift

had occurred. In no instance did we need to compensate for drift during

measurements. During O2 measurements in May 2014 we also recorded pH (pH-

Temp2000 Madgetech, Warner, NH, USA)and conductivity (HOBO U24-001)

continuously in surface waters. All pH electrodes were calibrated before deployment

and controlled for drift after deployment. A close linear relationship between parallel

direct measurements of carbonate alkalinity (= HCO3- + 2 CO3

2-) and conductivity

normalized to 20 oC has previously been established for the lake (Andersen et al.

2015a,b). This enabled calculations of total dissolved inorganic carbon (DIC) and

CO2 from carbonate alkalinity, pH, temperature and conductivity according to

Mackereth et al. (1978).

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Previous measurements on a similar small lake dominated by charophytes

(Christensen et al. 2013) have shown that wind-based models of air-water oxygen

exchange rate such as those in Jähne et al. (1987) and Cole and Caraco (1998)

overestimated exchange rates and produced erratic model predictions. We, therefore,

made a series of direct gas exchange measurements to account for the characteristics

of this particular lake (e.g., size, depth and charophyte cover) and the sitespecific

effects of wind velocity and fetch across the lake. Air-water gas exchange was

measured as CO2 exchange in free-floating flux chambers resembling those applied

by Raymond et al. (2012). CO2 was measured continuously in the flux chamber by a

portable IRGA (LiCor 840, Lincoln, Ne, USA) and CO2 in the water was determined

by continuous measurements of pH and temperature and frequent measurements of

DIC (dissolved inorganic carbon). Calculations of CO2 in the water and gas exchange

rates followed the procedures used by Sand-Jensen and Staehr (2012) and Alnoee et

al. (2015) and the precision and reproducibility were as high as in their

measurements. To convert measured gas exchange rates for CO2 to O2, we corrected

for chemical enhancement according to the measured temperature and pH (Bade &

Cole 2006) and the basic difference in exchange rate between the two gases

according to molecular weight (Wanninkhof 1992, Sand-Jensen & Staehr 2012). For

every location of the flux chamber we made three consecutive measurements and

used the average in further evaluations. Overall, we determined air-water exchange

rate at 21 locations, wind velocities and fetches. The air-water exchange rate (k600)

was positively related to mean wind speed, mean wind gust speed and to fetch at the

particular location of the flux chamber according to wind direction. The best

relationship to k600 (y, cm h-1) found by multiple regression using bidirectional

stepwise selection was for mean wind gust speed (x, m s-1) averaged for 2 hours prior

to measurements and mean wind speed grouped below and above 2 m s-1 during the

same period. At mean wind speed below 2 m s-1 the relationship was: y = 0.14 x +

0.04 (R² = 0.60, n=6). Above 2 m s-1 the relationship was: y = 0.87 x – 2.10 (R² =

87

0.76, n=15). The oxygen flux between the atmosphere and the water (F) was

calculated in time steps of 10 minutes as: F = k600 (O2surface - O2sat), where O2surface is

the measured concentration in surface waters and O2sat is the concentration in water at

equilibrium with the atmosphere at ambient water temperature and atmospheric

pressure. The flux was calculated for each time step using one of the two equations

for k600 depending on average wind speed and wind gust speed.

Oxygen metabolism

The vertical series - In measurements in May 2014 the O2 pool through the water

column was weighted according to the depth and water volumes represented by each

of the five sensors. For calculation of the air-water flux only temperature and O2

concentration in the surface water were used.

The horizontal series - Measurements in June 2013, using O2 concentrations and

temperature from the horizontally dispersed sensors, were averaged and NEP rates

normalized to surface area determined. Ecosystem rates for the entire lake was

determined after weighting according to the surface area represented by each sensor.

The sensor position was plotted on the map created and half of the distances to

neighbouring sensors were used as boundaries of the area represented by the sensor.

Boundaries were extrapolated onto the shore. The area covered by each sensor could

be calculated using the georeferenced map and weighted against the total lake area.

Ecosystem Respiration and GPP - Respiration rates in the lake during the night were

measured directly as the decline of O2 concentration in the water corrected for gas

exchange with the atmosphere. Respiration rates were integrated for the entire night

defined by incident photon irradiance below 1.3 µmol m-2 s-1 (PAR). Nocturnal

respiration rates were also determined for the initial 30 minutes (RIni) and the final 90

minutes (REnd) of the night by calculating the mean rate of O2 decline for 3 and 9

pairs of O2 measurements, respectively. These data allowed us to test whether

respiration rates declined from early to late during the night for temperature-

88

uncorrected rates and rates normalized to 20 oC applying a general Q10 value of 2.0.

Thus, the decline of temperature-corrected respiration rates during the night can be

represented by the quotient REnd (corr.)/RIni (corr) with values below 1.0 suggesting a

depletion of respiratory organic substrates.

Daytime respiration rates were calculated from the initial rate of O2 decline during

the first 30 minutes of the following night (RIni). This initial dark respiration rate was

assumed to be equal to the daytime respiration rate as organic carbon limitation of

respiration can be expected to be small and of the same magnitude. Daytime

respiration was corrected to ambient temperature applying the Q10 value of 2. GPP

was calculated for each ten minute step as the increase of O2 concentration corrected

for atmospheric exchange and with respiration added.

Averages of NEP and GPP were calculated for the entire measuring period, but only

including measurements for entire days. Standardized diurnal courses (Fig. 2 and 3)

were calculated as the mean of measurements in each time interval on the different

days. Values of NEP-day and NEP-night (= nocturnal respiration) were calculated for

the daytime and the night period as described above, and mean hourly respiration

rates were calculated by dividing cumulated NEP-night by the duration of the night

for comparison with sediment respiration rates.

Sediment respiration - Five sediment cores representing the different regions of the

small lake were retrieved in late May for measurements of aerobic sediment

respiration at ambient temperature (15 oC). Cores were made of Acrylic plastic of low

gas permeability, were 40 cm long, had an inner diameter of 5.2 cm and were closed

with rubber stoppers. Cores were incubated in a temperature-controlled water bath

filled with anoxic water preventing significant O2 influx to the sediment cores while

O2 was gradually depleted by sediment consumption over 4 days. Water within the

cores was kept homogeneous by a magnetic stirrer bars fastened to the upper rubber

stopper and driven by a large slowly-rotating magnet placed in the center and

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surrounded by the five sediment cores. Oxygen concentrations were continuously

recorded in each core by a Firesting Pyroscience fiber optic sensor. Oxygen readings

showed constant sediment respiration rates during the first three days of incubation,

then used for calculation, and a decline on the fourth day.

Charophyte production - Charophyte production rates and the influence of gradual

self-limitation by depletion of CO2 and enrichment of O2 in the water during

photosynthesis were measured in situ in early June by incubating small apical shoots

of Chara aspera in 50 ml glass bottles placed immediately above the charophyte

canopy. Four replicate bottles were filled with surface water and four additional

replicate bottles were filled with bottom water at 2 pm on a sunny day with profound

density stratification typical of summer (Andersen et al. 2015a). Three blanks without

charophytes were incubated with either surface or bootom water and showed

negligible O2 changes. Before incubation, surface water had been enriched in O2 (520

µM, 206% saturation) and depleted in CO2 and DIC (1.37 mM) by photosynthesis

before noon, while bottom water located in the shade below the canopy had been

depleted in O2 (95 µM, 35% saturation) and enriched in CO2 and DIC (2.37 mM)

forming a distinct contrast and test for afternoon depression of photosynthesis in

surface water because of O2 accumulation and DIC depletion by ongoing

photosynthesis. Net photosynthesis was calculated as the release of O2 from 2 to 4 pm

relative to plant dry mass.

Results

Environmental conditions - The sky was clear and the incident irradiance followed a

regular sinusoid course on day 1, 2 and 5 in late May (Fig. 1). The water column

underwent strong daytime surface heating and vertical stratification at about 20 cm

depth on day 1 and 2 (Andersen et al. 2015a). Day 5 was colder and surface heating

and stratification were much weaker than on day 1 and 2 (Fig. 1 and Table 1). The

90

weather was partly overcast, colder and windy on day 3 and 4 and no vertical

stratification developed. The water column underwent convective mixing by surface

cooling every night (Andersen et al. 2015a). Dissolved O2 and pH in surface waters

exhibited profound and regular sinusoid diurnal courses on day 1 and 2, while diurnal

amplitudes were smaller and less regular on the other days. Photosynthesis during the

day depleted CO2 in surface waters below air saturation to only 0.42-2.92 µM late in

the afternoon on the five days, while O2 rose above air saturation to 430-509 µM. The

molar quotient of CO2 to O2 dropped to only 0.0009-0.0063.

Vertical daytime stratification was accompanied by strong DIC depletion by

photosynthesis and CaCO3 precipitation from surface waters at high pH, while DIC

was replenished by respiration and dissolution of CaCO3 at high CO2 concentration at

low pH in bottom waters during the day and continued throughout the night restoring

the DIC pool for photosynthesis on the following day (Andersen et al. 2015b). DIC

replenishment in bottom waters was weaker on day 3-5, because daytime vertical

mixing prevented the build-up of high CO2 concentrations in bottom waters

conducive to CaCO3 dissolution, leading to a marked decline of the mean DIC

concentration before noon from 1.30 mM on day 2 to only 0.84 mM on day 5 (Table

1).

Surface and vertically integrated metabolism - Average diurnal patterns of GPP and

NEP for the week in late May based on the surface sensor at 2 cm depth and all five

sensors dispersed through the water column at a mid-site were both highly regular

(Fig. 2) compared to the more irregular course of NEP estimated from the surface

sensor during individual days (Fig. 1). The average course of GPP reached a

maximum between 8 and 12 o’clock, while rates were lower in the afternoon. GPP

was slightly higher when based on measurements in surface waters alone than for the

entire water column (Fig. 2). Rates of NEP were positive from sunrise to the early

afternoon, close to zero in the late afternoon and negative during the evening and the

night. Nocturnal respiration declined from sunset to sunrise. Overall, diurnal patterns

91

of NEP were the same when based on measurements in surface waters or the entire

water column except that dark respiration was about 50 % higher when calculated

with data for the entire water column rather than surface waters alone.

