Oxygen dynamics and plant-sediment interactions in isoetid ...

Oxygen dynamics and plant-sedimentinteractions in isoetid communitiesfollowing organic enrichment

F A C U L T Y O F S C I E N C EU N I V E R S I T Y O F C O P E N H A G E NB i o l o g i c a l I n s t i t u t eF 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 yPhD thesisClaus Lindskov MøllerOxygen dynamics and plant-sediment interactionsin isoetid communities following organic enrichmentPrincipal Supervisor: Kaj Sand-JensenSubmitted: 25/05/11

PrefaceThe content of this thesis includes part of the work I have been involved in during the last three year as aPhD-student at the Freshwater Biological Laboratory. Five scientific manuscripts are presented forevaluation. Two are already published in New Phytologist and Plant Signaling and Behavior. The otherthree manuscripts are ready for submission. One additional article published in the Danish magazineURT is also included.In addition to the papers selected for evaluation I am a co-author of two peer reviewed articles and fourarticles published in magazines during my time as a PhD-student. One article addresses the rare slow growingcyanobacterium, Nostoc zetterstedtii (submitted to Limnology & Oceanography), where in-situ growth ratesproved to be the slowest recorded for any aquatic macrophyte. Three other articles arose from our yearly fieldtripsto Öland working with the remarkably inhabitants of the small alvar ponds an environment with constantlychanging temperatures and water levels. One of these articles is published in Svensk Botanisk Tidskrift, theremaining ones in URT. The last article is also published in URT addressing effects of organic enrichment ofsediments on root morphology and plant anchorage.

Content9 Abstract11 Dansk resumé13 Introduction18 Aim of thesis19 Thesis summary25 Conclusions and Implications27 Paper 1 Oxygen, carbon and iron dynamics in natural and organically enrichedsediments from oligotrophic lakes inhabited by isoetid vegetation39 Paper 2 High sensitivity of Lobelia dortmanna to sediment oxygen depletion followingorganic enrichment53 Paper 3 Higher gas permeability of leaves provides greater tolerance of Littorellauniflora than Lobelia dortmanna to sediment organic enrichment69 Paper 4 Organic enrichment of sediments decreases arbuscular mycorrhizal fungicolonization of the aquatic macrophytes Lobelia dortmanna and Littorellauniflora85 Paper 5 Outstanding Lobelia dortmanna in iron armor91 Paper 6 Planterødders overlevelse i vanddækket bund99 Acknowledgements

AbstractAbstract● Isoetids occupy littoral zones of the most pristine oligotrophic softwater lakes due to their uniqueadaptations. Intrinsic low growth rates, use of sediment CO 2 for photosynthesis and mycorrhizalsymbiosis enable them to grow where other plants cannot. Eutrophication of many formeroligotrophic lakes has caused a dramatic decline in the occurrence and distribution of isoetidsthroughout northern Europe and North America within the last 100 years since degradable organicmatter builds up in sediments creating a hostile environment for isoetids. The objective of this thesiswas to investigate effects of changes in sediment biogeochemistry following organic enrichment ofsediments on growth and survival of isoetid species with focus on O 2 dynamics, plant nutrition andmycorrhizal associations.● A combination of laboratory and in-situ experiments was performed to investigate plant andsediment response to organic enrichment. Pore-water samples were used to track changes insediment biogeochemistry while O 2 electrodes were used to measure O 2 availability and dynamics inplants and sediment. Plant morphology and nutrition were used as stress indicators. The response ofmycorrhizal fungi to organic enrichment was also addressed.● The studies showed that addition of even small amounts of labile organic matter to sediments hadprofound and long-lasting effects on sediment biogeochemistry and affected isoetid growth andsurvival. Moderate additions sometimes resulted in increased growth and no apparent plant stress,but higher additions resulted in widespread anoxia in sediments and plants, decreased plant nutritionand photosynthesis and accumulation of Fe 2+ , NH 4 + and dissolved inorganic carbon in pore-water ofsediments. Addition of organic matter also resulted in reduced arbuscular mycorrhizal fungicolonization of roots.● Sediment composition is therefore a key issue in rehabilitation of isoetid populations, since hostilesediments cause reduced growth and survival. Littorella was able to cope with higher organicadditions than Lobelia agreeing with its wider distribution presumably because it avoids tissueanoxia during the night by O 2 diffusion across permeable leaves from the water column whereasLobelia experiences many hours of anoxia and low energy yield in anaerobic respiration. Littorellacould due to higher eutrophication tolerance, higher growth rates and higher oxygenation capacity ofsediments be used in restoration projects to creating suitable sediment conditions for less tolerantspecies such as Lobelia.9

Dansk ResuméDansk resumé● Isoetider er den dominerende vegetationstype i bredzonen af næringsfattige blødvandede søer.Deres lave vækstrate, brug af CO 2 fra sedimentet til fotosyntese og bedre næringsforsyning gennemsymbiose med mykorrhizasvampe muliggør vækst, hvor andre planter er ude af stand til at klare sig.Eutrofiering har imidlertid forårsaget en voldsom tilbagegang af isoetiddominerede søer iNordeuropa og Nordamerika. En del af forklaringen er øget sedimentation af omsætteligt organiskstof, som ændrer sedimentbiogeokemien ved at øge iltforbruget og akkumulering af fytotoksiner. Pågrund af den langsomme omsætning i anaerobe sedimenter forbliver sedimentforholdene dårlige ilang tid uanset vandkvaliteten i søen. Formålet med dette phd-projekt var at undersøge isoetidersrespons og overlevelse i organisk berigede sedimenter med særlig fokus på ilttilgængelighed ogsammenspil mellem planter og sediment.● Effekter af berigning blev undersøgt i kontrollerede laboratorieforsøg og i naturlige populationer ifelten. Porevandsprøver blev brugt til at følge ændringer i sedimentprocesser, mens iltelektroder blevbenyttet til at måle ilttilgængelighed i planter og sedimenter under varierende forhold.Plantemorfologi, næringsindhold og fotosyntetisk kapacitet blev brugt som indikatorer forplantestress. Mykorrhizasvampes respons på organisk berigning blev også undersøgt.● Undersøgelserne viste, at selv små tilsætninger af organisk stof til mineralske sedimenter ændrersedimentbiogeokemien, og høje tilsætninger forårsager iltmangel, alkalinisering og akkumulering afammonium og reduceret jern i porevandet. Små tilsætninger har en positiv effekt på isoetiders vækstmens høje tilsætninger forårsager iltstress, nedsat rodudvikling, nedsat fotosyntese og dårlignæringsstofforsyning, hvis iltforbruget i sedimentet overskrider planternes iltningskapacitet.Tilsætning af organisk stof nedsætter ligeledes mykorrhizasvampes evne til at kolonisere planternesrødder.● For at bevarre isoetidvegetationen i søer må iltforbruget forbundet med sedimentering af organiskstof ikke overskride deres iltningskapacitet, da det stresser isoetiderne, mens næringsstoffer blivertilgængelige for andre hurtigtvoksende plantearter. Af de to undersøgte arter var Strandbo mestmodstandsdygtig overfor sedimentberigning i overensstemmelse med dens større udbredelse.Årsagen er sandsynligvis, at Strandbo undgår iltstress om natten via O 2 diffusion overbladoverfladerne fra vandfasen, mens Tvepibet Lobelie med sine gasimpermeable blade udsættes forlange iltfrie perioder i plantevævet, når iltforbruget i sedimentet stiger. Strandbos størreforureningstolerance kan forvente at sætte den i stand til, som en af de første, at genetablere sig irestaurerede søer, og den kan eventuelt bruges til genetablering af søer via udplantninger.11

IntroductionIntroductionOligotrophic soft-water lakes are characterizedby waters low in nutrients (phosphorus andnitrogen) and dissolved inorganic carbon (DIC)because they receive most of their water throughprecipitation or as ground water from carbonateand nutrient poor catchments (Smolders et al.2002). The low concentrations of CO 2 (≈18µM), bicarbonate (

Introductionroot surfaces. This radial oxygen loss (ROL)from the roots generates an oxygenatedrhizosphere (Sand-Jensen et al. 1982; Pedersenet al. 1995; Møller & Sand-Jensen 2011). Unlikemore nutrient-rich organic sediments, microbialrespiration is so low in oligotrophic sandysediments that the entire sediment is fullyoxygenized to several centimeters depth duringday time in dense isoetid stands (Pedersen et al.1995; Møller & Sand-Jensen 2011). In thewinter when respiration in the sediments isslowed down by low temperatures, isoetidsbuild up a sufficiently large pool of O 2 in thesediment during the day to cover the respiratorydemand of sediment and roots throughout thenight (Pedersen et al. 1995). However, increasedrespiration during summer at highertemperatures deprives the sediment of O 2 for ashort period late in the night (Møller & Sand-Jensen 2011).While most isoetids have gas permeableleaves (Sand-Jensen et al. 1982), Lobeliadortmanna has its leaves covered with a thickcuticula almost impermeable to gasses resultingin anoxia in roots and leaf lacuna when thesediment turns anoxic (Møller & Sand-Jensen2011). Impermeable leaves of Lobeliadortmanna is probably an adaptation to avoidloss of CO 2 from the sediment pore-water to thewater column during night where CO 2 diffusingfrom the sediment cannot be used for plantphotosynthesis. Most other species of isoetidshave permeable leaves, but avoid excessive CO 2loss to the water column due to crassulaseanacid metabolism (CAM), where CO 2 is stored inthe plants as malate in the dark for later use forphotosynthesis in the subsequent light period(Madsen 1985; Keeley 1998).Sediment biogeochemistry: By oxidizing thesediment isoetids promote degradation oforganic matter and prevent formation of reducedcompounds in the pore-water (Wium-Andersen& Andersen 1972b; Farmer et al. 1985; Møller& Sand-Jensen 2011). Under these conditionsphosphorus is strongly adsorbed to sedimentparticles and co-precipitates with highlyinsoluble oxidized metals (e.g. Fe and Mnoxides), hence, largely inaccessible to plants(Christensen & Andersen 1996). Furthermore,shifting from fully oxic in the daytime to anoxiain some parts of the rhizosphere during nightpromotes coupled nitrification-denitrificationleading to loss of N from the sediment(Christensen & Sørensen 1986; Ottosen et al.1999). Isoetids can thereby prevent nutrientsfrom entering the water column by promoting Nloss to the atmosphere and P is adsorbed orprecipitated in sediments which is important tomaintain oligotrophic conditions.Mycorrhizal associations: Isoetids are the onlyaquatic plants with frequent and highcolonization of arbuscular mycorrhiza fungi(AMF). This phenomenon was first discoveredin aquatic plants by Søndergaard & Laegaard(1977) and since then confirmed by numerousothers (Farmer et al. 1985; Beck-Nielsen &Madsen 2001; Nielsen 2004). The chronicallyslow growth of isoetids can result in strongnutrient depletion zones around roots causingdecreased uptake (Smith & Read 2008). Byinvesting in AMF the sediment volume andsurface area for absorption and sediment contactare grossly increased which is important foruptake of especially P (Smith & Read 2008).AMF are involved in uptake of P from solid facefractions and are of great importance for isoetids(Smolders et al. 2002). It has been shown that Pcontent of isoetid leaves, unlike non mycorrhizlaaquatic plants, are correlated to total P insediment rather than available P in the pore-14

Introductionwater (Wigand et al. 1998). Very few studieshave addressed the effect of AMF on isoetidnutrition and this calls for further attention sinceisoetids might be obligate mycorrhizal-plantswhen growing in nutrient depleted sediment(Smolders et al. 2002).The isoetids are, in summary, highly adapted tothe most oligotrophic conditions. They arehighly valued indicators of the most pristineaquatic environments. Their intimate contactwith the sediment enables them to grow andmaintain oligotrophic conditions by oxidizingthe sediment and exploiting elevated CO 2concentrations in sediments, but this intimatecontact also means that isoetids face severalproblems when nutrients and inorganic carbonlevels are increased in the environment due toanthropogenic impact.Eutrophication and threats: Many formeroligotrophic lakes have received high amountsof nutrients and DIC through runoff andgroundwater from agricultural and urban areas(anthropogenic activities) and organic matterfrom forest clearing in the upland (Arts 2002;Smolders et al. 2002; Jeppesen 2005).Furthermore, deposition of sulfate and nitratethrough precipitation has caused acidification.Acidification increases CO 2 availability in thewater column and increases sedimentmineralization leading to increased DIC contentof sediments and surface water (Smolders et al.2002) with devastating consequences for thesepristine environments (Roelofs 1983; Smolderset al. 2002; Pedersen et al. 2006; Geurts et al.2008). Surface waters rich in nutrients and DICpromote growth of algae and fast-growingelodeid species of aquatic plants shifting thesystems towards competition for light rather thatnutrients and CO 2 in sediments (Smolders et al.2002).Isoetids by their intrinsic low growthrate and short leaves therefore receive limitedlight restricting them to the shallowest parts oflakes (Jeppesen et al. 2005). Furthermore,increased production in the water column leadsto increased sedimentation of labile organicmatter speeding up decomposition in sediments,hence, creating a higher O 2 demand. Isoetidstherefore face multiple problems with decreasedphotosynthesis caused by shading andincreasing O 2 demand depleting the sedimentand plants of O 2 (Møller & Sand-Jensen 2011)which in turn causes build-up of potential toxiccompounds in the pore-water (Smolders et al.2002). Plants species normally found ineutrophic sediments with high O 2 demand formroot barriers by incorporation of lignin andsuberin in the outer epidermis to avoid ROL andanoxia (Visser et al. 2000; Colmer 2003) butisoetids are unable to form such barriers and aretherefore more susceptible to sediment anoxia(Smith et al. 1990; Møller & Sand-Jensen2008).Restoration projects have reducednutrient levels in the water column resulting inimproved light conditions in many lakes but theisoetids have rarely returned (Jeppesen et al2005). The reason seems to be changes insediment conditions (Sand-Jensen et al. 2008).Sedimentation of organic matter from highproduction in the water column builds up in thesediment during periods of eutrophication.Degradation in sediment is slowed down by lackof O 2 leading to inefficient anaerobicdegradation. Sediments therefore develop amuddy character with low redox potentials, lowO 2 content and low sediment density whichstrongly inhibit growth of isoetids.15

IntroductionIn sediments of high O 2 demand isoetidsroots are restricted to the uppermost sedimentlayer where O 2 supply to root tips is possible(Sand-Jensen et al. 2005; Raun et al. 2010).Short roots offer low anchorage and is furtherweakened in organic sediments by low densityincreasing the risk of uprooting and loss ofpopulations (Pulido et al. 2010). Furthermore,anoxia in roots of isoetids leads to reducedtranslocation of nutrients with decreasedphotosynthesis and low tissue content ofnutrients as a result (Sorrell 2004; Møller &Sand-Jensen 2011). To promote the return ofisoetids to their former growth sites improvedunderstanding of how isoetids respond and copewith changes in sediment biogeochemistry isneeded. This was the main aim of this thesis.ReferencesArmstrong W. 1979. Aeration in higher plants. Advancesin Botanical Research 7: 225–332.Arts GHP. 2002. Deterioration of Atlantic soft watermacrophyte communities by acidification, eutrophicationand alkalinisation. Aquatic Botany 73: 373-393.Beck-Nielsen D, Madsen TV. 2001. Occurrence ofViscicular-arbuscular mycorrhiza in plants from lakes andstreams. Aquatic Botany 71: 141-148Christensen PB, Sørensen J. 1986. Temporal variationof denitrification activity in plant-covered, littoralsediment from Lake Hampen, Denmark. Applied andEnvironmental Microbiology 51: 1174-1179Christensen KK, Andersen FØ. 1996. Influence ofLittorella uniflora on phosphorus retention in sedimentsupplied with artificial porewater. Aquatic Botany 55:183-197Colmer TD. 2003. Long-distance transport of gases inplants: a perspective on internal aeration and radialoxygen loss from roots. Plant Cell Environment 26: 17-36.Farmer AM. 1985. The occurrence of vesiculararbuscularmycorrhiza in isoetid-type submerged aquaticmacrophytes under naturally varying conditions. AquaticBotany 21: 245-249.Geurts JJM, Smolders AJP, Verhoeven JTA, RoelofsJGM, Lamers LPM. 2008. Sediment Fe:PO 4 ratio as adiagnostic and prognostic tool for the restoration ofmacrophyte biodiversity in fen waters. FreshwaterBiology 53: 2101–2116.Jeppesen E, Søndergaard M, Jensen JP, Havens KE,Anneville O, Carvalho L & others. 2005. Lake responseto reduced nutrient loading – an analysis of contemporarylong-term data from 35 case sturies. Freshwater Biology50:1747-1771Keeley JE. 1998. CAM photosynthesis in submergedaquatic plants. The botanical Review 64:121-175.Madsen TV. 1985. A community of submerged aquaticCAM plants in Lake Kalgaard, Denmark. Aquatic Botany23: 97-108Murphy KJ. 2002. Plant communities and plant diversityin softwater lakes of northern Europe. Aquatic Botany 73:287-324Møller CL, Sand-Jensen K. 2008. Iron plaques improvethe oxygen supply to root meristems of the freshwaterplant, Lobelia dortmanna. New Phytologist 179: 848-56.Møller CL, Sand-Jensen K. 2011. High sensitivity ofLobelia dortmanna to sediment O2 depletion followingorganic enrichment. New Phytologist 190: 320-331.Nielsen KB, Kjøller R, Olsson PA, Schweiger PF,Andersen FØ, Rosendahl S. 2004. Colonization andmolecular diversity of arbuscular mycorrhizal fungi in theaquatic plants Littorella uniflora and Lobelia dortmannain southern Sweden. Mycological research 108: 616-625.Ottosen LDM, Risgaard-Petersen N, Nielsen LP. 1999.Direct and indirect measurements of nitrification anddenitrification in the rhizosphere of aquatic macrophytes.Aquatic Microbial Ecology 19: 81-91.Pedersen O, Andersen T, Ikejima K, Hossain MZ,Andersen FØ. 2006. A multidisciplinary approach tounderstanding the recent and historical occurrence of thefreshwater plant, Littorella uniflora. Freshwater Biology51: 865-877.Pedersen O, Sand-Jensen K, Revsbech NP. 1995. Dielpulses of O 2 and CO 2 in sandy lake-sediments inhabitedby Lobelia dortmanna. Ecology 76: 1536–1545.Raun AL, Borum J, Sand-Jensen K. 2010. Influence ofsediment organic enrichment and water alkalinity ongrowth of aquatic isoetid and elodeid plants. FreshwaterBiology 55: 1891-1904.Roelofs JGM. 1983. Impact of acidification andeutrophication on macrophyte communities in soft watersin the Netherlands I. Field observations. Aquatic Botany17: 139-155.Sand-Jensen K, Borum J, Binzer T. 2005. Oxygenstress and reduced growth of Lobelia dortmanna in sandylake sediments subject to organic enrichment. FreshwaterBiology 50:1034-1048.Sand-Jensen K, Pedersen NL, Thorsgaard I, MoeslundB, Borum J, Brodersen KP. 2008. 100 Years ofvegetation decline and recovery in Lake Fure, Denmark.Journal of Ecology 96: 260-271Sand-Jensen K, Prahl C. 1982. Oxygen exchange withthe lacunae and across leaves and roots of the submergedvascular macrophyte, Lobelia dortmanna L. NewPhytologist 91: 103–120.16

IntroductionSand-Jensen K, Prahl C, Stokholm H. 1982. Oxygenrelease from roots of submerged aquatic macrophytes.Oikos 50:1034-1048.Sand-Jensen K, Riis T, Vestergaard O, Larsen S. 2000.Macrophyte decline in Danish lakes and streams over thepast 100 years. Journal of Ecology 88:1030-1040.Smith SE, Read DJ. 2008. Mycorrhizal symbiosis.Elsevier Academic Press, San Diego, CA, USA.Smits AJM, Laan P, Thier RH, Vandervelde G. 1990.Root aerenchyma, oxygen leakage patterns and alcoholicfermentation ability of the roots of some nymphaeid andisoetid macrophytes in relation to the sediment type oftheir habitat. Aquatic Botany 38: 3–17.Smolders AJP, Lucassen ECTET, Roelofs JGM. 2002.The isoetid environment biogeochemistry and threats.Aquatic Botany 73: 325-350.Sorrell B. 2004. Regulation of root anaerobiosis andcarbon translocation by light and root aeration in Isoetesalpines. Plant, Cell and Environment 27:1102-1111.Søndergaard M, Laegaard S. 1977. Vesciculararbuscularmycorrhiza in some vascular plants. Nature268: 232-233.Wigand C, Andersen FØ, Christensen KK, Holmer M,Jensen HS. 1998. Endomycorrhizae of isoetids along abiogeochemical gradient. Limnology and Oceanography43: 508-515.Visser EJW, Colmer TD, Blom CWPM, VoesenekLACJ. 2000. Changes in growth, porosity, and radialoxygen loss from adventitious roots of selected monoanddicotyledonous wetland species with contrastingtypes of aerenchyma. Plant, Cell & Environment 23:1237–1245.Wium-Andersen S, Andersen JM. 1972a. Carbondioxide content of the interstitial water in the sediment ofGrane Langsø, a Danish Lobelia lake. Limnology andOceanography 17: 943–947.Wium-Andersen S, Andersen JM. 1972b. The influenceof vegetation on the redox profile of the sediment ofGrane Langsø, a Danish Lobelia lake. Limnology andOceanography 17:948-952.17

Aim of thesisAim of thesisThe aim of this thesis was 1) to investigate response of isoetids to sediments undergoing organic enrichment2) to establish thresholds for sediment conditions where isoetid species can grow and 3) improve ourunderstanding of the complex interactions between isoetids and sediment under both oligotrophic andeutrophic conditions. Sediment conditions were investigated by measuring O 2 availability in sediments and byanalyzing pore-water for potential phytotoxins and reduced compounds to clarify main biogeochemicalprocesses. Plant response and interactions with sediments were investigated by measuring O 2 dynamics withmicro O 2 electrodes and analyzing plant content and morphology after exposure to hostile sediment conditions.18

Thesis summaryThesis summaryAddition of labile organic matter dramaticallychanged biogeochemistry of sedimentsinhabited by Lobelia dortmanna and Littorellauniflora and affected growth and survival of theplants. In paper 1 changes in sedimentbiogeochemistry, fluxes from sediments andretention of carbon and nutrients was followedin intact sediment turfs subjected to addition ofdifferent amounts of labile organic matter in thelaboratory. Sediment biogeochemistry wasmonitored by frequent extraction of pore-watersamples and O 2 availability in sediments wasmeasured by O 2 mini-electrodes.Organic enrichment caused decreased O 2availability since O 2 consumption fordegradation of added organic matter exceededthe oxygenation capacity of the plants and O 2flux from the water (Fig 1). The lack of O 2resulted in accumulation of NH +4 and use ofalternative electron acceptors causingaccumulation of Fe 2+ in the pore-water lastingFig. 1. Depth of O 2 penetration in Lobelia sediments as afunction of time after addition of different amounts oflabile organic matter (per sediment DW) (○=control;●=0.1%; □=0.2%; ■=0.4%; ◊= 0.8%; ♦=1.6%).Measurements were made 10–12 h into the 12 h lightperiod. Measurements could not extend deeper than 40mm into the sediment, when O 2 penetration in controlsediments was described as > 40 mm. Values are mean oftwo (0–170 d) and three (194 d) measurements ± SD.From Møller & Sand-Jensen 2011.Fig 2. Pore-water concentrations of Fe 2+ and NH 4 + insediments inhabited by Lobelia dortmanna (a & b) orLittorella uniflora(c & d) following enrichment withdifferent amounts of labile organic matter (% ofsediment dry weight; ○=control; ●=0.1%; □=0.2%;■=0.4%; ◊= 0.8%; ♦=1.6%).throughout the experiments and being morepronounced with increasing organic additions(Fig 2). Therefore, addition of even smallamounts of labile organic matter has longlasting effects on sediment biogeochemistry inisoetid sediments. In anoxic sediments oxidizediron becomes increasingly important fordecomposition processes; hence, iron reductionconstituted a larger fraction of DIC formation athigh organic additions than at lower additionsbut although sediments at high additionsremained anoxic for 200 days only 14% of the+iron pool was reduced. Accumulation of NH 4can be explained by nitrification and the lack ofshifts from oxic to anoxic conditions leading tolittle denitrification. In the anoxic sedimentsnitrogen released from degradation thereforeexceeds denitrification rates and loss to the-overlying water. Since isoetids prefers NO 3over NH + 4 as a source of N and that other fastergrowing plants and algae prefers NH + 4 this is apotential problem for preserving isoetidcommunities. Phosphorus (P) was opposite+NH 4 efficiently retained in the sediments19

Thesis summaryadsorbed to particles and precipitated withmetals (89-93% of added P) so isoetidsediments can counteract some P loading byefficiently retaining it in sediments. Regardlessof the amount of organic matter added,sediments inhabited by Littorella were alwaysmore “oxidized” that Lobelia sediments. This isthe result of higher plant density along withhigher photosynthetic rates and greater leaf gaspermeability of Littorella (see paper 3)increasing O 2 supply to sediments.The non-destructive sampling of smallpore-water volumes used in this experimentallowed monitoring of both spatial and temporalchanges in sediment biogeochemistry fordetermining degradation intensity and mainsediment processes. This method can, therefore,be a useful tool for further research especially ifcombined with measurements of release fromsediments to the water column.O 2 demand of sediments) leads to prolongedanoxia in Lobelia (Fig 3). Furthermore, additionof organic matter resulted in reduced rootlengths, low leaf nutrient and chlorophyllcontent and low photosynthetic rates of Lobelia.Prolonged anoxia occurred already 7-9 daysafter addition and reduced photosynthetic rateswere measured after 18 days and were furtherenhanced over the 200 days long laboratoryexperiment (Fig 4).Paper 2 investigates the response of Lobeliadortmanna to sediment O 2 depletion andchanges in sediment biogeochemistry followingorganic enrichment. O 2 dynamics in plants andsediment were measured by O 2 micro and minielectrodes (50 µm tip diameter for plants and500 µm for sediments) and plant response wasmeasured on several occasions by sacrificingplants and measuring leaf content, rootdevelopment and maximum photosyntheticrates. Laboratory experiments were coupledwith in-situ measurements of O 2 dynamics innatural populations.In-situ measurements showed that lacunasystems of Lobelia dortmanna and sedimentsare depleted of O 2 for some hours during thenight in natural populations at summertemperatures (>16o C) and in laboratoryexperiments organic enrichments (increasing theFig. 3. Diurnal changes of O 2 partial pressure (PO 2 ) inleaf lacunae of Lobelia (solid line), sediment porewater at 10 mm depth (dashed line) and water phase(dotted line) in laboratory experiments in the control(a), 0.4% (b) and 1.6% (c) treatments after 4, 9 and 7days of enrichment, respectively. The diurnal tracestarted with a shift from 12 h light to 12 h darknessfollowed by a shift back to 12 h light. Values are singlemeasurements.20

