nostoc specii

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INVITED REVIEW

Ecophysiology of gelatinous Nostoccolonies: unprecedented slow growth

and survival in resource-poor and harsh environments

Kaj Sand-Jensen*

Freshwater Biological Laboratory, Biological Institute, University of Copenhagen, Universitetsparken 4,2100 Copenhagen, Denmark

* [email protected]

Received: 17 February 2014 Returned for revision: 6 March 2014 Accepted: 1 April 2014

Background The cyanobacterial genusNostocincludes several species forming centimetre-large gelatinous col-onies in nutrient-poor freshwaters and harsh semi-terrestrial environments with extended drought or freezing.These

Nostoc species have filaments with normalphotosynthetic cells and N2-fixing heterocysts embedded in an extensivegelatinousmatrixof polysaccharides andmany otherorganicsubstancesproviding biologicaland environmental pro-tection. Large colony size imposes constraints on the use of external resources and the gelatinous matrix representsextra costs and reduced growth rates.Scope Theobjective of this review isto evaluate themechanisms behindthe lowrates of growth andmortality,pro-tection against environmental hazards and the persistence and longevity of gelatinous Nostoccolonies, and theirability to economize with highly limiting resources.ConclusionsSimple models predict the decline in uptake of dissolved inorganic carbon (DIC) and a decline in thegrowth rate of spherical freshwater colonies of N. pruniforme and N. zetterstedtii and sheet-like colonies of

N. commune in response to a thicker diffusion boundary layer, lower external DIC concentration and higherorganic carbon mass per surface area (CMA) of the colony. Measured growth rates of N. commune and

N. pruniforme at high DIC availability comply withgeneralempirical predictions of maximum growth rate (i.e. doub-ling time 1014 d) as functions of CMA for marine macroalgae and as functions of tissue thickness for aquatic andterrestrial plants, while extremely low growth rates ofN. zetterstedtii (i.e. doubling time 23 years) are 10-foldlower than model predictions, either because of very low ambient DIC and/or an extremely costly colony matrix.DIC uptake is limited by diffusion at low concentrations for all species, although they exhibit efficient HCO 3

uptake, accumulation of respiratory DIC within the colonies and very low CO2 compensation points. Long lightpaths and light attenuation by structural substances in largeNostoccolonies cause lower quantum efficiency and as-similationnumberand higher lightcompensationpointsthanin unicellsand other aquaticmacrophytes.Extremelylow

growth and mortality rates ofN. zetterstedtiireflect stress-selected adaptation to nutrient- and DIC-poor temperatelakes, whileN. pruniforme exhibits a mixed ruderal- and stress-selected strategy with slow growth and year-long sur-vivalprevailing in sub-Arctic lakes andfastergrowth andshorterlongevity in temperate lakes.Nostoccommune anditsclose relativeN. flagelliformehave a mixed stressdisturbance strategy not found among higher plants, with stressselection to limiting water and nutrientsand disturbance selection in quiescentdry or frozen stages.Despite profoundecological differencesbetween species, activegrowthof temperate specimens ismostlyrestricted to thesame tempera-ture range(0 35 8C; maximum at25 8C). Future studies should aimto unravel the processes behind the extremeper-sistence and low metabolism ofNostocspecies under ambient resource supply on sediment and soil surfaces.

Key words: Gelatinous colonies, cyanobacteria,Nostoc commune,Nostoc flagelliforme,Nostoc pruniforme,Nostoczetterstedtii, carbon use, carbon concentrating mechanisms, photosynthesis, light use, growth, long-lived, survival,desiccation tolerance, nutrient-poor.

I NT RODUCT I ON

The cyanobacterial genusNostocincludes many species that arehighly diversewith respect to morphology, functionalproperties,biotic relations and habitat distribution (Dodds et al., 1995).Nostocspecies have filaments with normal photosynthetic cellsand N2-fixing heterocysts and they periodically form resistantakinetes for survival and short motile filaments (hormogonia)for reproduction (Mollenhaueret al., 1994). Some species arefree-living and many species engage in loose or obligate cooper-ation with land plants and fungi (e.g. lichens; Mollenhauer,1988; Kaasalainen et al., 2012). A third, fascinating Nostoctype forms large gelatinous colonies of variable shape and

structure in rice fields, freshwater lakes, ponds and streams and

on alternating wet and dry soils or rock surfaces (Doddset al.,1995;Gao et al., 2012). The large gelatinous species requirespecial adaptations to obtain sufficient light, nutrients and dis-solved inorganic carbon (DIC) (Sand-Jensenet al., 2009b) inwater and to survive the extreme variations in temperature,water supply and irradiance on naked soils and rock surfaces(Tamaruet al., 2005;Liet al., 2011;Yu and Liu, 2013). Whilephototrophs in resource-rich environments have been treated innumerous original papers, reviews and books (Larcher, 2003;Lamberset al., 2008), studies on the adaptations of phototrophsto resource-poor and physically extreme environments are rare.The objective of this review is to evaluate the mechanisms

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Annals of Botany114: 17 33, 2014

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behind the extremely low rates of growth and mortality and highpersistence, longevity and protection against environmentalhazards of gelatinousNostoc colonies and their ability to econo-mize with highly limiting resources.

Thereview focuses on four colonialNostoc species: the sheet-like Nostoc commune, the closely related N. flagelliforme and

two species that form spherical colonies, N. pruniforme andN. zetterstedtii. All four species resemble each other byforming centimetre-large gelatinous colonies, but they differ indistribution, habitat preference, growth rate, environmental tol-erance and appearance (Fig.1).Nostoc commune is a globallywidespread terrestrial and semi-aquatic species forming inactivecrusts when dry, but rapidly changing to 15-mm thick sheet-like gelatinous structures when wet (Scherer et al., 1984;Marsh et al., 2006; Novis et al., 2007).Nostoc communehas un-precedented tolerance of environmental extremes (Satoh et al.,2002;Tamaruet al., 2005;Sand-Jensen and Jespersen, 2012).It tolerates desiccation during dry seasons and can survive for.100 years on a museum shelf (Cameron, 1962). It toleratesfreezing to 60 8C in Antarctic deserts (Davey, 1989; Novis

et al., 2007) and to 2698C in liquid helium in the laboratory(Sand-Jensen and Jespersen, 2012), but rapidly resumes metab-olism when rewetted at physiologically suitable temperaturesbetween 0 and 30 8C(Schereret al., 1984;Tarantoet al., 1993;Sand-Jensen and Jespersen, 2012;Mlleret al., 2014).Nostocflagelliforme is also widely distributed in arid and semi-aridareas throughout the world (Gaoet al., 2012;Ye et al., 2012).It has a thread-like appearance but resembles N. commune inits genetics, growth habitat, colony composition and extreme tol-erance to and rapid recovery from desiccation, freezing and saltstress (Gao and Zou, 2001;Qiuet al., 2004;Huanget al., 2005;Yong-Hong et al., 2005; Liu etal., 2010; Arima et al., 2011). Wehave observed Nostoc with the typical morphology of bothN. commune andN. flagelliformeon the Great Alvar on Oland,