Horizontal variability and integrated lake metabolism - Mean diurnal patterns of GPP

and NEP for the week in early June 2013 resembled each other between the seven

different positions of the O2 sensors and the volume-weighted average for the entire

lake (Fig. 3). Diurnal patterns also resembled those already described for the mid-site

in late May (Fig. 2). As expected, GPP was close to zero during the night, reached a

daytime maximum before noon, was somewhat lower in the afternoon and then

dropped steeply in the late afternoon and early evening. The lower GPP in the

afternoon than before noon took place at higher temperatures, O2 concentrations and

pH and lower availability of CO2 and DIC than before noon as described before (Fig.

1). Afternoon depression was more profound for NEP than GPP reflecting the much

higher respiration rate at high temperature and O2 concentration in the afternoon than

at lower temperature and O2 before noon. Positive values of NEP were typically

recorded for 12 hours between 5 and 17 o’clock and maximum rates between 8 and

11 o’clock. At night, respiration rates dropped from sunset to sunrise as temperature

and O2 declined similar to the nocturnal course in late May (Figs. 1 and 2).

Mean daily NEP (mmol O2 m-2 d-1) for the week was almost the same (-5 to -9) for

the different horizontal positions and the integrated average of the lake (Table 2). The

minimum (-7 to – 14) and maximum daily NEP rates (6 to 13) also resembled each

other among locations. Rates of GPP and R were both about 20-fold higher than the

small difference (NEP) between them. Mean rates of GPP and R varied about 2-fold

between different horizontal positions (e.g. GPP: 125-266 mmol m-2 d-1) and tended

to be smaller in very shallow water with a short charophyte canopy (e.g., location E)

than in deeper water with a taller canopy (locations B and D).

Ecosystem and sediment respiration – During all 11 nights in late May and early

June, ecosystem respiration rates declined significantly as the night progressed.

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Respiration rates during the last 90 minutes of the night were 28-84% (avg. 58%) of

the rate during the first 30 minutes immediately after sunset (Table 2). After

correction for falling temperature during the night, respiration before sunrise still

remained 29-90% (avg. 63%) of the rate immediately after sunset.

The charophyte canopy was more active in terms of GPP and nocturnal respiration in

late May than in early June although temperature was higher in June (Tables 1 and 2).

Thus, the mean nocturnal respiration was 7.9 mmol m-2 h-1 in late May and 6.8 mmol

m-2 h-1 in early June (Table 1). The mean respiration rate after sunset corrected to a

common temperature of 20 oC was significantly higher in late May (15.7±1.8) than

in early June (8.2±1.4 mmol m-2 h-1, mean ± 95% C.L).

Sediment respiration in late May at 15 oC was 0.70±0.15 mmol m-2 h-1 (mean ± 95%

C.L.) and only about 10% of total ecosystem respiration. Because respiration in the

transparent, oligotrophic water was negligible, the dense charophyte canopy

accounted for about 90% of total ecosystem respiration.

On a diurnal basis sediment respiration averaged 16.8 mmol m-2 d-1 which is of the

same order of magnitude as the mean diurnal NEP in late May (5 mmol m-2 d-1) and

early June (- 7 mmol m-2 d-1). When sediment respiration was accounted for in the

ecosystem balance, mean net production of the charophyte canopy was slightly

positive in late May and early June (21.8 and 9.8 mmol m-2 d-1, respectively.

Regulation of daily and short-time variability of GPP and NEP – Rates of GPP and

NEP can be influenced by day-to-day and and short-time variability of irradiance,

temperature, DIC, CO2 and O2 concentrations through their impact on photosynthesis

and respiration. Daily GPP and NEP were not significantly correlated to day-to-day

variability of daily irradiance and temperature during the two measuring weeks

(Table 1). Though too few days were examined to test the relationship between daily

GPP and NEP and available DIC and CO2 in late May, data pointed at distinct

impacts. On day 5 with very low DIC and CO2 concentrations before noon, daytime

NEP was extremely low and GPP was 2 times lower than rates on day 1 and 2 when

93

DIC and CO2 were much higher (Table 1). The data suggest that availability of DIC

and CO2 represents a stronger constraint on daily GPP and NEP than irradiance and

temperature.

We evaluated further the influence of irradiance and pH on short-time variations og

GPP and NEP during 10-minutes intervals. pH was used as a proxy for inorganic

carbon availability relative to O2 because progressively higher pH during the day

accompanied depletion of DIC and CO2 and accumulation of O2 (Fig. 1, Andersen et

al. 2015b). We examined production rates as a function of irradiance and pH

separately before and after noon (Fig. 4). We only examined the patterns on sunny

days with clear skies where irradiance, temperature, O2 and pH follow regular

courses. It was apparent that GPP as a function of incident irradiance above 50 µmol

m-2 s-1 followed almost the same pattern before and after noon with approximate

saturation being attained above 500 µmol m-2 s-1. In contrast, rates of NEP were a

positive function of irradiance before noon, while rates were much lower and close to

zero and virtually independent of irradiance during the afternoon supporting that

other environmental variables constrained NEP. Rates of GPP were more similar

before and after noon than those of NEP because respiration rates are higher in the

afternoon at higher temperatures and GPP is the sum of NEP and respiration.

The relationship of GPP and NEP to pH between 8.0 (typical late morning pH) and

9.3 (typical late afternoon pH) was examined at irradiances above 500 µmol m-2 s-1 to

ensure light saturation of photosynthesis (Fig. 4) The relationship of GPP to

increasing pH was slightly negative, while that of NEP to pH was strongly negative

reaching zero at about pH 9.3. In the relationships of GPP and NEP to pH, afternoon

measurements took place at systematically higher temperature than before noon

conducive to higher afternoon respiration and, therefore, particularly low rates of

NEP compared with GPP.

Test of afternoon depression – We performed a direct test of afternoon depression of

charophyte photosynthesis as a result of DIC depletion and O2 accumulation in

94

surface waters by comparing rates of O2 release in surface waters (DIC/O2 molar

quotient of 2.7) with those obtained in DIC- enriched and O2-depleted bottom waters

(DIC/O2 quotient of 29). Net photosynthesis of apical shoots at high irradiance were

stimulated almost 4-fold by incubation in water collected near the bottom (151±52)

relative to shoots incubated in surface waters (40± 28 µmol g-1 DM h-1, mean±95

C.L.) confirming strong afternoon depression of photosynthesis by the build-up of O2

and the drawdown of DIC and CO2.

Discussion

Diurnal course of metabolism – Net ecosystem metabolism (NEP) exhibited two

profound features: afternoon depression and falling respiration during the night at all

locations and during all days of measurements. Positive NEP was mainly restricted to

the period from sunrise to shortly after noon , while NEP was close to zero during the

later afternoon and strongly negative during the evening until midnight. From the

morning to the afternoon the marked rise of temperature, O2 and pH and the decline

of CO2 and DIC can probably account for the distinct afternoon depression of NEP.

This explanation was supported by the steep decline of NEP with rising pH at high

irradiance and confirmed by in situ experiments showing almost 4-fold higher

photosynthesis of apical charophyte shoots incubated in DIC-enriched and O2-

depleted bottom waters compared with DIC-depleted and O2-enriched surface waters.

While NEP represents direct estimates from changes of concentrations and

atmospheric exchange of O2, GPP is the sum of NEP and R and is, therefore,

dependent on the selected temperature coefficient for respiration and the assumption

that daytime respiration rates is best represented by respiration rates immediately

after sunset, which is supported by most measurements (Markager & Sand-Jensen

1994). Thus, a lower estimate of the basic respiration rate (e.g. the average for the

night instead of the early night) and a lower temperature coefficient of respiration

than the selected Q10 of 2.0 would reduce respiration and, thus, depress GPP further in

95

the afternoon relative to rates before noon.Temperature rose 2-12 oC from sunrise to

late afternoon during our measurements. For a Q10 of 2.0, respiration would increase

0.4-2.4-fold. Because GPP and R are high relative to NEP (= GPP – R), the increase

of respiration contributed to the shift from maximum NEP values before noon to very

low NEP values during late afternoon. As a result afternoon depression is more

prominent for NEP than GPP.

The daytime depletion of DIC by photosynthesis and carbonate precipitation and the

even stronger decline of CO2 due to rising pH and temperature will increase inorganic

carbon limitation of photosynthesis (Madsen & Sand-Jensen 1991, Pedersen et al.

2013). Maximum inorganic carbon concentrations in surface waters in the morning

were 32-337 µM CO2 and 0.75-1.26 mM HCO3-, while minima late in the afternoon

were 0.39-2.7 µM CO2 and 0.62-0.82 mM HCO3-. Charophytes are capable of using

both HCO3- and CO2 for photosynthesis, but the affinity is higher for CO2 than HCO3

-

(Lucas 1985). Earlier in situ incubation experiments at light saturation showed that

net photosynthesis of communities of the charophyte, Chara virgata in these small

lakes declined almost 2-fold when exposed to higher pH (9.5) and lower CO2 (1 µM)

characteristic of the afternoon relative to the rate under early morning conditions

(e.g., pH 7.5 and 150 µM CO2; Christensen et al. 2013). While the latter experiment

reflects the direct impact on photosynthesis of the charophyte community of higher

pH and lower CO2 at the same concentrations of DIC and O2, the stronger almost 4-

fold stimulation of net photosynthesis in the incubations of small charophyte shoots

in surface waters versus bottom waters reported here can be explained by the

additional constraints by lower DIC and higher O2 accompanying higher pH and

lower CO2 in surface waters during the afternoon. Lower DIC and higher O2

concentrations in itself and added lower molar quotients of DIC/O2 and CO2/O2

restrict photosynthesis and enhance photorespiration of submerged plants and algae

(Sand-Jensen & Frost-Christensen 1998) and high water temperatures enhance

mitochondrial respiration and photorespiration even further (Beardall et al. 2003).