Thesis summaryFig. 4. Chlorophyll content (a) and maximum netphotosynthesis (b) of Lobelia leaves after increasingaddition of labile organic matter (% of sediment DW).Single measurements were made after 18 d (○) and 59 d(●) and triplicate measurements (± SD) after 194 d (□) ofexperiments in the laboratory.The long anoxic periods in plants duringthe night is the most plausible reason for theobserved plant stress since low yieldinganaerobic respiration can deplete Lobelia ofcarbon resulting in insufficient carbon supply toroots. This will cause root malfunction andosmotic stress constraining transfer of nutrientsto leaves. However, Lobelia can cope withanoxia which is a recurring phenomenon innatural populations during the summer.Paper 3 addresses differences in plantmorphology and response to organic addition ofLobelia and Littorella. Measurements from turfexperiments in the laboratory and in-situexperiments with mixed populations were usedto evaluate effects of enrichment. O 2 loss fromleaf surfaces of both species to hypoxic waterwas used to investigate leaf gas permeability.Addition of organic matter dramaticallydecreased photosynthetic rates of Lobelia whileLittorella was able to cope with additions inlaboratory experiments. This is partly explainedby higher plant density in Littorella turfs, butmarked differences were observed between O 2dynamics in Lobelia and Littorella where O 2concentration in leaves was unaffected inLittorella regardless of O 2 availability insediments while Lobelia faced long anoxicperiods during nighttime. Littorella was able tomaintain nutrient levels and chlorophyll contentin leaves at higher organic additions thanLobelia. The observed difference in O 2dynamics is the result of 13-16 times higher gaspermeability of Littorella leaves compared toLobelia (Table 1).In-situ measurements confirmed thatLobelia was more subjected to stress thanLittorella when mixed populations weresubjected to organic enrichments, hence,experiencing exactly the same conditions sincethe higher oxygenation capacity of Littorellaalso benefits Lobelia in mixed populations. Inthis experiment Littorella was more stressedthan in the laboratory experiments. The higherresponse of both species over the much shorterexperiment (90 days) could be due to highertemperatures (Mean 20.6 o C, maximum 35 o C) inthe in-situ experiment, then the laboratoryTable 1. O 2 flux across leaf surface to hypoxic water withbasal leaf lacunae in air contact and evaporation to drystill air of Lobelia dortmanna and Littorella uniflora.SpeciesTemperature( o C)Flux(n mol O 2 m -2 s -1 )Lobelia 5 30 ± 7 a15 48 ± 3 aLittorella 5 482 ± 199 b15 608 ± 144 bValues are means ± SD of 3 (O 2 flux) or 10 (Evaporation)replicates. Different letters show significant differences(2-way or 1-way ANOVA).21

Thesis summaryexperiment (15 o C), speeding up decompositionprocesses and respiration in both plants andsediment, hence, increased temperature isprobably an extra stress factor. The ability tomaintain oxic conditions in plant tissue canexplain the broader occurrence of Littorella inlakes undergoing eutrophication. Hence,Littorella only needs sufficient light to sustaincarbon production to cover aerobic respirationof the plant tissue. In eutrophic lakes Lobeliawill suffer from both decreased production andlow energy output under anaerobic respirationwhich could be the main reason for loss ofLobelia from many former growth sites.Although widespread anoxia occurred inLobelia at summer temperatures, O 2 diffusionacross leaves may be sufficient to satisfy therespiratory demand during winter when snowcovered ice prevents photosynthesis and lowtemperature slow down respiration.Paper 4 focus on occurrence of arbuscularmycorrhizal fungi associations in isoetids andsediments and the responses to organicenrichment. Mixed field populations were usedto compare AMF colonization of both specieswhile experiments with intact sediment turfs ofLobelia or Littorella with indigenous AMFassociations were used to investigate responseof AMF colonization and hyphal density insediment to organic enrichment. Furthermore,experiments with non-colonized Littorellaintroduced to sediments with AMF and differentorganic additions were performed to investigatecolonization under more eutrophic conditions.In nutrient poor sediments Littorellahave higher AMF colonization than Lobeliawhen growing in mixed populations undernatural conditions. This result is consistent withthe two times higher growth rate of LittorellaFig 5. Root length colonized by AM fungi (%) in rootsof Lobelia dortmanna (upper panel) and Littorellauniflora (lower panel) after experimentation withaddition of labile organic matter to sediments.Colonization frequencies of roots produced during theexperiment (○) and roots most likely established priorexperimentation (●) are presented. Values are means ±SD.than Lobelia. i.e., more nutrients are required tosustain higher growth. The investigation alsoshowed that hyphal density in the aquaticsediments were high and comparable withterrestrial systems, with hyphal surfacesexceeding the area of roots several times andscavenging a much larger sediment volume.Hyphal density was well correlated to colonizedroot length in the sediments.Addition of organic matter to sedimentsdecreased colonization of roots of plants alreadycolonized by AMF and was mainly caused bydecreased colonization of roots produced whilegrowing in more reduced sediments (Fig 5).Almost no colonization occurred in initiallynon-colonized Littorella at low additions whileAMF colonized roots in low-organic controlsediments. The stress of Lobelia and Littorellaobserved at high organic additions could not be22

Thesis summaryascribed to lack of AMF and AMF should beseen as an advantage under the extremelynutrient poor conditions that characterize thegrowth habitat of isoetids.Paper 5 is a short communication (addendum)addressing effects of organic enrichment ofsediments on O 2 dynamics and survival ofisoetid species. Radial O 2 loss (ROL) from rootsof isoetids was compared to other rootedmacrophytes and effects of Fe-plaques on rootsurfaces was discussed.Species that normally grow in reducedsediments form barriers to prevent O 2 loss fromroots whereas isoetids are believed to lack thisability. However, Fe-plaques form when Fe 2+from anoxic pore-water enters an oxygenatedroot rhizosphere where it precipitates as varioushighly insoluble crystalline oxidized ironcompounds. An investigation of ROL fromroots of Lobelia has shown that Fe-plaquesconstitute a barrier to ROL (Fig 6) resulting inhigher downward O 2 supply to root meristems.This could improve internal oxygenation ofisoetids when eutrophication leads to increasedO 2 demand of sediments. However, uponenrichment roots of isoetids are dramaticallyshortened and offer week anchorage.Furthermore, a recent investigation showed thatLobelia is depleted of O 2 when grown insediments added labile organic matter eventhough Fe-plaques occurred on roots (Paper 2).Whether Fe-plaques benefit isoetids growing inorganically enriched sediments is still to beshown.Fig 6. Radial oxygen loss measured with platinum sleeveelectrodes along the length of roots in an anoxic medium.The basal part of the roots was in contact withatmospheric air. Lobelia dortmanna had either none orthick iron coatings of the root surface (0.09 ± 0.05 and 30± 3 mmol Fe m-2 root surface respectively), whilePhragmites australis (from Armstrong & Armstrong2001) and the seagrass, Zostera marina (Unpublished)were without iron coatings.scientific language to communicate findings ofseveral investigations to a broader audience. O 2supply to root systems via rapid diffusionthrough aerenchyma or pressurized convectivegas-flow in stems and gas impermeable rootbarriers preventing excessive O 2 loss from rootsto sediment are discussed in regard to sedimentO 2 demand.Paper 6 is a Danish article addressingmorphological adaptations of aquatic andemergent plants to life in waterloggedsediments. The article is written in a popular23

Conclusions and implicationsConclusions- In natural populations of Lobelia and Littorella the entire rhizosphere is oxidized by extensive oxygenloss from roots during daytime whereas anoxia occurs in sediments late in the night at summertemperatures.- Oxygen loss from roots prevents formation of reduced compounds in the pore-water and facilitates lossof nitrogen, use of inorganic carbon and precipitation of P in the sediment, hence, maintainingoligotrophic conditions.- Symbiosis with arbuscular mycorrhizal fungi mediates nutrient supply to isoetids by increasing thesurface area for uptake and sediment contact in oligotrophic sediments.- Addition of even small amounts of organic matter to sediments has long-lasting effects on sedimentbiogeochemistry and affects growth of isoetid species. Isoetids seem to benefit from small enrichmentsdue to increased nutrient and CO 2 availability but high organic additions lead to plant stress whensediment O 2 demand exceeds root oxygenation capacity.- High organic additions leads to anoxia, ankalinization and accumulation of NH 4 + and Fe 2+ in the sedimentpore-water and increased efflux of inorganic carbon and NH 4 + to the water column, hence, favoringfaster growing plant species and phytoplankton.- When organic enrichment deprives sediments of O 2 Lobelia experience prolonged tissue anoxia duringnighttime forcing this low productive species to use low energy yielding anaerobic metabolism to coverthe respiratory demand. Littorella avoids plant anoxia due to higher gas permeability of leaves whichmediates O 2 diffusion from the water column across leaf surfaces during the night.- Organic addition also results in low arbuscular mycorrhizal colonization in roots of isoetids but isprobably not responsible for the low nutrient content and plant stress observed at high additions.Implications- Isoetids counteracts eutrophication and maintain oligotrophic sediment conditions by controlling thesediment biogeochemistry. Oxidation of sediments prevents accumulation of organic matter andmediates denitrification and precipitation of P in the sediments unless O 2 demand of sediments exceedsthe oxygenation capacity of the isoetids. However, when isoetids are lost from lakes, hostile sedimentscan prevent re-colonization.- O 2 availability in sediments dictates the sediment biogeochemistry and is thereby the single mostimportant parameter since permanent anoxic sediments hamper growth of isoetids.- To prevent further loss of isoetid populations it is important to keep sedimentation of labile organicmatter low and water transparency high since isoetids otherwise lose control of sedimentbiogeochemistry.- Littorella can by its higher tolerance to eutrophication be used for restoration by improving sedimentconditions for less tolerant isoetid species such as Lobelia and preventing P release from sediments.25

Paper 1Oxygen, carbon and iron dynamics in natural andorganically enriched sediments from oligotrophiclakes inhabited by isoetid vegetationPhoto: Ole Pedersen

Paper 1Oxygen, carbon and iron dynamics in natural and organically enriched sedimentsfrom oligotrophic lakes inhabited by isoetid vegetation.Claus Lindskov Møller and Kaj Sand-JensenFreshwater Biological Laboratory, Biological Institute, Helsingørsgade 51, DK-3400 Hillerød, Denmark.Key words: Sediments,isoetids, oligotrophic lakes,iron, iron reduction, oxygenand carbon dynamicSummary● Nutrient-poor sandy sediments inhabited by small isoetid species, Lobeliadortmanna and Littorella uniflora contain O 2 to more than 40 mm depth in thelight because of high photosynthetic O 2 release from gas permeable rootsadapted to use sediment CO 2 . We examined the biogeochemical consequencesof variable organic sediment enrichment (0.1-1.6% of sediment dry weight) in c.200-days long experiments.● To track changes in sediment chemistry small pore-water samples werefrequently extracted at different depths and analyzed for NH + 4 , Fe 2+ andinorganic carbon (DIC). Mini O 2 electrodes were used to measure changes in O 2availability. Fate of added organic matter and changes in sediment pools wasassessed from sediment composition after c. 200 days and integrated sedimenteffluxes calculated from pore-water concentrations.● Upon organic enrichment, O 2 penetration dropped immediately to a few mmand concentrations of DIC increased up to 10-fold. Anaerobic Fe 3+ reductionwas induced after several days delay and did not peak until after 60 days.Cumulative anaerobic Fe 3+ reduction over 200 days increased relative to DICformation with higher organic enrichment, but molar proportions remained lessthan 15% according to sediment accumulation and estimated effluxes. Higher O 2release to Littorella than to Lobelia sediments because of 3 times higher leafbiomass and 1.3 times higher mass-specific photosynthesis permitted recoveryof O 2 penetration depth and decline of DIC, Fe 2+ and NH + 4 to control levels athigher organic enrichment of Littorella (0.1-0.4%) than Lobelia sediments(0.1%). Added phosphorus in organic matter was efficiently retained (89-93%)in the sediments compared to added organic carbon (21-55%).● We conclude that non-destructive measurements of sediment depth profiles ofO 2 accurately picture changes in oxic and anoxic zones over time, while Fe 2+reflects anaerobic Fe 3+ reduction in permanent anoxic zones and DIC thecombined aerobic and anaerobic respiration.IntroductionOligotrophic, softwater lakes inhabited by smallisoetid species typically have coarse-grainedsediments low in organic matter, dissolvedinorganic carbon (DIC) and nutrients (N and P)but high in Fe (Sand-Jensen and Søndergaard1979, Geurts et al. 2008). Sediments are welloxygenateddue to extensive radial O 2 releasefrom isoetid roots and low organic degradationrates and oxidized Fe 3+ concentrations are highrelative to phosphorus (P; Møller and Sand-Jensen 2011a,b). High P-binding to Fe 3+strongly constrains growth and development offast-growing, competitive C- and R-selectedelodeid species (sensu Grime 1977, Farmer andSpence 1986). In contrast, rare S-selectedisoetids thrive because of their inherently slowgrowth, perennial-evergreen nature andcapability to effectively extract P from an29

Paper 1extensive sediment volume by symbiosis withfungi (Sand-Jensen and Søndergaard 1978,Wigand et al. 1998, Andersen and Andersen2006).Organic enrichment of sedimentsaccompanying higher algal growth byeutrophication or greater terrestrial input,however, has been a serious threat to thepersistence of transparent lake waters andmineral sediments inhabited by isoetids duringthe last 100 years (Sand-Jensen et al. 2000,Smolders et al. 2002). We have recentlyconfirmed the critical consequences of sedimentanoxia for plant performance and survivalfollowing organic enrichment (Møller and Sand-Jensen 2011a,b), but the profound alterations ofsediment chemistry have not been analyzed. Wehere explore changes in sedimentbiogeochemistry by repeated measurements ofpore-water chemistry over ca. 200 days insediments inhabited by the two common isoetidspecies, Lobelia dortmanna and Littorellauniflora including both initial phases ofmarkedly increased organic degradation ratesand later recovery phases after the most labileorganic matter has vanished.O 2 dynamics of sediments is regulatedby O 2 production (photosynthesis), consumption(root and bacterial respiration) and physicalexchange with lake waters. Addition of organicmatter is expected to enhance degradation rates,accelerate O 2 consumption, increase+accumulation of DIC and NH 4 and induceanoxic Fe 3+ -reduction to soluble Fe 2+ . Theaccumulation of DIC should reflect thecombined O 2 respiration and anaerobicrespiration by alternative electron acceptors(NO - 3 , Mn 4+ , Fe 3+ and SO 2- 4 ). NO - 3 respiration isrestricted by low N-content of the sediments andlow nitrification rates once O 2 disappears(Christensen and Sørensen 1986, Risgaard-Petersen and Jensen 1997). Likewise, SO 2- 4 andMn 4+ reduction are constrained by lowconcentrations leaving Fe 3+ as the most potentoxidation agent due to very high sediment pools(Christensen and Sand-Jensen 1998). We hereanalyze the temporal course of sediment porewaterFe 2+ to follow the induction, rise anddecline of anaerobic respiration over time anduse the vertical profiles to estimate Fe 2+ effluxesderiving from anaerobic Fe 3+ reduction. Fe 2+flux estimates are sensitive measures ofanaerobic Fe 3+ respiration in response to organicenrichment and effluxes of Fe 2+ relative to DICprovides a proxy of anaerobic Fe reductionrelative to total aerobic and anaerobicrespiration.Input of organic matter to sedimentsincreases pools and release of organic matter tolake waters. In contrast to inorganic C releasedby organic decomposition, we foresee thatinorganic N and P, in particular, will be moreeffectively retained in the sediment due toincorporation in microbial biomass anddevelopment of higher N:C and P:C ratios inorganic sediment pools. Moreover, NH +4 isbound to organic compounds and sedimentparticles with negative charges, while PO 3- 4 isstrongly adsorbed to all particle surfaces andforms insoluble complexes and minerals with Aland Fe. Thus, when leaf concentrations of N andP and photosynthesis of isoetids dropdramatically upon high organic sedimentenrichment (Møller and Sand-Jensen 2011a,b),despite increasing sediment N and Pconcentrations, the likely reason is poor rootdevelopment and performance (Raun et al.2010, Møller and Sand-Jensen 2011a,b) andimpaired intra-plant translocation of organicsolutes and nutrients as a result of sediment andtissue anoxia (Sorrell 2004).Our objectives here were: (i) todetermine the temporal course and coupling of+O 2 , DIC, Fe and NH 4 and aerobic andanaerobic respiration processes with increasingorganic enrichment of Lobelia and Littorellasediments; (2) to use estimates of Fe 2+ and DICeffluxes as a proxy for the relative changes ofanaerobic respiration to total sedimentrespiration; and (iii) to evaluate C, N and Pretention of added organic matter to sediments.This study present original data for sediment30

Paper 1biogeochemistry, while O 2 penetration depth insediments (Møller and Sand-Jensen 2011b) isincluded to evaluate where and when anoxicFe 3+ reduction should take place and what therelationship is to Fe 3+ reduction rates.Materials and methodsSediment turfs in culture facilitySix intact sediment turfs inhabited by Lobeliadortmanna and six with Littorella uniflora werecollected from homogeneous plant stands inologotrophic, softwater Lake Värsjö, SWSweden as described before (Møller and Sand-Jensen 2011a,b). Turfs were 17 cm long, 15 cmwide and 12-14 cm deep to ensure intact rootsystems of about 20 Lobelia and 150 Littorellaplants with a mean biomass of 47 and 142 g dryweight (DW) m -2 , respectively. Turfs werebrought fully submerged to the laboratory forexperiments. One of the six turfs was used as acontrol, while gradually longer rod-shaped drypellets (5 mm in diameter, 1-15 mm long) ofcommercially available pasture grass (DLG,Copenhagen, Denmark) were added to the otherfive turfs forming a gradient of added organicmatter equivalent to 0 (control), 0.1, 0.2, 0.4, 0.8and 1.6% organic matter of DW. Organic pelletscontained (% of DW) 91% organic matter,46.9% organic carbon, 2.3% total nitrogen (TN)and 0.27% total phosphorus (TP). Pellets wereinserted with pincers at 4 cm sediment depthwith a fixed horizontal distance of 2 cm. Theturfs were incubated at 14-16 o C in 80 l aquariain a 12 : 12 h light : dark cycle exposed to aphotosynthetic active radiation (PAR) of 110µmol photons m -2 s -1 . The water was renewedevery second week to keep nutrients low andavoid algal growth and it was bubbled slowlywith atmospheric air to ensure mixing and airsaturation. Because large water volumes wereneeded we used filtered water from nearby LakeEsrom diluted 20 times with demineralizedwater to obtain a chemical compositionresembling that of Lake Värsjö (details inMøller & Sand-Jensen 2011a).Pore-water chemistry and fluxesPore-water samples for analyzing DIC, pH, Fe 2+and NH + 4 were extracted at intervals during theexperiment from 1 and 4 cm depth and from 1,2, 4, 6 and 8 cm depth at termination of the c.200-days long experiments. Water samples wereanalyzed simultaneously. Sediment sampleswere always taken at two (during theexperiment) or three (at termination)representative sites distributed evenly across theturf area. Pore-water was sampled by insertingthin capillary glass tubes (1 mm in diameter) inthe sediment, keeping the upper end above thewater. Capillary force and pressure differencefilled the capillary tube with water from thedesired sediment depth within a few minutes.Quantities of 50-100 µl of pore-water werewithdrawn with minimum air contact from thecapillary tube and immediately analyzed. DICwas analyzed by injecting a minute volume into3% HNO 3 in a bubble chamber purged with N 2gas carrying evolved CO 2 into an Infrared GasAnalyzer (ADC-225-MK3, Hoddesdon, UK;Vermaat and Sand-Jensen 1987). pH wasmeasured by a flat membrane electrode(LoT403-M8-S7/120, Mettler Toledo,Greifensee, Switzerland) positioned close to aglass surface allowing measurements on smalldroplets injected between the electrodemembrane and the glass surface. Free CO 2 andHCO - 3 were calculated from pH, ionic strengthand temperature (Rebsdorf 1972). Fe 2+ wasanalyzed spectrophotometrically by the+phenanthroline method (Eaton et al 1995). NH 4was measured spectrophotometrically bydiluting 100 µl pore-water with 900 µldemineralized water and adding phenol andhypochlorite reagents according to amicroversion of Solórzano (1996). After theexperiment duplicate cores were analyzed forwet and dry weight in depth strata of 0-1 cm, 1-3 cm, 3-5 cm and 5-8 cm. Dried homogenizedsamples were then analyzed for TN and organicC on a CHN Ea1108-elemental analyzer (CarloErba Instruments, Milan, Italy), TP by the31

Paper 1method of Andersen (1976) and total Fe (TFe)by the modified phenantroline method (Møllerand Sand-Jensen 2008).Effluxes of DIC (DIC eff ) during the course ofthe experiment were calculated from depth-gradients as the sum of CO 2 and HCO 3 effluxesby integration and linear interpolation betweenmeasuring days. Fe 2+ efflux (Fe 2+ eff) wascalculated from the Fe 2+ concentration gradient.Effluxes were estimated as the product of: 1) theconcentration gradient from 10 mm depth to thesediment surface (assuming that the latter hadthe same concentration as the lake water), 2) thediffusion coefficient of the element at 15 o C (Liand Gregory 1974), 3) the porosity of thesediment and 4) the inverse of turtuositypreviously measured for these sediments(Pedersen et al. 1995). Accumulation of DICand Fe 2+ (DIC acc and Fe 2+ acc) in the pore-waterwas calculated by the increase of depthintegratedsediment concentrations from beforeto termination of the experiment. Effluxes plusaccumulations are minimum estimates ofprocess rates because exchangeable ions werenot included and this pool can be considerablefor Fe 2+ . Moreover, we did not necessarily findthe maximum concentration gradient. Finally,effluxes of CO 2 from the sediment to the watervia the plant and effluxes of Fe 2+ to andprecipitation of Fe 3+ on root surfaces were notincluded either.Data treatment and statistical analysisData were processed in Excel 2007 andstatistical analysis and graphs were made inGraph Pad Prism 5. Data are presented as means± SD of pseudo replicates from the same turfwhen possible. Experiments were a gradientstudy with no replication of treatments butsuited for regression/correlation analysis acrossthe treatment gradient. Pore-waterconcentrations were measured in duplicate atdifferent time points and in triplicate attermination of the experiment.ResultsFig. 1. Changes in sediment biogeochemistry during c. 200days following organic enrichment of Lobelia sediments(left column) and Littorella sediments (right column) at 0%(○), 0.1% (●), 0.2% (□), 0.4% (■), 0.8% (Δ) and 1.6% (▲)of sediment dry weight. First panel: O 2 penetration depth;second panel: DIC concentration at 1 cm depth; thirdpanel: Fe 2+ concentration at 1 cm depth; fourth panel: NH 4+concentration at 1 cm depth. Mean values of triplicates. SDomitted for clarity.Concentrations of O 2 , DIC, Fe and NH 4 NaturalFig. 1. Changes in sediment biogeochemistry during c.200 low-organic days following Lobelia organic and enrichment Littorella of sediments Lobeliasediments maintained (left deep column) O 2and penetration Littorella sediments depth (>(right40column) at 0% (○), 0.1% (●), 0.2% (□), 0.4% (■), 0.8%mm), low DIC concentrations and negligible(Δ) and 1.6% (▲) of sediment dry weight. First panel: O 2penetrationFe 2+ anddepth;NH + 4 secondat 40-mmpanel:depthDIC concentrationthroughoutatthe1cm 200-days depth; third long panel: experiments Fe 2+ concentration (Fig. 1). at 1 Increasing cm depth;fourth addition panel: NH of +4 concentration organic matter 1 cm depth. stimulated Meanvalues of triplicates. SD omitted for clarity.degradations rates and rapidly diminished O 2penetration depths to just a few mm andincreased DIC concentrations up to 5-20 timesrelative to control sediments. DIC accumulationpeaked only 5-15 days after organic additionand a second peak appeared after some 100days. Fe 2+ appeared in the pore-water after arelatively long delay (ca. 10 days) and slowly32

Paper 1Fig. 2. Depth profiles ofDIC (a and d), Fe 2+ (b and e)+and NH 4 (c and f) insediments at termination ofc. 200-days longexperiments with Lobelia(upper panels: a-c) andLittorella (lower panels: d-f)with organic enrichments of0% (○), 0.1% (●), 0.2% (□),0.4% (■), 0.8% (Δ) and1.6% (▲) of sediment dryweight. Mean values ± SDof triplicates.built up maximum concentrations after about 60days suggesting that first anoxia and then apopulation of Fe 3+ reducing bacteria had to beformed before Fe 2+ formation peaked. At theend of the experiment significantly higherconcentrations of Fe 2+ were present in Lobeliathan in Littorella sediments (Linear regression,slopes different, p