Sweden (Sand-Jensen et al., 2010). Nostoc flagelliforme hasbeen classified as a variety ofN. commune in arid regions of

Spain (Aboalet al., 2010) and recent DNA studies group thosetwo species together with N. sphaeroidesand N. punctiformein a common clade (Arima et al., 2011; Gao et al., 2011),whichmakes species identity dubious. Nonetheless, we maintainthe distinction betweenN. commune andN. flagelliforme here inaccordance with most available literature.

Nostoc pruniforme forms a beautiful, dark green, sphericalcolony with a smooth surface like a plume. It is more commonand widely distributed both geographically and ecologically inoligo- and mesotrophic freshwaters in temperate and sub-Arctic regions than its rare freshwater cousin, N. zetterstedtii(Dodds et al., 1995;Raun et al., 2009). Danish alkaline lakeshousing N. pruniforme contain .0.7 mM DIC (Sand-Jensenet al., 2009b). In temperate lakes, N. pruniforme appears togrow in diameter from0.2 cmin latespring to2 3 cm inmid-summer and it can rapidly die off, supposedly because of attacksby viruses or bacteria at high summer temperatures (K. Sand-Jensen, unpubl. data). In contrast, in a cold (4 8C), nutrient-poorspring in Oregon, USA,N. pruniforme reached a handball size of1517 cm in diameter and weighed 2.6 kg after sustained slow

growth for 914 years (Dodds and Castenholz, 1987). Verylarge colonies are also found in cold transparent lakes inGreenland (K. Sand-Jensen, unpubl. data). In addition to theirlarger size and persistence, they differ from Danish temperatespecimens by having a more solid surface and a denser gel.Two additional species, N. calcareous and N. sphaericus(Mollenhaueret al., 1994) resembleN. pruniforme but are notdiscussed further here.

The fourth species,N. zetterstedtii, is a rare, highly persistentorganism whose colonies resemble firm blackberries. It lives inonly 50100 nutrient-poor soft-water lakes in Sweden and in afew other lakes in neighbouring countries (Bengtsson, 1986,1995;Mollenhaueret al., 1999). Soft-water lakes usually haveDIC concentrations of 0.020.3 mM and concentrations of

total phosphorus ,0.1 0.3 mM in open waters (Sand-Jensenet al., 2009b). Among the freshwater phototrophic speciesstudied so far, Nostoc zetterstedtii has unprecedented lowgrowth and mortality rates (Sand-Jensen and Mller, 2011). Inoligotrophic Lake Varsjo, Sweden, it takes 3 years forN. zetterstedtii to double its colony mass and 30 years toreach the recorded maximum diameter of 7 cm (Sand-Jensenand Mller, 2011). There is no apparent mortality of grazersand pathogenic bacteria and viruses (Sand-Jensen and Mller,2011). The persistence of N. zetterstedtii is confirmed by itsability to survive for 14 months in the dark at 5 8C with intactcolony structure and unaltered fresh mass (K. Sand-Jensen,unpubl. data).

All mentionedNostocspecies must have common structural

and functional properties linked to the large size and extensivematrix of the colonies. However, the species differ with respectto metabolism, growth, mortality, tolerance to environmentalstress and habitat preference. In this review, I first examinesome of the main structural properties of the three mainspecies (N. commune, N. pruniformeand N. zetterstedtii) withsupplementary dataforN. flagelliforme relatedto the centimetre-large size and gelatinous matrix of the colonies. Second, I evalu-ate theoretically the diffusive flux of DIC to the colony surfacefor a set of environmental constraints on boundary layer thick-ness and external concentrations. Third, I examine the functionalconsequencesofthelargecolonysizefortheuseofDICandlight

F IG . 1 . Images of colonies of rehydratedNostoc commune(upper left),Nostoczetterstedtii (upper right, usually more spherical) and N. pruniforme (lower

middle).

Sand-JensenEcophysiology ofNostoccolonies18


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in photosynthesis and growth. Fourth, I compare the tolerance ofNostocspecies to environmental stresses related to temperature,freezing and desiccation. Fifth, I evaluate the growth, mortalityand longevity of the three Nostoc species and the ecologicalimplications of these properties. The final section points outthat costs and benefits of colony formation should be quantified

in future studies. For this comparative analysis of the eco-physiology of gelatinous Nostoccolonies, I use published dataand include supplementary unpublished datato ensure a compre-hensive comparison.

S I Z E , S HAP E AND COM P OS I T I ON

Centimetre-thick colonies with photosynthetic filaments embed-ded in a gelatinous matrix exert large physical constrains onfunctional properties.

Size and shape

Largesize of phototrophsimposes constraints ontheuse of light

and uptake of inorganic carbon and nutrient ions from the sur-rounding water (Nielsen and Sand-Jensen, 1990;Agusti et al.,1994). For a body of a given shape, surface area (SA L2)scales to the second power of the linear dimension (L), volume(V L3) to the third power, and surface area:volume ratio(SA:V L1) to the inverse of the linear dimension. These allo-metric relationships reflect that the supply rates of light andexternal resources are scaled to the outer surface while therequirements for production and maintenance of the cells andthe gelatinous matrix are mainly scaled to the volume.

The spherical shape adopted by N. pruniforme andN. zetterstedtii is a simple shape of low SA:V ratio (3 r1,where r is the radius of the sphere). Individual filaments ofNostocembedded in the gelatinous matrix and free-living fila-

ments of cyanobacteria and algae are cylindrical and maintaina high SA:Vratio inversely scaled to the transverse radius ofthe filament (2 r1) but independent of filament length. TheSA:V ratio of the flat sheet-like structure ofNostoc commune,multicellular algae and leaves of mosses and higher plants is in-versely scaled to thickness (Lth) of the structure (SA:V 2Lth

1)but is independent of its surface area, volume and total mass aslong as tissue thickness remains constant.