96

These general phenomena will apply to charophytes, though their specific responses

have not been studied. The inhibition of net carbon fixation depends on the ability of

charophytes to increase CO2 in the cells by active carbon concentrating mechanisms

(Lucas 1985, McConnaughey 1991). The CO2/O2 quotient at the site of Rubisco

activity is a main regulator of the balance between photosynthetic CO2 assimilation

and CO2 loss by photorespiration because of competitive inhibition between

carboxylase and oxygenase activity of Rubisco (Sand-Jensen & Frost-Christensen

1998, Beardall et al. 2003). Incubation experiments in bottles in the lake with the

dominant Chara species confirmed that net photosynthesis dropped from the morning

to almost zero in the afternoon when O2 within incubation bottles approached 700-

800 µM (3-fold supersaturation) and CO2 declined below 1 µM (Andersen, Kragh and

Sand-Jensen pers, comm, 2015). Our field measurements also confirmed that NEP

reached zero in the afternoon when O2 approaced 400 µM and CO2 dropped to 0.5-5

µM in surface waters. All together these measurements stress the strong self-

limitation of net photosynthesis of the dense charophyte stands in the shallow water.

As predicted, respiration rates declined progressively during the night. On average,

respiration rates in the lake before sunrise were 58% of the rate immediately after

sunset and 63% when respiration rates were corrected for falling nocturnal

temperatures from sunset to sunrise. Because charophytes are responsible for 90% of

ecosystem respiration, we suggest that diminishing pools of respiratory substrates due

to ongoing consumption during the evening and the night can account for the

declining rates following temperature correction. Declining respiratory rates during

the night have been observed in other field studies in oligotrophic lakes and streams

of low organic productivity (Sadro et al. 2011b, Solomon et al. 2013, Alnoee et al.

2015), but also in dense communities of microalgae (Gibson 1975, Markager &

Sand-Jensen 1989). The falling respiratory rates during the night can be correlated to

consumption of the main pools of soluble carbohydrates and neutral lipids (Gibson

1975, Lacour et al. 2012).We cannot exclude that synchronization of metabolic

97

activity involved in different synthetic pathways during diurnal cycles can also

influence the course of nocturnal respiration. In whole-system measurements

endogenous rhytms in synthetic pathways cannot be separated from the influence of

variable respiratory substrate pools.

Resemblance of GPP and R – Mean daily rates of GPP and R integrated across the

entire lake during the week in early June (198 and 191 mmol O2 m-2 d-1) were close to

each other such that NEP shifted from slightly negative to slightly positive rates

between days (-15 to 10 mmol m-2 d-1), though with an overall negative average for

the week (-7 mmol m-2 d-1). Because of the complete cover of the lake bottom by

charophytes they were responsible for the entire gross production and 90% of

respiration in the lake. Sediment respiration was mainly fueled by degradation of

dead charophyte debris, the surrounding terrestrial landscape being very oligotrophic,

sparsely vegetated and having thin soils (Baastrup‐ Spohr et al. 2015). Low sediment

respiration rates can be explained by the low organic content in sediments (about

10% of DM) and regular exposure of shallow sediments to atmospheric air during

summer drought (Christensen et al. 2013, Sand-Jensen et al. 2015) resulting in

enhanced decomposition of organic material with access to the atmosphere and no O2

consumption from the lake water.

Net production of the charophyte community was slightly positive during the two

weeks in late May and early June (about 22 and 10 mmol m-2 d-1, respectively). These

low rates are expected when the charophyte community has obtained the observed

high density at which a net increase of the biomass and an associated positive daily

O2 balance for the charophyte community is prevented by profound self-shading,

depletion of inorganic carbon and accumulation of O2 during the day. Slightly

negative daily NEP rates have been observed before in similar lakes dominated by

dense charophyte communities during mid-summer, when GPP and NEP reached

about 200-300 mmol O2 m-2 d-1, while positive NEP rates (about 40 mmol m-2 d-1)

have been measured during active growth of the charophyte biomass in spring and in

98

late summer after refilling of the shallow waterbody following mid-summer drought

(Christensen et al. 2013).

Reproducible metabolic estimates across the lake – Trust in the reported metabolic

features is strengthened by the calculations of reproducible diurnal courses from

single surface sensors at different locations and integrated responses from many

sensors along the vertical profile and across the lake. The variability of estimated

rates between sensors was also relatively modest. For example, nocturnal respiration

exhibited a characteristic decline from sunset to sunrise during all 11 nights. Also, all

seven O2 sensors located across the lake showed almost the same average rates

(mmol m-2 d-1) of NEP during a week measurements (-5 to – 9), while GPP (125 to

266) and R (-118 to – 256) varied 2-fold. Some of the shallow sites had a shorter

charophyte canopy and a thinner sediment on the limestone (e.g. location E and G)

which may account for the lower rates of GPP and R than deeper sites with taller

charophytes.

The reproducible patterns are probably due to the continuous charophyte cover across

the small shallow lake. The short vertical and horizontal distances and the recurring

nocturnal mixing will tend to homogenize the water masses, the O2 signals and the

estimated metabolism despite vertical stratification of temperature and O2 at the

deepest sites during summer days. In addition, we made a special effort to directly

determine gas exchange with the atmosphere as a function of wind speed as part of

the metabolic estimates permitting us to perform running corrections for the air-water

O2 exchange. Outside the summer, continuous mixing of the entire water mass should

make the O2 signal from a single mid-station sensor in this small lake sufficiently

reliable for estimating whole-system metabolism.

In many other lakes, metabolic activity and physical dispersion of O2 signals are

much more complex and several sensors and more sophisticated models of air-water

gas exchange are needed to obtain reliable metabolic rates(Lauster et al. 2006, Van

de Bogert et al. 2007, Sadro et al. 2011a, Van de Bogert et al. 2012). This is the

99

situation in lakes with a high spatial physical and biological complexity because of a

shoreline with variable development of benthic phototrophs and heterotrophs on

different sediment types and partly separated surface and bottom waters of variable

volumes and diverging metabolic activity (Staehr et al. 2012b). However, even in

lakes with a regular shape and bathymetry, variability of metabolic estimates between

sensors can be astonishingly high; e.g. GPP from - 132 to 250 mmol O2 m-2 d-1 during

ten days in Sparkling Lake (Van de Bogert et al. 2012). Using four randomly placed

oxygen sensors increased the precision of daily metabolism estimates 4-fold over

single-location measures in Van de Bogert et al.’s study. The generality and possible

reasons for the high variability of metabolism estimates should be checked in future

studies because the results sincerely question the reliability of free-water approaches

to estimate metabolism in large and medium-sized lakes. This high within-lake

variability could explain why long-time series of metabolism estimates may show

extensive day-to-day variation that is only partially explainable by known driving

variables such as irradiance, temperature, vertical mixing and desiccation-refilling

(Sand-Jensen & Staehr 2007, Coloso et al. 2011, Christensen et al. 2013).

Alternatively, if there are time-delays between driving variables (e.g. mixing) and

metabolism, or important driving variable (e.g. nutrient availability) are not

accounted for in model analyses, metabolism estimates could still reflect the actual

conditions.

In the present multiple-sensor study and a previous single-sensor study of metabolism

in small charophyte-dominated lakes (Christensen et al. 2013), we found

reproducible diurnal patterns and long-term patterns closely related to environmental

characteristics known to regulate photosynthesis and respiration (e.g., irradiance,

temperature, DIC, pH, O2, and phototroph biomass). To confirm the effect of these

variables it is essential, that the free-water approach are combined with controlled

experiments in the field or in the laboratory on field samples retrieved for immediate

analysis. Such experiments allowed us to confirm afternoon depression of charophyte

100

photosynthesis by elevated O2 and pH and declining DIC and CO2. It is

recommended to perform controlled experiments with field material concurrent with

free-water measurements (Alnoee et al. 2015). Controlled chamber experiments

coupled to open-water measurements help identifying the main processes and

mechanisms that are driving ecosystem metabolism and its photosynthetic and

respiratory processes.

References

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Solomon, C. T., Bruesewitz, D. A., Richardson, D. C., Rose, K. C., Van de Bogert, M. C., Hanson, P. C., Kratz, T. K., Larget, B., Adrian, R., and Babin, B. L. 2013. Ecosystem respiration: Drivers of daily variability and background respiration in lakes around the globe. Limnol. Oceanogr 58:849-866. Staehr, P. A., Baastrup-Spohr, L., Sand-Jensen, K., and Stedmon, C. 2012a. Lake metabolism scales with lake morphometry and catchment conditions. Aquatic Sciences 74:155-169. Staehr, P. A., Christensen, J. P., Batt, R. D., and Read, J. S. 2012b. Ecosystem metabolism in a stratified lake. Limnology and Oceanography 57:1317-1330. Staehr, P. A., Testa, J. M., Kemp, W. M., Cole, J. J., Sand-Jensen, K., and Smith, S. V. 2012c. The metabolism of aquatic ecosystems: history, applications, and future challenges. Aquatic Sciences 74:15-29. Vadeboncoeur, Y., Kalff, J., Christoffersen, K., and Jeppesen, E. 2006. Substratum as a driver of variation in periphyton chlorophyll and productivity in lakes. Journal of the North American Benthological Society 25:379-392. Vadeboncoeur, Y., Vander Zanden, M. J., and Lodge, D. M. 2002. Putting the Lake Back Together: Reintegrating Benthie Pathways into Lake Food Web Models. BioScience 52:1. Van de Bogert, M. C., Bade, D. L., Carpenter, S. R., Cole, J. J., Pace, M. L., Hanson, P. C., and Langman, O. C. 2012. Spatial heterogeneity strongly affects estimates of ecosystem metabolism in two north temperate lakes. Limnology and Oceanography 57:1689. Van de Bogert, M. C., Carpenter, S. R., Cole, J. J., and Pace, M. L. 2007. Assessing pelagic and benthic metabolism using free water measurements. Limnology and Oceanography: Methods 5:145-155. Wanninkhof, R. 1992. Relationship between wind speed and gas exchange. J. Geophys. Res 97:7373–7382.