Paper 1Table 3. Retention of added C, N and P to sediments at termination of the c. 200-days longexperiments with Lobelia (Lob) and Littorella (Lit).C (mol m -2 ) TN (mol m -2 ) TP (mol m -2 )Treatmen Added LeftLobLeftLitAdded LeftLobLeftLitAdded LeftLobLeftLit0 % 0 0 0 0 0 0 0 0 00.1 % 5.8 4.6 -2.9 0.24 0.16 0.15 0.013 0.017 0.0080.2 % 11.6 3.5 5.8 0.49 0.24 0.60 0.026 0.024 0.0480.4 % 23.1 13.3 6.9 0.97 1.00 -0.10 0.051 0.047 0.0320.8 % 46.2 26.6 13.3 1.94 1.31 1.39 0.103 0.095 0.0631.6 % 92.4 58.9 39.3 3.89 4.02 1.98 0.206 0.121 0.149the two species, while retention wassignificantly smaller for C (44±35%), and alsotended to be lower for N (77±26%) than P (ttest;Table 3). Retentions were not significantlydifferent between Lobelia and Littorella.DiscussionIn natural low-organic sediments of low O 2consumption rates, high O 2 release frompermeable roots of isoetids builds-up O 2concentrations above air saturation in the porewaterin the light (Møller and Sand-Jensen2011a,b) and O 2 penetrates to more than 40 mmdepth in the sediments. Lobelia has leaf surfacesof low gas permeability and releases mostphotosynthetic O 2 in the light from the roots tothe sediment and takes up O 2 by the roots in thedark until the pore-water and the plant tissueturn anoxic (Møller and Sand-Jensen 2011a).Littorella’s leaf surfaces have 12-14 timeshigher permeability than Lobelia’s (Møller andSand-Jensen 2011b) and releases about 30% ofphotosynthetic O 2 from the roots in the light(Sand-Jensen et al. 1982), while O 2 uptake fromlake water in the dark ensures O 2 concentrationsin the leaf lacunae above 5-10 Pa (Møller andSand-Jensen 2011b), downstream transport toroots and release to sediments at about 20-40%of the rate in the light (Christensen et al. 1994).The more rapid recovery of O 2 penetrationdepths and disappearance of Fe 2+ after 0.1-0.4%organic enrichment of Littorella but not Lobeliasediments and the small efflux andaccumulation of Fe 2+ relative to DIC ofLittorella sediments suggest that O 2 supply ishigher to Littorella sediments than to Lobeliasediments. Three times higher leaf biomass, 1.3-1.5 times higher mass specific photosynthesis(Møller and Sand-Jensen 2011b) andappreciable dark release of Littorella(Christensen et al. 1994) can account for anestimated higher O 2 release to sediments withLittorella (20-40 mmol m -2 d -1 ) than Lobelia(10-20 mmol m -2 d -1 ).Well oxygenated conditions ofsediments with no or low organic enrichment(0.1%) ensure that aerobic respiration is theprominent respiration mode. O 2 depth profiles inthe light show one peak a few mm below thesediment surface due to photosynthesis ofbenthic microalgae and a second peak at 20 mmdue to O 2 release from isoetid roots (Pedersen etal. 1995, Sand-Jensen et al. 2005). High O 2concentrations permit conversion of NH +4 toNO - 3 that can drive denitrification during anoxiain two zones below a surface and a deeper zoneof maximum O 2 concentrations at depthintegratedrates of 500-700 µmol m -2 d -1 in fieldmeasurements on Littorella and laboratoryexperiments with Lobelia (Christensen andSørensen 1986, Risgaard-Petersen and Jensen1997). The experiments with Lobelia showedthat denitrification was stimulated by a factorgreater than six when compared to baresediments and this enhanced activity was due to+root mediated oxic stimulation of both NH 4-release and NO 3 formation driving35

Paper 1denitrification in the lower anoxic zone(Risgaard-Petersen and Jensen 1997). Thisstimulation of nitrification and denitrificationcan also develop in cylindrical zones withincreasing distance from individual roots.It may seem paradoxical that Lobelia byrelease of O 2 introduces bacterial competitorsfor inorganic nitrogen in the rhizosphere.However, the special anatomy and morphologyof Lobelia has evolved as an adaptation toutilize CO 2 from the rhizosphere and the rootrelease of O 2 stimulating N loss bydenitrification is simply a side effect of havinghighly gas permeable roots. We can argue thatLobelia due to its highly stress-selected traitscan better tolerate N loss than possible plantcompetitors and that it by depriving thesediments of N and reducing the availability ofP due to strong binding to oxidized Fecompounds generate nutrient-poor sedimentconditions that it can better tolerate than anyother species. Although the isoetid sedimentswere strongly enriched by addition of organicmatter, they maintained the ability to retain most(avg. 91%) of the added P and dissolved ortho-Pcould not be detected in the pore-water except inthe most enriched sediments (Møller and Sand-Jensen 2011a,b).Estimated Fe 3+ -reduction is negligible (c.9-18 µmol m -2 d -1 ) in low-organic controlsediments of both species relative todenitrification rates, but high Fe 3+ reductionrates (1050-3500 µmol m -2 d -1 ) develop insediments of 0.4-1.6% organic enrichment forLobelia and 0.8-1.6 % for Littorella, whereasdenitrification should be strongly inhibited by-the decline of NO 3 following reduction ofsediment O 2 penetration to just a few mm thicksurface layer and loss of the deep denitrificationzone.Oxidized Mn is more soluble thanoxidized Fe and although Mn pools in thesediments are 10 times smaller than Fe pools,Mn exceeds Fe concentrations in root plaques incontrol and low-organic sediments (Christensenand Sand-Jensen 1998). In contrast, Feconcentrations are 10-fold higher in the thickroot plaques formed in high-organic sedimentsof intense anaerobic degradation (Christensenand Sand-Jensen 1998). These results suggestthat oxidized Mn contributes significantly toanaerobic respiration at low organic enrichmentand in the early phases following higher organicenrichment, while oxidized Fe takes over withtime at high organic enrichment because thesmaller Mn pools are exhausted. Oxidized Feforms such large pools in the oligotrophicisoetid sediments that even high Fe reductionover 200 days at 1.6% organic enrichmentconsumes less than 11% of the total Fe pool.Bulk measurements of inorganicelements and redox potential are often used infreshwater sediments as relatively unspecificmeasures of the characteristics and the processesoccurring there. We demonstrate here thatspecific pore-water measurements ofconcentrations at high depth resolution by theaid of microelectrodes and capillary samplesand estimates of fluxes of O 2 , DIC, Fe 2+ and+NH 4 can be made over extended periodswithout disturbing the sediment. Thus, O 2microelectode measurements are extremelyprecise descriptors of the spatial and temporalvariation of oxic and anoxic zones that can evenbe applied in the field and Fe 2+ is a highlysensitive measure of whether strictly anoxicconditions have existed for some time at thespecific sampling depth. DIC concentrations andefflux estimates are suitable quantifications ofthe combined CO 2 formation by aerobic andanaerobic respiration over time. In the isoetidsediments, it is evident that Fe reduction is agradually increasing contributor to microbialrespiration following higher organic enrichmentbut that its molar magnitude relative to theformation and efflux of DIC remains relativelymodest (< 15%). The biphasic temporal patternof DIC concentrations suggests that a pool ofhighly labile organic matter (e.g. simplecarbohydrates, organic acids and amino acids inthe organic pellets) is rapidly degraded duringthe first one-two weeks whereas more36

Paper 1recalcitrant organic pools are later degradedleading to secondary peaks after more than 100days when the sufficient microbial biomasseshave been established. The late peak of Fe 3+reduction rates after about 60 days suggests thatit takes a while before Mn pools have beenexhausted and populations of Fe-reducingbacteria have been fully established. The timecourse of DIC release and Fe reduction is highlytemperature sensitive because it is markedlyshortened by higher temperatures (28 o C) andprolonged by lower temperatures (8o C,Hammer 2010). If pulses of organic matter arereceived by the sediments, isoetid plants may,therefore, be more capable of coping with thisimpact at low temperatures because anoxicstress is less severe and concentrations ofreduced compounds are lower though of moreprolonged duration because degradation ratesare impeded.AcknowledgementsWe thank the Center for Lake Restoration(CLEAR) a Willum Kann Rasmussen center ofexcellence for funding.ReferencesAndersen JM 1976. An ignition method fordetermination of total phosphorus in lake sediments.Water Research 10: 329-331.Andersen FØ, Andersen T 2006. Effects of arbuscularmycorrhizae on biomass and nutrients in the aquatic plantLittorella uniflora. Freshwater Biology 51: 1623-1633.Christensen KK, Sand-Jensen K 1998. Precipitated ironand manganese plaques restrict root uptake of phosphorusin Lobelia dortmanna. Canadian Journal of Botany 76:2158-2163.Christensen PB, Sørensen J 1986. Temporal variation ofdenitrification in plant-covered, littoral sedimentfromLake Hampen, Denmark. Applied and EnvironmentalMicrobiology 51: 1174-1179.Christensen PB, Revsbech NP, Sand-Jensen K 1994.Microsensor analysis of oxygen in the rhizosphere of theaquatic macrophyte Littorella uniflora (L.) Aschers. PlantPhysiology 105: 1174-1179.Eaton AD, Clesceri LS, Greenberg AE 1995. Standardmethods for the examination of water and wastewater.Washington DC, USA: American Public HealthAssociation.Farmer AM, Spence DHN 1986. The growth strategiesand distribution of isoetids in Scottish freshwater lochs.Aquatic Botany 26: 247-258.Geurts JJM, Smolders AJP, Verhoeven JTA, RoelofsJGM, Lamers LPM 2008. Sediment Fe:PO 3 ratio as adiagnostic and prognostic tool for the restoration ofmacrophyte biodiversity in fen waters. FreshwaterBiology 53: 2101-2116.Grime JP 1977. Plant Strategies and VegetationProcesses. John Wileys and Sons, New York.Hammer KH 2010. Plante-sediment samspil hos Lobeliadortmanna ved stigende temperaturer. BC-Thesis,University of Copenhagen, Denmark.Li Y-H, Gregory S 1974. Diffusion of ions in sea waterand in deep-sea sediments. Geochimica et CosmochimicaActa 38: 703-714.Møller CL, Sand-Jensen K 2008. Iron plaques improvethe oxygen supply to root meristems of the freshwaterplant, Lobelia dortmanna. New Phytologist 179: 848-856.Møller CL, Sand-Jensen K 2011a. High sensitivity ofLobelia dortmanna to sediment oxygen depletionfollowing organic enrichment. New Phytologist 190:320-331.Møller CL, Sand-Jensen K 2011b. Higher gaspermeability of leaves provides greater tolerance ofLittorella uniflora than Lobelia dortmanna to sedimentorganic enrichment. (Paper 3).Pedersen O, Sand-Jensen K, Revsbech NP 1995. Dielpulses of O 2 and CO 2 in sandy sediments inhabited byLobelia dortmanna. Ecology 76: 1536-1545.Raun AL, Borum J, Sand-Jensen K 2010. Influence ofsediment organic enrichment and water alkalinity ongrowth of aquatic isoetid and elodeid plants. FreshwaterBiology 55: 1891-1904.Rebsdorf A 1972. The carbon dioxide system infreshwater. Freshwater Biological Laboratory,Copenhagen.Risgaard-Petersen N, Jensen K 1997. Nitrification anddenitrification in the rhizosphere of the aquaticmacrophyte Lobelia dortmanna L. Limnology andOceanography 42: 520-537.Sand-Jensen K, Borum J, Binzer T. 2005. Oxygenstress and reduced growth of Lobelia dortmanna in sandylake sediments subject to organic enrichment. FreshwaterBiology 50: 1034-1048.Sand-Jensen K, Prahl C 1982. Oxygen exchange withthe lacunae and across leaves and roots of the submerged37

Paper 1vascular macrophyte, Lobelia dortmanna L. NewPhytologist 91: 103-120.Sand-Jensen K, Prahl C, Stokholm 1982. Oxygenrelease from roots of submerged aquatic macrophytes.Oikos 38: 349-354.Sand-Jensen K, Riis T, Vestergaard O, Larsen SE2000. Macrophyte decline i Danish lakes and streams overthe past 100 years. Journal of Ecology 46: 269-280.Sand-Jensen K, Søndergaard M 1978. Growth andproduction of isoetids in oligotrophic Lake Kalgaard,Denmark. Internationale Vereiningung füt theoretischeund angewandte Limnologie, Verhandlungen 9: 659-666.Sand-Jensen K, Søndergaard M 1979. Distribution andquantitative development of aquatic macrophytes inrelation to sediment characteristics in oligotrophic LakeKalgaard, Denmark. Freshwater Biology 9: 1-11.Sand-Jensen K, Søndergaard M 1997. Plants andenvironmental conditions in Danish Lobelia-lakes. In:Freshwater Biology. Priorities and Development inDanish Research (Eds. K Sand-Jensen & O Pedersen), pp54-73. GEC Gad Publishers, Copenhagen.Smolders AJP, Lucassen ECHE, Roelofs JGM 2002.The isoetid environment: biogeochemistry and threats.Aquatic Botany 73: 325-350.Solórzano L 1969. Determination of ammonia in naturalwater by the phenolhypochlorite method. Limnology andOceanography 14: 799-801.Vermaat, J, Sand-Jensen, K 1987. Survival, metabolismand growth of Ulva lactuca under winter conditions: alaboratory study of bottlenecks in the life cycle. MarineBiology 95: 55-61.Wigand C, Andersen FØ, Christensen KK, Holmer M,Jensen HS 1998. Endomycorrhizae of isoetids along abiogeochemical gradient. Limnology and Oceanography43: 508-515.38

Paper 2High sensitivity of Lobelia dortmanna to sedimentoxygen depletion following organic enrichmentPhoto: Ole Pedersen

ResearchPaper 2NewPhytologistHigh sensitivity of Lobelia dortmanna to sedimentoxygen depletion following organic enrichmentClaus Lindskov Møller and Kaj Sand-JensenFreshwater Biological Laboratory, Biological Institute, University of Copenhagen, Helsingørsgade 51, DK-3400 Hillerød, DenmarkSummaryAuthor for correspondence:Claus Lindskov MøllerTel: +45 3532 1909Email: clmoller@bio.ku.dkReceived: 7 October 2010Accepted: 5 November 2010New Phytologist (2011) 190: 320–331doi: 10.1111/j.1469-8137.2010.03584.xKey words: eutrophication, Lobeliadortmanna, organic enrichment, oxygen,anoxia, photosynthesis, sedimentbiogeochemistry, stress.• Lobelia dortmanna thrives in oligotrophic, softwater lakes thanks to O 2 andCO 2 exchange across roots and uptake of sediment nutrients. We hypothesizethat low gas permeability of leaves constrains Lobelia to pristine habitats becauseplants go anoxic in the dark if O 2 vanishes from sediments.• We added organic matter to sediments and followed O 2 dynamics in plants andsediments using microelectrodes. To investigate plant stress, nutrient content andphotosynthetic capacity of leaves were measured.• Small additions of organic matter triggered O 2 depletion and accumulation ofNH 4 + ,Fe 2+ and CO 2 in sediments. O 2 in leaf lacunae fluctuated from above airsaturation in the light to anoxia late in the dark in natural sediments, but organicenrichment prolonged anoxia because of higher O 2 consumption and restricteduptake from the water. Leaf N and P dropped below minimum thresholds for cellfunction in enriched sediments and was accompanied by critically low chlorophylland photosynthesis.• We propose that anoxic stress restricts ATP formation and constrains transfer ofnutrients to leaves. Brief anoxia in sediments and leaf lacunae late at night is arecurring summer phenomenon in Lobelia populations, but increased input oforganic matter prolongs anoxia and reduces survival.IntroductionLobelia dortmanna L. inhabits sandy beaches of the mostpristine oligotrophic, softwater lakes in Europe and NorthAmerica (Sculthorpe, 1967). It maintains an evergreenbiomass and displays the lowest metabolism and the slowesttissue turnover of any temperate aquatic plant (Moeller,1978; Sand-Jensen & Søndergaard, 1978; Nielsen & Sand-Jensen, 1991). Lobelia has small, thick leaves in a rosettefrom a short stem and numerous roots with a higher surfacearea than the leaves (Sand-Jensen & Prahl, 1982). Largecontinuous air lacunae run through leaves and roots, a thickleaf cuticle reduces the permeability of O 2 and CO 2 , whilethe roots have no barriers to gas exchange with the sedimentpore water (Møller & Sand-Jensen, 2008). Thus, Lobeliahas efficient intraplant transport of O 2 and CO 2 betweenleaves and roots and rapid gas exchange between roots andsediment. This plant–sediment coupling is enforced byextensive root symbiosis with arbuscular mycorrhiza fungi(Wigand et al., 1998). Although these features help Lobeliato acquire sediment CO 2 and nutrients in nutrient-poorsoftwater lakes, they could also make the plant particularlysusceptible to depletion of O 2 and reduced conditions insediments following higher sedimentation of labile organicmatter (Sand-Jensen et al., 2005a; Raun et al., 2010). Ourgeneral objective was, therefore, to determine how O 2dynamics, sediment processes and Lobelia’s performancerespond to enrichment of sediments with different amountsof labile organic matter.Eutrophication of numerous Lobelia lakes has led todecline of Lobelia and other isoetid species (Sand-Jensenet al., 2000; Smolders et al., 2002; Geurts et al., 2008).The efficient intraplant transport of gases and the lack ofdiffusive barriers across the root surfaces mean that O 2 isreadily lost to the sediment. At high sediment O 2 consumption,steep diffusive gradients result in large root O 2 effluxes(Christensen et al., 1994; Sand-Jensen et al., 2005b) andproblems in maintaining sufficient internal O 2 transportalong the roots to the tips (Armstrong et al., 2000; Møller& Sand-Jensen, 2008). Other aquatic plants often havediffusive root barriers to prevent radial O 2 loss or producebarriers to maintain sufficient O 2 transport to the root tips320 New Phytologist (2011) 190: 320–331www.newphytologist.com41Ó 2010 The AuthorsNew Phytologist Ó 2010 New Phytologist Trust

NewPaper 2Phytologist Research 321when sediments become reduced (Colmer, 2003). Lobeliadoes not form root tissue barriers because it requires CO 2for photosynthesis from the sediment (Møller & Sand-Jensen, 2008). As a result, roots of Lobelia and other isoetidspecies become shorter when grown in sediments with highoxygen demand (Sand-Jensen et al., 2005a; Raun et al.,2010), but shorter roots also have smaller root surfacesfor absorption of mineral nutrients. Solute transport andmineral nutrition can be further compromised by O 2deprivation of roots and symbiotic root fungi because ofcessation of ATP production by oxidative phosphorylationand thus a severe energy deficit (Colmer & Flowers, 2008).Anaerobic carbohydrate catabolism provides some ATPduring anoxia, albeit only 3–35% of the rate of energyproduction in aerobic cells (Gibbs & Greenway, 2003;Greenway & Gibbs, 2003), but takes a higher toll on carbohydrates.Lobelia could be particularly sensitive to anoxiabecause of the observed low photosynthesis and carbohydrateproduction, high proportions by weight of nonphotosyntheticstem and root tissue and low capability of root tissueto speed up anaerobic fermentation and ATP production(Sand-Jensen & Søndergaard, 1979; Nielsen & Sand-Jensen,1989; Smits et al.,1990).O 2 availability is the main determinant of sediment andplant processes. We therefore made a special effort to measureO 2 continuously during light and dark periods in lakewater, sediments and leaf lacunae to establish if, where andfor how long hypoxia or anoxia occurred in laboratory andfield populations subjected to gradually increasing organicenrichment and O 2 consumption rates. Because Lobeliagrows slowly and sediment processes respond slowly, wefollowed the responses for up to 194 d after organic enrichmentto make sure that plant stress was fully expressed andrecovery was possible.Materials and MethodsLaboratory experiments with organic enrichmentSix intact sediment turfs were collected in mid-Octoberfrom shallow water in a homogeneous Lobelia dortmannapopulation in oligotrophic Lake Värsjö, south-west Sweden.The low-organic sandy turfs (0.60 ± 0.05% (mean ± SD)organic matter of sediment DW) were 17 cm long and15 cm wide and had a sediment depth of 12–14 cm thatensured intact root systems of c. 20 plants. Turfs werebrought fully submerged to the laboratory for experiments.One turf was left as a control, while gradually longer rodshapeddry pellets (5 mm in diameter, 1–15 mm long) ofcommercially available pasture grass (Dlg, Copenhagen,Denmark) were added to the other five turfs forming a gradientof added labile organic matter equivalent to 0 (control),0.1, 0.2, 0.4, 0.8 and 1.6% organic matter of DW. Organicpellets contained (as a percentage of DW) 91 ± 1% (n =4)organic matter, 46.9 ± 0.4% organic carbon (C), 2.3 ±0.15% total nitrogen (TN) and 0.27 ± 0.01% total phosphorus(TP). This composition is equivalent to weightproportions of 156 C, 8.5 N and 1.0 P, which is richer innutrients relative to C and richer in P relative to N thanLobelia tissue (Sand-Jensen et al., 2005a). Pellets wereinserted with pincers at 4 cm sediment depth with a fixedhorizontal distance of 2 cm equivalent to 71 pellets per turf.Pincers were stuck into control sediments to keep physicaldisturbance constant. Previous experiments have documentedthat aerobic O 2 consumption rates of surfacesediments increase linearly with addition of this type oforganic matter, generating an increasing O 2 stress on plantroots (Raun et al., 2010). The turf specimens were incubatedat 14–16°C in 80 l aquaria with a 12 : 12 h light : darkcycle at an irradiance of 110 lmol photons m )2 s )1 (photosyntheticallyactive radiation, PAR) and exposed to the samewater to eliminate possible effects of elevated carbon andnutrient availability in the water resulting from release fromenriched sediments. The water was renewed every secondweek to keep nutrient concentrations low and avoid algalgrowth and it atmospheric air was bubbled slowly through itto ensure mixing and air saturation. Because large watervolumes were needed we used filtered water from nearbyLake Esrum diluted 20 times with demineralized water toobtain a chemical composition closely resembling that ofLake Värsjö. After dilution mean values were: 0.13 mMinorganic carbon (DIC), 0.3 lM plant available P (ortho-P), 0.4 lMNO ) 3 and 0.3 lMNH + 4 .Pore-water chemistry and sediment characteristicsRepeated pore-water samples from 1, 2, 4, 6 and 8 cm depthwere extracted from three representative sites distributedevenly across the turf area at the end of the 194-d-long experimentand analysed for dissolved Fe 2+ , DIC, pH, NH + 4 andortho-P. Pore-water was sampled by inserting thin capillaryglass tubes (1 mm) in the sediment, keeping the upper endabove the water. Capillary forces and pressure differenceslowly and steadily filled the capillary tube with water fromthe desired sediment depth within a few minutes. Quantitiesof 50–100 ll of pore-water were withdrawn with a glasssyringe from each of five capillary tubes at a certain sedimentsite to yield an integrated sample for 0–8 cm depth withminimum air contact and these were analysed immediately.Reduced Fe 2+ was measured spectrophotometrically accordingto the phenanthrolin method (Eaton et al., 1995). DICwas determined by injecting a minute pore-water volumeinto 3% HNO 3 in a bubble chamber purged with N 2 gascarrying evolved CO 2 into an Infrared gas analyzer (IRGA,ADC-225-MK3, Hoddesdon, UK) as previously described(Vermaat & Sand-Jensen, 1987). pH was measured by aflat-membrane pH electrode (LoT403-M8-S7 ⁄ 120, MettlerToledo, Greifensee, Switzerland) positioned close to a glassÓ 2010 The AuthorsNew Phytologist Ó 2010 New Phytologist Trust42New Phytologist (2011) 190: 320–331www.newphytologist.com

322 ResearchPaper 2NewPhytologistsurface allowing pH measurements on small droplets injectedbetween the electrode membrane and the glass surface. FreeCO 2 was calculated from DIC, pH, ionic strength andtemperature (Rebsdorf, 1972). NH 4 + was measured spectrophotometricallyon 100 ll pore water by diluting with900 ll distilled water and adding 100 ll phenol and 100 llhypochlorite reagents according to a microversion ofSolórzano (1969). Analysis of dissolved ortho-P according toStrickland & Parsons (1968) initially caused analytical problemsas a result of very low P concentrations and high blanksresulting from nonremovable colloidal organic matter.Measurements succeeded when pore water from each sedimentand depth without reagents served as blanks for parallelsamples with reagents added.After the experiment, sediment composition was characterizedin duplicate sediment cores by analysing wet and dryweight in samples from depth intervals of 0–1 cm, 1–3 cm,3–5 cm and 5–8 cm. Dried homogenized samples were thenanalysed for TN and organic carbon (C) on a CHN EA1108-elemental analyser (Carlo Erba Instruments, Milan, Italy), TPby the method of Andersen (1976), total iron (TFe) by thephenanthrolin method slightly modified by Møller & Sand-Jensen (2008) and organic content as loss on ignition at 550°C.Plant morphology and performancePlant morphology and metabolism were measured on oneplant of each treatment 18, 59 and 109 d into the experiment,while three plants were killed after 194 d. In general,plants within the same treatment looked very similar.Therefore, representative plants could easily be selected.Plants were gently removed from the sediment to minimizedisturbance. Total leaf and root lengths were measured oneach plant by a ruler. Two replicates for measurements ofphotosynthesis and respiration were prepared from eachharvested plant. Two leaves for each replicate were split longitudinallythrough the lacunae to facilitate CO 2 supplyand avoid the influence of O 2 storage in the extensive lacunaeand then transferred to gas-tight glass bottles containingalkaline water adjusted to pH 6.9 to provide a high saturatingCO 2 concentration of 1000 lM for photosynthesis.The bottles were transferred to a rotating wheel in anincubator at 16°C and exposed to a saturating irradianceof 340 lmol photons m )2 s )1 (PAR). Net photosynthesiswas calculated as the increase in O 2 concentration after 2 hin the light. O 2 concentration was measured with a Clarktype OX500 mini-electrode (Unisense, Århus, Denmark).Photosynthesis was normalized to leaf DW determined after24 h of freeze-drying. Photosynthetic capacity measuredunder these standardized experimental conditions is suitablefor evaluating the physiological well-being of leaves, butdoes not directly reflect photosynthesis in the growthexperiments because of different supplies of sediment CO 2to Lobelia between treatments.Chlorophyll was measured on leaves used for photosyntheticexperiments by ethanol extraction for 24 h andspectrophotometric analysis according to Christoffersen &Jespersen (1986). TN, TP and TFe were measured on leavesfrom three to five different plants at different times duringthe experiments and analysed by methods already describedfor sediments.O 2 dynamics and penetration depthO 2 dynamics in plants and sediment were measured on fouroccasions in different plants during the laboratory experimentusing Clark-type O 2 micro- and mini-electrodes (Ox50, tip diameter 50 lm for leaf lacunae and Ox 500, sturdytype applicable for sediments with a tip diameter of500 lm) advanced by micromanipulators (Unisense). O 2was recorded using a picoamperometer connected to acomputer enabling continuous logging of signals. Likewise,temperature was measured continuously using thermocouples(type K) connected to the computer through a DC10converter and used to correct the electrode signal fortemperature changes. Electrodes were inserted into lacunaeof mature leaves number 3–4 in the rosette and into sedimentsat the desired depth and left for a full 12 : 12 hlight : dark cycle. Electrodes were calibrated before andafter experiments in water bubbled with atmospheric airand in O 2 -free water obtained by adding dithionite.According to the manufacturer, the detection limit ofmicroelectrodes for O 2 is 0.03 kPa. However, because oftemperature variations in our measurements, determinationsunder confirmed anoxic conditions suggest that theoperational detection limit is 0.1 kPa. Nonetheless, we stilluse the term anoxia for sediments and leaf lacunae in thedark below 0.1 kPa, because O 2 concentrations declinedsteeply before reaching the steady minimum O 2 signal,stressing that O 2 consumption rates exceeded O 2 supplyrates to such a great extent that it is unlikely that hypoxiacharacterized by trace amounts of O 2 (i.e. < 0.1 kPa)existed.O 2 penetration depth was measured in duplicate in eachsediment turf on nine occasions during the course of experiments,and in triplicate at termination with the sameequipment used for measuring sediment O 2 dynamics.Measurements were always made 10–12 h into the lightperiod when O 2 penetrated deepest. The O 2 electrode wasmoved downwards in the sediment by a micromanipulatorin steps of 500 lm until anoxia occurred or the depthrange of the manipulator prevented deeper measurements(> 40 mm).Field experiments with organic enrichmentField experiments were performed in summer–autumn inthe same shallow homogeneous Lobelia population in LakeNew Phytologist (2011) 190: 320–331www.newphytologist.com43Ó 2010 The AuthorsNew Phytologist Ó 2010 New Phytologist Trust