The gelatinous colonies ofN. commune, N. pruniforme andN. zetterstedtii all have relatively thick tissues (0.117 cm)and very low SA:V ratios (0.420 cm1) compared with thethinNostoctrichomes within the gel (SA:V40008000 cm1)and the 0.01- to 0.3 cm-thick submerged leaves of mosses andangiosperms (SA:V702000 cm1) (Table1).Nostoc commune

sheets typically vary in thickness from 0.1 to 0.5 cm when wetand have SA:Vratios of 220 cm 1. The smooth spherical col-onies ofN. pruniforme vary from a diameter of0.2 cm andan SA:Vratio of 30 cm 1 to an astonishing maximum diameterof 17 cm and an exceedingly low SA:Vratio of only 0.36 cm1.Small colonies ofN. zetterstedtii are smooth, have a diameterof0.2 cm,just likeN. pruniforme, while large colonies,reach-ing a recorded maximum diameter of 7 cm, have a granulatedsurface that can be regarded as consisting of small hemispheres(exposed area 2 pr2 and planform area pr2) distributed all overa large spherical surface. The granulated surface area has a2-fold larger surface area relative to a smooth surface and the

SA:Vratio is only 1.7 cm

1

for the largestN. zetterstedtiicol-onies. The granulated surface could, in theory, double thediffusion-limited uptake of external solutes for N. zetterstedtiiby doubling the surface area compared with smooth coloniesof N. pruniforme of the same diameter (see section Sizeeffects, diffusive supply and loss of solutes). The granulatedsurface could also contribute to the formation of thinner diffu-sion boundary layers (DBL) around the surfaces ofN. zetterstedtiiwhen the granules increase micro-turbulence(Boudreau and Jrgensen, 2001). The micro-topography of thegranulated surface can result in thinner DBLs at the protrudingtips of the granules and thicker DBLs in the crevices betweenthe granules (Carpenter and Williams, 1993;Jrgensen, 2001)so the diffusiongeometry is difficult to interpret. Although diffu-

sive fluxesremain experimentally unexplored, theyare, nonethe-less, highly relevant considering that N. zetterstedtii usuallygrows in lakes with lower concentrations of DIC, N and P thanN. pruniforme(Sand-Jensenet al., 2009b).

Composition of colony matrix

A high proportion ofNostoccolonies is composed of theextra-cellular matrix, primarily of polysaccharides of high viscosityand molecular weight. The hydrolysed polysaccharides fromN. flagelliforme were mainly composed of glucose (43 %),galactose (30 %), xylose (21 %), mannose (6 %) and smallamounts of glucuronic and uronic acids (Jia et al., 2007;Pereira etal.,2009).Muchthesamemainsugarswerehydrolysed

from hot water extracts of polysaccharides from field samples ofN. commune,N. flagelliformeandN. sphaeroides, though smallamounts of arabinose appeared inN. flagelliforme and rhamnoseand fucose inN. sphaeroides(Huanget al., 1998). The compos-ition of extracellular compared with intracellular polysacchar-ides was suggested to be quite different in N. commune andN. flagelliforme, perhaps dueto a selectivemechanism forexcre-tion of polysaccharides (Mehta and Vaidya, 1978). Differentstudies ofN. commune have shown the complex chemistry ofthe hydrolysed matrix, which includes six to nine monosacchar-ides, uronic acid, deoxy-sugars, pyruvate, acetate and peptides(Pereira etal.,2009). Scanning electronmicroscopy has revealed

TA B L E 1. Shape, range of linear dimensions and surfacearea:volume ratio (SA:V ) of three species of Nostoc colonies,

Nostoc trichomes, submerged leaves and single phototrophicunicells

Species andphototrophic type

Shape Diameter/ thickness (cm)

SA:V(cm1)

N. commune Flat structure 0.1 10 to 200.5 2 to 4

N. pruniforme Sphere 0.2 3017.0 0.36

Nostoc zetterstedtii Sphere,granulated surface

0.2 307.0 0.851.7

N. trichomes Cylinders 5 104 800010 104 4000

Submerged leaves Flat structure 1 103 200030 103 70

Single-celled algae orcyanobacteria

Sphere 2 104 300002 103 3000

Sand-JensenEcophysiology ofNostoccolonies 19


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a net-like matrix of polysaccharides around Nostoc filaments(Cupac and Gantar, 1992), suggesting that their ultrastructurecould influence colony shape. The chemical structure, the 3-Dnetwork and the presence of special peripheral groups, such asnosturonic acid, are probably essential for the outstandingmoisture absorption and moisture retention capacity ofNostoc

polysaccharides. These properties are highly interesting inbiomedical applications involving living cells and tissue(Yuet al., 2010;Liet al., 2011).

When the gelatinous matrix ofN. commune,N. flagelliformeand N. pruniforme is hydrated its water content is very high,leading to a soft structure that is easy to cut with a finger nail,as opposed to the very solid structure ofN. zetterstedtii, whichcan only be cut, with great force, using a scalpel. Summer col-onies ofN. pruniforme from mesotrophic Lake Esrum with afresh mass (FM) of 3.5 g have a mass specific density of1.01 gFM cm3 while colonies ofN. zetterstedtii from oligotrophicLake Varsjo have a specific density of 1.13 g FM cm3

(Raun et al., 2009; Sand-Jensen et al., 2009b). Both specieshave gradually decreasing mass specific density for larger col-

onies, which tend to be hollow and water-filled in the centre ofN. pruniforme and possess a massive, though less dense, struc-ture without trichomes inN. zetterstedtii (Table2). The carbonmass per surface area [CMA, equivalent to leaf mass persurface area for plants (LMA)] is closely scaled to structuraland functional properties of macroalgal thalli (Markager andSand-Jensen, 1994, 1996) and plant leaves (Nielsen andSand-Jensen, 1989, 1991; Lambers and Poorter, 1992, 2004;Poorteret al., 2012). The CMA increased by a power of0.25relative to colony FM for N. zetterstedtii and only 0.05 forN. pruniforme, because the first species maintains massive col-onies and the second species tends to develop hollow colonieswith increasing size (Table 2). Thus, in two summer collections,CMAwas 8-fold higher forN. zetterstedtii thanN. pruniforme

for colonies of 0.1 g FM, while the difference was 14-fold forcolonies of 3.5 g FM.