Legends

Fig. 1. Diurnal course of incident irradiance and surface temperature (upper panel),

dissolved oxygen and pH in surface waters (middle panel), and GPP and NEP (lower

panel) based on measurements of a single oxygen sensor in surface waters in the

middle of the 0.31-m deep lake during six days in late May 2014.

Fig. 2. Mean diurnal patterns of GPP and NEP based on measurements during six

days in late May 2014 from two oxygen sensors near the water surface (2 and 9 cm,

upper panel) and a total of five sensors placed along the vertical profile (2 to 30 cm,

lower panel) at a 31-cm deep site in the middle of the lake.

Fig. 3. Mean diurnal patterns of GPP and NEP based on measurements during seven

days in early June from seven oxygen sensors (A-G) placed at 8-12 cm depth below

the surface at different locations in a small charophyte-dominated lake. The mean

volume-weighted GPP and NEP for the entire lake based on the seven oxygen sensors

was determined. Depth contours and location of sensors are shown on the map.

103

Fig. 4. Rates of GPP and NEP as a function of irradiance and pH before noon and in

the afternoon on three days in late May with a clear sky and regular sinusoid diel

changes in irradiance, temperature, oxygen and pH. Rates are based on measurements

of dissolved oxygen every 10 minutes from five oxygen sensors placed across the

vertical profile (2 to 30-cm depth) at a 31-cm deep site in the middle of the lake.

Measurements in the left panels were restricted to irradiances above 50 µmol m-2 s-1,

while measurements in the right panels were restricted to irradiances above 500 µmol

m-2 s-1.

104

-20

0

20

40

NEP

GPP

Day 1 Day 2 Day 3 Day 4 Day 5 Day 6

GP

P a

nd

NE

P

(mm

ol m

-2 h

-1)

0

1000

2000

0

10

20

PAR

Temp

PA

R (

µm

ol m

-2 s

-1)

Tem

p (C

)

0

100

200

300

400

500

600

7

8

9

O2

pH

Oxyg

en

(µ

M)

pH

105

0 4 8 12 16 20 24

-10

0

10

20

30

NEP

GPP Water column

GP

P a

nd

NE

P (

mm

ol m

-2h

-1 )

Time of day

-10

0

10

20

30 Surface waters

106

-10

0

10

20 A

A

-10

0

10

20

B

B

-10

0

10

20

GP

P a

nd

NE

P (

mm

ol m

-2h

-1)

C

C

0 4 8 12 16 20 24

-10

0

10

20

Time of day

D

D

-10

0

10

20

-10

0

10

20

-10

0

10

20

GP

P a

nd

NE

P (

mm

ol m

-2h

-1 )

0 4 8 12 16 20 24

-10

0

10

20

Time of day

E

E

F

FG

G

Mean

107

-10

0

10

20

30

GP

P (

mm

ol m

-2h

-1)

8 9 10

Before noon

Afternoon

pH

0 500 1000 1500-10

0

10

20

30

NE

P (

mm

ol m

-2h

-1)

Irradiance (µmol m-2

s-1

)

108

Table 1. Daily incident photon irradiance (PAR), surface temperature, surface inorganic carbon

concentrations (DIC and CO2) and integrated daytime NEP, nocturnal NEP (= nocturnal respiration)

and diurnal NEP during 5-6 days in late May and early June in a small lake. Photon irradiance and

temperature are mean values for the entire day, while DIC and CO2 are concentrations at noon.

Mean duration of the night was 6.7 hours in late May and 5.7 hours in early June resulting in a

mean nocturnal ecosystem respiration rate of 7.87 and 8.23 mmol O2 m-2 h-1 in late May and early

June, respectively.

109

Table 2. Nocturnal respiration rates in the small lake for 5-6 days in late May and early June during

the first 30 minutes (RIni) and the last 90 minutes (REnd) of the night. Respiration rates and the

quotient REnd/RIni are shown both uncorrected and corrected for temperature changes (Q10 = 2)

during the night.

Table 3. Mean daily rates of GPP, NEP and R derived from continuous oxygen measurements at

seven different positions (A to G) and the overall mean of all measurements in a small lake during a

week in early June. Daily minimum and maximum daily rates are in parenthesis.

110

Paper 4 - Whole-stream metabolism in nutrient-poor calcareous streams on Öland, Sweden

111

Whole stream metabolism in nutrient-poor calcareous streams on Öland,

Sweden

Anette B. Alnoee 1), Tenna Riis1), Mikkel R. Andersen2), Annette Baattrup-Pedersen1) and Kaj

Sand-Jensen2)

1) Department of Bioscience, Aarhus University, Ole Worms Alle 1, 8000 Aarhus C, Denmark

2) Department of Biology, Freshwater Biology, University of Copenhagen, Universitetsparken 4,

2100 København Ø, Denmark

Keywords: seasonal metabolism, primary production, ecosystem respiration, surface irradiance,

headwater streams

Aquatic Sciences 2015, 77(2), 207-219

112

Abstract

We studied whole-stream metabolism in three headwater non-forested stream reaches on the island

of Öland, Sweden in order to characterize the metabolism of this unusual ecosystem and to compare

it with other stream ecosystems in NW Europe. Gross primary production (GPP) was generally low

(< 4 g O2 m-2 d-1) with the lowest GPP recorded in the most upstream, shallow reach draining the

thin soils of the limestone Alvar plains. Here, completely flooded terrestrial plants could account for

the whole primary production at baseflow. Ecosystem respiration (ER) increased several fold with

agricultural impact, resulting in heterotrophic stream conditions downstream and higher light

requirements for photosynthesis to outweigh respiration. A strong relationship between daily GPP

and ER was found at the two most nutrient-poor sites. Temperature corrected instantaneous ER rate

was highest in the beginning of the night, but decreased at the end of the night at the same reaches,

indicating that dark respiration depleted photosynthetic products and became limited by organic

substrates. The broad-scale comparison of open NW European streams showed a 1:1 relationship,

indicating a tight link between daily GPP and ER during summer (April-August) but not during

winter. This study has extended the range of GPP and ER measurements to include nutrient-poor

NW European streams, thereby increasing the knowledge on stream metabolism in this, otherwise,

highly agricultural impacted region. It also documented a strong relationship between GPP and ER

in streams, ranging from extremely nutrient poor to moderately nutrient rich conditions during

spring and summer.

113

Introduction

Land use has proven to be an important factor determining whole stream metabolism.

Agricultural and urban streams receive additional nutrients and are more productive than natural

streams which are relatively unaffected by human activities (Bernot et al. 2010). Most open streams

in north-temperate lowland regions of Europe drain deep, fertile agricultural soils, they are rich in

dissolved nutrients and support high biomasses of benthic microalgae and submerged macrophytes

(e.g. Sand-Jensen et al. 1988; 1989; Kristensen and Hansen 1994; European Environment Agency

2005). Gross primary production (GPP) of benthic algae and macrophytes is high under well-

illuminated conditions from spring to autumn, and ecosystem respiration (ER) is relatively high

year-round due to degradation of allochthonous and autochthonous material (Edwards and Owens

1962; Kelly et al. 1983). The open lowland streams are heterotrophic (ER > GPP) on an annual

basis, though GPP may exceed ER during periods in spring-summer with intense phototrophic

growth (Sand-Jensen 1997). Therefore, the magnitude and temporal pattern of GPP and ER reflect

both the intensity and timing of phototrophic production and organic decomposition which, in turn,

are influenced by light availability, nutrient richness and input of easily degradable organic matter

(Simonsen 1974; Mulholland et al. 2001).

Streams with very low nutrient availability due to thin, slowly mineralizing or well-

leached soils in the catchment supposedly have low in-stream growth of benthic phototrophs and

heterotrophic communities. Daily GPP and ER should be low and in approximate balance provided

that the external input of easily degradable organic matter is low and less than the autochthonous

material, and that the temporal and spatial coupling between production and decomposition is close

(Odum 1971; Solomon et al. 2013). Low daytime photosynthesis may also lead to extensive organic

carbon limitation of respiration by phototrophs and bacteria during the subsequent night period and

gradually lower respiration rates as the night progresses and respiratory organic pools are

consumed, as previously shown for pelagic algal communities (Gibson 1978; Markager and Sand-

Jensen 1989). In open shallow clear-water streams, the incident solar UV-flux during the day could

also disrupt the organic aromatic compounds and facilitate coupled bacterial degradation during the

early part of the night. We propose that close coupling of daily GPP and ER and gradually

decreasing night-time respiration rates may prevail for small, shallow nutrient-poor streams

draining the thin soils on the open Ordovician limestone pavements of the great Alvar plain on the

island of Öland, SW Sweden (Ekstam and Forshed 2002). The streams on Öland are small and

shallow, have low slopes and low current velocities, facilitating the coupling between production

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and decomposition. The small ephemeral and permanent ponds on the Alvar have highly transparent

waters and concentrations of organic matter and dissolved inorganic nutrients are notably low

(Sand-Jensen et al. 2010; Christensen et al. 2013). We expect that water chemistry is similar in the

streams on the Alvar plain and that this may explain why there is little visible growth of algae on

the stream bottom.

Here we present a case study including three stream reaches located on Öland,

Sweden in order to include hitherto unexplored nutrient-poor streams in a comparison of the

metabolism of open, NW European temperate streams to evaluate the range of metabolic rates and

the coupling between GPP and ER. The three stream reaches included an upstream reach, reach I,

draining the natural sparsely vegetated grassland on the thin soils of the great Alvar plain, reach II,

impacted by agricultural land use over short distances, and reach III draining deeper soils and

impacted by mixed forest and agricultural fields over longer distances. From reach I to reach III, our

predictions were that (i) the biomass of benthic algae would increase from very low to higher

values, (ii) GPP, and in particular ER, would increase from very low to higher values, generating

stronger heterotrophy, and (iii) the coupling between GPP and ER would decrease.