NewPaper 2Phytologist Research 323Värsjö as used for collection of sediment turf for laboratoryexperiments to make sure that patterns observed in laboratoryexperiments resembled natural sediment and plantresponses in the field.A long-term organic enrichment experiment was madeover 80 d in the field (10 July–1 October) in which sedimentchemistry and plant performance were comparedbetween triplicate controls and treatments (20 · 20 cm perplot) enriched with organic matter to 0.8% DW asdescribed for the laboratory experiment. At the end of theexperiment, pore-water concentrations and sediment compositionwere measured on sediment cores (5 cm indiameter, 10 cm in depth) taken from each plot andbrought back to the laboratory in Perspex tubes with rubberstoppers preventing disturbance of sediment and porewater. Cores were stored in the laboratory at 15°C ina12 : 12 h light : dark cycle for 1–4 d before pore water wasextracted and analysed as already described. Likewise, netphotosynthesis was measured in the laboratory incubator onleaf samples from each plot. Leaf samples derived fromharvested field plants were transported to the laboratory insealed moist plastic bags and stored at 8°C and used forexperiments within 2 d.O 2 dynamics was measured continuously over a full 24 hlight–dark cycle in the field in late August in duplicated experimentsby inserting microelectrodes into leaf lacunae of twoplants and inserting mini-electrodes at 1 cm depth in thesediment next to these two plants using the same type of equipmentas for laboratory experiments. HOBO data loggers wereused to measure water temperature and irradiance (OnsetComputer Corporation, Bourne, Massachusetts, USA).Data treatment and statistical analysisData were processed in Excel 2007 and statistical analysesand graphs were made in Graph Pad Prism 5. Data arepresented as means ± SD. P < 0.05 was considered significant.The in situ experiment was a block design in triplicatesuitable for t-test analysis, whereas the laboratory experimentwas a gradient study with six amounts of added organicmatter to single sediment turfs inhabited by 20 plants(pseudo-replicates) suited for regression ⁄ correlation analysis.Values from the laboratory experiment are presented asmeans ± SD of usually three replicates from each sediment turf.ResultsSediment processes and chemistryOrganic enrichment of sediments in long-term laboratoryexperiments stimulated O 2 use for degradation of organicmatter and reduced O 2 depth penetration (Fig. 1). O 2 penetrationdepth exceeded 40 mm in control sediments butdeclined to < 3 mm after 25 d in sediments fertilized withFig. 1 Depth of O 2 penetration in Lobelia sediments as a functionof time after addition of different amounts of labile organic matter(per sediment DW) (open circles, control; closed circles, 0.1%; opensquares, 0.2%; closed squares, 0.4%; open diamonds, 0.8%; closeddiamonds, 1.6%). Measurements were made 10–12 h into the 12 hlight period. Measurements could not extend deeper than 40 mminto the sediment, when O 2 penetration in control sediments wasdescribed as > 40 mm. Values are mean of two (0–170 d) and three(194 d) measurements ± SD.‡ 0.4% organic matter. In sediments receiving only 0.1 and0.2% organic matter, O 2 penetration depth had onlydeclined to 21 and 10 mm, respectively, 25 d after fertilization.As degradation of organic matter progressed overtime, O 2 gradually penetrated deeper into the sediments.However, only at the lowest organic dose did O 2 penetrationrecover to the same value (> 40 mm) as in the controlsediment within the 195 d of experiments. At the end ofthe experiment, O 2 penetration depth was significantlyand negatively correlated to the magnitude of addition(Spearman’s r, P < 0.001). Thus, organic enrichment hadprofound and long-lasting effects on O 2 availability anddecomposition processes.This persistent enrichment effect was evident in sedimentchemistry even at the lowest enrichment after 194 d inthe laboratory experiment (Table 1). Across the gradient,organic content, TN and TP increased two- to threefoldand they were significantly correlated to the amount oforganic matter added (linear regression, P < 0.0001), whilewater content and TFe did not change significantly.Approximately 14.5 mg organic matter, 368 lg TN and43 lg TP (all g –1 dry sediment) were added with the highest1.6% organic enrichment and the treated sediment stillcontained an extra 10.2 mg organic matter, 382 lg TNand 25.5 lg TP relative to the control sediment after194 d. Likewise, the 0.4% organic treatment received anadditional 3.6 mg organic matter, 92 lg TN and 9.8 lgTP per g dry sediment and still contained extra 2.3 mgorganic matter, 96 lg TN and 9.8 lg TP after 194 d,stressing that organic matter was lost by degradation butmost N and P remained in the sediment.Ó 2010 The AuthorsNew Phytologist Ó 2010 New Phytologist Trust44New Phytologist (2011) 190: 320–331www.newphytologist.com

324 ResearchPaper 2NewPhytologistTable 1 Organic content (%), water content (%), total nitrogen (TN), total phosphorus (TP), total iron (TFe) all normalized to sediment DW and mean depth-integrated concentrations ofFe 2+ +,NH 4 , dissolved inorganic carbon (DIC) and CO2 measured in sediments after 195 d of experimentation in the laboratory across a gradient of added labile organic matter (0–1.6% DW)Treatment Sediment composition Pore-water concentrationOrganic matter added (%) Organic content (%) Water content (%) TN (lg g )1 ) TP(lg g )1 ) TFe (mg g )1 ) Fe 2+ (lM) NH4 + (lM) CO2 (mM) DIC (mM)0 0.60 ± 0.05 31.1 ± 0.5 170 ± 20 24.1 ± 2.3 1.97 ± 0.13 0003 ± 3 4.1 ± 0.8 0.52 ± 0.09 1.99 ± 0.150.1 0.68 ± 0.02 29.7 ± 0.3 186 ± 10 27.6 ± 0.3 1.91 ± 0.01 0070 ± 44 22.6 ± 11 0.67 ± 0.02 2.34 ± 0.270.2 0.66 ± 0.05 28.7 ± 0.3 193 ± 20 29.1 ± 1.8 1.93 ± 0.00 0510 ± 117 200 ± 13 1.03 ± 0.03 4.53 ± 0.120.4 0.83 ± 0.16 31.1 ± 0.4 266 ± 14 33.9 ± 2.2 1.87 ± 0.04 0979 ± 13 590 ± 76 1.78 ± 0.06 7.01 ± 0.020.8 1.06 ± 0.16 30.7 ± 1.0 294 ± 8.9 44.0 ± 2.1 2.02 ± 0.06 1989 ± 107 850 ± 95 2.18 ± 0.09 7.89 ± 0.571.6 1.62 ± 0.37 30.9 ± 1.7 552 ± 241 49.6 ± 3.3 1.79 ± 0.14 2700 ± 152 568 ± 203 4.14 ± 0.17 7.72 ± 0.50r 2 = 0.61,P < 0.0001r 2 = 0.96,P < 0.0001r 2 = 0.45,P < 0.001r 2 = 0.94,P < 0.0001r 2 = 0.11,nsr 2 = 0.90,P < 0.0001r 2 = 0.80,P < 0.0001r 2 = 0.03,nsCorrelation r 2 = 0.96,P < 0.0001r 2 and significance levels of linear regressions between the dependent variable and % organic matter added are shown.Mean ± SD, n = 2 (sediment composition) or n = 3 (pore water). ns, not significant.In sediment pore water examined at the end of experiments,DIC and CO 2 increased four- to eightfold and NH 4 + -Nincreased > 100-fold with the 1.6% organic enrichmentreflecting the enhanced organic decomposition (Table 1). A1000-fold increase of soluble Fe 2+ is the result of use of Fe 3+by microorganisms (i.e. use of alternative electron acceptors)and it is one of the main reasons for the increase of HCO 3)contained in DIC (Lucassen et al., 2009). Dissolved ortho-Pconcentrations significantly different from zero could not bedetected in sediments with 0–0.8% added organic matter, butin the 1.6% organic treatment high concentrations wererecorded (i.e. 328 ± 176 lg Pg )1 sediment DW at 4 cmdepth).In field experiments, pore-water chemistry responded as inlaboratory experiments. Significantly higher pore-waterconcentrations of DIC (4.8 ± 0.95 mM) and Fe 2+ (1.03 ±0.54 mM) were found in 0.8% organic treatments after 80 dcompared with 1.0 mM DIC and 0.02 mM Fe 2+ in controlsediments (t-test, P < 0.01), reflecting stimulation of organicdecomposition, sediment anoxia and alkalinization by Fereduction (Table 2).O 2 dynamics in plant lacunae and sedimentsIn leaf lacunae of Lobelia on nonenriched sediments inlaboratory experiments, O 2 changed from 27–33 kPa(130–160% saturation) late in the light period to anoxialate in the dark period (Fig. 2a). Diurnal O 2 changes insediment pore water at 10 mm depth tracked these changesvery closely with a 1 h time lag upon changes to light ordarkness (Fig. 2a). By contrast, leaf lacunae rapidly becameanoxic in the dark in Lobelia grown for 9 or 7 d on sedimentsenriched with 0.4 (Fig. 2b) and 1.6% organicmatter, respectively (Fig. 2c). Pore water at 10 mm depthwas already permanently anoxic 9 d after enrichment with0.4% organic matter. The 1.6% organic treatment still containedsome O 2 in the sediment pore water in the light 7 dafter organic enrichment because O 2 production by photosynthesiswas initially very high according to the steep riseof O 2 in the leaf lacunae upon illumination. Photosynthesisis probably stimulated by high sediment CO 2 accompanyingfaster organic decomposition (Fig. 2c). However, in alllater incubations, sediments enriched with 0.4 and 1.6%organic matter remained permanently anoxic at 10 mmdepth, whereas the control treatment had similar sedimentO 2 traces as first observed (data not shown).During the 194-d-long laboratory experiment, thediurnal fluctuation of O 2 in the leaf lacunae remainedalmost the same in Lobelia growing on unfertilized sediments(Fig. 3). O 2 increased to c. 30 kPa during daytime photosynthesisand declined to anoxia late at night. Dependingon the specific experiment, maximum daytime O 2 concentrationsin leaf lacunae of Lobelia on sediments enrichedwith 0.4% organic matter ranged from 15 to 43 kPa andNew Phytologist (2011) 190: 320–331www.newphytologist.com45Ó 2010 The AuthorsNew Phytologist Ó 2010 New Phytologist Trust

NewPaper 2Phytologist Research 325Table 2 Maximum net photosynthesis (NP), chlorophyll (chl), total phosphorus (TP) and total nitrogen (TN) content of Lobelia leaves inrelation to DW, sediment organic content and pore-water concentrations of Fe 2+ and dissolved inorganic carbon (DIC) at 4 cm depth after80 d of experiments with sandy sediments receiving 0 and 0.8% labile organic matter in Lake Värsjö from 10 July to 1 OctoberTreatment Photosynthesis Leaf contentSedimentcompositionPore waterOrganic matteradded (%)NP (lmolO 2 g )1 DW h )1 )Chl (mgg )1 DW)TP (mgg )1 DW)TN (mgg )1 DW)Organiccontent (%) Fe 2+ (lM) DIC (mM)0.0 282 ± 18 2.47 ± 0.34 1.62 ± 0.26 23.2 ± 4.32 0.66 ± 0.10 16.0 ± 5.61 1.03 ± 0.540.8 64 ± 17 0.99 ± 0.15 0.89 ± 0.02 11.3 ± 2.21 0.81 ± 0.03 1204 ± 381 4.78 ± 0.95Mean ± SD, n =3.(a)(b)(c)they rapidly went anoxic and stayed anoxic for several hoursin the dark. Daytime O 2 in leaf lacunae of Lobelia on sedimentsenriched with 1.6% organic matter never exceeded20 kPa after 40 d into the experiment and O 2 disappearedmore rapidly in the dark than in the 0.4% organic treatment.The duration of leaf anoxia during the 12 h dark periodwas significantly positively correlated to % organic matteradded (Spearman’s r, P < 0.01, joint analysis of all measurements;Fig. 4). As root morphology changed anddegradation of organic matter progressed towards the endof the experiment, the anoxic period in leaf lacunae in thedark declined to 1.8 h on control sediments and to 5.9 and6.6 h on sediments enriched with 0.4 and 1.6% organicmatter, respectively (Fig. 4). The decline of the anoxicperiod with time of the experiment was systematic in alltreatments, although not statistically significant.Field experiments with Lobelia populations in lateAugust under the same temperatures, a 13 : 11 h light :dark cycle and higher daytime irradiance displayed the samediurnal O 2 course in leaf lacunae and sediment pore waterat 1 cm depth as in laboratory experiments (Fig. 5). Thus,O 2 in the sediment reached 20–23 kPa (close to 100%saturation) late in the afternoon and vanished for 1–6 h latein the night. In the leaf lacunae, O 2 peaked at 22–31 kPalate in the afternoon and vanished for 1–3 h late in thenight.Fig. 2 Diurnal changes of O 2 partial pressure (PO 2 ) in leaf lacunaeof Lobelia (solid line), sediment pore water at 10 mm depth (dashedline) and water phase (dotted line) in laboratory experiments in thecontrol (a), 0.4% (b) and 1.6% (c) treatments after 4, 9 and 7 d ofenrichment, respectively. The diurnal trace started with a shift from12 h light to 12 h darkness followed by a shift back to 12 h light.Values are single measurements.Chlorophyll content and photosynthesisChlorophyll content and photosynthesis at saturating lightand CO 2 changed in concert across organic enrichments andover time in laboratory experiments (Fig. 6). Chlorophyllcontent and maximum photosynthesis were highest and atapproximately the same level among organic enrichments18 d into the experiment. A small increase of both chlorophyllcontent and photosynthesis sometimes occurred atlow organic enrichment (0.1 and 0.2%); however, whenomitting the control treatment from the dataset, both variablessubsequently declined significantly (linear regression,Ó 2010 The AuthorsNew Phytologist Ó 2010 New Phytologist Trust46New Phytologist (2011) 190: 320–331www.newphytologist.com

326 ResearchPaper 2NewPhytologist(a)(b)Fig. 4 Duration of anoxia during the 12 h dark period in leaf lacunaeof Lobelia as a function of time after addition of different amountsof labile organic matter (circles, control; squares, 0.4%; diamonds,1.6% of added organic matter to sediments). Different plants fromeach treatment were used for measurements over time (n = 1).(c)(d)P < 0.05) across the range of organic enrichments. Photosynthesisdropped to very low amounts in the 1.6% organicenrichment.For all leaf samples among treatments and over time,chlorophyll content was significantly positively related totissue concentrations of TN and almost so to TP (linearregression, P = 0.06, Table 3). Chlorophyll was significantlynegatively related to TFe because some leaves fromthe 1.6% organic treatment had surface plaques of Fe. TPand TN were also significantly interrelated. Photosynthesiswas highly significantly related to chlorophyll, TP and TNin the leaves, while it was significantly negatively related toTFe (Table 3). Also in field experiments, photosynthesis,chlorophyll, TN and TP declined significantly with 0.8%organic enrichment (t-test, P < 0.01; Table 2).Fig. 3 Diurnal changes in O 2 partial pressure (PO 2 ) in leaf lacunaeof Lobelia with increasing time into the experiment (a, 4–9 d; b, c.40 d; c, c.73d;d,c. 150 d (control and 1.6%) and 111 d (0.4%))for plants growing in the laboratory in sediments with 0% (control,solid line), 0.4% (dashed line) and 1.6% addition (dotted line) oflabile organic matter per sediment DW. Data in (a) are derived fromFig. 2. The diurnal trace started with a shift from 12 h light to 12 hdarkness followed by a shift back to 12 h light. Different plants fromeach treatment were used for measurements over time, n =1.Root development and leaf nutrientsMaximum root length declined from c. 7.1 ± 1.6 cm incontrol sediments to only 4.3 ± 0.5 cm in the organicallyrichest sediments deprived of O 2 in laboratory experiments.Across the organic enrichment gradient, the ratio of leaflength to root length increased fourfold from 0.53 ± 0.1in the control to 1.89 ± 0.1 in the 1.6% organic mattertreatment (Table 3).Total phosphorus and TN in leaf tissue changed profoundlyamong treatments and over time in both laboratoryand field experiments (Fig. 7, Table 2). The highest andmost constant leaf concentrations occurred in plants fromcontrol sediments, but progressively lower TP and TNconcentrations occurred with duration of the experimentand higher addition of organic matter despite increasingnutrient concentrations in the sediments (Tables 1, 2).New Phytologist (2011) 190: 320–331www.newphytologist.com47Ó 2010 The AuthorsNew Phytologist Ó 2010 New Phytologist Trust

NewPaper 2Phytologist Research 327(a)(a)(b)(b)(c)Fig. 6 Chlorophyll content (a) and maximum net photosynthesis (b)of Lobelia leaves after increasing addition of labile organic matter(% of sediment DW). Single measurements were made after 18 d(open circles) and 59 d (closed circles) and triplicate measurements(± SD) after 194 d (squares) of experiments in the laboratory.DiscussionFig. 5 Diurnal changes of O 2 partial pressure measured at 10 mmdepth in sandy sediments next to two different plants (a, solidline), in leaf lacunae of two plants (b, solid line) and in the waterphase (a and b, dotted lines) in Lobelia populations in Lake Värsjöin late August. Irradiance (photosynthetically active radiation,400–700 nm; c, solid line) and water temperature (c, dashed line)are also shown.Tissue TP remained close to the common critical thresholdfor maximum biomass yield of submerged macrophytes incontrol sediments and in sediments enriched with only 0.1and 0.2% organic matter (Fig. 7, Table 2). However,depletion of TP was more extreme and concentrationsdropped below common critical threshold for maximumgrowth rate, maximum biomass yield and even minimumthreshold to sustain growth of submerged macrophytes at0.4–1.6% organic enrichment (Fig. 7, Table 2). Depletionof TN in leaf tissue occurred later during the experimentand at higher additions of organic matter, and TN concentrationsonly dropped below the critical threshold formaximum biomass yield at the highest organic treatmenttowards the end of experiments.O 2 dynamics in sediments and plantsLaboratory experiments reflected the natural processes inthe field. We observed the same profound O 2 fluctuationsfrom above air saturation in the light to anoxia in the darkin the sandy sediments and also the same plant responses inthe two instances. It was surprising, however, that leaf lacunaetracked O 2 conditions in the sediment pore water soclosely and that they went anoxic in the dark shortly afterO 2 had disappeared from the sediment. In a Novemberexperiment at 4°C in the same Lobelia population, O 2remained in leaf lacunae (9 kPa), root lacunae (6 kPa) andin the sediment (3 kPa) in the dark because plant and sedimentrespiration is reduced at low winter temperature(Sand-Jensen et al., 2005b). Leaf and root anoxia is, however,a recurring phenomenon during summer nights infield populations of Lobelia despite 100% air saturation inlake water and large retention capacity for O 2 in the extensiveair lacunae (Sand-Jensen & Prahl, 1982). The impededgas exchange across leaf surfaces as a result of very lowpermeability (Sand-Jensen & Prahl, 1982; Pedersen &Sand-Jensen, 1992) implies that air lacunae and plant tissuego anoxic only 1–2 h after O 2 has disappeared from theÓ 2010 The AuthorsNew Phytologist Ó 2010 New Phytologist Trust48New Phytologist (2011) 190: 320–331www.newphytologist.com

328 ResearchPaper 2NewPhytologistTable 3 Summary of linear regressions between dependent variable(y) and independent variable (x)(a)y x Slope Intercept r 2 P nL : R Org 0.78 )0.64 0.83 < 0.0001 18Chl TFe )3.84 1.03 0.51 0.0001 23Chl TP 0.76 )2.63 0.16 0.0595 23Chl TN 0.11 )6.14 0.34 0.0026 24TN TP 7.68 )1.31 0.68 < 0.0001 23NP Chl 43.1 0.01 0.59 < 0.0001 72NP TFe )181 1.01 0.27 0.0105 23NP TP 90.4 )0.37 0.55 < 0.0001 23NP TN 8.80 3.57 0.51 < 0.0001 24Ratio of leaf to root length (L : R) to sediment enrichment (org, %DW) after 195 d and relationship between chlorophyll content (Chl,mg g )1 DW), net photosynthesis (NP, lmol O 2 g )1 DW h )1 ), TNcontent (mg g )1 DW), TP content (mg g )1 DW) and TFe content(mg g )1 DW) of Lobelia leaves retrieved after different experimentalperiods (18–194 d) in the laboratory from sediments of differentorganic enrichment. Slopes, intercepts, significance levels (P),r 2 -values, and number of data points (n) used for calculations arepresented [correction added after online publication 27 January2011: some values in the columns headed ‘Slope’ and ‘Intercept’have been altered from negative values to positive values by theremoval of minus signs; additionally within the Probability ‘(P)’column, some significance levels have been altered by the removalof less than (

NewPaper 2Phytologist Research 329oligotrophic habitat. Lobelia’s special structure can insteadbe interpreted as an adaptation to use CO 2 in the sedimentpore water as a much richer carbon source for photosynthesisthan the surrounding water. Pore water in natural sandysediments contained 0.60 mM CO 2 (Table 1), which is 40times higher than CO 2 concentrations in air-saturated lakewater (c. 0.015 mM). Lobelia can only use CO 2 for photosynthesisand air-saturated concentrations are too low tosupport positive net photosynthesis (Winkel & Borum,2009). To ensure high CO 2 supply from the sediment toleaf photosynthesis, root surfaces must be large and highlypermeable and air lacunae must be wide and short throughroots and leaves. Moreover, to maintain high CO 2 concentrationsin plants growing on sediments of low decompositionrates, plants must prevent CO 2 loss to the water via theintraplant gas transport route and this requires a lid (adiffusion barrier) on leaf surfaces.The diffusion barrier on Lobelia’s leaves also reducesevaporation and ensures survival when plants regularlybecome exposed to the air following drawdown of the watertable during summer (Pedersen & Sand-Jensen, 1992).Most submerged aquatic plants dry out upon exposure toair and several species, including the common isoetidLittorella uniflora (Nielsen et al., 1991), survive by producingnew aerial leaves with stomata. Lobelia does not investin a new set of leaves, which would be costly and perhapsimpossible considering its low intrinsic growth rate andnutrient-poor habitat. Thus, special structural and physiologicaladaptations serve several purposes and need to beviewed in regard to the entire plant life.Environmental changes and plant stressSediment chemistry was very susceptible to addition ofmodest amounts of easily degradable organic matter. O 2disappeared from most of the sediment in the light withaddition of only 0.1 or 0.2% organic matter and it took100 d for O 2 to resume penetration deeper than 40 mm atthe lowest dose. Even after 194 d, pore-water concentrationsof DIC, CO 2 , NH + 4 and Fe 2+ were elevated relative tocontrol values, reaching the substantial 200 lM NH + 4 and510 lM Fe 2+ at 0.2% organic addition (Table 1). Theselow organic treatments did not reduce leaf nutrients,+chlorophyll and photosynthesis, although such high NH 4concentrations stress other macrophytes when leaves aredirectly exposed (Smolders et al., 1996).Higher additions of organic matter (0.4–1.6%) clearlyimpeded photosynthesis and incorporation of TP and TNin the leaves. The most parsimonious explanation for thestress is prolonged anoxia in leaves and roots in the darkreducing ATP production by oxidative phosphorylation tosustain uptake, transport and incorporation of mineral ionsand organic solutes in cell products (Aguilar et al., 2003;van Dongen et al., 2003). A crucial role of TP and TN inleaf tissue for forming the photosynthetic apparatus issupported by significant positive correlations between TP,TN, chlorophyll and photosynthesis. According to highNH + 4 concentrations in all organic amendments and highortho-P concentration in the 1.6% treatment, it is not alack of N and P in the sediment that restricts leaf nutrients,but insufficient uptake from the sediment and transfer toleaf tissue.Anoxia initiates the accumulation of NH +4 and theformation of Fe 2+ and other reduced compounds in thesediment (e.g. Mn 2+ , sulphides and small fatty acids), whichmay contribute to plant stress (Gibbs & Greenway, 2003).These compounds accumulate as a consequence of anoxiaand do not have the same fundamental physiological influenceas many hours of anoxia in the plant tissue. Also,insufficient ATP formation and gradual depletion of carbohydratereserves can account for the inability to incorporateN and P in leaf tissue despite high sediment availability.Moreover, high concentrations of NH + 4 and Fe 2+ withoutany apparent stress on photosynthesis developed in sedimentwith 0.2% organic addition, i.e., concentrations onlytwo to five times lower than observed at greater organicamendments. This result supports the notion that O 2 deprivationis the overriding stress factor.Phosphorus in leaf tissue declined below the general minimumthreshold to support photosynthesis and growth ofsubmerged macrophytes at organic amendments of 0.4–1.6% (legend to Fig. 7). Although the critical threshold isan average for many macrophytes and Lobelia may havelower requirements than most other species (Moeller, 1978;Demars & Edwards, 2007), leaf P, chlorophyll and photosynthesisare so low in the 1.6% organic treatment thatLobelia can just barely survive. These results imply that Plimitation in Lobelia represents a strong additional stress oforganic enrichment and O 2 deprivation as a result of one ormore mechanisms. First, inorganic P remains adsorbed tosoil particles or gradually thicker Fe-coatings formed onroot surfaces in reduced sediments (Christensen & Sand-Jensen, 1998). Second, and probably most significantly,reduced root uptake, translocation and leaf incorporation ofP are the result of O 2 stress and insufficient ATP production(Gibbs & Greenway, 2003). Thirdly, widespreadanoxia is proposed to reduce uptake and translocation of Pby mycorrhiza fungi (Wigand et al., 1998). TN depletionin leaf tissue is less severe, perhaps because of continued diffusiveuptake of NH + 4 from high pore-water concentrations(Marschner, 1995), which does not require active rootmembrane uptake or transfer by fungi.In summary, anoxia in sediments and leaf lacunae late atnight was a surprising recurring summer phenomenon inpristine Lobelia populations on nutrient-poor sandy sediments.Small additions of labile organic matter drasticallyreduced O 2 depth penetration and prolonged leaf anoxiabecause impermeable leaf surfaces prevented O 2 supplyÓ 2010 The AuthorsNew Phytologist Ó 2010 New Phytologist Trust50New Phytologist (2011) 190: 320–331www.newphytologist.com