The typical differences in CMA values for 3.5 g FM coloniesof N. zetterstedtii (1400 mmol organic C cm2) andN. pruniforme (100 mmol organic C cm 2) have importantimplications. Assuming the same net photosynthesis relative to

colony surface area of both species and the same proportion ofnet photosynthesis being incorporated into new biomass, theturnover rate of the colony mass will be 14-fold lower forN. zetterstedtiithanforN. pruniforme. Withthe sameassumptionof constant photosynthesis per surface area independent ofcolony size, biomass turnover and relative growth rate should

decline with colony weight in proportion to the increase inCMA; i.e. an 8- to 9-fold decline across the range of colonyweights ofN. zetterstedtii from 0.1 to4 g FM. We haverecordeda decline in the relative growth rate and the surplus of photosyn-thesis minus respiration per colony mass with increasing colonysize inN. pruniforme, but did not find the same strong size de-pendence for N. zetterstedtii (Sand-Jensen and Mller, 2011).Weproposethatallocation to extracellularproductsand theirdif-fusive loss to the environment may decline with colony size andthat these processes are relatively more important for the meta-bolically less active N. zetterstedtii. Reduced loss of exudateswith higher colony size could counteract the expected declinein growth rate in larger colonies (see section Size effects, diffu-sive supply and loss of solutes).

As emphasized, a very high proportion of the colony volumeand fresh mass is made up of the water-rich colony matrix com-pared with the denser Nostocfilaments. Still very high propor-tions of the carbon content of the colony are bound in thematrix. InN. commune, polysaccharides in the matrix constitute.60 % of the colony dry mass (Hill etal., 1997). Among spher-ical Nostoc colonies, filaments are confined to an outer shell,which occupies a falling proportion of the volume with increas-ing colony size, such that the contribution of the matrix to thetotal organic content should also increase. If we assume thatthe organic carbon:chlorophyll a mass quotient in Nostocfila-ments has a typical range of 3050 at 158C and moderate irra-diances (Geider, 1987; his Fig. 13), we estimate that the matrixconstitutes 1845 % of the organic carbon mass in the smallest

colonies ofN. pruniforme, while it is 3762 % in large coloniesof 3.5 g FM. The proportion of organic carbon in the matrix ismuch higher inN. zetterstedtii, being 5070 % in small col-onies weighing a few milligrams and 90 % in large coloniesweighing 3.5 g FM.The smaller proportion of livingNostoc fila-ments inthe colony massinN. zetterstedtii than inN. pruniformewill, as already mentioned, result in less photosynthetic produc-tionof neworganic materialrelative to colony massand thereforemuch lower growth rates for N. zetterstedtii than forN. pruniforme.

Functional properties of colony matrix

The gelatinous matrix ofNostoc colonies is composed of a

complex mixture of polysaccharides (Bertocchi et al., 1990;Yousimura et al., 2012), which is responsible for forming andmaintaining the colonyshape andprotecting thecells against en-vironmental hazards and pathogens (Tamaru et al., 2005;Knowles and Castenholz, 2008). It is fascinating, yetunexplored,how the different colony shapes come about. A variety of disac-charides,includingsucroseand non-reducing trehalose, alsopar-ticipate in the protection of cells and matrix (Caiolaet al., 1996;Hillet al., 1997;Potts, 1994,1999,2000). Poly- and disacchar-ides protect membranes and macromolecules against freezing,heating, desiccation and salt stress (Tamaru et al., 2005;Sakamotoet al., 2009;Yoshida and Sakamoto, 2009).

TA B L E 2. Organic carbon content per surface area (CMA, mmolC cm 2) relative to colony fresh mass (FM,g) of three Nostoc

species

Species Relationship Source

N. commune CMA

184+47(mean+ s.e.)

K. Sand-Jensen, unpubl. data

N. pruniforme Log CMA 0.0474 logFM + 1.88

C. Mller and K. Sand-Jensen,unpubl. data

CMA for 0.1 gFM colony 69CMA for 4.0 gFM colony 81

N. zetterstedtii Log CMA 0.26 logFM + 3.13

Sand-Jensenet al.(2009)Sand-Jensen and Mller (2011)

CMA for 0.1 gFM colony 587CMA for 4.0 gFM colony 1531



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The formation and accumulation of trehalose inN. flagelliformeand N. punctiforme are under strict regulationin response to water limitation (Yong-Hong et al., 2005;Yoshida and Sakamoto, 2009), presumably because trehaloseis an essential, though costly, product that diverts organiccarbon away from growth processes. The functional importance

of the gel is supported by the observation that strains ofN. commune with no gel and strains where the gel has been phys-ically removed from the filaments become susceptible to injury(Tamaru et al., 2005). Addition of exogenous polysaccharidesto endolithicNostocsp. andChlorellasp. increases their toler-ance to desiccation (Knowles and Castenholz, 2008), probablyby retaining a water layer around the cells and preventing mem-branes from cracking and macromolecules from undergoing de-structive conformational changes. The main polysaccharidesobserved in the colony gel ofN. commune have a strong capacityfor in vitro moistureabsorptionand water retention, permitting a.10-fold increase in mass when desiccated colonies becomerehydrated (Liet al., 2011;Sand-Jensen and Jespersen, 2012).

The gel has a strong adsorption capacity for heavy metal ions

that are toxic to cyanobacteria in free ionic form (Bender et al.,1994; De Philippis etal., 2007; Pereira etal., 2009). Non-reducingsugars, UV-screening pigments and water stress proteins alsoassist in the environmental protection of Nostoc colonies (Hillet al., 1997;Potts, 1999). In response to increasing intensity andduration of UVexposureofN. flagelliforme, contentsof protectivescytonemin and mycosporine-like amino acids increase (Budelet al., 1997;Yu and Liu, 2013). A large number (573) of thegenesthatwereprobed(6903)inN. punctiformerespondedsignifi-cantly to UVA exposure, 473 genes being up-regulated andonly 100 being down-regulated (Soule et al., 2013). As expected,genes encoding antioxidant enzymes and the sunscreen scytone-min were up-regulated, while many genes involved in thesynthesis of photosynthetic pigments were down-regulated.

Also, Nostoc polysaccharides are capable of reducing oxidativedamage by scavenging superoxide anions and hydroxyl radicals(Liet al., 2011).