Material and methods

Study site description

The study was conducted in two stream systems in separate catchments originating on

the great Alvar plain on the large Swedish island of Öland located in the southern Baltic Sea. These

Alvar streams experience approximately the same climatic conditions as the nutrient-rich Danish

and United Kingdom streams draining fertile agricultural soils (Edwards and Owens 1962;

Simonsen 1974). Annual mean precipitation is around 500 mm and mean temperature is -1°C in

January and 15°C in July (www.smhi.se). The great Alvar plain is a 260 km2, genuine nature

reserve (UNESCO World Heritage) having a species-rich, unproductive grassland vegetation with

very few deciduous trees occurring only in fissures in the horizontal, almost impermeable,

limestone plates and small areas with thicker soil layers covering the limestone surfaces. As the

streams leave the Alvar plain and pass through 0.5-4 km wide strips of agricultural land towards the

Baltic Sea located in the East, they become deeper and more influenced by bank shading and

agriculture.

The upstream reach I is supplied by rain water and aquifers close to the surface of the

Alvar plain and regularly dries out for 2-4 months between May and August, and occasionally also

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in September-October (Leberfinger and Hermann, 2011). Reach II and III receive some water from

deeper cultivated moraine soils and may retain small discharges during summer. Therefore, to allow

measurement and comparison, we conducted all measurements in May 2010 and May and October

2011 when all three reaches were transporting water.

Reach I (56° 32.473’N, 16° 34.037’E) was located in River Frösslundabäcken close to

the spring on the Alvar plain and 6.5 km from the Baltic Sea (Leberfinger and Hermann, 2011, their

Fig. 1). Reach II (56° 32.695’N, 16° 36.644’E) was located 4 km downstream of reach I in River

Frösslundabäcken after the stream had passed about 2 km of open Alvar and meadow and 2 km of

cultivated grain fields. Reach III (56° 35.680’N, 16° 39.165’E) was located in the lower part of

River Åbybäcken in an agricultural area east of Gårdby. This reach was 5 km from the spring on the

Alvar plain and 3.5 km from the sea. Reach I and II are in one catchment while reach III is in

another. The proportion of catchment occupied by Alvar plain ranged from 100% at reach I, to 40%

at reach II to 15% at reach III. In contrast, agricultural land use occupied 30% and 40% at reach II

and III, respectively, as determined by GIS analysis of maps showing land use.

The length of the study reaches varied from 130 to 246 m. To describe the physical

conditions and vegetation composition and abundance, transects were placed across the stream

every 10 m along the reaches. In each transect, stream width was measured and water depth,

substrate type and macrophyte species occurrence were determined at five points at equal distances.

In each reach, the proportions of run, riffle and pool flow types were estimated by visual assessment

at 10 m intervals. Average current velocity and travel time of water for the whole reach were

measured by injecting a pulse of NaCl and recording conductivity over time downstream of the

reach (White 1978). Discharge was calculated as the product of mean current velocity and mean

cross-sectional stream area for the whole reach. The slope of the reaches was determined during the

first visit using standardized leveling equipment to measure change in the surface water level along

the stream reach. Reach III had a much lower discharge in October 2011 than in May 2011, and

slope measurements were repeated.

In-stream plant cover on the reach scale was calculated by dividing the number of

points in transects with plant observations by the total number of points examined. The relative

frequency of species occurrence was calculated by dividing all observations per species by the total

number of plant observations.

Two water samples were collected at each reach and during each sampling period, and

analyzed in the laboratory for NO3- and NH4

+ using a flow injection analyzer (Lachat, QuikChem

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FIA+, 8000series, Method 10-107-04-1-C and Method 10-107-06-3-D, respectively). Detection

limits are 0.01 mg N L-1 for NO3- and 0.005 mg N L-1 for NH4

+. Soluble reactive phosphorus (SRP)

and total phosphorus (TP) were measured using the methods described by Brix and Schierup (2001)

based on spectrophometry, and total nitrogen (TN) and particulate organic carbon (POC) was

determined on a TOC-VCPH analyzer (Shimadzu, TNMU). Detection limits were 1 µg P L-1 for TP

and SRP, 0.1 mg N L-1 for TN, and 0.56 mg C L-1 for POC. Water samples for NO3-, NH4

+ and SRP

were filtered before analyses. Alkalinity was determined by acidimetric end point titration (pH 3.7)

using 0.05 M HCl (Rebsdorf 1972). From alkalinity and pH, we determined free CO2 content using

Table 3 in Pedersen et al. (2013).

Stream reach metabolism

Stream reach metabolism was measured by the upstream-downstream two-station

oxygen change technique described by Odum (1956) following improvements by Marzolf et al.

(1994, 1998) and Young et al. (1998). We used YSI 6600 V2-2, Multiparameter Water Quality

Probes to measure dissolved O2 (DO, mg L-1), pH and temperature every 10 minutes. Surface

irradiance was measured with a LiCor (Li-1400) quantum sensor (Lincoln, Nebraska) placed on the

bank. The probes were calibrated at 100% saturation in calibration caps before they were deployed

in the streams. To correct for drift of O2, the probes were placed together in the stream before and

after measurements for at least half an hour. Drift in the O2 sensors was assumed to be linear over

time, and any difference between sensors over time was corrected accordingly. Average drift

between two sensors was 0.08 mg O2 L-1 day-1.

We measured reaeration rate based on measurements of gas exchange velocity (min-1)

for gas exchange over the air-water interface using cylindrical chambers of the same type as

described by Sand-Jensen and Staehr (2012). Bott et al (2006) recommends to measure reaeration

using propane gas, but a gas chromotograph (GC) could not be brought for the remote fieldwork, so

the chamber method was used. The chambers permit a natural, undisturbed water flow below a

water-air interface area of 0.39 m2 inside the chamber holding an air volume of 47.9 L. The

chamber was placed at the water surface and flushed with N2-gas from a pressure tank.

Subsequently, the influx rate of CO2 from the water to the enclosed air volume was recorded at 3

second intervals using an infrared gas analyzer (LI-COR Environmental Li-840) connected to a

laptop computer. The gas exchange was recorded for 10 minute intervals and repeated three times at

each location. CO2 concentrations in the chamber increased linearly over time (r2: 0.994-0.999), and

the mean coefficient of variation of triplicate calculation of evasion rates (CV: SD/xmean) was only

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0.08. The calculated values for reaeration was normalized to 20 ˚C, which corresponds to a Schmidt

number of 600 (k600), to allow comparisons between gas exchange velocities measured at ambient

temperatures. The reaeration speeds was normalized according to the equation by Raymond et al.

(2012):

(1)

n

gas

gas

gas

gas

Sc

Sc

k

k

2

1

2

1

which is based on work done by Jähne et al. (1987) and Wanninkhof (1992), where kgas1 and kgas2 is

the reaeration rate at ambient temperature and 20˚C respectively, Sc is Schmidt number for the two

gasses and n is a constant expressing the mixing regime. In streams, n is set to 0.5 according to

Benson et al. (2014). The same equation was used to convert reaeration rates for CO2 to reaeration

rates for O2. Schmidt numbers for CO2 and O2 at different temperatures are available in

Wanninkhof (1992). The reaeration rate was measured for the different flow types (run, riffle and

pool). At each site, we measured reaeration three times in a row and the measurements were

averaged. Relative cover of flow types was multiplied by the respective reaeration rates and

summed to attain a whole-reach flow-weighted reaeration. In May 2010, reaeration rates were

measured at two runs at reach I, and at two runs and one riffle at reach II. In May 2011 at reach II,

we measured reaeration at two runs (one slow and one fast) and at two riffles. No pools were

present in the reach, but one of the runs had deep slow flowing water. At reach III, we measured

reaeration at two runs (one slow and one fast). In October 2011, we measured reaeration rates at

two runs and one riffle (the only one on the reach) at reach I. At reach II, we measured reaeration at

two riffles (one slow and one fast), at one run and one pool, and at reach III, we measured

reaeration rate at two runs (one slow and one fast).

Metabolism was measured on sunny days, and therefore, measured GPP (g O2 m-2 d-1)

is considered as maximum values for the reaches and periods. Net O2 changes corrected for

reaeration were calculated at 10-min intervals during the 24 hours. Metabolism calculations

followed Bott (2006). Daily net ecosystem production (NEP, g O2 m-2 d-1) was determined from one

hour after sunrise to one hour before sunset (according to timeanddate.com). ER (g O2 m-2 d-1) was

determined from changes in metabolism in the dark period between 0 and 3 a.m. and multiplied by

24 to attain daily ER. GPP was calculated as the sum of NEP during the photic period plus hourly

ER rates multiplied by the photic hours. NEP was converted to NEP g O2 m-2 min-1 from the 10

minute measurements and plotted against surface irradiance to compare the potential production

between the three reaches. Here we omitted measurements from the afternoon in reach I and

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measurements before noon in reach II and III due to shading from bank vegetation to be able to

compare measurements made in full sun between the reaches. During night-time, the progressive

changes in dark respiration were determined after correcting the metabolism (R15) for temperature

changes according to an Arrhenius equation (2) with a Q10 of 2.0 as being a general Q10 for

biological processes:

(2) R15 = Ra * Exp[(15-T a)*0.0693]

where, R15 is the calculated metabolism at 15 °C, Ra (g O2 m-2 min-1) is the measured respiration

rate at ambient temperature (Ta) and 0.0693 (˚C-1) is a constant temperature coefficient

corresponding to Q10 of 2 (Sand-Jensen et al. 2005).

Chlorophyll a (g chl. a m-2) was measured on four sediment types (mud, sand, gravel and stone).