330 ResearchPaper 2NewPhytologistfrom the lake water. Prolonged anoxia and insufficient ATPproduction to sustain root uptake and translocation canaccount for the decline of TP below critical thresholds tosustain optimum photosynthesis, growth and cell function.Elevated NH 4 + , Fe 2+ and other reduced compounds inenriched sediments can further enhance plant stress, butprolonged anoxia alone can account for the observed poorperformance of Lobelia.AcknowledgementsWe thank The Willum Kann Foundation for financial supportto this study through The Centre of Excellence forResearch on lake Restoration (CLEAR). We thank BirgitKjøller and Ole Pedersen for technical assistance and threeanonymous referees for constructive comments.ReferencesAguilar EA, Turner DW, Gibbs DJ, Armstrong W, Sivasithamparam K.2003. Oxygen distribution and movement, respiration and nutrientloading in banana roots (Musa spp. L.) subjected to aerated and oxygendepletedenvironments. 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Regulation of photosynthetic rates ofsubmerged rooted macrophytes. Oecologia 81: 364–368.Nielsen SL, Sand-Jensen K. 1991. Variation in growth-rates of submergedrooted macrophytes. Aquatic Botany 39: 109–120.Pedersen O, Sand-Jensen K. 1992. Adaptations of submerged Lobeliadortmanna to aerial life form – morphology, carbon sources and oxygendynamics. Oikos 65: 89–96.Pedersen O, Sand-Jensen K, Revsbech NP. 1995. Diel pulses of O 2 andCO 2 in sandy lake-sediments inhabited by Lobelia dortmanna. Ecology76: 1536–1545.Raun AL, Borum J, Sand-Jensen K. 2010. Influence of sediment organicenrichment and water alkalinity on growth of aquatic isoetid andelodeid plants. Freshwater Biology 55: 1891–1904.Rebsdorf A. 1972. The carbon dioxide system in freshwater. A set of tables foreasy computation of total carbon dioxide and other components of thecarbon dioxide system. Copenhagen, Denmark: Freshwater BiologicalLaboratory, University of Copenhagen.Sand-Jensen K, Borum J. 1991. Interactions among phytoplankton,periphyton, and macrophytes in temperate freshwaters and estuaries.Aquatic Botany 41: 137–175.Sand-Jensen K, Borum J, Binzer T. 2005a. Oxygen stress and reducedgrowth of Lobelia dortmanna in sandy lake sediments subject to organicenrichment. Freshwater Biology 50: 1034–1048.Sand-Jensen K, Pedersen O, Binzer T, Borum J. 2005b. Contrastingoxygen dynamics in the freshwater isoetid Lobelia dortmanna andthe marine seagrass Zostera marina. Annals of Botany 96: 613–623.Sand-Jensen K, Prahl C. 1982. Oxygen exchange with the lacunae andacross leaves and roots of the submerged vascular macrophyte, Lobeliadortmanna L. New Phytologist 91: 103–120.Sand-Jensen K, Riis T, Vestergaard O, Larsen SE. 2000. Macrophytedecline in Danish lakes and streams over the past 100 years. Journal ofEcology 88: 1030–1040.Sand-Jensen K, Søndergaard M. 1979. 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NewPaper 2Phytologist Research 331characteristics in oligotrophic Lake Kalgaard, Denmark. FreshwaterBiology 9: 1–11.Sand-Jensen K, Søndergaard M. 1978. Growth and production of isoetidsin oligotrophic Lake Kalgaard, Denmark. Verhandlungen InternationaleVereinigung der Theoretiche Limnologie 20: 659–666.Sculthorpe C. 1967. The biology of aquatic vascular plants. London, UK:Edward Arnold.Smits AJM, Laan P, Thier RH, Van der Velde G. 1990. Rootaerenchyma, oxygen leakage patterns and alcoholic fermentation abilityof the roots of some nymphaeid and isoetid macrophytes in relation tothe sediment type of their habitat. Aquatic Botany 38: 3–17.Smolders AJP, Den Hartog C, Van Gestel CBL, Roelofs JGM. 1996. Theeffects of ammonium on growth, accumulation of free amino acids andnutritional status of young phosphorus deficient Stratiotes aloides plants.Aquatic Botany 53: 85–96.Smolders AJP, Lucassen ECHET, Roelofs JGM. 2002. The isoetidenvironment: biogeochemistry and threats. Aquatic Botany 73: 325–350.Solórzano L. 1969. Determination of ammonia in natural waters byphenolhypochlorite method. Limnology and Oceanography 14:799–801.Strickland J, Parsons T. 1968. A practical handbook of seawater analysis.Ottawa, ON, Canada: Fishery Research Board of Canada.Vermaat JE, Sand-Jensen K. 1987. Survival, metabolism and growth ofUlva lactuca under winter conditions – a laboratory study of bottlenecksin the life-cycle. Marine Biology 95: 55–61.Wigand C, Andersen FO, Christensen KK, Holmer M, Jensen HS. 1998.Endomycorrhizae of isoetids along a biogeochemical gradient. Limnologyand Oceanography 43: 508–515.Winkel A, Borum J. 2009. Use of sediment CO 2 by submersed rootedplants. Annals of Botany 103: 1015–1023.Ó 2010 The AuthorsNew Phytologist Ó 2010 New Phytologist Trust52New Phytologist (2011) 190: 320–331www.newphytologist.com

Paper 3Higher gas permeability of leaves provides greatertolerance of Littorella uniflora than Lobeliadortmanna to sediment organic enrichmentPhoto: Claus Lindskov Møller

Paper 3Higher gas permeability of leaves provides greater tolerance of Littorella uniflorathan Lobelia dortmanna to sediment organic enrichmentClaus Lindskov Møller and Kaj Sand-JensenFreshwater Biological Laboratory, Biological Institute, University of Copenhagen, Helsingørsgade 51, DK-3400 Hillerød,DenmarkKey words: Permeability,isoetids, eutrophication,biogeochemistry, anoxia,oxygen, stress.Summary● Lobelia dortmanna and Littorella uniflora are prominent in nutrientpoor,softwater lakes because of efficient root exchange of CO 2 and O 2 .We hypothesize that higher gas permeability of Littorella than Lobelialeaves ensures O 2 uptake from water and greater tolerance to organicenrichment and anoxia of sediments.● We studied O 2 dynamics in plants and sediments and leaf photosynthesisupon organic sediment enrichment in long-term laboratory and fieldstudies.● Addition of organic matter triggered O 2 depletion and accumulation ofNH 4 + , Fe 2+ and CO 2 in sediments. O 2 in Lobelia’s leaf lacunae fluctuatedfrom air saturation in the light to anoxia late at night on natural sediments,but organic enrichment prolonged night anoxia. Littorella’s leaf surfaceswere 13-16 times more permeable and O 2 in the lacunae remained above10 kPa on anoxic sediments due to uptake from the water. Leaf N, P andphotosynthesis dropped to critically low levels under prolonged sedimentanoxia for Lobelia but not for Littorella.● High gas permeability of Littorella leaves improves performance andsurvival on organically enriched, anoxic sediments compared with Lobelia.Littorella changes between gas impermeable aerial leaves and permeableaquatic leaves whereas Lobelia uses the same impermeable leaves in airand water which is cost-effective in ultra-oligotrophic environments.IntroductionOligotrophic, softwater lakes in Europe andNorth America are inhabited by a taxonomicallydiverse group of small isoetid species all havingleaf rosettes, large root systems and extensiveaerenchyma as adaptations to acquire CO 2 andnutrients from sediments (Smolders et al. 2002).The isoetids are key species in internationalevaluations of pristine nature quality such asEU’s Habitat Directive and Water FrameworkDirective (Stelzer et al. 2005) and oligotrophic,softwater lakes are named Lobelia-lakes afterthe conspicuous flowering species, Lobeliadortmanna. Despite the same growth form andrequirement of relatively unpolluted habitats ofall isoetids, the species vary substantially inanatomy and physiology which may account fordifferences in their distributions (Sand-Jensen &Søndergaard 1979). For example, Littorellauniflora has a wider ecological range andtolerates greater eutrophication than Lobeliadortmanna which has gone extinct in many NWEuropean lakes and it is now confined to a fewpristine oligotrophic lakes (Arts et al. 1989;Sand-Jensen et al. 2000; Pedersen et al. 2006).We here test if Littorella is more tolerant thanLobelia to organic enrichment and anoxia of55

Paper 3sediments and, if so, what the structural andfunctional reasons for these differences couldbe.Both Littorella and Lobelia use sedimentCO 2 for photosynthesis (Wium-Andersen 1971;Søndergaard & Sand-Jensen 1979) and releaseso much O 2 from the roots during the day that asubstantial O 2 pool can build up in surfacesediments of low degradation rate and be usedin night-time respiration (Christensen et al.1994; Pedersen et al. 1995). However, if thesediment O 2 demand becomes high, such asoccurs in eutrophied lakes (Sand-Jensen et al.2005), isoetids may be unable to maintain oxicconditions in roots and even in leaves and, as aconsequence, die.Recent experiments show that a smallorganic enrichment of very oligotrophicsediments stimulates growth of Littorella andLobelia because of release of nutrients and CO 2from organic degradation (Raun et al. 2010;Møller & Sand-Jensen 2011). Higherenrichment with labile organic matter, however,reduces root development and leafphotosynthesis and can diminish plant survivalbecause of widespread sediment anoxia and,perhaps, additional plant stress fromaccumulation of reduced ions (Fe, Mn and S)and organic acids (Armstrong et al. 2000;Colmer 2003; Møller & Sand-Jensen 2011).Both Lobelia and Littorella have highlypermeable root surfaces to CO 2 and O 2exchange and will consequently face extensiveradial O 2 loss in anoxic sediments and higherrisk of root and plant mortality (Møller & Sand-Jensen 2008). Several features suggest thatLobelia may be more susceptible to O 2deprivation than Littorella. Lobelia has thinnerroots, higher root/leaf mass ratio andproportionally greater root O 2 release (virtually100%, Sand-Jensen & Prahl 1982) ofphotosynthetic O 2 than Littorella (about 30%,Sand-Jensen et al. 1982) suggesting that Lobeliashould have greater difficulties than Littorellamaintaining downward O 2 supply to root tipsand taking up sufficient O 2 for dark respirationby the leaves from the water column oncesediments go anoxic. We propose that lower gaspermeability of leaf surfaces and higher capacityfor intra-plant gas transport in continuouslacunae would leave Lobelia even moresusceptible to anoxia in root and leaf tissuesthan Littorella when sediments becomedeprived of O 2 .New experiments have supported theproposition that the threshold of sedimentorganic enrichment and O 2 consumptioninducing plant stress is systematically lower forLobelia than Littorella (Raun et al. 2010). Theseexperiments were performed with plantedindividuals that may be more susceptible toorganic enrichment and O 2 stress because newroots were formed and this requires extra energyof species with chronically low metabolism andgrowth (Sand-Jensen & Borum 1991).Therefore, we wanted to determine theresponses of undisturbed Lobelia and Littorellapopulations to changes of sedimentbiogeochemistry by organic enrichment.Because O 2 availability is the main determinantof many sediment and plant processes, we madea special effort to measure O 2 continuouslyduring light-dark cycles in lake water, sedimentsand leaf lacunae to establish if, where, and forhow long hypoxia or anoxia occurred inpopulations subjected to increasing sedimentorganic enrichment.Biogeochemical changes andphysiological responses of Lobelia have beenstudied recently (Møller & Sand-Jensen 2011).56

Paper 3Here, we perform a parallel experimental studyof Littorella and expand the experimentalstudies of both species exposed to the sametemperature, light-dark cycle and organicenrichment of sediments under laboratory andfield conditions. We tested three specifichypotheses: (i) Both species experienceprofound changes of sediment biogeochemistryand plant stress upon sediment organicenrichment, (ii) Sediment anoxia deprivesLobelia of O 2 but not Littorella leaves whichtake up O 2 from the lake water because of highgas permeability compared with Lobelia, and(iii) higher gas permeability of Littorella leavesmeans that nutrient content, photosynthesis andgrowth are better sustained than in Lobelia withsediment enrichment.Materials and methodsLaboratory experiment with intact sedimentturfsSix intact sediment turfs with monocultures ofLobelia dortmanna and six with Littorellauniflora were collected in mid-October fromshallow homogeneous populations inoligotrophic Lake Värsjö, SW Sweden. Turfswere transferred to a laboratory growth facilitysubmerged in lake water. In the laboratory turfswere used for experimentation for ca. 200 dayssubmerged (see below) at 14-16 o C in a 12-hlight: 12-h dark cycle at an irradiance (400-700nm, PAR) of 110 µmol photons m -2 s -1 aspreviously described (Møller & Sand-Jensen2011). Experimental water was filtered (1µmfilter) water from a nearby lake diluted indemineralized water to match the chemistry ofLake Värsjö (see Møller & Sand-Jensen 2011for details). Water was slowly bubbled withatmospheric air, mixed by a submersible pumpand renewed every second week to prevent algalgrowth. Sediments were exposed to anenrichment gradient by adding increasingamounts of labile organic matter held in drypellets of pasture grass equivalent to 0, 0.1, 0.2,0.4, 0.8 and 1.6% enrichment per dry weight ofsediment. Pellets were inserted in the sedimentat 4 cm depth with an equi-distance of 2 cm.Pellets contained per dry weight (DW): 91± 1% organic matter (of which 46.9 ± 0.4% wasorganic carbon), 2.3 ± 0.15% total nitrogen(TN) and 0.27±0.01% total phosphorus (TP).Sediment characteristics, pore-waterchemistry, O 2 dynamics in sediments and leaflacunae, leaf nutrients and net photosynthesiswere measured as previously described (Møller& Sand-Jensen 2011). Here we present O 2penetration depth in sediments of both speciesduring the course of experiments and light-darkcycles of O 2 in leaf lacunae and sedimentsmeasured midway into the experiments.Concentrations of Fe 2+ , DIC, NH + 4 , NO - 3-3 , PO 4and pH were measured in the pore-water waterand DW, organic DW, organic C, TN and TPwere measured in the bulk sediment at 1, 2, 4, 6and 8 cm at termination of the experimentpermitting calculation of mean depth-integratedsediment concentrations in the root zone (0-8cm). Leaf chlorophyll, N, P and netphotosynthesis at light and CO 2 saturation at 15o C were measured at the end of the experimentsusing methods previously described (Møller &Sand-Jensen 2011).In situ experimentA gradient study was established in Lake Värsjöat a wind-exposed site with mineral sediments at0.4 m water depth from May to August. Thesame site was used for collecting sedimentsplantturfs for the laboratory experiment. Nine57

Paper 315*15 cm plots homogenously vegetated withboth Lobelia dortmanna and Littorella uniflorawere subjected to organic enrichments withorganic pellets in the sediments as described forthe laboratory experiment over a gradientincluding 0, 0.2 and 0.8 % added organic DW.During the experiment temperature andirradiance were measured every 5 minutes at 0.4m depth at the site by a combined Lux andtemperature logger (Onset ComputerCorporation, Bourne, Massachusetts, USA).Averages for the 90-day long experiment were:20.6 o C (range 12-34), 217 µmol m -2 s -1 (PAR)and a day length of 17.4 hours.At the end of the in situ experiment anintact sediment core was sampled from eachplot in a Perspex tube (5 cm diameter, 30 cmhigh) equipped with rubber stoppers as bottomand lid. The cores were brought back to thelaboratory and stored at 16 o C in a 12-h light: 12-h dark cycle until measurements of pore-waterand sediment characteristics within 1-3 days.All remaining plants from each plot were rinsedand brought to the laboratory in sealed plasticbags holding some water and plenty of air toensure water and O 2 supply. In the laboratory,shoot number of each species, wet weight (WW)and DW of aboveground biomass weredetermined. Leaf samples from each species andplot were then analysed for chlorophyll, TN, TPas described in Møller & Sand-Jensen (2011).Pore-water concentrations of DIC, NH + 3-4 , PO 4and Fe 2+ and sediment content of organicmatter, TN and TP were also determined aspreviously described (Møller & Sand-Jensen2011).O 2 and H 2 O exchange and across leaf surfacesLeaf O 2 exchange for a standard O 2 gradientwas measured as O 2 loss from leaf surfaces tohypoxic water surrounding them with the baseof leaf lacunae exposed to atmospheric air. Sixleaves were cut off and mounted in a PVCcylinder (volume 45.3 ml) through small holesin the lid leaving the most basal 1 mm of theleaf outside the cylinder and leaf surfacesexposed to water in the cylinder. Holes orcracks between leaves, lid and cylinder werecarefully sealed with silicone grease. An O 2electrode (Ox 500, Unisense, Århus, Denmark)connected to a computer for continuouslylogging was introduced to follow O 2concentration in the water of the cylinder. Amagnetic stirrer provided a slow, steady mixing.Measurements were performed undertemperature constant conditions at 5.0 and 15.0o C by submerging the PVC cylinder in water butleaving the exposed leaf bases with air contact.Prior to measurements, the cylinder was flushedwith anoxic water and left for 20 minutes toequilibrate before the linear rate of increase ofO 2 was measured for 10 minutes. Allmeasurements were preformed with a low O 2partial pressure of 1-2 kPa in the watersurrounding the leaves. Similar measurementswithout leaves were used to correct for physicalleakage of O 2 into the PVC cylinder not causedby transport over the leaves. Physical leakagewas very constant (1.45±0.03 and 0.43 ± 0.05nmol O 2 h -1 at 5 and 15 o C respectively). Afterexperiments, leaf dimensions were measured at45 x magnification with a dissectionmicroscope. Three measurements of leaf widthand thickness were made along the length ofevery leaf to calculate surface area. Lobelia hasapproximately rectangular leaves in crosssection while Littorella has cylindrical leaves.Fluxes of O 2 were normalized to leaf surfacesarea and time for a mean gradient of about 19kPa between lacunae at the leaf base and O 258

Paper 3Fig. 1. O 2 penetration depth in sediments inhabited by Lobelia dortmanna (left panel from Møller & Sand-Jensen 2011)and Littorella uniflora (right panel) in laboratory experiments at different time points after addition of different amountsof labile organic matter (0% (○), 0.1% (●), 0.2% (□), 0.4% (■), 0.8% (Δ) and 1.6% (▲) of sediment dry weight).Measurements were made 10-12 hours into the 12 hour light period when O 2 oxygen penetration depth was highest.Values are means ± SD, n = 2 (0-170 days) or n =3 (end of experiment).depleted water surrounding the outer leafsurfaces. The O 2 flux is assumed to be of similarmagnitude but in opposite direction for a reversegradient with air-saturated water around theleaves and anoxia at the base of leaf lacunae.Water loss across surfaces of newlydetached leaves to dry, still air was measured ina desiccator at 20 o C. Each leaf was placedupright with the cut base in a small drop ofsilicone grease in a weighing-boat andrepeatedly weighed on a 5-decimal precisionbalance over time. The initial, constant weightloss by evaporation was normalized to time andleaf surface area measured as described above.Ten replicate leaves of both species were tested.Statistical analysisGraphs and statistical analyses were performedin Graph Pad Prism 5. The laboratoryexperiment was a dose response with sixenrichment levels of organic matter to sedimentsappropriate for correlation or regressionanalysis. The in situ experiment was a blockdesign with three levels of organic addition intriplicate appropriate for ANOVA analysis.Differences in O 2 and H 2 O flux across leafsurfaces were examined by one-way ANOVAfor the influence of temperature and species.Data were square root transformed to meet testrequirements when needed. When transformeddata failed to meet test requirements nonparametrictests were used. Probabilities above95% were considered significant. Data arepresented as mean ± 1 Standard Deviation (SD).ResultsSediment biogeochemistryO 2 penetrated to more than 40-mm depth in unenrichedsediments of both species whenmeasured 10-12 hours into the light period inlaboratory experiment. Addition of labileorganic matter increased O 2 consumption in thesediments and significantly lowered O 2penetration depth (Spearmans r, p

Paper 3Table 1. Sediment and pore-water chemistry at the termination of about 200-days long laboratoryexperiments with pure natural stands of Lobelia dortmanna (Lobelia experiment) and Littorella uniflora(Littorella experiment) and 90-days long field experiments in stands of both species (Mixed fieldexperiment). Different amounts of labile organic matter (%) were initially added to the sediments.Experiment/treatmentTP(µg P g -1DW)SedimentTN(mg N g -1DW)Orgcontent(% of DW)DIC(mmol l -1 )Pore-waterNH 4+(µmol l -1 )Fe 2+(µmol l -1 )Lobelia experiment0 24.1±2.2 170±20 0.60±0.05 1.89±0.11 4.1±0.8 2.9±3.20.1 27.6±0.3 186±10 0.68±0.02 2.15±0.25 23±11 70±440.2 29.1±1.9 193±20 0.66±0.05 4.26±0.11 200±13 506±1170.4 33.9±2.2 266±14 0.83±0.16 6.78±0.07 590±76 979±90.8 44.0±2.1 294±8.9 1.06±0.16 7.59±0.42 850±95 1990±1071.6 49.6±3.3 552±241 1.62±0.37 7.71±0.54 568±203 2700±152Littorella experiment0 30.9±1.7 326±40 1.11±0.15 1.42±0.19 9.4±2.1 3.2±3.50.1 32.6±1.4 341±26 1.06±0.03 1.64±0.18 8.2±2.8 4.4±4.30.2 41.0±4.3 383±48 1.21±0.06 2.75±0.49 125±22 54±390.4 37.5±5.0 317±71 1.23±0.10 2.06±0.19 70±25 73±540.8 44.1±5.3 458±20 1.34±0.16 4.75±0.25 240±28 175±361.6 62.2±2.2 514±120 1.79±0.03 10.1±0.57 1099±140 1480±73Mixed field experimentno plants 15.9±1.8a 133±27a 0.43±0.07a 1.00±0.02a 72±16a 53±36ab0.0% 26.5±5.2ab 183±45a 0.63±0.17a 0.58±0.11b 31±4a 16±10ab0.2% 25.8±3.0ab 162±3a 0.73±0.21a 1.01±0.07a 56±27a 0.3±3.0b0.8% 33.2±0.7b 235±136a 0.62±0.04a 2.34±0.35c 178±144a 328±203aMeasurements are means ± SD, n=3. One-way ANOVA was performed in field experiments with differentletters showing significant differences among treatments.enrichment of Littorella sediments over ca. 200days, but only in the 0.1% organic enrichmentof Lobelia sediments. At the highest 1.6%organic enrichment, sediments remained anoxicbelow 5-6 mm depth. Reestablishment ofoxygenated surface sediments was only partialin higher organic treatments, but it was alwaysstronger in Littorella than Lobelia sediments.DIC concentrations increased 4-7 timesin the organically enriched sediments in thelaboratory as a result of higher organicdegradations rates (Table 1). The increase ofNH 4 + concentrations was even more profound(> 100 times, Table 1). Anoxia inducedextensive anaerobic Fe 3+ reduction resulting inabout 500-1000 times higher Fe 2+concentrations in high-organic (1.6 %) thancontrol sediments (Table 1). Concentrations ofNO 3 - and PO 4 3- were negligible.In field experiments, all added organicmatter had been degraded and lost after 3summer months (Table 1). DIC concentrationswere still significantly higher in sedimentsreceiving 0.2-0.8 % organic matter, and meanconcentrations of NH 4 + and Fe 2+ were alsohigher at 0.8 % organic enrichment, althoughnot significantly relative to control sediments.Sediments without plants had higher pore-water60

Paper 3Table 2. O 2 flux across leaf surface to hypoxic waterwith basal leaf lacunae in air contact and evaporation todry still air of Lobelia dortmanna and Littorella uniflorameasured at 5 and 15 o C.o CSpeciesFlux Evaporation(n mol O 2 m -2 s -1 ) (µl m -2 s-1)Lobelia 5 30 ± 7 a15 48 ± 3 a 1.20 ± 0.83aLittorella 5 482 ± 199 b15 608 ± 144 b 14.5 ± 8.9bValues are means ± SD of 3 (O 2 flux) or 10(Evaporation) replicates. Different letters showsignificant differences (p