Antibiotic production has been recorded in many Nostocspecies (de Cano et al., 1986; Bloor and England, 1991;Gromov et al., 1991), including gelatinous colonies (Hameedet al., 2013). When N. zetterstedtii was homogenized inwater and mixed 1:10 v/v with natural lake water containingbacteria, DNA synthesis stopped immediately (Sand-Jensenet al., 2009a). The polysaccharide nostoflan, isolated fromN. flagelliforme, has in vitro and in vivo antiviral effects on avariety of enveloped viruses, including influenza A virus(Kanekiyo et al., 2005, 2007, 2008). Symbiotic Nostoc in agreat variety of lichens either contains toxic microcystins or

has the genes for producing them in 12 % of 803 lichen speci-mens collected worldwide (Kaasalainenet al., 2012). Micro-cystins occurred in 52 different variants in the 803 lichenspecimens (Kaasalainenet al., 2012); they are small cyclic pep-tides andare known to be highlytoxicto eukaryotes by inhibitingprotein phosphatases (MacKintoshet al., 1990). Microcystinsare active at multiple sites with gradually declining effects onnitrogenase activity, respiration, photosynthesis and growth ofcompeting cyanobacteria. Three different classes of peptidecompounds (anabaenopeptin, cryptophycin and nostocyclopep-tides) could protect the toxin-producing cyanobacteria againstbacteria, viruses, fungi, fish and ducks and could be deadly to

mice and dogs around paddy fields (Nowruzi et al., 2012).Theirmost important ecological effects, however, involvethe in-hibition of pathogenic viruses and bacteria, potential grazers andcompeting phototrophs, not mice and dogs.

SIZE EFFECTS, DIFFUSIVE SUPPLY AND LOSSOF S OL UT E S

As organisms increase in size, diffusion gradually becomes a lessefficient means of supplying external dissolved substances.

Size effects

Because resource uptake in phototrophic organisms scales tosurface area while resource demand scales to volume and mass,a first principle dictates that relative growth rate scales to SA:Vand CMA (Nielsen and Sand-Jensen, 1990; Lambers andPoorter, 1992, 2004). Studies on N. pruniforme andN. zetterstedtii showthat chlorophyll content and photosyntheticcapacity relative to surface area are approximately independentof colonysize because theNostoc trichomes are mainly confinedto the 12 mm-thick outer periphery of the spherical colony,while its dark central part is mostly devoid of trichomes andphotosynthetic activity (Raun et al., 2009;Sand-Jensen et al.,2009b). If Nostoc colonies produce hollow spheres with anouter shell of uniform thickness and a water-filled centralcavity, CMA would be independent of colony size, just as inthe uniformly thick thalli of many macroalgae, plant leavesand the sheet-like structure ofN. commune. This is also almostthe situation for N. pruniforme in summer collections inDanish lakes, where CMA increases with colony mass to apower of only 0.05, corresponding to a 1.3-fold increase inCMA for a 100-fold increase in colony FM (Table 2).

Spherical colonies could in this case maintain an almost un-alteredgrowthratewithincreasingsizeprovidedtheextentofdif-fusive supply of limiting DIC and nutrients remained constantrelative to surface area, or that this diffusive flux was not rate-limiting. Because colonies of N. zetterstedtii are massive,CMA is high and increases by a power of 0.75 with respect tocolony size and thereby should contribute to a low and declininggrowth rate in larger colonies.

For growth performance, it is therefore crucial how resourcefluxes and mass density respond to changes in environmentalconditions and colonysize and how photosynthates are allocatedbetween new cells, colony matrix and protective organiccompounds.

Resource supply by diffusion and turbulence

The diffusive flux (Fa) of a dissolved substance to a unitsurface of a spherical colony is determined by colony size (e.g.radius r), thickness of the DBL surrounding the surface (d),the concentration gradient across the boundary layer and the dif-fusion coefficient (D), according to eqn 1 (Table 3). The fluxis highly dependent on size for small colonies, whereas forlarge colonies (r d) the flux gradually approaches the two-dimensional situation for diffusion to a flat plate where the fluxrelative to surface area is independent of colony radius (eqn 2in Table3).



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The flux of HCO3 was calculated to a unit surface of smooth

spherical colonies like those ofN. pruniforme (eqn 1) of variablesize for realistic thicknesses (0.3, 1 and 3 mm) of the DBL inmoving and quasi-stagnant water and differences in concentra-tions of 0.1, 0.5 and 2.0 mM (mM mmol L1 mmol cm3)between the external water and the colony surface representative

of what would be expected in lakes of low,intermediate and highHCO3 concentrations, respectively(Fig. 2). Profiles of dissolved

oxygenmeasured withmicroelectrodes above thetop surfaceof aNostoc parmelioides colony as a function of water velocity mea-sured 1 mm above the colony suggested DBL thicknesses of

0.1 mm at 610 cm s1, 0.20.3 m m a t 1 c m s 1 and.0.4 mm in quasi-stagnant conditions (Fig. 3 in Dodds et al.,1995). The lower part of the colony resting on the surface hasgreater DBL thicknesses.Nostoc zetterstedtiicould be expectedto mostly experience concentration differences of 0.10.3 mMHCO3

andN. pruniforme 0.72 mM. When N. commune(and

N. flagelliforme) is supplied with rainwater the DIC concentra-tion is very low. However, when water with dissolved HCO3 is

receivedfrom the carbonate-richsoils of the typical growth habi-tats, the expected DIC range is 0.42 mM (Sand-Jensenet al.,2010; Christensen et al., 2013). Because of the granulatedsurface ofN. zetterstedtii, the flux is perhaps doubled relativeto the smooth surface ofN. pruniforme, while the flux to thesheet-like colonies ofN. communeis much lower than the fluxto small spherical colonies, but only slightly lower than thevalues for large colonies ofN. pruniforme, because they physic-allyapproach the two-dimensional diffusion geometry. Only thediffusiveflux of HCO3

hasbeen calculated, because HCO3 con-

stitutes .95 % of the DIC pool in alkaline water (.0.5 mM) and85 % in soft water (0.1 mM) at air saturation with 0.015 mM