Five replicates were collected on mud, sand and gravel and 10 on stone (five stones from run and

five from riffles). Sand and mud were sampled using a small cylindrical core (area = 6.7 cm2) and

gravel with a large core (area = 22.2 cm2). Chlorophyll was extracted from a 1 cm (sand and mud)

and a 3 cm (gravel) surface sediment core. Chlorophyll on stones was measured by scrubbing the

stones thoroughly and filtering the particles onto glass fiber filters (GFC) for ethanol extraction.

Chl. a was measured in triplicate in May and October 2011 by filtering stream water onto glass

fiber filters. All chlorophyll extractions were made in 90% ethanol for 24 hours, and chlorophyll

was measured in a spectrophotometer (Shimadzu, UV-1800) and calculated according to

Lichtenthaler (1987).

Organic matter (ash-free dry mass, AFDM) in sediment samples was also measured at

all reaches and for all sediment types using the same number and type of samples as for chlorophyll

determination. Organic matter was measured as loss on ignition at 550 °C for 24 hours of 60 °C

dried samples.

For chl. a and organic matter, we calculated habitat-weighted chl. a (mg chl. a m-2)

and organic matter (g AFDM m-2) by multiplying chl. a biomass and organic matter content with

the proportional cover of the different sediments and adding all values together.

Photosynthesis experiment

For the dominant in-stream plant species (Alopecurus geniculatus L., Carex flacca

Schreb., Galium palustre L., Mentha aquatic L.) and algae species (Spirogyra sp. and Cladophora

sp.) at reach I and II, a photosynthetic experiment was performed in May 2010 to estimate the

possible contribution of plants to in-stream primary production and respiration. Before the

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experiment, the oxygen electrode (OX500; Unisense, Aarhus, Denmark) was calibrated in water

with 0% and 100% air saturation obtained by bubbling with N2 and atmospheric air, respectively.

Separate plant leaves or algal filaments were incubated in triplicate in ambient stream water in 50

mL glass-stoppered glass bottles, placed on a rotating wheel in a temperature-constant incubator (15

oC) and illuminated with 450 µmol m-2 s-1 for 2 hours, without reaching supersaturation.

Subsequently, the bottles were transferred to the dark for 20 hours to measure respiration. Ambient

stream water without plants was used in four blank bottles. Oxygen concentration was measured in

samples and blanks after light and dark incubation. Plants and algae were then freeze-dried and

weighed to calculate the photosynthetic rate (Pambient, mg O2 g DW-1 d-1) and dark respiration (R, mg

O2 g DW-1 d-1) relative to dry weight (DW).

To estimate the contribution from the different plants and algae to the metabolism of the entire

reach, we multiplied net photosynthesis and respiration relative to DW with the biomass in the field

expressed as DW m-2 d-1, thereby getting GPP (g O2 m-2 d-1) and R24 (g O2 m

-2 d-1). Hourly rates

were converted to daily rates by multiplying with day length. Photosynthetic rates will, therefore,

overestimate in situ rates because the calculation assumes that tissues are fully light saturated

throughout the day, even in dense stands, which is unlikely to be fulfilled. Furthermore, the

measurements did not include respiration from roots, thus, the photosynthetic calculations represent

maximum capacity under ambient conditions rather than realized rates. In the initial survey, we

determined the coverage (25, 50 or 75% cover) of the following plants: A. geniculatus, C. flacca, G.

palustre, and M.aquatica, and of two macroalgae: Spirogyra sp. and Cladophora sp.. We sampled

the different species in triplicate (bottom area = 22.2 cm2) within the 25, 50 and 75% cover, dried

the samples and measured DW, after which the data obtained were used to calculate in-stream plant

DW m-2 stream bed.

Comparison of metabolism among streams

To identify variability and regulating factors for the metabolic parameters (GPP, ER,

GPP/ER), we compared daily values for stream reaches on Öland with those reported for other open

reaches in North European lowland streams (Edwards and Owens 1962; Simonsen 1974; Kelly et

al. 1983; Riis et al. 2012; 2014; Alnoee unpubl. data). We examined the relationship between daily

GPP and ER for the spring-summer period (April-August) when irradiance is high and the autumn-

winter period (September-March) when irradiance is low. Furthermore, we compared GPP/ER with

surface irradiance for open reaches from all over the world where these values have been measured

simultaneously (Edwards and Owens 1962; Simonsen 1974; Fellows et al. 2001; Mulholland et al.

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2001; Acuña et al. 2011; Rasmussen et al. 2011). This relationship was constructed to evaluate if

Öland streams separate from other streams due to strong nutrient limitation. If the streams are

nutrient limited, one should expect that the GPP/ER ratio would be lower than in streams in

nutrient-rich areas at the same irradiance.

Results

Physics and chemistry

All three stream reaches on Öland were small, had low downstream slopes and low

current velocities (Table 1). Reach I and II ran close to the riparian ground surface, whereas reach

III was incised and more shaded by emergent plants on the south bank. Travel time varied from 41

to 256 min between reaches.

Alkalinity was consistently high (3.01-4.88 meq L-1, Table 2), and the concentrations

of TP (4.89-14.88 µg P L-1, Table 2) and SRP were extremely low (<0.01-7.73 µg P L-1). Reach I

on the Alvar plain was only weakly supersaturated (about two-fold) with CO2 (30 µmol L-1), while

saturation was about 20-fold higher in reach III (250-380 µmol L-1). Water pH declined with

increasing concentrations of CO2. Oxygen concentrations were close to air saturation in reach I and

II and consistently undersaturated in reach III.

While NO3- was under the detection limit and NH4

+ was very low (0.01-0.03 mg N L-

1) in reach I, both NO3- (0.05-0.24 mg N L-1) and NH4

+ (0.02-0.08 mg N L-1) increased in reach II

after passage through an agricultural section (Table 2). The influence of agriculture and more fertile

soils on NO3- concentrations (0.70-1.99 mg N L-1) was even stronger in reach III.

Sediment, vegetation and organic matter

Sediment cover varied between sampling periods and reaches (Table 3). The main

sediment types were thin deposits of sand and gravel on the limestone pavement in reach I, bare

limestone pavement and thin organic deposits on the limestone pavements in reach II, and thicker

sand deposits in reach III. The in-stream plant species were A. geniculatus, C. flacca, G. palustre,

M. aquatica and Potentilla acaulis L. covering 73-78 % of reach I, Menyanthes trifoliata L., Berula

erecta (Huds.) Coville, Sium latifolium L. and M. aquatica covering 27-29% of reach II, and B.

erecta, S. latifolium and Phragmites australis (Cav.) Trin. ex Steud covering 43-69% of reach III.

These species were amphibious plants able to live submerged as well as emerged. Obligate

submerged macrophytes were only represented by filamentous green algae (Cladophora sp. and

Spirogyra sp.) covering 5-17% of reach II.

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Habitat-weighted benthic chlorophyll a varied from 32 to 398 mg chl. a m-2 with

reach II having higher content than reach I and III (Table 3). The chl. a content was consistently

low in the water at the three reaches (0.6 to 1.1 μg chl. a L-1). Compared to benthic chl. a, the chl. a

content in pelagic microalgae relative to stream bed area was negligible (i.e. < 0.5 mg chl. a m-2).

Habitat-weighted organic matter (AFDM) varied from 338 g m-2 to 1221 g m-2 (Table 3). Values

varied the most between sampling periods at reach II and the least at reach III. There was no

tendency of reach III having higher organic matter content on the stream bed (Table 3) or suspended

in the water (Table 2).

Stream metabolism

Daily GPP was generally low but reach II attained moderately high values in May

2010 (Fig. 1, App. 1). Daily ER increased several fold from reach I to reach III in both May and

October. As a result, GPP/ER declined downstream mainly due to the increase of ER. In May,

GPP/ER was close to 1.0 in reach I and II, but lower in reach III. In October, all GPP/ER values

were below 0.32. Variation in GPP, ER and GPP/ER was small between days within sampling

periods (Fig. 1).

NEP during the day exhibited a hyperbolic saturation response to surface irradiance

for measurements conducted before noon in reach I and in the afternoon in reach II and III when

shading by bank vegetation was minimal (Fig. 2). NEP required gradually higher incident

irradiances to reach zero further downstream. Thus, 80% of the maximum NEP rate was obtained at

1370 µmol PAR m-2 s-1 in reach I, whereas reach II and III had not reached this level at 2500 µmol

PAR m-2 s-1. Temperature-corrected dark respiration rates were very low and decreased as the night

progressed in reach I and II, except for October in reach II (Fig. 3). In reach III, respiration rates

were much higher and changed less during the night compared to reach I and II (Fig. 3).

The contribution of submerged plants to the overall stream metabolism showed that

the photosynthetic rates varied seven-fold among species, with the filamentous macroalgae,

Cladophora sp. and Spirogyra sp., being the most productive (Table 4). The habitat-weighted

metabolism of the different species derived from the laboratory measurements showed that they

could contribute substantially to the in situ rates of GPP (1.2 to 14.1 g O2 m-2 d-1, corresponding to

52% to 210%; Table 4) and R24 (0.2-0.9 g O2 m-2 d-1, corresponding to 3% to 43%). Adding up the

estimates of Pambient to GPP from the different individual plant species, yielded high values for reach

I (6.1 g O2 m-2 d-1) and reach II (15.7 g O2 m

-2 d-1), suggesting that in-stream plants, in theory, could

account for the measured whole-system rates in reach I (2.3 g O2 m-2 d-1, Table 4, Fig. 1, App. 1)

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and reach II (6.7 g O2 m-2 d-1, Table 4, Fig. 1, App. 1). The sum of habitat weighted dark respiration

rates of plants yielded rates (3.0 in reach I and 1.1 g O2 m-2 d-1 in reach II, Table 4) that were within

the range of the in situ measurements recorded in reach I (2.1 g O2 m-2 d-1, Fig. 1) and substantially

lower than the rates in reach II in May 2010 (6.4-7.8 g O2 m-2 d-1, Fig. 1).