Paper 3In sediments enriched with 0.4 or 1.6%organic matter, anoxia in the dark was markedlyprolonged in Lobelia’s leaf lacunae compared toplants on un-enriched control sediments (Fig. 2).In contrast, anoxia never developed inLittorella’s leaf lacunae and the diurnal coursehere typically varied between 25-30 kPa in thelight and 5-15 kPa in the dark with no differencebetween control and enriched sediments.Plant performance and critical leaf nutrientsBoth Lobelia and Littorella reduced thedevelopment of roots relative to leaves withincreasing sediment enrichment (Fig. 3). Lengthof leaves relative to roots increased significantlyas a function of sediment organic matter andsignificantly more for Lobelia than Littorella(Linear regression, slope difference, p

Paper 3Fig. 4. Leaf chlorophyll of Lobelia dortmanna andLittorella uniflora (○ upper panel) and Littorella uniflora(●, lower panel) after 200 days in the laboratory insediments receiving increasing amounts of labile organicmatter. Means ± SD, n = 4-6. Regression lines are alsopresented. Chlorophyll values from control treatmentswere removed from the regression of Lobelia since asmall enrichment had a positive effect.field experiments (one-way ANOVA, p

Paper 3Fig. 5. Net photosynthesis at light and CO 2 saturation (y)as a function of leaf chlorophyll content x) of Lobeliadortmanna (upper panel) and Littorella uniflora (lowerpanel). Measurements were made with leaves fromcontrol sediments (open symbols) and sedimentsreceiving increasing amounts of labile organic matter(0.1-1.6% DW; gradually darker symbols).Littorella sediments, whereas Lobelia sedimentswent anoxic. The higher oxygenation ofLittorella sediments can be explained by thehigher biomass, photosynthesis and darktransport of O 2 from the lake water through theplant to the sediment. High O 2 permeability ofLittorella’s leaf surfaces can account for thesubstantial O 2 content (10 kPa) in the leaflacunae in the dark despite anoxic sediments,whereas Lobelia’s leaf surfaces are almostimpermeable and lacunae go anoxic in the dark.The higher resistance to O 2 release from leafthan root surfaces of Lobelia accords with thepredominant (90-100%) release of O 2 from rootsurfaces during photosynthesis, whereasLittorella releases more O 2 from leaves (72%)than roots (28 %; Sand-Jensen et al. 1982, Sand-Jensen & Prahl 1982). Thus, whereas Lobeliaturns anoxic entirely when in anoxic sediments,Littorella’s leaves remain oxic and rootscontinue to receive O 2 from the lake waterthrough leaf surfaces and intra-plant downwardtransport. The longest Littorella roots,nonetheless, face O 2 deficiency on highlyreducing sediments according to observed FeSprecipitates at the root tips and the roots growshorter to ensure sufficient downward O 2transport to the active meristmatic root zone(Colmer 2003, Raun et al. 2010). Lobelia ismuch more susceptible to sediment anoxia andreduces root development earlier and moreprofoundly than Littorella upon organicenrichment of the sediment (Fig. 4, see alsoRaun et al. 2010).The almost gas impermeable leaf surfaceof Lobelia is responsible for extensive anoxia inall plant tissue once the O 2 supply from thesediment vanishes. A few hours of anoxia late atnight is a natural recurring phenomenon even onnutrient-poor, low-organic sandy sedimentsduring summer because the species has almostno O 2 uptake from the water (Møller & Sand-Jensen 2011). Even a modest increase of O 2consumption in the sediments due to supply oflabile organic matter, therefore, increases theduration of night anoxia in Lobelia in contrast toLittorella leaves which remain permanentlyoxic. It is unlikely that the leaf anatomy ofLobelia has been selected to optimize O 2dynamics because it strongly increases the riskof anoxia and several associated stress reactions.Thus, low leaf permeability must havealternative advantages in order to have becomeselected.Firstly, the gas impermeable Lobelialeaves reduces the passive loss of CO 2 from the64

Paper 3high internal concentrations in the leaf lacunaeto the low air-equilibrium CO 2 concentrations inthe lake water. Because CO 2 formation isextremely low in pristine, oligotrophic Lobelialakesit is essential to maintain intimateexchange between the plant and the sedimentboth during photosynthesis and respiration andminimize CO 2 losses to the lake water becauseCO 2 concentrations needed to saturatephotosynthesis (~ 3000 µM) or just tocompensate respiration (~ 60 µM) are muchhigher than air-saturated levels (~ 15 µM; Sand-Jensen 1987, Pedersen et al. 1995, Winkel &Borum 2009). High permeability of Littorellaleaves could result in much greater CO 2 loss tothe lake water in the dark, but Littorellapossesses a special physiological feature whichallows temporary incorporation of leaf CO 2 byPEP-carboxylase into malate in the dark, anddecarboxylation and CO 2 incorporation vianormal C-3 photosynthesis in the following lightperiod, thus lowering plant-mediated CO 2release from the sediment via the aerenchyma(Madsen 1985). These CO 2 fluxes through theplants have not been quantified as yet.Secondly, the diffusion barrier onLobelia leaves effectively reduces evaporationand ensures survival of leaves when theyregularly become exposed to the air followingdrawdown of the water table during summer inthe seepage lakes (Pedersen & Sand-Jensen1992). Most submerged aquatic plants,including Littorella with 12-fold fasterevaporation rates than Lobelia, dry out upon airexposure and these species either die or survive,as in the case of Littorella, by producing newaerial, less permeable leaves with cuticle andfunctional stomata (Nielsen et al. 1991). Lobeliadoes not need to invest in a new set of leavesfollowing alternating submergence and airexposure which is costly and perhaps impossibleconsidering its low intrinsic growth rate andvery nutrient-poor habitat. In contrast, Littorellafaces this extra cost, but also has a fasterintrinsic growth rate and prefers to inhabitnutrient-richer sediments (Sand-Jensen &Søndergaard 1978, 1979). Thus, particularstructural and physiological adaptations fulfillseveral purposes and need to be viewed inregard to the entire plant life and the complexityof environmental conditions.Organic additions had more profoundand lasting effects on sediment chemistry inlaboratory than in field experiments. The muchfaster loss of organic matter and produced DIC,NH 4 + and Fe +2 in the field can be explained byhigher field temperatures promoting degradationrates, longer days with higher irradiancesstimulating oxygenation and, thereby, organicdegradation and, very likely, better physicalexchange by pressure waves or groundwaterflow between pore-water and lake water.Anoxic stress, nutrient supply and plantperformanceLobelia experienced extensive anoxia in bothleaves and roots by organic enrichment, whereasLittorella leaves always remained oxic andcould supply O 2 to the roots. More widespreadanoxia in Lobelia will impede effective ATPformation by oxidative phosphorylation, exhaustenergy resources (Greenway & Gibbs 2003a,b)and, in turn, restrict ion uptake and transportfrom roots to shoots (Colmer & Flowers 2008;Møller & Sand-Jensen 2011). Catabolicprocesses to sustain the photosyntheticapparatus in the leaves can also be impededbecause less metabolic energy and fewernutrients are available. This may account for thedecline of nutrients, chlorophyll and65

Paper 3photosynthesis of Lobelia leaves at all organicenrichments whereas TN, chlorophyll andphotosynthesis of Littorella leaves are notsignificantly reduced by any organic enrichmentand only TP is reduced at 0.8 and 1.6 % organicmatter.TP declined more significantly than TNin the leaves by organic sediment enrichmentand TP in Lobelia in several instances droppedbelow the minimum average threshold ofsubmerged macrophytes to sustain growth (0.08% P; Colman et al. 1987, Demars and Edwards2007) both in laboratory and field experiments.3-While PO 4 remained below analyticaldetection (0.2 µM) in the pore-water oforganically enriched sediments due to strongadsorption, NH +4 reached high concentrationsand can be supplied to the plants by passivediffusion (Marchner 1986). P-supply to roots viaVA-fungi is also believed to be impeded bysediment anoxia (Wigand et al. 1998) andinfection of new roots is grossly reduced inorganically enriched anoxic sediments (Møller,Kjøller & Sand-Jensen, pers. comm.).Our results, therefore, imply that low gaspermeability of leaf surfaces is essential for theexcellent ability of Lobelia to thrive onextremely low-organic, nutrient-poor mineralsediments by ensuring an intimate O 2 and CO 2exchange between plant and sediment andpermitting the same leaves to function in air andunder water. In contrast, even small enrichmentof sediments with labile organic matter led toprogressive problems for Lobelia’s performanceand survival by inducing sediment and plantanoxia, impeding P uptake and, finally, stoppingvirtually all photosynthesis and growth.Littorella by possessing gas permeable leafsurfaces is less likely to experience anoxia andmandatory nutrient supply, photosynthesis andgrowth and can much better cope and evenprofit from sediment enrichment. The maindisadvantage for Littorella is that it must investin new leaves upon air exposure or submergenceputting an extra burden on the species undervery nutrient-poor conditions but permitting it toadapt and grow faster under richer conditions.Thus, a distinct anatomical trait, i.e., leaf gaspermeability appears to have heavyconsequences for the fundamental and realizedniche of the two species and, therefore, for theirdistribution and survival in an increasinglyeutrophied environment.AcknowledgementsWe thank Tim D. Colmer for commenting on themanuscript and Birgit Kjøller and Annette Adamssonfor technical assistance. This project was funded bya grand from the Willum Kann RasmussenFoundation to Center of Lake Restoration (CLEAR).ReferencesArmstrong W, Cousins D, Armstrong J, Turner DW,Beckett PM 2000. Oxygen distribution in wetland plantroots and permeability barriers to gas-exchange with therhizonsphere: a microelectrode and modeling study withPhragmites australis. Annals of Botany 54: 177-198.Arts GHP 2002. Deterioration of Atlantic softwatermacrophyte communities by acidification, eutrophicationand alkalinisation. Aquatic Botany 73: 273-393.Christensen PB, Revsbech NP, Sand-Jensen K 1994.Microsensor analysis of oxygen in the rhizosphere of theaquatic macrophyte Littorella uniflora (L.) Aschers. PlantPhysiology 105: 1174-1179.Colman JA, Sorsa K, Hofmann JP, Smith CS,Andrews JH 1987. Yield-derived and photosynthesisderivedcritical concentrations of tissue phosphorus andtheir significance for growth of Eurasian Water Milfoil,Myriophyllum spicatum L. Aquatic Botany 29: 111-122.66

Paper 3Colmer TD 2003. Long-distance transport of gases inplants: a perspective on internal aeration and radialoxygen loss from roots. Plant, Cell and Environment 26:17-36.Colmer TD, Flowers TJ 2008. Flooding tolerance inhalophytes. New Phytologist 179: 964-974.Demars BOL, Edwards AC 2007. Tissue nutrientconcentrations in freshwater aquatic macrophytes: highinter-taxon differences and low phenotypic response tonutrient supply. Freshwater Biology 52: 2073-2086.Gerloff GC, Krombholz PH 1966. Tissue analysis as ameasure of nutrient availability for the growth ofangiosperm aquatic plants. Limnology and Oceanography11: 529-537.Gibbs J, Greenway H 2003. Mechanisms of anoxiatolerance in plants. I. Growth, survival and anaerobiccatabolism. Functional Plant Biology 30: 1-47.Greenway H, Gibbs J 2003. Mechanisms of anoxiatolerance in plants. II- Energy requirements formaintenance and energy distribution to essentialprocesses. Functional Plant Biology 30: 999-1036.Madsen TV 1985. A community of submerged aquaticCAM plants in Lake Kalgaard, Denmark. Aquatic Botany23: 97-108.Marschner H. 1986. Mineral nutrition of higher plants.Academic Press, London.Nielsen SL, Gacia E, Sand-Jensen K 1991. Land plantsof amphibious Littorella uniflora (L.) Aschers. maintainutilization of CO 2 from sediments. Oecologia 88: 258-263.Møller CL, Sand-Jensen K 2008. Iron plaques improvethe oxygen supply to root meristems of the freshwaterplant, Lobelia dortmanna. New Phytologist 179: 848-856.Møller CL, Sand-Jensen K 2011. High sensitivity ofLobelia dortmanna to sediment oxygen depletionfollowing organic enrichment. New Phytologist 190: 320-331.Pedersen O, Andersen T, Ekejima K, Hossain MZ,Andersen FØ 2006. A multidisciplinary approach tounderstanding the recent and historical occurrence of thefreshwater plant Littorella uniflora. Freshwater Biology51: 865-873.Pedersen O, Sand-Jensen K, Revsbech NP 1995. Dielpulses of O 2 and CO 2 in sandy sediments inhabited byLobelia dortmanna. Ecology 76: 1536-1545.Pedersen O, Sand-Jensen K 1992. Adaptations ofsubmerged Lobelia dortmanna to aerial life form:morphology, carbon sources and oxygen dynamics. Oikos65: 89-96.Raun AL, Borum J, Sand-Jensen K 2010. Influence ofsediment organic enrichment and water alkalinity ongrowth of aquatic isoetid and elodeid plants. FreshwaterBiology 55: 1891-1904.Sand-Jensen K 1987. Environmental control ofbicarbonate use among freshwater and marinemacrophytes. In RM Crawford (ed), Ecology andPhysiology of Intertidal plants, pp. 99-112. Blackwell,Oxford.Sand-Jensen K, Borum J 1991. Interactions amongphytoplankton, periphyton and macrophytes in temperatefreshwaters. Aquatic Botany 41: 137-175.Sand-Jensen K, Borum J, Binzer T 2005. Oxygen stressand reduced growth of Lobelia dortmanna in sandy lakesediments subject to organic enrichment. FreshwaterBiology 50: 1034-1048.Sand-Jensen K, Riis T, Vestergaard O, Larsen SE2000. Macrophyte decline in Danish lakes and streamsover the past 100 years. Journal of Ecology 88: 1030-1040.Sand-Jensen K, Prahl C 1982. Oxygen exchange withthe lacunae and across leaves and roots of the submergedvascular macrophyte, Lobelia dortmanna L. NewPhytologist 91: 103-120.Sand-Jensen K, Prahl C, Stokholm H 1982. Oxygenrelease from roots of submerged aquatic macrophytes.Oikos 38: 349-354.Sand-Jensen K, Søndergaard M 1978. Growth andproduction of isoetids in oligotrophic Lake Kalgaard,Denmark. Internationale Vereiningung füt theoretischeund angewandte Limnologie, Verhandlungen 9: 659-666.Sand-Jensen K, Søndergaard M 1979. Distribution andquantitative development of aquatic macrophytes inrelation to sediment characteristics in oligotrophic LakeKalgaard, Denmark. Freshwater Biology 9: 1-11.Smolders AJP, Lucassen ECHE, Roelofs JGM 2002.The isoetid environment: biogeochemistry and threats.Aquatic Botany 73: 325-350.Stelzer D, Schneider S, Melzer A 2005. Macrophytebasedassessment of lakes – a contribution for theimplementation of the European Water Framework67

Paper 3Directive in Germany. Internationale Revue ofHydrobiologie 90: 223-237.Søndergaard M, Sand-Jensen K 1979. Carbon uptakeby leaves and roots of Littorella uniflora (L.) Aschers.Aquatic Botany 6: 1-12.Wigand C, Andersen FØ, Christensen KK, Holmer M,Jensen HS 1998. Endomycorrhizae of isoetids along abiogeochemical gradient. Limnology and Oceanography43: 508-515.Winkel A, Borum J 2009. Use of sediment CO 2 bysubmersed rooted plants. Annals of Botany 103: 1015-1023.Wium-Andersen S 1971. Photosynthetic uptake of freeCO 2 by the roots of Lobelia dortmanna. PhysiologiaPlantarum 25: 245-248.68

Paper 4Organic enrichment of sediments reduces arbuscular mycorrhizalfungi colonization of the aquatic macrophytes Lobelia dortmannaand Littorella unifloraPhotos: Claus Lindskov Møller

Paper 4Organic enrichment of sediments reduces arbuscular mycorrhizal fungicolonization of the aquatic macrophytes Lobelia dortmanna and Littorellauniflora.Claus Lindskov Møller 1 , Rasmus Kjøller 2 and Kaj Sand-Jensen 11 Freshwater Biological Laboratory, Biological Institute, University of Copenhagen, Helsingørsgade 51, DK-3400Hillerød, Denmark.2 Terrestrial Ecology, Biological Institute, University of Copenhagen, Øster Farimagsgade 2B.SummaryKey words: Isoetids, arbuscularmycorrhizal fungi, colonization, hyphaldensity, eutrophication, oxygen,anoxia, organic enrichment.● Arbuscular mycorrhizal fungi (AMF) commonly colonize isoetid speciesinhabiting the littoral zone in oligotrophic lakes but are absent in other aquaticplants. We hypothesize that organic enrichment of oligotrophic sedimentsreduces AMF colonization and hyphal growth because O 2 depletion, highernutrient availability and low carbon supply from stressed host plants.● We added organic matter to sediments inhabited by isoetids and measured O 2availability with mini O 2 electrodes, pore-water content of inorganic carbon,Fe 2+ and NH 4 + , colonization intensity of roots and hyphal density after exposure.We also report hyphal densities relative to root density and colonizationintensity.● Addition of organic matter reduced AMF colonization of roots of Lobelia andLittorella and high additions clearly stressed the plants. Even small additionsalmost stopped AMF colonization of initially un-colonized Littorella. Hyphaldensities in sediments were low in the upper 1 cm but otherwise high in the rootzone and positively related to root density. Hyphal surface area exceeded rootsurfaces 1.7-3.2 times.● We conclude that AMF efficiently colonize isoetids in oligotrophic sedimentsand mediate nutrients supply to plants by their extensive hyphal net-works.However, even small organic additions to sediments with no apparent plantstress reduce AMF colonization of roots.IntroductionMore than 80% of all plant species arecolonized by arbuscular mycorrhizal fungi(AMF; Smith & Read 2008). Although AMF isvery common on land, high AMF colonizationamong aquatic plants is only found in smallrosette species (so-called isoetids) inhabitingoxygenated sediments in oligotrophic freshwaterlakes (Søndergaard & Laegaard 1977, Beck-Nielsen & Madsen 2001, Nielsen et al. 2004). Ineutrophic lakes plants are rarely colonized andin streams only emergent species have beenfound to be mycorrhizal (Beck-Nielsen &Madsen 2001).71

Paper 4Surveys on aquatic plants report fallingAMF colonization in plants growing insediments of high contents of organic matter andnutrients and low redox potential (Tanner &Calyton 1985; Wigand et al 1998; Beck-Nielsen& Madsen 2001). However, no experiment hasdirectly addressed the response of AMFcolonization of roots to O 2 depletion andchanges in sediment biogeochemistry followingorganic enrichment of aquatic sediments. Herewe tested this aspect by enriching aquaticsediments with labile organic matter andquantifying AMF root colonization, sedimenthyphal density and plant nutrition of the isoetidspecies Lobelia dortmanna and Littorellauniflora.Isoetids are small slow-growing plantsdominating the littoral zones of oligotrophicsoft-water lakes due to their unique adaptationsto take up sediment CO 2 for photosynthesisacross permeable root surfaces and ensure fastintra-plant diffusion through aerenchyma to theleaves (Sand-Jensen et al. 1982; Møller & Sand-Jensen 2008). These features also result in largeproportions of photosynthetic O 2 being releasedfrom the roots and generating an oxygenatedrhizosphere (Sand-Jensen et al. 1982; Pedersenet al. 1995; Møller & Sand-Jensen 2001b) whichis believed to be required by AMF (Le Tacon etal. 1983; Beck-Nielsen & Madsen 2001). Mostisoetids are reported with mycorrhiza(Søndergaard & Laegaard 1977; Wigand et al.1998; Beck-Nielsen & Madsen 2001) and couldbenefit from the symbiosis because they: (i)inhabit the most nutrient-poor aquaticenvironments of low P availability (Christensen& Andersen 1996; Christensen & Wigand 1998;Smolders et al. 2002), (ii) have relatively thickroots and lack roots hairs to provide highsediment contact (Søndergaard & Laegaard721977; Beck-Nielsen & Madsen 2001; Farmer1985), and (iii) have slow root growthincreasing the risk of strong nutrient depletionzones around roots (Sand-Jensen & Søndergaard1978). AMF associations increase both thesurface area and the sediment volume fornutrient uptake and hyphae extend beyond thedepletion zones surrounding the roots (Smith &Read 2008). We report here distribution andextension of the mycorrhizal mycelia in relationto plant density of isoetids in lakes for the firsttime.Most studies on AMF in aquatic plantshave focused on AMF colonization underdifferent natural conditions (Beck-Nielsen &Madsen 2001; Søndergaard & Laegaard 1977;Wigand et al. 1998), and on plant morphologiesassociated with high AMF colonization(Søndergaard & Laegaard 1977; Beck-Nielsen& Madsen 2001). AMF benefits aquatic plantsby increasing P uptake and P content of leaves(Tanner & Clayton 1985; Wigand & Stevenson1997) and plant biomass (Andersen & Andersen2006). Among different populations of Lobeliadortmanna plants with low tissue P content hadhigher AMF colonization (Wigand et al. 1998).Occurrence and extension of hyphae in aquaticsediments are largely unknown in contrast tonumerous terrestrial investigations (Smith &Read 2008). Only Beck-Nielsen and Madsen(2001) reported hyphal densities in aquaticsediments inhabited by Littorella uniflora, butthe lack of measurements of plant densityprevented conversion to densities of hyphae pervolume or surface area of sediments needed forcomparison with terrestrial communities. Herewe made a special effort to measure densities ofhyphae and roots for comparison with terrestrialcommunities.

Paper 4Organic enrichment of lake sedimentscan result from eutrophication and enhancedprimary production and sedimentation oforganic matter (Smolders et al. 2002). A smallorganic increment can stimulate isoetid growthdue to higher sediment concentrations of CO 2and nutrients (Sand-Jensen et al. 2005; Raun etal. 2010; Møller & Sand-Jensen 2011).However, high organic additions lead to anoxiain sediments and root systems during the nightand subsequently reduced nutrient translocationand photosynthesis (Sand-Jensen et al. 2005;Raun et al. 2010; Møller & Sand-Jensen 2011).Nutrient stress in isoetids is ascribed toinefficient transport of inorganic nutrients fromroots and photosynthates from leaves underanaerobic conditions causing root die-off(Sorrell 2004; Møller & Sand-Jensen 2011).Similarly AMF colonization has been found todecrease with increasing organic content ofsediments, but relations has not been evaluatedin regard to plant stress. Hence, lack of AMFassociations could contribute to lower plantnutrition.Two experiments were carried out toaddress the response of plants and AMF toorganic sediment enrichment. In the firstexperiment Littorella uniflora runners with noinitial AMF colonization were grown in naturalsediments without or with addition of organicmatter to determine root colonization rates andplant response. In the second experiment naturalsediment turfs with indigenous arbuscularmycorrhiza fungi inhabited with mono-speciesstands of Lobelia dortmanna or Littorellauniflora were subjected to organic enrichment todetermine the response of AMF in roots andsediments and changes in plant fitness.73Materials and methodsColonization of Littorella unifloraLittorella uniflora without AMF colonizationwas supplied by the aquatic plant manufactureAnubias (France). Prior to the experiment, sixLittorella plants were stained and tested formycorrhizal colonization and none were foundto be colonized (see below). For the experimentnatural sediment with a high density ofLittorella uniflora (ca. 15,000 plants m -2 ) fromoligotrophic, softwater Lake Värsjö, SWSweden was collected. Plants were removed andthe sediment was thoroughly mixed and dividedinto three portions. One portion was used as acontrol, while the other two portions receivedfeeding pellets made of pasture grass equivalentto 0.2% and 0.8% organic matter of sedimentdry weight (DW), respectively. Pellets contained91±1% (n=4) organic matter (47± 0.4% organicC) 2.3 ±0.15% total nitrogen (TN) and 0.27 ±0.01% total phosphorus (TP). Sediments werethen left for two weeks under waterlogging toallow new sediment conditions to establishbefore sediments were transferred to 5 Perspexcylinders (6.5 cm diameter and 15 cm deep) foreach treatment and one AMF-free Littorellauniflora of uniform mean size of 0.23-0.29 gwet weight in the treatments (One-way Anova, p< 0.05) was planted in each cylinder. Cylinderswith plants were placed in 15 separate 9 Laquaria for 135 days in the laboratory at 15 o C ina 12: 12 hour light: dark cycle with aphotosynthetic available radiation (PAR) of 110µmol photons m -2 s -1 . Plants were growncompletely submerged in experimental water ofsimilar composition as water from Lake Värsjö(see Møller & Sand-Jensen 2011a for details)and slowly bubbled with atmospheric air to