CO2. All tested Nostoc colonies can use HCO3

actively (seelater).With an HCO3

gradient of 2 mMthrough a 1-mm-thick DBL,the potential flux is 1440 nmol C cm 2 h1 for colonies with aradius of 1 mm (5 mg FM) and about half that value (790nmol C cm2 h1) for colonies with a 10-fold higher radius of10 mm (4.5 g FM) (Fig. 2). The potential flux for the two-dimensional diffusion geometry to a sheet-like structure likethat ofN. communeis 720 nmol C cm2 h1 and is independentof size. Fluxes decline 20-fold when the HCO3

gradient is20-fold smaller (i.e. 0.1 mM across the DBL). In contrast, athinner DBL of0.3 mm increases theflux most forlarge coloniesbecause they are more restricted by diffusion geometry thansmall colonies. Thus, for a gradient of 2 mM, the calculated

flux increases to 3120 nmol C cm2

h1

for a small sphericalcolony (radius 1 mm), to 2470 nmol C cm 2 h1 for a large

TA B L E 3. Equations 1 5 describe the diffusive flux relative tosurface area (Fa), volume (Fv), shell volume (Fvs) and entire colony(Fcol) of smooth spherical colonies with a given radius (r) of a solutewith diffusion coefficientDacross a DBL (d) with the concentrationgradientCmCo, whereCm andCo are concentrations in the bulkmedium and at the colony surface, respectively. Equation 6 is for a

hollow sphere with an outer shell of thicknessLs. Equations 7 and 8describe the concentration of a solute at the colony surface (Cr)

produced at a rate ofPv per unit volume of a massive (eqn 7) orhollow colony (eqn 8) and lost by diffusion to stagnant external

water of zero concentration

No. Equation

1 Fa r2 (r(r+ d)d1)D(Cm Co) r

1 (1 + r/d)D(Cm Co)2 Fa d

1D(Cm Co); (r d)3 Fa r

1D Cm; (Co 0)

4 Fcol 4p r D Cm; (Co 0)5 Fv 3r

2D Cm; (Co 0)

6 Fvs r1Ls

1D Cm; (Co 0)7 C(r) 1/3PvD

1r2; (Cm 0)

8 C(r) PvLsr D1; (Cm 0)

Equations are derived fromNobel (1983)andDenny (1993).

10000

20, 03

20, 10

20, 30

05, 03

05, 10

05, 30

01, 03

01, 10

01, 30

1000

Carbon

flux

(nmo

lDIC

cm2h1)

100

1001

Colony radius (cm)

1 10

F IG . 2. Calculated diffusive fluxof HCO3 [J r2 [rr(r r) D (Cm Co)]; Nobel, 1983] asa functionofradius (r) forsphericalcoloniesexposedto three ambient

concentrationlevels (Cm: 0.1, 0.5and2.0 mM HCO3) andthree levelsof boundarylayer thickness(DBL:d r r; 0.3, 1.0and3.0 mm). The combinations are indi-

catedin thekeyin thefigure,with Cm first anddsecond. The moleculardiffusioncoefficient (D)issetat1.0 109 m2 s1 (Zeebe,2011) andtheHCO3

concentrationat the colony surface is assumed to be zero.



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spherical colony (radius 10 mm) and to 2400 nmol C cm 2 h1

for a sheet-like structure.What do these fluxes imply in terms of potential growth rate

and biomass turnover? The answer is different forN. pruniforme, with carbon mass relative to surface area of100mmolCcm 2 for largecolonies (radius 10 mm) comparedwith 1400 mmol C cm2 for N. zetterstedtii of similar size.Assuming an inward flux during 12 daytime hours, a largeHCO3

gradient of2 mM acrossa DBL thickness of1 mm, the po-tential turnover time of the biomass would be 11 d forN. pruniforme and 147 d for N. zetterstedtii, not accounting

for night-time respiration, while a small gradient of 0.1 mMcould only permit a potential turnover time of 210 d forN. pruniformeand 2940 d for N. zetterstedtii emphasizing theprofound influence on carbon turnover of higher CMA forN. zetterstedtii compared with N. pruniforme. This differenceshould be enhanced by N. pruniformes preference for hard-waterlakes, ensuring a higherflux, andN. zetterstedtiis predom-inant existence in soft-water lakes. Thus, typical turnover timesofN. pruniforme could be ofthe order of 11d under suitable con-ditions in hard-water lakes containing 2 mM DIC for coloniesmeasuring 10 mm in radius as opposed to 2940 d (8 years) forN. zetterstedtii of the same size growing in soft-water lakeswith only 0.1 mM DIC available. If we account for the possibilityof a 2-fold higher inward HCO3

flux forN. zetterstedtiibecause

of thegranulated colonysurface, the turnover time would drop to4 years, which is close to the observed values forN. zetterstedtii(3 years) in thesoft-waterLake Varsjo(Sand-Jensenand Mller,2011).Also,the turnovertime of thebiomass ofN. pruniforme inlaboratory experiments at 1525 8C and in the shallow water ofthe hard-water Lake Esrum (2.5 mMDIC) during early summerwas 1014 d (Mller et al., 2014; C. Mller and K. Sand-Jensen, unpubl. data) and resembled the theoretical estimate of11 d mentioned above. These calculations suggest that account-ing for DIC concentrations, DBL thickness and CMA permitssound predictions of growth and biomass turnover of Nostoccolonies.

In addition to DIC, the metabolism and growth ofNostoccol-onies could be constrained by the supply rate of dissolved inor-ganic phosphorus (DIP). Nitrogen can be fixed as elementaryN2and is less likely to be limiting, although growth stimulationby high external concentrations may occur, particularly for ter-restrial species with unrestricted access to atmospheric CO2

(see later). Lakes and ponds with Nostoc species mostly haveDIP concentrations in the water column ranging from undetect-able by standard chemical methods to 0.3 mM (Sand-Jensenetal.,2009b),though higherconcentrations canoccurat thesedi-ment surface. For a DIP gradient of 0.3 mMacross a 1 mm thickDBL, theestimated influx was0.22nmolPcm 2 h1 for smallcolonies (radius 1 mm) and 0.12 nmol P cm 2 h1 for large col-onies(radius10 mm).These fluxesshould permit a turnovertimeof the P pool within 6 d for small and 72 d for large colonies ofN. zetterstedtii, supporting the hypothesis that growth is morelikelyto be constrained by thesupply rateof DICacrossgradientsof 0.1 mM HCO3

. These calculations agreed withthe observationthat growth wasunaffectedby P richness in thesediments of soft-water Lake Varsjo(Sand-Jensen and Mller, 2011).