Metabolic variability among Northern European streams

There was a strong positive relationship between GPP and ER in the April-August

measurements in NW European open lowland streams (r2 = 0.78, Fig. 4a). GPP values for the most

nutrient-poor reach, reach I, on the Alvar plain were 5-10-fold lower than most values from

nutrient-rich streams in the United Kingdom and Denmark. GPP and ER values for reach I and II on

Öland were close to the 1:1 line, while the deviation was higher for reach III (Fig. 4a). The

regression line including all points in figure 4a did not have a significantly different slope (p = 0.61)

or intercept (p = 0.47) from the 1:1 line. During autumn-winter (September-March, Fig. 4b), GPP

rates were low and typically lower than the ER rates. In accordance with these findings, the ratio of

GPP to ER in a range of stream reaches increased with mean surface irradiance, reaching values

above 1.0 at 43 mol PAR m-2 day-1 and a mean value of about 1.2 for even higher irradiances (Fig.

5). The shift from heterotrophy to autotrophy at 43 mol PAR m-2 d-1 can take place from April to

September (5 months) in Denmark and South-Sweden following the seasonal change in irradiance

at these latitudes.

Discussion

Downstream gradients in chemistry, biomass and metabolism

We found a generally low GPP and ER in the upstream extremely nutrient-poor reach I. GPP

peaked in reach II moderately affected by agriculture and ER peaked with very high values in reach

III. SRP remained low along the streams from the Alvar plain to the reaches located 4-6 km

downstream after passage of cultivated moraine soils, whereas NO3- gradually increased from

below detection limits in reach I to relatively high concentration in reach III. Therefore, growth of

benthic algae should be severely constrained by P availability at all reaches (Bothwell 1985;

Kjeldsen et al. 1996), although we expect that the P input to the stream increases downstream due to

more agricultural activity in the catchment. The lower P concentration at reach III compared to

reach II in May 2010 and from reach I to downstream stations in October 2011 could be due to

concomitant P uptake by P-limited benthic algae and bacteria.

As originally predicted, the chl. a content increased from reach I to II but decreased

from reach II to reach III along with more agricultural impact. This decline in chl. a could support

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the presence of P-limitation on benthic chl. a or be explained by less available light reaching the

benthic algae in the deeper and more shaded reach III, which, in contrast to the shallower and less

shaded reach I and II, is incised and shaded by emergent plants and tall banks. Thus, no obligate

submerged plants were present in reach III.

As a consequence of changes in nutrient availability, benthic chl. a and light

availability, GPP peaked in reach II and was lower in reach I and III. There was also a high cover of

in-stream obligate macrophytes dominated by filamentous macroalgae at reach II. It is most likely

that benthic microalgae had a strong effect on GPP, because GPP in May 2010 was almost three-

fold higher and chl. a was four-fold higher in reach II than in reach I. The same pattern was found

in October where reach I and III had a lower chl. a content and GPP than reach II. In October, the

highest GPP for all three reaches was only 1.67 g O2 m-2 d-1, probably due to lower daily irradiance

and temperature. GPP was generally low in our study, but comparable with the findings of other

European studies conducted at oligotrophic conditions (Kelly et al. 1983; Von Schiller et al. 2008).

In the extremely nutrient-poor Alvar stream (reach I), GPP was only 2-3 g O2 m-2 d-1 during sunny

summer days with high irradiance and favorable temperatures. GPP between 2-3 g O2 m-2 d-1 is low

compared to other more nutrient-rich unshaded streams with similar irradiance and temperature

levels (Kelly et al. 1983), and our study thus increases the range of measured GPP in European

streams. However, our measurements were higher than those reported by Bunn et al. (1999) for

some shaded Australian forest streams (0.33-0.53 g O2 m-2 d-1) and by Fellows et al. (2006) for

some American montane and forest streams (0.05-1.4 g O2 m-2 d-1). Our result is consistent with

results found by Bernot et al. (2010) which showed that natural streams have a lower GPP than

agriculture and urban streams.

We found a close coupling between GPP and ER in the most nutrient-poor reach I and

II in May. This is consistent with the highly oligotrophic conditions and algae being the primary

source for heterotrophic metabolism, and it suggests low external input of degradable organic

matter at these two reaches and low influence of the hyporheric zone. A much lower GPP than ER

in October in reach I and II suggested supplementary decomposition of terrestrial material supplied

by senescing plants in autumn (Roberts et al. 2007). The high respiration but low primary

production in reach III indicates high influence of the hyporheic zone and high input of

allochthonous degradable material as also reported by Graeber et al. (2012), although we could not

measure higher content of organic matter in surface sediment or seston. The sediments in reach I

were solid limestone pavements with no or thin sand deposits, the sediment in reach II was bare

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limestone pavements covered by thin layers of organic matter, while the sediment in reach III was

mainly deeper sand deposits such that a substantial respiration may take place in the hyporheic zone

deeper down below the surface sediment. It is known that the hyporheric zone may account for 40-

93% of ER (Fellows et al. 2001). Reach III was always strongly heterotrophic and had a metabolism

comparable to that measured in Sonoran desert streams in Arizona (Uehlinger et al. 2002), which

showed GPP/ER values below 0.17. This pattern is accompanied by gradually higher irradiances

needed for photosynthesis to balance ecosystem respiration and for photosynthesis to become light

saturated. While the rates of ER in reach I were 5-10-fold below the summer values recorded in

nutrient-rich Danish streams with the same climate (7.3-22.9 g O2 m-2 d-1, Simonsen, 1974), the

rates in reach III approached similarly high levels.

Rates of photosynthesis and ecosystem respiration were particularly low in reach I and

II where dark respiration appeared to deplete photosynthetic products during the night to the extent

that respiration rates declined and approached zero as the night progressed. In contrast, in reach III

respiration rates and supposedly the supply rates of organic substrates were higher and respiration

remained approximately constant during the night. Such a decline in night-time respiration has to

our knowledge only been demonstrated a few times in streams (Tobias et al. 2007; Hotchkiss and

Hall 2014), but the mechanism is also known from phytoplankton communities (Gibson 1978;

Markager and Sand-Jensen 1989). Future studies should test whether the gradual respiration decline

during night is a general phenomenon for oligotrophic streams fuelled by daytime photosynthesis.

The importance of the Alvar vegetation

In-stream plant coverage was high in all three stream reaches and during all three sampling periods,

reach I having the highest cover (about 75%). By calculating the potential contribution of plants to

whole-stream metabolism using habitat-weighted estimates in reach I, we found that respiration was

less than 1 g O2 m-2 d-1 for all plants and that GPP varied from 1.2 to 14.1 g O2 m

-2 d-1. The

estimated whole stream rates of GPP and R24 in reach I were 6.1 g O2 m-2 d-1 and 3.0 g O2 m

-2 d-1,

respectively, and thus the plants dominated by terrestrial grass species, could account for the whole

stream metabolism. In reach II, respiration of submerged macrophytes dominated by filamentous

macroalgae could only account for one fifth of the total respiration, whereas they could account for

the entire production. Acuña et al. (2011) also found that macroalgae habitats in streams were able

to account for 30-90% of GPP at the Pampean investigated reach but only for 2-20% of ER. Our

results suggest that in-stream terrestrial plants may have a strong influence on whole-stream

metabolism in intermittent reaches where this plant type may cover large parts of the stream bed.

125

The terrestrial in-stream plants in May 2010 in reach I remained photosynthetically

active despite the restricted availability of 30 µmol L-1 of CO2 in the water. All former experiments

have shown that these plant types are unable to exploit the abundant source of HCO3- in the water

(Sand-Jensen et al. 1992; Maberly and Madsen 2002). However, some species (e.g. Carex flacca

and Alopecurus geniculatus) retain a thin gas film on the leaves under water, allowing stomata to

operate, gases to bypass the resistant cuticle and higher rates of photosynthesis and respiration to

take place than is the case for leaves devoid of a gas film (Colmer and Pedersen 2007). Inorganic

nutrients were mostly undetectable in the stream water in reach I, which suggests strong constraints

of the chl. a development and productivity of benthic algae. In contrast, vascular plants can enhance

the dissolution of phosphorus from the calcareous soils by root release of organic acids (Tyler and

Ström 1995; Ström et al. 2005). After decomposition of plant tissue and terrestrial detritus, nutrients

are transferred to the streams during high-flow periods and become available to benthic algae. The

intermittent flow in the Alvar streams increases the likelihood that dissolved nutrients are exported

during the early phase of resumed flow before benthic algae have had the time to develop an

appreciable biomass for nutrient uptake. Intermittent flow supposedly restricts benthic biomass and,

in particular, prevents development of perennial species, which contrasts the situation in permanent

ponds on the Alvar plain where a gradual build-up of a high charophyte biomass occurs via

exploitation of sediment rather than water nutrient resources (Christensen et al. 2013). Such

charophyte beds in Alvar ponds attain a high GPP and ER (about 10 g O2 m-2 d-1) at the biomass

peak in July (Christensen et al. 2013). Thus, provided a long time period and availability of

alternative sediment resource, slow-growing species can establish a high biomass and substantial

rates of GPP, although they do not reach the peak values recorded in nutrient-rich lowland streams

(e.g. 25 g O2 m-2 d-1, Fig. 4).

In conclusion, we found generally low GPP in the Öland streams and some six-fold

lower rates than the maximum rates found in shallow streams in agricultural landscape of NW

Europe. A strong positive relationship between daily GPP and ER was found at the two most

nutrient-poor sites with very thin sediments on limestone pavements and low influence from the

hyporheric zone. Temperature corrected instantaneous ER rate was highest in the beginning of the

night, but decreased at the end of the night at all three reaches, indicating that dark respiration

depleted photosynthetic products and became limited by organic substrates. This study has extended

the range of GPP and ER measurements in NW Europe by including a very nutrient poor stream,

increased the knowledge on stream metabolism in this, otherwise, highly agricultural impacted

126

region, and documented a strong relationship between GPP and ER in streams ranging from

extremely nutrient poor to moderately nutrient rich conditions during spring and summer.