Paper 4ensure mixing and air saturated conditions.Water was changed weekly.Pore-water content and O 2 penetrationMinute pore-water samples were extracted after135 days of experiments by small glass tubesinserted in 4 cm depth in the sediments (Møller& Sand-Jensen 2011) and analyzed fordissolved inorganic carbon (DIC), reduced Fe 2+ ,ortho-phosphate (O-P) and NH + 4 . DIC wasdetermined by injecting 5-50 µl samples into3% HNO 3 in a bubble chamber purged with N 2gas carrying evolved CO 2 into an Infrared gasanalyzer (IRGA, ADC-225-MK3, Hoddesdon,UK; Vermaat & Sand-Jensen, 1987). Fe 2+ wasmeasured on 100 µl samples diluted in 900 µl0.1 M HCL according to Eaton et al. (1995). O-P was measured according to a modified versionof Strickland & Parsons (1968) as previouslydescribed in Møller & Sand-Jensen (2011).NH +4 was measured on 100 µl pore-waterdiluted in 900 µl distilled water added 100 µlphenol and 100µl hypochlorite reagentsaccording to Solórzano (1969). O 2 penetrationdepth was measured by a mini O 2 electrodes(Ox 500, Unisense, Århus, Denmark) movedand positioned by a micromanipulator between0 and 40 mm depth in the sediment. O 2penetration was measured within the last twohours of the light period to ensure nearmaximum O 2 penetration.Plant morphology and myzorrhizal colonizationPlants were gently removed from the sedimentand rinsed in water after the 135-days longcolonization experiment. Number of leaves wasrecorded and plants were split into above-(leaves) and below-ground (stem and roots)biomass. Freeze-dried leaves were analyzed forTP, TN and chlorophyll content. TP wasmeasured according to Andersen (1976), TN bya CHN EA1108-elemental analyzer (Carlo ErbaInstruments, Milan, Italy) and chlorophyll byextraction in ethanol for 24 hours andspectrophotometrical analysis (Christoffersenand Jespersen (1986). Total root length of eachplant was determined by the grid interceptmethod (Newman (1966). Roots were cleared in10% KOH and stained for AMF with tryphanblue(Kormanik & Mosse 1980) and stored inlactoglycerol. AMF colonization frequency wasdetermined at 20 x magnification as the averageoccurrence of hyphae, arbuscles or vesicles in 2x 100 intercepts between grit and roots(Giovannetti & McGraw 1982). Supplied AMFfreeplants had been grown emerged with rootsin an agar medium exposed to light resulting inlateral root systems with chlorophyll. This madeit possible to determine that all roots had beenreplaced at the end of the experiment since onlyvertically oriented roots without chlorophyllremained. Similarly, the initial air leaves (thinand long) had been shed and renewed by aquaticleaves (shorter and thick) at the end of theexperiment.Experiment with indigenous AMF associationsSix intact sediment turfs (15 cm long, 17 cmwide and 13 cm deep) inhabited by densepopulations of Lobelia dortmanna and six withLittorella uniflora with indigenous AMFcolonization and normal hyphal density werecollected at a sandy site in Lake Värsjö. Turfswere brought back to the laboratory submergedin lake water and subjected to organicenrichments of 0, 0.1, 0.2, 0.4, 0.8, 1.6%organic matter per sediment DW by insertingfeeding pellets of variable length at 4 cm depth74

Paper 4in the sediment at a fixed equi-distance of 2 cm,thereby keeping the AMF-plant association asundisturbed as possible (Møller & Sand-Jensen2011 for further information). Turfs were grownin the laboratory facility in two 80 liter aquariafor about 200 days in the same experimentalwater and under the same daily irradiance,temperature and aeration regimes as describedfor the colonization experiment.Pore-water concentrations of DIC, Fe 2+and NH +4 were measured at the end of theexperiment in 1, 2, 4, 6 and 8 cm depth and arepresented here as depth-integrated means.Plant morphology and leaf content ofTP, TN and chlorophyll were determined onrandomly drawn plants (three Lobelia and sixLittorella). Leaf content was analyzed asdescribed for the colonization experiment whileroot and leaf length were measured by a ruler. Itwas possible to divide Lobelia roots into old andnewly set roots at harvest since new rootsemerge further up the stem and did not have thesame thick iron-plaques as old roots at highorganic enrichments. However, the exact rootage is unknown and may vary amongtreatments. When calculating colonized rootlength of Lobelia new and old roots wereassumed to have the same length. Thisdistinction between old and newly set roots wasnot possible for Littorella. Root surface areawas calculated from the measured root lengthsand literature values for root diameters ofLobelia (mean=0.728 mm) and Littorella (1.248mm; Raun et al. 2010). Root density wascalculated from root lengths of investigatedplants multiplied by measured plant density atthe end of the experiment. Sediment content oforganic matter was determined on dried mixedsediment samples (24 hours at 105 o C) as losson ignition at 550o C. TP and TN were75determined as described for leaves on mixedsediment samples from 1 to 10 cm depth.Hyphal density in sediments wasdetermined in duplicate for each sediment turfon sediment cores (1 cm diameter 10 cm depth)sliced for every 1 cm from 0 to 10 cm depthaccording to Kjøller and Rosendahl (2000). Inbrief, sediment samples were suspended in 50ml distilled water whereafter hyphae werecollected on a 38 µm sieve. Hyphae were thentransferred to a test-tube and suspended in 30 mlwater shaken vigorously and two 5 mlsubsamples were transferred to a filter, stainedwith tryphan-blue and conserved inlactoglycerol. Hyphal density was determinedby counting intercepts between grid and hyphaon 20 views for each subsample at 200 xmagnification according to Giovannetti &McGraw (1982). Hyphal diameter wasmeasured on 6 hyphae from 4 random views ofeach treatment.In natural homogeneous plantpopulations in shallow sandy areas in LakeVärsjö, AMF colonization, plant biomass andnutrient content of both Lobelia and Littorellawere determined in triplicate 15 x 15 cm plots.Plants were gently rinsed from sediment andbrought to the laboratory in sealed plastic bags.In the laboratory 4 random Lobelia and 5Littorella plants were removed for determiningroot length and AMF colonization. Theremaining plants were separated into above- andbelow-ground biomass of each species. Dryweight, TP, TN and chlorophyll content ofleaves and root length and AMF colonizationfrequency were determined as previouslydescribed for the colonization experiment.Data treatment and statistical analysis

Paper 4ResultsFig 1. O 2 -penetration depth in intact sediment turfsinhabited by Lobelia dortmanna (○) or Littorella uniflora(●) after about 200 days and in sediments from thecolonization experiments with Littorella uniflora (■) after135 days enrichment with labile organic matter. O 2 -penetration deeper that 40 mm could not be measured.Values are mean ± SD (n=3 for turf experiments, n=5 forcolonization experiments).Statistical analysis was performed in GraphPadprism 5 (software info). The colonizationexperiment was a gradient study with threelevels of 5 replicates appropriate for ANOVAanalysis. When data failed to meet testrequirements for variance-homogeneity, datawere square-root transformed prior to analysis.The experiment with intact sediment turfs was agradient study with six levels withoutreplication for Lobelia and Littorella turfs anddifferences were tested by linear regression orSpearmann´s correlation analysis. Differencesbetween Lobelia and Littorella from mixed insitupopulations were investigated by t-test.Probabilities above 0.95 were consideredsignificant. Data are presented as mean ± 1standard deviation (SD) of true replicates for thecolonization experiment and mixed in-situpopulations while SD of pseudo-replicates ispresented for the turf experiment when possible.Sediment biogeochemistryAddition of organic matter caused O 2 depletionand accumulation of DIC, Fe 2+ , NH 4 + and NH 4+in sediments throughout both experiments (Fig1, Fig 2ab & Table 1). O 2 penetrated to morethan 40 mm in control sediments but organicadditions significantly reduced O 2 penetrationdepths (Spearman´s r, p

Paper 4Fig 2. Pore-water content of dissolved inorganic carbon(DIC, ○) reduced iron (Fe 2+ , ●), AMF root colonizationfrequency in old roots (□) and new roots (■; in c data isfrom the colonization experiment) and chlorophyll (Δ)and TP (▲) content of leaves as a function of addedorganic matter to intact sediment turfs with indigenousAMF inhabited by Littorella uniflora (a,c,e) or Lobeliadortmanna (b,d,f) after about 200 days.Plant growth, morphology and leaf contentLeaves and roots were completely replacedwithin the 135-days long colonizationexperiment with Littorella. Average root lengthdecreased significantly with increasing additionof organic matter (One-way ANOVA, p

Paper 4Table 2. Individual leaf biomass, mean root length, total root length per plant and two measures of AMF colonization (% ofroot length colonized and root length colonized per leaf biomass) in field and laboratory experiments with Littorella andLobelia. Relations between root length colonized and leaf biomass are also shown. Experimental treatment was addition oflabile organic matter in different percentages of sediment dry weight. Measurements were taken after 135 days in AMFcolonization experiments and after about 200 days in turf experiments.Treatment(addition oforganicmatter)Mean rootlength (cmroot -1 )Total rootlength (mplant -1 )Colonization(% of rootlength)Root lengthcolonized(m plant -1 )Root lengthcolonized:Leaf biomass (mg -1 DW)Leaf biomass(mg DW plant -1 )Colonization experiment with Littorella unifloraControl 9.7±2.4 a 1.18±0.13 a 30 ± 17 a 0.37 ± 0.24 a 14.9±8.3 23.8±3.90.2% 5.3±2.0 b 1.24±0.35 a 5.1 ± 6.5 b 0.048±0.042 b 1.2±1.3 31.3±4.40.8% 2.2±0.4 c 0.22±0.03 b 3.2 ± 6 b 0.007±1.46 b 0.5±1.0 15.3±8.5Littorella turfsControl 4.1 ±1.1 0.40 ±0.17 91 ±5.7 0.36 41.5 8.7 ±2.10.1% 3.4 ±0.6 0.32 ±0.12 93 ±1.4 0.29 23.8 12.2 ±1.30.2% 3.1 ±0.7 0.28 ±0.07 86 ±3.5 0.24 20.8 11.5 ±2.60.4% 4.2 ±0.6 0.42 ±0.13 63 ±18 0.26 17.6 14.8 ±5.70.8% 3.3 ±0.5 0.29 ±0.07 36 ±3.5 0.10 6.8 14.8 ±4.41.6% 2.9 ±0.3 0.16 ±0.04 54 ±6.0 0.09 7.2 12.5 ±3.9r 2 (p) 0.33 (0.23) 0.62 (0.06) 0.55 (0.09) 0.79 (0.02) 0.62 (0.06) 0.16 (0.42)Lobelia turfsControl 4.0 ± 0.5 0.65 ±0.29 * 53 ±0.1 0.34 * 14.4 24 ±60.1% 5.2 ±0.6 1.32 ±0.28 51 ±0.9 0.68 15.7 43 ±70.2% 4.2 ±0.5 1.03 ±0.10 59 ±6.8 0.61 12.7 48 ±70.4% 2.8 ±0.4 0.61 ±0.22 39 ±13 0.23 3.4 68 ±220.8% 4.1 ±0.8 0.70 ±0.21 43 ±14 0.33 4.7 70 ±141.6% 1.7 ±0.2 0.30 ±0.09 16 ±22 0.05 1.6 31 ±10r 2 (p) 0.61 (0.07) 0.75 (0.06) 0.85 (0.09) 0.74 (0.06) 0.68 (0.04) 0.00 (1.0)Mixed populations from VärsjöLittorella Nd 0.25±0.01 a 77.5±4.4 a 0.20±0.01 a 231±66 a 0.89±0.26 aLobelia Nd 0.81±0.25 b 49.7±3.1 b 0.41±0.12 b 48.9±13.7 b 8.6±2.5 bValues are means ± SD of true replicates in colonization experiments and mixed populations and of pseudo-replicates inLobelia and Littorella turfs, n=3-5. Superscripted letters show significant differences in colonization experiments and mixedin-situ populations respectively (One-way ANOVA, p

Paper 4Table 3. Leaf content of total phosphorus (TP), total nitrogen (TN) and chlorophyll from monocultures of Lobelia dortmannaand Littorella uniflora grown in the laboratory after about 200 days in experiments with addition of different amounts oflabile organic matter.LobeliaLittorellaTreatment TPTN Chlorophyll TPTN(mg P g -1 (mg N g -1 (mg g -1 (mg P g -1 (mg N g -1 Chlorophyll(mg g -1 DW)DW) DW) DW)DW) DW)0.0% 1.47 ± 0.19 22.0 ± 3.92 1.79 ± 0.29 1.30 ± 0.33 24.9 ± 2.8 5.17 ±0.890.1% 0.88 ± 0.24 14.3 ± 3.01 2.74 ± 0.39 1.21± 0.15 20.3 ± 0.8 4.32 ±0.540.2% 0.89 ± 0.25 18.8 ± 3.66 3.15 ± 0.97 0.93 ± 0.15 22.5 ± 1.2 5.30 ±1.210.4% 0.69 ± 0.18 18.1 ± 3.62 2.46 ± 0.57 1.23 ± 0.33 23.0 ± 4.9 5.97 ±1.320.8% 0.53 ± 0.17 12.6 ± 2.52 1.90 ± 0.31 0.62 ± 0.12 15.3 ± 2.7 4.79 ±0.811.6% 0.61 ± 0.13 13.0 ± 3.33 0.75 ± 0.13 0.81 ± 0.20 23.0 ± 6.7 4.67 ±1.94Lin reg. p p=0.09 p=0.11 p

Paper 4Fig. 4. Root density (○) and hyphal density (●) insediments of Littorella uniflora (upper panel) and Lobeliadortmanna (lower panel) in experiments with addition oflabile organic matter to sediment with indigenous AMFcolonization and sediment density. Values for hyphaldensity are mean ± SD whereas root density is derivedfrom average root length of harvested plants, plant densityand sediment depth.root surface area 1.7 times for Littorella and 3.2times for Lobelia in control and 0.1 and 0.2%organic enrichments.DiscussionOrganic enrichment and AMF colonizationAMF colonization declined by addition of labileorganic matter and even small additions with noapparent plant stress resulted in very lowcolonization of initially un-colonized Littorellauniflora. This finding is consistent with surveysfrom wetlands and lakes of colonizationdecreasing with higher organic content and80lower redox potentials of sediments (Beck-Nielsen & Madsen 2001; Wigand et al. 1998).Reduced colonization upon organic enrichmentwas also observed in Lobelia dortmanna andLittorella already colonized by AMF althoughthe effect was less pronounced. The slowreplacement of roots of isoetids (Sand-Jensen &Søndergaard 1978) and the fact that establishedroots remain colonized under suboptimalconditions (Filer & Boardfood 1968) are themost plausible explanations for the smallresponse in already colonized plants. Thisinterpretation is also supported by the muchlower colonization of new roots of Lobeliaformed under high organic treatment whereascolonization remained high on new roots underlow organic treatment.Reduced root colonization could be dueto lack of AMF species acclimatized to thenewly established sediment conditions, butsimilar patterns have been reported for naturalpopulations (Wigand et al. 1998). The diversityof AMF in isoetid lakes is high and comparableto terrestrial habitats and AMF can survive longunder unfavorable periods as spores (Miller2000; Nielsen et al. 2004). It therefore seemsmore likely that AMF are unable to thrive underreduced conditions in organically enrichedsediments.Investigations have shown that AMFcolonization decreases with addition of P toterrestrial soils (Abbott et al. 1984) and aquaticsediments (Tanner & Clayton 1985). P wasadded here contained in the organic matter, butO-P in the pore-water remained low due tostrong binding and P content remained low inleaves, implying that plants could still benefitfrom increased P uptake through AMF.Hyphal density of sediments

Paper 4Hyphal densities of isoetid sediments arecomparable to terrestrial soils having typicallengths of hyphae between 2 and 40 m per cm -3(Smith and Read 2008). Densities were low inthe upper 1 cm of the isoetid sediment probablydue to disturbance by wave exposure andbioturbation and pH-changes due tophotosynthesis of microalgae (Jasper et al. 1991;Van Aarle et al. 2002). In the root zone, hyphaldensity was high and correlated positively withroot density. With lengths of hyphae relative toroots of 700 and conservative estimates ofhyphal surface area exceeding that of rootsurface areas by 1.7- 3.2 times in low-organicsediments the AMF-isoetid symbiosis results ina large increase of sediment contact for nutrientretrieval. Lengths of hyphae relative to infectedroots were also comparable to terrestrial potexperiments with Trifolium subterraneuminoculated with Glomus fasciculatum (450-900,Abbott et al. 1984) suggesting quantitativesimilarity to well-oxygenated nutrient-poorisoetid sediments.In contrast to AMF root colonizationpercentages, no decrease was observed inhyphal density in sediments following organicenrichment. In fact, addition of organic mattertended to increase hyphal density (Fig 4). Thisresult could, however, be due to staining of bothliving and dead hyphae because degradation ofdead hyphae can be slow in anaerobicsediments.Beck-Nielsen & Madsen (2001) havereported hyphal densities from aquaticsediments inhabited by Littorella at 3-20 timeslower density than in the present investigation.They found hyphal densities to decrease withlower redox potential of sediments. However,different redox potentials were found bysampling in sediments differing in plant densityand, therefore, relations could alternatively arisefrom differences in root densities which werehighly correlated to hyphal density in thepresent investigation. Sanders et al. (1998)reported of increased hyphal growth interrestrial soils under elevated CO 2 levels andsimilarly Andersen & Andersen (2006) foundincreased AMF colonization of Littorella grownsubmerged at 10 times CO 2 air saturation in thewater. Addition of organic matter and thefollowing increase in CO 2 could be responsiblefor the observed tendencies of higher hyphaldensities upon organic enrichments but coupledinvestigations of hyphae growth and AMFcolonization are needed to address this questionfurther.Sediment biogeochemistryAMF colonization decreased when organicadditions deprived sediments of O 2 and Fe 2+accumulated in the pore-water. Redox potentialswere not measured, but in sediments at normalpH levels (≈7) thermodynamics favor theoccurrence of Fe 2+ as the primary form below 0mV, while the presence of O 2 leads to rapid Fe 2+oxidation to Fe 3+ (half-life of minutes; Canfieldet al. 2005). With anoxic sediments andcertainly low redox potentials following organicenrichment the results are consistent with thoseof Beck-Nielsen & Madsen (2001) showingreduced AMF colonization in roots at redoxpotentials below 250 mV. Recent experimentsby Møller & Sand-Jensen (2011) showed thatanoxia occurred in oligotrophic isoetidsediments during nighttime, and anoxia evendeveloped in root and leaf aerenchyma ofLobelia late at night. This result implies thatAMF can cope with anoxia for at least some81

Paper 4hours every night although low O 2 tension hasbeen shown to prevent AMF development (LeTacon et al. 1983).Plant growth, stress and nutritionPlants were clearly stressed by high organicaddition and this was always coupled with lowerAMF colonization. In experiments with intactsediment turfs, P levels in leaves tended todecrease at the same organic addition (0.4%)causing AMF colonization to drop (Fig. 2; Table3). In contrast, Littorella was able to grow andmaintain unaltered nutrient and chlorophylllevels and biomasses with very low AMFcolonization at low organic enrichments (0.2%in the colonization experiment) as plants withhigh AMF colonization in control sediments(Table 1). There is, therefore, no clear evidenceof AMF being responsible for the observedstress. It has been shown for the isoetid Isoetesalpinus that root anoxia stops translocation ofphotosynthates from leaves to roots (Sorrell2004) which can lead to root malfunction anddecreased plant nutrition (Møller & Sand-Jensen2011) and this is the most plausible reason forthe observed stress. Furthermore, under thesesediment conditions shading of the isoetids byfaster growing plants or filamentous algae islikely to occur (Sand-Jensen 2000; Arts 2002).It is, therefore, more reasonable to regard AMFas an advantage under very nutrient-poorconditions whereas transport of photosynthatesto AMF under more nutrient-rich sedimentconditions can be spared because plants cansustain sufficient nutrient uptake without thepresence of AMF (Smith & Read 2008).This investigation showed that isoetidswith high AMF colonization have extensivehyphal networks increasing sediment contactand surface area for nutrient uptake in nutrientpoorsediments. AMF colonization in plantsfrom mixed populations in Värsjö was similar tothat in long-term laboratory experiments withmono-specific populations in Littorella andLobelia turfs collected in the field. Littorellahad higher root colonization and a higher AMFcolonization per leaf weight than Lobelia and isknown to have two times higher growth rateunder field conditions (Sand-Jensen &Søndergaard 1978; Bosten & Adams 1989).Thus, Littorella needs a higher nutrient uptakeper biomass than Lobelia. Higher colonizationof Littorella roots being replaced more rapidlythan Lobelia roots requires higher colonizationrates. This higher colonization rate of AMFcould be supported by higher downward O 2supply to Littorella roots and sediments duringthe night whereas Lobelia even at moderatetemperatures (≈16 o C) under natural conditionsexperiences anoxia in the roots (Møller & Sand-Jensen 2011a). Furthermore, higherphotosynthesis of Littorella than Lobelia couldpromote higher organic carbon supplies to thesymbionts.AcknowledgementsWe thank Anubias (France) and Jan OlePedersen for providing non-mycorrhizalLittorella uniflora and Helene Rasmussen forprocessing and counting hyphal lengths insediment samples. We thank The Willum KannFoundation for financial support to this studythrough The Centre of Excellence for Researchon Lake Restoration (CLEAR).References82

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Paper 5Outstanding Lobelia dortmanna in iron armorPhoto: Ole Pedersen

[Plant Signaling & Behavior 3:10, 882-884; October 2008]; ©2008 Landes BiosciencePaper 5Article AddendumOutstanding Lobelia dortmanna in iron armorKaj Sand-Jensen, Claus Lindskov Møller* and Ane Løvendahl RaunFreshwater Biological Laboratory; Biological Institute; University of Copenhagen; Hillerød DenmarkKey words: isoetids, Lobelia dortmanna, iron, ROL, sediment oxygen, iron plaquesLobelia dortmanna leads a group of small, highly-valued rosettespecies that grow on coarse, nutrient-poor soils in temperate softwaterlakes. They acquire most CO 2for photosynthesis by rootuptake and efficient gas transport in large air channels to the leaves.Lobelia is the only species that releases virtually all photosyntheticoxygen from the roots and generates profound day-night changes inoxygen and CO 2in the sediment pore-water. While oxygen releasefrom roots stimulates decomposition and supports VA-mycorrhizafungi, the ready gas exchange presents a risk of insufficient oxygensupply to the distal root meristems as sediments accumulate organicmatter from lake pollution. So the plant with the greatest oxygenrelease from roots is also the most sensitive to oxygen depletion insediments and it dies or losses anchorage by shortening the rootsfrom 10 to 2 cm at even modest contents (2.4%) of degradableorganic matter. Coatings of oxidized iron on roots in organicallyenriched sediments reduce radial oxygen loss and, thereby, increaseinternal concentrations and supply of oxygen to root tips. Oxidizediron is also a redox buffer which may prevent the ingress of sulfidesand other reduced toxic solutes during nights. Controlled experimentsare under way to test if iron enrichment can help survival ofrosette species threatened by lake pollution or whether removal oforganic surface sediments is required.Inhospitable SedimentsAquatic sediments and waterlogged soils are usually anoxic a fewmm below the surface. 1,2 Aquatic and wetland plants, therefore,need to supply oxygen to the roots by rapid intra-plant gas transportthrough large air channels running from the green shoot in contactwith atmospheric air or aerated water to the roots deeply buried inanoxic environments. 3,4 To ensure oxygen transport to the root tipin competition with radial oxygen loss to the anoxic hydrosoil, itshould be advantageous for roots to be thick, short and impermeableto radial oxygen loss. 5,6 Thick roots, however, are costly to producerelative to their capacity to take up nutrients so this morphology is*Correspondence to: Claus Lindskov Møller; Freshwater Biological Laboratory;Biological Institute; University of Copenhagen; Helsingørgade 51; Hillerød DE-3400Denmark; Email: clmoller@bio.ku.dkSubmitted: 06/26/08; Accepted: 06/26/08Previously published online as a Plant Signaling & Behavior E-publication:http://www.landesbioscience.com/journals/psb/article/6500Addendum to: Møller CL, Jensen KS. Iron plaques improve the oxygen supply to rootmeristems of the freshwater plant, Lobelia dortmanna. New Phytol 2008; 179:848–56; PMID: 18513220; DOI: 10.1111/j.1469-8137.2008.02506.x.not a general solution to cope with anoxic hydrosoils. Short roots,likewise, have a reduced nutrient uptake capacity and also a pooranchorage. Thus, the third option—having roots relatively impermeableto radial oxygen loss—appears to be a suitable solution. Indeed,this property is common among wetland plants, 6 it is present in themarine seagrass, Zostera marina and probably widespread amongmarine and freshwater plants (Fig. 1).Small freshwater rosette species, however, have highly gas permeableroot surfaces as an adaptation to take up free CO 2forphotosynthesis from the CO 2-rich hydrosoil in the nutrient-poorsandy sediments of softwater lakes. 1,7 These rosette species have thickentire leaves from a short stem, many roots and well-developed airchannels facilitating transport of oxygen and CO 2between leavesand roots. Utilizing CO 2from the hydrosoil for photosynthesis, theroots are also highly permeable to radial oxygen transport 8 and thisproperty has recently been confirmed by quantification of oxygenloss and root wall permeability for every 5 mm along the roots ofLobelia dortmanna. 9 The question therefore arises: how do thesespecies supply oxygen to root tips in sediments undergoing organicenrichment and oxygen depletion?Coping with Sediment Oxygen DepletionLobelia dortmanna is the only known species that releases virtuallyall photosynthetic oxygen from the roots to the sediments thanksto high root surface permeability and low leaf surface permeabilityand it, therefore, generates profound day-night fluctuations in poolsand penetration depth of oxygen in sandy sediments of low oxygenconsumption. 1,2 Sediment CO 2varies opposite to oxygen due totheir complementary roles in photosynthesis and respiration. 1 Otherrosette species also exchange much CO 2and oxygen via the roots, 8but leaf exchange is more important than in Lobelia.The ready oxygen exchange across Lobelia roots and the oxicconditions in nutrient-poor sediments have been regarded as anadvantage for the nutrient supply due to stimulation of organicdecomposition by aerobic sediment bacteria and mycorrhiza fungi. 1This situation is reversed, however, in sediments undergoing organicenrichment by input from the catchment or from phytoplankton-richlake water. Organically enriched sediments of higher oxygen demandoffer a threat to survival of rosette species due to higher radial oxygenloss and insufficient oxygen supply to root tips during the night whenphotosynthetic oxygen production is switched off and the lake wateris the only oxygen source to plant and sediment respiration. 7,9Organic enrichment of sandy Lobelia sediments leads to a declinein root length from about 10 cm to 2 cm (Fig. 2). While growth and882 87Plant Signaling & Behavior 2008; Vol. 3 Issue 10