DIC supply is also important in dense epilithic communitiesgrowing on inertsubstrata and coveredby relatively thickbound-ary layers (Sand-Jensen, 1983). Thus, short epilithic algal com-munities in marinewaters have DBLthicknessessimilar to thoseapplied forNostoc colonies and they appear to be constrained bytheDIC fluxto photosynthesis despite high HCO3

concentration(2 mM) in seawater.Larkumet al. (2003)showed that DBLs ofepilithic algal communities covering dead coral surfaces were0.180.59 mm thick under moderate flow (8 cm s 1) and.2 mm under quasi-stagnant conditions, and they generatedhigh resistance to DIC influx, limiting primary production.Independent experiments with epilithic algal communities inothercoral reefenvironments appeared to support the conclusionthat DIC is a main limiting factor, as elevated nutrient levels had

no effect on primary production and growth (Larkum and Koop,1997; Miller et al., 1999), while large macroalgae in suchnutrient-poor waters were stimulated by nutrient amendment(Lapointeet al., 1987).

Resource supply solely by diffusion

The flux toNostoc colonies may be controlled entirely by dif-fusion in a stagnant water layer overlying the sediment. A stag-nant surface layer may exist almost permanently in deep waterwith restricted turbulence and periodically in shallow waterduring calm weather. The colonies could be buried in a viscousboundary layer a few centimetres thick overlying the sediment(Boudreau and Jrgensen, 2001). When the surrounding water

is virtually stagnant and the solute concentration is reduced tozero atthe colonysurface, theflux perunitsurface area isinverse-ly scaled to colony radius, emphasizing the immense advantageofbeing small (eqn 3 in Table 3).The calculated flux is720 nmolC cm2 h1 forsmallcolonies(radius 1 mm)in watercontaining2 mM HCO3

or half the flux thatoccurs when DBL is1 mm thick(eqn 1). For large colonies (radius 10 mm), however, the flux instagnantwateris only72 nmolC cm2 h1, or 11-fold lower thanin stirred waterwith a DBLof 1 mm.The potentialbiomassturn-over time forlarge coloniesas describedabove would then be120dforN. pruniforme understagnant conditionsin hard-water lakesand 32 350 d (89 years) for N. zetterstedtii in soft-water lakes.

25

20

DIC

up

take

(

mo

lg1

co

lony

h1)

15

10

05

0

050 05

Oxygen release (mol g1colony h1)

10 15 20 25

F IG . 3. DIC exchange ofN. zetterstedtii with water (open symbols) and withboth water and the colony volume (filled symbols) as a function of oxygen ex-change with water and colony volume during 18 h of photosynthesis in thelight. The dotted line represents the 1:1 molar exchange of DIC and oxygen.Colonies were incubated at three DIC levels containing initially 0.99 mM(circles), 0.29 mM (triangles) and 0.10 mM(squares). After Sand-Jensen et al.(2009b); reproduced with permission of the Association for the Sciences of

Limnology and Oceanography, Inc.



8/17

Both values are very high, stressing that the water overlying thesediment is either not entirely stagnant or is enriched with DIC(CO2 and HCO3

) from sediment decomposition. Maberlys(1985)measurements 10 cm above the sediments in a shallowbaycoveredbythemossFontinalisantipyreticashowedelevatedCO2concentrations relative to surface waters between 0.05 and

0.26 mM on 14 differentdates. Concentrations are higher directlyon thesediment surface and a near-bed CO2 concentration of, for

example, 0.3 mM CO2 could permit a 6-fold higher influx of CO2thanaHCO3

gradientof0.1 mM because thediffusioncoefficientofCO2 is approximately twice that of HCO3

(Denny, 1993). Theresulting turnovertime could then be 6.3 yearsfor large coloniesof N. zetterstedtii if we assume that the granulated surfacedoubles the influx and 3.2 years if the inward flux continues inboth light and darkness. Thus, we cannot exclude that large col-onies ofN. zetterstedtii could survive in an entirely diffusive en-vironment provided available concentrations of CO2and HCO3

were markedly higher than 0.1 mM. Small colonies a few milli-metres in diameter can perform well by diffusive fluxes aloneand are likely to be fully embedded in a diffusive boundary

above the sediment surface.

Resource supply by diffusion relative to colony volume

The diffusive flux throughstagnant water to a smooth, spheric-al colony follows thewell-known formula often used forbacteria(eqn 4 in Table3;Fenchelet al., 1998) because the colonies areusually so small relative to the thickness of DBL that the supplyrate is entirely determined by diffusion. When metabolic activityand the demand for external resources are evenly distributedthroughout the colony volume, the flux relative to unit volume(Fv)willbeinverselyscaledtothesecondpowerofradius,empha-sizing the likelihood of extreme limitation as the organisms growin size (eqn 5 in Table3). The solutions to cope with this rapidly

increasing risk of resource limitation with larger size would beeither to reduce volume-specific requirements for metabolism(in the case of N. zetterstedtii) or develop a hollow spherewhere all activity is restricted to an outer shell of thickness Lsand volume 4/3p(r3(r Ls)

3) 4p(r 0.5Ls)2 Ls. Whenr

is large relative toLs,volumeiscloseto4p r2Ls andthe fluxrela-

tive to volume of the outer shell (Fvs) declines linearly withcolony radius (eqn 6 in Table3).

Thus, a 10-fold increase in colony radius will reduce the fluxrelative to the surface area and volume of a hollow sphericalcolony by the same order of magnitude, while the flux relativeto the volume of a homogeneous,solid spherical colony declines100-fold,emphasizingthat if large colonies are indeedsolidthentheircentral partsmust be composed of recalcitrant matrix mater-

ial and either have no cells or cells of low metabolism. BecauseN. zetterstedtiicolonies with a diameter of 1020 mm absorb96 % of incident irradiance from the surface to the centre(Sand-Jensen etal., 2009b), there is no scope for photosynthesisof Nostoc trichomes in the centre, but there is organic matterpresent here that could potentially be used by heterotrophic bac-teria. The colony matrix is persistent and protected by bacteri-cides (Sand-Jensenet al., 2009a), however, implying that thereare no or very few active heterotrophic bacteria living withinN. zetterstedtiicolonies. The year-long persistence and low res-piration rates of the colonies in complete darkness support thisimplication (see below).