Acknowledgements

The authors acknowledge The Danish Council for Independent Research, Carlsberg Foundation

and Villum Kann Rasmussen Foundation to "Centre of Excellence for Lake Restoration" (grants for

T. Riis and K. Sand-Jensen). We also thank Peter Anton Staehr for comments to the paper, Anne

Mette Poulsen for valuable editorial comments and four anonymous reviewers.

127

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Table 1 Mean physical conditions in reach I, II and III during the three sampling periods.

Further details on study sites are found in Methods

Table 2 Water chemistry, oxygen concentration (O2, mg L-1) and temperature in reach I, II and III

for the three sampling periods

Table 3 Sediment cover (%), macrophyte cover (%), habitat-weighted benthic chl. a (mg chl. a m-2)

and habitat-weighted organic matter (g AFDM m-2) in reach I, II and III for the three sampling

periods

Reach I Reach II Reach III

May 2010 Oct 2011 May 2010 May 2011 Oct 2011 May 2011 Oct 2011

Sand (%) 48.1 50.6 26.2 19.0 24.0 63.6 68.1

Gravel (%) 41.2 32.6 9.6 23.5 15.6 20.3 18.9

Stone and limestone plates (%) 10.7 16.8 64.2 57.5 60.4 16.1 13.0

Obligate water plants (%) 0.9 1.7 16.6 5.0 6.4 0 0

Amphibious plant (%) 77.7 73.0 27.6 28.6 25.5 42.5 69.2

Benthic chl. a (mg chl. a m-2) 99.6 31.9 398.1 194.4 215.3 108.8 169.9

Organic matter (g AFDM m-2) 939.4 840.6 1220.7 690.6 337.8 557.9 525.5

Reach I Reach II Reach III

May 2010 Oct 2011 May 2010 May 2011 Oct 2011 May 2011 Oct 2011

Slope (m km-1) 0.23 - 0.23 - - 0.07 0.04

Velocity (cm s-1) - 1.4 3.7 2.8 4.9 6.0 1.3

Discharge (L s-1) - 0.3 22 11 24 130 23

Width (m) 2.21 2.39 3.85 3.24 3.34 4.56 4.01

Depth (m) 0.11 0.10 0.15 0.13 0.15 0.47 0.33

Travel time (min) - 255.5 130.3 148.9 84.2 40.58 183.9

Reaeration rate (min-1) 0.021 0.011 0.015 0.008 0.021 0.005 0.009

Reach I Reach II Reach III

May 2010 Oct 2011 May 2010 May 2011 Oct 2011 May 2011 Oct 2011

pH 8.43 8.38 8.15 8.01 8.02 7.48 7.64

Alkalinity (meq L-1) 3.01 3.07 3.74 3.74 4.12 4.24 4.88

CO2 (μmol L-1) 30 30 60 100 90 380 250

POC (mg C L-1) 4.53 5.55 5.22 2.44 3.68 3.69 4.68

TP (μg L-1) 4.89 10.74 14.88 13.23 11.66 11.34 10.93

SRP (μg L-1) <0.01 3.43 5.82 7.73 <0.01 2.72 <0.01

Org-N (mg L-1) 0.29 0.31 0.41 0.07 0.17 0 0.1

NH4+ (mg L-1) 0.01 0.03 0.02 0.08 0.04 0.03 0.05

NO3- (mg L-1) <0.01 <0.01 0.15 0.24 0.05 1.99 0.7

O2 max (mg O2 L-1) 12.1 13.7 13.1 13.1 12.1 9.1 7.0

O2 min (mg O2 L-1) 8.9 11.2 8.0 8.3 10.1 6.2 5.5

Temperature (˚C) 14.39 5.63 12.48 11.97 6.51 6.47 6.73

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Table 4 In-stream plant cover (%) in May 2010 in Reach I and II, dry weight (DW, g m-2), leaf

photosynthesis (Pambient, mg O2 g DW-1 d-1), leaf respiration (R, mg O2 g DW-1 d-1), and habitat-

weighted and reach scale GPP (mg O2 m-2 d-1) and R24 (mg O2 m

-2 d-1) for the five dominant

submerged plants, obtained by ex situ experiments

Alopecurus

geniculatus

Carex

flacca

Galium

palustre

Mentha

aquatica

Spirogyra and

Cladophora

n 4 4 1 3 1

Plant cover (%) Reach I 38.3 23.5 17.4 17.4 -

Reach II - - - 17.5 34.0

DW (g m-2) 72.0 116.3 72.0 116.3 317.9

Pambient (mg O2 g DW-1 d-1) 30.5 ±6.9 20.4 ±9.9 70.9 58.3 ±48.3 136.1

R (mg O2 g DW-1 d-1) 30.0 ±5.2 17.4 ±3.6 66.2 45.9 ±39.5 2.4

Habitat-weighted metabolism Total Reach

scale

GPP (g O2 m-2 d-1) Reach I 1.6 1.2 1.7 1.6 - 6.1 2.3

Reach II - - - 1.6 14.1 15.7 6.7

R24 (g O2 m-2 d-1) Reach I 0.8 0.5 0.8 0.9 - 3.0 2.1

Reach II - - - 0.9 0.2 1.1 7.1

133

Fig. 1 Mean daily GPP (g O2 m-2 d-1), ER (g O2 m

-2 d-1) and GPP/ER in the three stream reaches in

May 2010 and 2011 and October 2011. Dashed line shows GPP/ER = 1. In May, n = 1 in reach I, in

reach II n = 2 in 2010 and n = 5 in 2011, n = 2 in reach III in 2011. In October, n = 2 in reach I and

II and n = 3 in reach III. Exact numbers are shown in appendix 1

Fig. 2 Relationships between surface irradiance (μmol m-2 s-1) and NEP (g O2 m-2 min-1) in the three

reaches. To reduce the influence from bank shading, data in reach I were from before noon in May

2010, and data in reach II and III were from the afternoon in May 2011. Overall significant

hyperbolic relationships are shown

Fig. 3 Night-time respiration rate (g O2 m-2 min-1) corrected for temperature variations in the three

stream reaches for three sampling periods. One night per sampling period is shown. Please note that

the scale for the y-axis of reach III differs from that of reach I and II

Fig 4 Relationships between daily GPP (g O2 m-2 d-1) and ER (g O2 m

-2 d-1) in different streams in

Northern Europe during two seasons; April-August (Spring-summer) and September-March

(Autumn). Overall significant linear regressions are shown, and the dashed line shows GPP/ER = 1.

Data were summarized from Edwards Owens (1962), Simonsen (1974), Kelly et al. (1983), Riis et

al. (2012;2014), Alnoee et al. (unpubl. data), and this study

Fig. 5 Relationships between GPP/ER and daily surface irradiance (mol m-2 d-1) in temperate

streams. An overall significant hyperbolic relationship is applied. Data were summarized from

Edwards and Owens (1962), Simonsen (1974), Fellows et al. (2001), Mulholland et al. (2001),

Rasmussen et al. (2011), Acuña et al. (2011), and this study

134

Figure 1

135

Figure 2

136

Figure 3

137

Figure 4

138

Figure 5

Appendix 1 Mean daily GPP (g O2 m-2 d-1), ER (g O2 m-2 d-1) and GPP/ER in the three stream reaches in May 2010 and 2011 and

October 2011. Values are shown in figure 1

Reach I Reach II Reach III

May 2010 Oct 2011 May 2010 May 2011 Oct 2011 May 2011 Oct 2011

n 1 2 2 5 2 2 3

GPP (g O2 m-2 d-1) 2.26 0.38 6.69 3.09 ±0.20 1.67 3.30 0.38 ±0.01

ER (g O2 m-2 d-1) 2.05 1.20 7.13 2.89 ±0.45 6.35 7.98 27.24 ±0.18

P/R 1.10 0.32 0.95 1.08 ±0.11 0.26 0.42 0.01 ±0.00

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Paper 5 - Caught Between Drought and Flooding on Ölands Great Alvar (in Swedish, English abstract)

© Ole Petersen

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Acknowledgements

First of all I want to thank my principal supervisor Kaj Sand-Jensen for taking me on this scientific

journey which has taught me so much! For your invaluable guidance and support, for taking time to

discuss matters even on the busiest of days and in weekends, for many fantastic days in the field,

“hunting” rare flowers and birds, for interesting talks and for almost coming to peace with the fact

that I don’t find grasses and half grasses interesting. I want to thank my co-supervisor Peter Stæhr

for always having an open door, for introducing me to the possibilities in international

collaborations, for great times in the field on all kind of projects and for technical guidance.

I am grateful to the VIPs at the department. Ole, Kirsten and Dean I have had the great pleasure to

teach courses with. Jens, dit skrummel, you are the heart, soul and entertainment of the department!

I want to thank Theis for technical guidance and many great hours on Öland.

I want to thank my old officemate Jesper for all the help and time you have given even in your free

time and for showing me how to relax in stressed situations. Lars I want to thank for many hours in

the field and for making the long days a little bit more fun.

I am grateful to have met so many fantastic people at the Freshwater Biological Laboratory: Trine,

Winkel, Anja, Ane, Claus Møller, Frandsen, Matteo, Laci, Stine, Petur, Søren, Søren, Jesper,

Magnus, Lasse, Dennis, Max, Jos, Iversen, Emilie, Anne, Emil and Kenneth. A special thanks to

kuttersøstrerne (our Kathrines) and Mikkel MØ who have all helped me in my field work, you make

our office a nicer place to work in.

I want to mention Eleanor, Bas, Biel and Rafa from NetLake, Anette and Tenna from Århus. In

England I met Ian, Iestyn, Alex and Stephen. Going there was a great experience.

The staff at Freshwater Biological Laboratory Anne, Finn, Allan, and their predecessors Birgit, Nils

and Flemming have all been incredibly helpful. Special thank goes to Ayoe, you are amazing, and

the department would never be as productive without you to kick the asses that need kicking or

without your incredibly helpful attitude when mountains of samples show up and need processing

“yesterday”.

Thanks and much love to my friends and family who have supported me all along.

Mange tak til jer alle!!