Outstanding Lobelia dortmanna in iron armortissue concentrations of N and P are stimulated by a small organicenrichment (e.g., from 0.3 to 0.6% of dry weight) due to highermineralization, a stronger organic enrichment (2.4%) stresses theplants and leads to loss of chlorophyll, poor growth and die off 4 .Organic enrichment is accompanied by deprivation of oxygen poolsand lower oxygen penetration depths (e.g., from 35 to 5 mm) in thesediments signaling that insufficient oxygen supply to the root tipsis a main reason for the reduction of root length and plant performance.4 Quantity and degradability of the organic matter input willobviously determine the severity of plant stress. 6Other rosette species such as Littorella uniflora and Isoetes lacustrisrespond similarly but are less sensitive to sediment anoxia presumablydue to lower radial oxygen loss from the roots. Littorella produces amixture of thin, short roots and thick, longer and probably lesssusceptible roots. Both species tolerate organic sediments and in deepcalm water you find Isoetes lacustris anchored with just the stem invery soft organic sediments, but with no roots and an obvious riskof vegetation loss during storms or release of methane bubbles fromthe sediments.Iron Plaques as a Protection?Wetland plants and probably most aquatic plants have continuouslayers of suberin or lignin just below the root surface as barriers toradial oxygen loss to the sediment. 6,11 In Phragmites australis thebarrier becomes stronger when the roots are exposed to anoxia andsmall fatty acids from anaerobic fermentation. 5 There is no sign thatthis diffusion barrier is present or inducible in Lobelia roots, whilethe response of other rosette species is unknown. 9 If the diffusionbarrier turns out to be inducible, it will impede root uptake of CO 2,but because sediment CO 2concentrations are elevated by organicenrichment 4 it could match a certain reduction of root permeabilityand still maintain the CO 2influx for photosynthesis.Figure 1. Radial oxygen loss measured with platinum sleeve electrodes alongthe length of roots in an anoxic medium. The basal part of the roots was incontact with atmospheric air. Lobelia dortmanna had either none or thickiron coatings of the root surface (0.09 ± 0.05 and 30 ± 3 mmol Fe m -2 rootsurface respectively), 9 while Phragmites australis 5 and the seagrass, Zosteramarina (Unpublished) were without iron coatings.For Lobelia, alteration of bio-geochemical processes accompanyingorganic enrichment of the sediment can offer additional protectionto radial oxygen loss from the roots and this mechanism has hithertobeen overlooked. As Lobelia’s roots become shorter by oxygen depletionof sediments, 1–2 mm thick coatings of oxidized iron precipitateon the root surfaces 4,10 (Fig. 2). Mobile dissolved Fe 2+ is producedfrom insoluble Fe 3+ by degradation of organic matter in the anoxicsediment and Fe 2+ is re-precipitated as Fe 3+ -oxyhydroxides at theroot surface when it meets the outward oxygen flux. Iron coatings onaquatic plant roots in organically rich sediments are common and theconsequences should be general.Figure 2. Roots become shorter and iron coatings thicker on Lobelia roots exposed from left to right to increasing levels of organic matter (from 0.2 to 3.4% of dry weight) and potential oxygen consumption rates (from 1.1 to 24.0 μg O 2 ml -1 sediment h -1 ) for 16 weeks. 10www.landesbioscience.com Plant Signaling & Behavior 883

Outstanding Lobelia dortmanna in iron armorPaper 5Iron coatings on Lobelia roots (30 mmol m -2 ) reduce 10-foldoxygen permeability of the root wall and two-fold radial oxygen loss(Fig. 1). Therefore, oxygen concentrations rise within the roots andcan better sustain root respiration including the distal meristem. 9While iron coatings only lead to c. 15% higher internal oxygenconcentrations in the root tips at low respiration rates at low temperatures,9 the influence will be stronger at higher respiration rates andat reduced oxygen supply from the leaves in the dark.Therefore, we anticipate that iron coatings should offer the strongestprotection to root anoxia and other toxic effects during warmsummer nights when oxygen consumption rates in the plants and thesediments peak, hence, no oxygen is produced by photosynthesis andoxygen supply from the bottom waters is reduced by falling oxygenconcentrations.Oxidized iron may offer additional protection by increasingthe diffusive resistance to toxic solutes across the root surfaces andproviding an oxidation capacity slowing the ingress of sulfide andreduced metals from the sediment. 5,9 Iron coatings formed byoxygen release from the roots during the day can, thereby, offerprotection during the night when oxygen release diminishes or isreversed. 1 Likewise, iron coatings formed during sunny days canoffer protection during dark days.Studies of marine sediments have clearly demonstrated howsurface pools of oxidized iron formed in winter and spring whenbottom waters are rich in oxygen can protect the anoxic bottomlayer during summer against the release of toxic sulfides from deepersediments. 12To test the protective effect of oxidized iron on or close to rootsurfaces independent of the influence of sediment anoxia and oxygensupply from the water, we shall expose Lobelia and other freshwaterspecies to controlled crossed gradients of degradable organic matterand iron in the sediments and set levels of dissolved oxygen in thewater and evaluate the resulting morphological and functional plantresponses.Iron and other important redox constituents vary extensively inamount and input by groundwater seepage through sediments fromdifferent lakes and wetlands as do the presence and well-being of thevegetation. Thus, if oxidized iron is generally ecological significantfor plant survival it will also be important for plant distribution andhistorical changes of the vegetation. Work is under way to evaluatethe importance of light availability and sediment contents of organicmatter and iron for the survival of the Red-listed rosette vegetation inthe diminishing numbers of oligotrophic lakes worldwide. 13References1. Pedersen O, Sand-Jensen K, Revsbech NP. Diel pulses of O 2 and CO 2 in sandy lake sedimentsinhabited by Lobelia dortmanna. Ecology 1995; 76:1536-45.2. Sand-Jensen K, Pedersen O, Binzer T, Borum J. Contrasting oxygen dynamics in the freshwaterisoetid Lobelia dortmanna and the marine seagrass Zostera marina. Ann Bot 2005;96:613-23.3. Justin SHFW, Armstrong W. The anatomical characteristics of roots and plant-response tosoil flooding. New Phytol 1987; 106:465-95.4. Sand-Jensen K, Borum J, Binzer T. Oxygen stress and reduced growth of Lobelia dortmannain sandy lake sediments subject to organic enrichment. Freshwater Biol 2005; 50:1034-48.5. Armstrong J, Armstrong W. Rice and Phragmites: effects of organic acids on growth, rootpermeability and radial oxygen loss to the rhizosphere. Am J Bot 2001; 88:1359-70.6. Colmer TD. Long-distance transport of gases in plants: a perspective on internal aerationand radial oxygen loss from roots. Plant Cell Environm 2003; 26:17-36.7. Sand-Jensen K, Prahl C. Oxygen exchange with the lacunae and across leaves and roots ofthe submerged vascular macrophyte, Lobelia dortmanna L. New Phytol 1982; 91:103-20.8. Sand-Jensen K, Prahl C, Stokholm H. Oxygen release from roots of submerged aquaticmacrophytes. Oikos 1982; 50:1034-48.9. Møller CL, Sand-Jensen K. Iron plaques improve the oxygen supply to root meristems ofthe freshwater plant, Lobelia dortmanna. New Phytol 2008; 179:848—56.10. Raun AL. Sediment organic matter influences growth and survival of submerged plants. MS.Thesis, Freshwater Biological Laboratory 2008; University of Copenhagen.11. Soukup A, Armstrong W, Schreiber L, Franke R, Votrubova O. Apoplastic barriers to radialoxygen loss and solute penetration: a chemical and functional comparison of the exodermisof two wetland species, Phragmites australis and Glyceria maxima. New Phytol 2007;173:264-78.12. Azzoni R, Giordani G, Viaroli P. Iron-sulphur-phosphorus interactions: implications forsediment buffering capacity in a Mediterranean eutrophic lagoon (Sacca di Goro, Italy).Hydrobiologia 2005; 550:131-48.13. Sand-Jensen K, Riis T, Vestergaard O, Larsen S. Macrophyte decline in Danish lakes andstreams over the past 100 years. J Ecol 2000; 88:1030-40.884 89Plant Signaling & Behavior 2008; Vol. 3 Issue 10

Paper 6Planterødders overlevelse i vanddækket bundFoto: Ane Raun, modified by Claus MøllerPhoto: Ole Pedersen

Planterødders overlevelse ivanddækket bundClaus Lindskov Møller og Kaj Sand-JensenIlttransport gennemluftvævAt planterødder kan have problemermed at overleve i envanddækket bund har somudgangspunkt en simpel fysiskforklaring. Luftmættet vand indeholdernemlig 20-25 gangemindre ilt end samme volumenluft, så den tilgængelige iltpuljeer lille. Endvidere er forsyningenmed ny ilt til denne iltpulje vedmolekylernes egenbevægelse(diffusion) ekstrem langsom ivand, faktisk 10.000 gange langsommereend i luft.I akvatiske sedimenter og vandmættedejorder forsvinder ilten derforsædvanligvis i få mm’s dybde selvved et beskedent iltforbrug, fordinytilførslen ovenfra går usædvanligtlangsomt. Skal planterødderne overleve,må ilttransporten i de flestetilfælde ske inde i selve planten fraskuddet i kontakt med ilt til røddernedybt nede i den iltfrie bund.Skal transporten gennem plantenvære effektiv, må den foregå iluft. De fleste sumpplanter ogvandplanter danner da ogsåsammenhængende luftkanalerfra blade og stængler til rodspidserne.Er der meget luftvæv,Paper 6Hvis kornmarken står under vand, rådner rødderneaf mangel på ilt og planterne dør. Mensom bekendt vokser sumpplanter og egentligevandplanter aldeles udmærket på en vanddækketbund. Hvordan formår netop de at forsyne røddernemed ilt? Det spørgsmål har en rækkespændende svar.bliver der samtidigt færre iltforbrugendeceller, så ilttransportenbedre kan dække behovet til respirationend hvis levende cellerudfyldte hele plantens indre.Plantearter, der kan vokse påen vandmættet bund, har derforkarakteristiske tilpasninger (figur1). De indeholder meget luftvævog de kan ofte øge mængdenmarkant, når de oplever et skiftefra en veldrænet iltholdig bundtil en vandmættet iltfri bund ogkan herved opretholde iltforsyningentil rødderne. Omvendthar planter, som er knyttet til enveldrænet bund, kun lidt luftvævog de formår heller ikke at øgeluftmængden nævneværdigt, hvisbunden vandmættes. Så derfordør deres rødder.Rødderne bliver tykkere,kortere og gastætteRøddernes vækstpunkt liggerlige bag rodspidsen. Her foregåralle celledelinger, respirationener høj og behovet for organiskstof og ilt særlig stort. Uden iltproducerer rødderne kun 2-4molekyler af det universelle energimolekyle,ATP, for hvertomsat glukosemolekyle, mensdet bliver til 38 molekyler medilt til stede. På grund af dennekæmpeforskel i energiudbytte erdet let at forstå, hvorfor iltenhelst skal frem til den energikrævenderodspids.Først og fremmest skal iltendiffundere ned gennem roden iluftfase (figur 2). Dernæst skaldiffusionen nedad være så effektivsom mulig i forhold til denFigur 1. Omfanget af luftvæv (%) er større blandt sumpplanter end tørbundsplanter.Sumpplanter er også i stand til at øge luftvævet mest ved et skiftefra en veldrænet til en vandmættet bund. Efter Justin & Armstrong (1987).54 URT 32:1 • februar 200893

Paper 6mangel på ilt eller af de stoffer(fx små organiske syrer), der produceresi bunden under iltfrie forhold.Effekten af aflejringerne erstor, idet de nedsætter rodoverfladensgaspermeabilitet til mindreend 1/10 af niveauet uden aflejringer.Ned mod rodspidsen falderiltindholdet i luftkanalerne gradvist,fordi ilten forbruges vedrespiration og der trods alt skeret radiært tab til den omgivendebund. Jo længere roden er, destovanskeligere er det derfor at forsynerodspidsen med ilt. Derforbliver rødderne kortere, hvis deenten vokser på en særlig stærktiltforbrugende bund, eller hvisforsyningen fra skuddet vanskeliggøres.Det sidste sker, hvis sumpplanteroversvømmes, så skuddetikke længere har kontakt med atmosfæriskluft, men må klare sigmed den mindre iltforsyning fraoversvømmende vand. De kortererødder forankrer plantendårligere, hvilket øger risikoenfor, at den rives løs. Den risikoforstærkes af, at rødderne fæstersig dårligere i en vandmættetbund, især hvis den ligner flydendemudder.Samlet set skaber en vandmættetbund derfor risiko for dårligereenergiudnyttelse, næringsoptagelseog fasthæftning afrødderne. Dog kan visse reduceredemetalioner af jern og manganvære mere tilgængelige underiltfrie forhold, men risikoenfor giftige effekter af disse ionersamt af sulfid og organiske syrerer samtidig større. Her sikrer degastætte rodoverflader formentligen vis beskyttelse.Figur 2. Tykke, luftfyldte rødder (A) og rødder med stor modstand modradiært ilttab (B) har lettere ved at forsyne rodspidserne med ilt end tynderødder med lille modstand mod radiært ilttab (C).konkurrerende diffusion ud afroden til den omliggende iltfribund. Effektiv transport nedadopnås bedst ved, at roden er tyk,så rodoverfladens areal, hvorigennemilttabet til omgivelsernesker, er lavt i forhold til luftkanalernestværsnitareal, der befordrertransporten til rodspidsen(figur 2). Men en tyk rod er dårligtil at optage næringsstoffer iforhold til sin vægt, så der er enklar grænse for, hvor tykke røddernekan blive og fortsat væreeffektive til at opsuge næringsstoffer.Det andet princip er derformere generelt anvendeligt: ”Rodoverfladenskal være så gastætsom muligt frem til rodspidsen”(figur 2). Det er da også et fællestræk for alle de sumpplanter ogvandplanter, der kan vokse på eniltfri vandmættet bund. Denneeffekt opnås med svært gennemtrængeligelignin- og suberinaflejringer,der ligger som en ubrudtcylinder umiddelbart under rodoverfladen(figur 3). Helt tæt pårodspidsen er aflejringerneendnu ikke udviklede. Aflejringerneinduceres og forstærkes afMassetransport gennemsumpplanters luftkanalerDen hidtidige beskrevne iltforsyningfra skuddet til røddernesker ved molekylær diffusion istillestående luft. Det er en effektivproces over korte afstande,men en ineffektiv proces overlange afstande. Hvis afstandenfra iltkilden i skuddet til rodspidserneer længere end en halv meter,så er diffusionen heltutilstrækkelig til at dække selv etbeskedent iltbehov.Her kræves derfor en megetmere effektiv massetransport afilt med en luftstrøm gennemluftkanalerne. En sådan indrevind blev opdaget af John Daceyi gul åkande tilbage i 1981.Gul åkande har en kraftig jordstængelog veludviklede rødder iden iltfrie mudderbund på 1-3meters dybde og kun flydebladeneer i kontakt med luften. I deyngste blade skabes et svagt overtryk(~250 pascal) i lys, som driveren indre vind gennem deluftfyldte bladstilke og jordstænglertilbage til atmosfæren via degamle blade. Overtrykket i deunge blade skyldes en kombinationaf solopvarmning, der fårluften i bladet til at udvide sig ogskabe tryk, og tilstedeværelse afmættet vanddamp i bladet, som94URT 32:2 • maj 2008 55

Paper 6Figur 3. Aflejringer af lignin og suberin (rødt efter farvning) i et cylindrisklag umiddelbart under rodoverfladen nedsætter det radiære ilttab til denomgivende iltfrie bund. Fotos af tagrør i Armstrong & Armstrong (2001) ogSoukup et al. (2007). Målestok 60 µm.udøver et separat overtryk påomkring 1000 pascal i forhold tilatmosfæren. De yngste blade harkontakt med atmosfæren via ultrasmåporer (< 0,1 µm), derikke tillader trykudligning, mengør det muligt for kvælstof og iltat diffundere ind i bladet til erstatningfor den luft, som strømmernedad i bladstilken. Tilstedeværelseaf et højere tryk afvanddamp inde i bladenes luftrumend i atmosfæren er afgørendefor, at partialtrykket afkvælstof og ilt er større i atmosfærenend i bladene, så erstatningsluftdiffunderer ind. Deældre blade har udvidet sig ogmistet den fine poreadskillelse oghar kun et marginalt overtryk iforhold til atmosfæren. Derforløber en luftstrøm på omkring1,5 liter i timen fra de yngsteblade via jordstænglerne i søbundentilbage til atmosfæren gennemde ældste blade. Typisk fal­der iltprocenten i luftstrømmenfra 20,7% i de yngste til 19,4% ide ældste blade, og dette iltfaldsikrer en iltforsyning, som er 20gange højere end den diffusionenmaksimalt kan levere. Da jordstængleni søbunden ventileres ogopnår en høj iltprocent, kan iltenherfra diffundere passivt ned i deret tykke rødder, som bliver 20-30 cm lange. På grund af respirationi planten og omsætning isøbunden indeholder udstrømningsluftenganske meget CO 2(~1,5%) og methan (0,3%).Det har vist sig, at flere almindeligesumpplanter såsom Tagrør,Dunhammer, Kogleaks,Sumpstrå og arter af Siv ogsåhar en ventileret massetransportdrevet af solopvarmning ogvanddampes tryk. I et studiumfra 1992 af Hans Brix varieredetrykopbygningen i luftrummenei sumpplanter mellem 20 og1300 pascal og luftstrømmen ermellem 10 og 600 ml i timen perskudsystem. Nogle gange kanvinden også skabe masse-strøm,hvis den blæser med forskellighastighed over forbundne plantedele.Øget vindhastighed skaberstørre trykfald og luftfyldte åbnestængler af forskellig højde hostagrør, vil derfor opleve det lavestetryk i de højeste og mest vindombrustestængler, der såledeskan trække en luftstrøm gennemjordstænglen fra de kortere,vindbeskyttede stængler.Da massetransport forudsætterventilering og strukturer,hvor gasser fra atmosfæren kankomme ind i planten ellerforlade den, så findes den ikkehos planter under vand. De måalene klare sig med diffusion ogde bliver derfor alle påvirkede afiltsvind i vandet.Ilttransport hos ægte undervandsplanterÅlegræs og Tvepibet LobelieFigur 4. Radiært ilttab fra rodspidstil basis hos Tagrør, Ålegræs ogTvepibet Lobelie. Efter Armstrong& Armstrong (2001) og Møller &Sand-Jensen (2008).56 URT 32:1 • februar 200895

Paper 6Figur 5. Rødderne bliver kortere og jernudfældningerne tykkere hos Lobelieopvokset med stigende indhold af iltforbrugende organisk stof i søbunden,vist fra højre mod venstre. Foto efter Raun (2008).repræsenterer to yderpoler, hvadangår levesteder og iltforsyningblandt undervandsplanterne. Ålegræsvokser i en havbund, der altovervejendeer iltfri og indeholderbetydelige mængder af sulfidog reduceret jern og mangan.De underjordiske jordstænglerog rødderne har da ogsåmarkante barrierer mod tab afilt fra de indre luftkanaler tilhavbunden (figur 4). Barrierernesikrer, at ilten normalt kan nåfrem til de yderste rodspidser.Det kræver selvfølgelig, at dereksisterer en iltkilde. I lys kanbladenes fotosyntese producereden nødvendige ilt. I mørke,hvor hele planten respirerer ogforbruger ilt, må vandet omkringbladene levere ilten. Det er derforkritisk for røddernes overlevelse,hvis der optræder langvarigeperioder med iltsvind ibundvandet, eller hvis planterneskygges af tætte algebestande.Nedsat iltforsyning til rødderneøger også risikoen for, at giftigsulfid trænger ind ved rodspidserne,hvor normal iltforsyningkan afgifte sulfiden ved at omdanneden til sulfat. At der ertale om en kritisk situation fremgåraf, at rodspidserne svitses afved lav lystilgang og dårlige iltforholdi bundvandet. Hvis iltforsyningentil selve vækstpunktet vedbasis af bladene svigter, dørplanten.De fleste ferskvandsplanter erformodentlig også ret gastætte ijordstængler og rødder for atsikre røddernes iltforsyning ogholde sulfid og reducerede metallerude, men der mangler målinger.Forholdene er helt anderledesfor Tvepibet Lobelie ogandre små rosetplanter somStrandbo og Brasenføde. Deoptager det meste af deres CO 2til fotosyntesen via rødderne isøbunden og de kan derfor ikkevære tætte for CO 2. De bliversamtidig piv utætte for ilt og detindsnævrer deres levemuligheder.Foreløbig ved vi fra eksperimentermed Tvepibet Lobelie,at den overhovedet ikke dannervævsbarrierer i roden mod ilttabover overfladen (figur 4).Rosetplanterne vokser især påen fattig mineralbund af sand ellergrus i de mest næringsfattigesøer. Faktisk er iltforbruget sålavt i disse søbunde, at Lobeliesiltfrigivelse fra rødderne sikrertilstedeværelse af opløst ilt tilflere cm’s dybde i bunden. Hervedomdannes sulfid og reduceretjern og mangan til iltede ufarligeforbindelser.Imidlertid opstår der problemer,når søbunden beriges mediltforbrugende organisk stofenten fra planktonproduktioneneller tilløb af spildevand fralandbrug eller husholdninger.Da lobelies rødder er utætteFigur 6. Det radiære ilttab overrodvæggen til søbunden falder i taktmed øget tykkelse af jernudfældningernepå lobelies rødder. EfterMøller & Sand-Jensen (2008).96URT 32:2 • maj 2008 57

Paper 6Vi har sat planterødders ve ogvel højt på vores forskningsagenda!Forfatternes adresseFerskvandsbiologisk Laboratorium,Biologisk Institut, Helsingørgade 51,3400 Hillerød. clmoller@bio.ku.dkFigur 7. Lobelierødder med jernbelægninger opretholder højere iltkoncentrationeri luftkanalerne end rødder uden belægninger. Efter Møller ogSand-Jensen (2008).tabes ilten i stor stil til søbunden,når dens ilt forbruges af dettilførte organiske stof. For atsikre forsyningen til rodspidsernebliver rødderne gradvistafkortet fra 10 cm til 2 cm ellerendnu kortere ved fortsat berigningmed organisk stof (figur5). Selv ved ret beskedne berigningermed organisk stof (~ 2%)ender lobelie med at dø ellermiste rodfæste.Vi har påvist, at jern kan medvirketil en vis beskyttelse, idetden i stor stil fælder ud på overfladenaf rødderne ved berigningaf søbunden med organiskstof. Forklaringen er den, at udfældetiltet jern (Fe+++) bidragertil nedbrydningen af organiskstof ude i den iltfrie søbundunder omdannelse til opløst reduceretjern (Fe++). Det reduceredejern diffunderer frem tilrodoverfladen, hvor iltet jerngendannes, fælder ud og dannerfaste belægninger ved kontaktmed ilten fra roden. Jernbelægningenligner kemisk set rust oghar vist sig at udgøre en barrierefor det radiære ilttab ud af roden(figur 6) og dermed øgerden faktisk iltindholdet i rodensluftkanaler og iltforsyningen tilrodspidsen (figur 7). Den beskyttermåske også mod indtrængningaf giftige stoffer fra den iltfrieomsætning i søbunden.Ved at eksperimentere medforskellige indhold af organiskstof og jern i søbunden er vinetop nu i færd med at afklarejernets betydning for lobeliesvækst og overlevelse.Søbundens betydning forplantebestandeneVi forsøger også at afklare, ihvilket omfang andre ferskvandsplantertåler fravær af ilt og tilstedeværelseaf potentielle giftigestoffer i søbunden ved at ændrerøddernes tykkelse, længde ogdiffusionsbarrierer. Røddernesstofskifte og følsomhed ændrersig måske også.Resultaterne bliver afgørendefor vores endelige svar på spørgsmålet:Hvor stort et problem eriltforbrugende organisk stof isøbunden for planternes vækstog overlevelse? Svaret skal giveos retningslinjer for, hvordan vikan restaurere søernes plantebestandeved enten at forbedre lysforholdenei vandet eller fjernesøbundens iltforbrugende mudder.Måske må vi gøre beggedele for at sikre rosetplanternesoverlevelse.LitteraturArmstrong, J., Armstrong, W. 2001. Riceand Phragmites: effects of organic acidson growth, root permeability, andradial oxygen loss to the rhizosphere.American Journal of Botany 88:1359-1370.Brix, H., Sorrell, B.K., Orr, P.T. 1992.Internal pressurization and convectivegas-flow in some emergent fresh-watermacrophytes. Limnology and Oceanography37: 1420-1433.Dacey, J.W.H. 1981. Pressurized ventilationin the yellow water-lily. Ecology62: 1137-1147.Justin, S.H.F.W., Armstrong, W. 1987.The anatomical characteristics ofroots and plant-response to soil flooding.New Phytologist 106: 465-495.Møller, C.L., Sand-Jensen, K. 2008.Iron plaques improve the oxygen supplyto root meristems of the freshwaterplant Lobelia dortmanna. Indsendt.Raun, A.L. 2008. Sediment organicmatter influences growth and survivalof submerged plants. Specialerapport,Københavns universitet, København,DK.Soukup, A., Armstrong, W., Schreiber,L., Franke, R., Votrubova, O. 2007.Apoplastic barriers to radial oxygenloss and solute penetration: a chemicaland functional comparison of theexodermis of two wetland species,Phragmites australis and Glyceriamaxima. New Phytologist 173: 264-278.58 URT 32:1 • februar 200897

AcknowledgementsAcknowledgementsNumerous people have supported me and contributed to my accomplishments over the past three years. Iwill start by thanking my girlfriend Ditte for moral support and coping with my changing mood whenapproaching deadlines. Thanks to all the people at Freshwater Biological Laboratory for contributing tothe relaxed but productive atmosphere at the lovely surroundings in Hillerød. A special thanks to Kaj ofinsightful supervision and for introducing me to the wonderful world of birdwatching. Thanks to JensBorum for scientific discussions and assistance when needed. I would probably still be in the laboratoryanalyzing samples without the help from Birgit Kjøller and Ayoe Lüchau so thank you very much forthat and assistance during field-work. Thanks to Lars Båstrup-Spohr for field assistance and the manyrelaxing hours spend at various shorelines trying to “catch a big one”; I am sure that we eventually willsucceed. Thanks to Anders, Cristina, Laci, Matteo, Stine, Mette, Ane, Mikkel and all the other currentand present students at FBL for great times. Also thanks to Tim, Tash and Sarah for making me feel athome during my visit to University of Western Australia and to Tim for comments and discussions ofmy scientific work. Finally I would like to thank Ole Pedersen for providing beautiful pictures(including the front cover) to “spice up” the layout of my thesis.Hopefully we will keep in touch in the future,Claus99