Efflux of solutes

While the large size of gelatinous Nostoccolonies constrainsthe influx of external resources, it will also limit the efflux ofvaluable dissolved products derived from metabolism withinthe colonies. The possible implications of variations in thisefflux have been overlooked so far. When the diffusive loss of

respiratory CO2 and inorganic nutrients (e.g. phosphate) fromthecolonyisconstrained,theycanbetterberetainedandrecycledwithin the colony (see section Size effects, diffusive supplyandloss of solutes). Organic compounds that are produced and to agreat extent released from the trichomes to form the extensivecolony matrix can also be lost to the surrounding water and thisloss can presumably be reduced in large colonies. Perhapsmore importantly, antibiotics and toxins produced byNostoctoreduce the risks of being attacked by harmful bacteria, viruses,mixotrophic algae and animals can be retained within thecolony and here reach highly effective concentrations for alimited rate of production. Thus, with increasing colony size aconstant production rate of the protective substance per colonyvolume will lead to increasing internal concentrations, or a con-

stant internal concentration can be attained despite a decliningproduction rate of the protective substance.

The formal analysis showsthat a soluteproduced by a massivesphericalorganismat a uniform specific rate perunit volume (Pv)and lost by diffusion to stagnant surrounding water with a zerobackground concentration will have an effective concentrationright at the surface of the colony that increases by the secondpower of the radius (eqn 7 in Table 3). If the concentration iskept constant, the production rate can decline by the secondpower of the radius. The mathematical formulas have beenderived fromDenny (1993), who treated an analogue biologicalexample.

Photosynthetic production is constant relative to the surfacearea ofN. pruniforme and N. zetterstedtii (Raun et al., 2009;Sand-Jensen et al., 2009b), in accordance with the fact thatphotosynthesis is confined to a surface shell of thickness Lshandan approximate volumeof 4p r2Lsh. Because protective sub-stances derive from photosynthetic products,they could have thesame scaling properties. Thus, inserting shell volume instead oftotal colony volume in eqn 7 predicts that the concentration onthe colony surface would increase in proportion to colonyradius (eqn 8 in Table 3). Alternatively, the concentrationwould remain constant if the production rate of protective sub-stances declined in proportion to increasing colony radius.

Therefore, the incorporation rate of inorganic carbon into newcells, colony matrix and extracellular products should all bedetermined as a function of colony size. This would permitdirect determination of turnover rates of cells and colonymatrix and evaluation of therelativecost of producingand releas-ing extracellular products. Despite extensive and elaborate workon the biochemistryofNostoccolonies,this analysis has not beenperformed as yet.

F UNCT I ONAL CONS E QUE NCE S OF T HE US EOF L I GHT AND I NORGANI C CARBON

Nostoc concentrates and circulates inorganic carbon efficientlywithin the large colonies, while the use of light suffers from



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extensive self-shading and absorption by non-photosyntheticelements.

Use of light

The large size, long internal light paths and high densities of

trichomes withinNostoccolonies imply that light absorptanceand internal self-shading are high. Mean light absorptanceranged from 55 % in N. commune to 96 % in N. zetterstedtii(Table4). Moreover, because the cells experience high O2andlow CO2 concentrations within the colonies during photosyn-thesis, maximum photosynthesis relative to cell chlorophyll(i.e. the assimilation number), maximum quantum efficiencyof photosynthesis and the ratio of photosynthesis to respirationshould be much lower than among free-living unicells. To theextent that photons are absorbed by non-photosynthetic pig-ments and other coloured substances inside the colonies,maximum quantum efficiency will decline further.

Quantum efficiency was indeed low forN. commune [average19.1 mmol O2 (mol absorbed photon)

1] and N. zetterstedtii

(11.239.9 in three series; Table4). A similarly low efficiency[27 mmol O2 (mol photon)

1] was calculated for the thick-walled, balloon-like green macroalga Codium bursa (Geertz-Hansenet al., 1994) and microbial mats with extensive absorp-tion (40 80 %) by structural components (Al-Najjar et al.,2012). In contrast,quantumefficiencies aremuch higher for free-living microalgae [70120 mmol O2 (mol photon)

1], macroal-gae and submerged plants [3779 mmol O2 (mol photon)

1](Frost-Christensen and Sand-Jensen, 1992).

Nostoc zetterstedtii had particularly low light-saturatedphotosynthesis relative to chlorophyll (average 0.50.7 mg O2mg1 chlorophyll a h1) and values were also low forN. commune (average 2.4) and N. pruniforme (average 2.1).Likewise, photosynthesis relative to dark respiration (NP:R)

was low for N. zetterstedtii (2.0 5.8) and N. commune(average 2.5), but not forN. pruniforme (average 10).Again free-living unicells have higher rates of photosynthesis relative tochlorophyll, typically between 2 and 20 (Harris, 1978) andNP:R ratios from 5 to 20 (Harris, 1978; Geider and Osborne,1992). High self-shading withinNostoc colonies is an importantconstraint because photosynthesis at high irradiance increased4-fold and NP:R increased 3-fold when self-shading wasreduced by cutting colonies of N. zetterstedtii into 12-mmpieces (Sand-Jensenet al., 2009b).

Light attenuation by coloured substances, structural sub-stances and dead or senescent cells within the colonies competes

with photosynthetic pigments for photons, and thereby lowersquantum efficiency and increases the light compensation point(where photosynthesis outweighs respiration) compared withfree-living unicells (Krause-Jensen and Sand-Jensen, 1998;Al-Najjar et al., 2012). Thus, light compensation points ofN. zetterstedtiiand N. commune (9.519.3 mmol photon m2

s1

) exceeded those of most unicells (typically 0.8 9 mmolm2 s1;Langdon, 1988) and thin thalli and leaves of macroal-gae and submerged plants (typically 2 12 mmol m2 s1;Sand-Jensen and Madsen, 1991). This comparison supports thehypothesis that inefficient light use inNostoc colonies combinedwith higher respiratory costs of producing and maintaining thecolony should lead to higher minimum light requirements forsurvival than forunicells,characeans and plants with thin photo-synthetic tissues. Observations of lower light availability at themaximum depth limits of characeans, filamentous algae andmosses (3.1 7.8 % of surface light) than ofN. pruniformeandN. zetterstedtii (9.412.5 % of surface light) in two Danishlakes support this hypothesis (Table 7 in Sand-Jensen et al.,2009b).

Use of inorganic carbon

Thediffusive supplyof DICfrom outside is constrained by thelarge size, low SA:Vratio and high density of filaments in the1 3-mm-thick outer shells of gelatinous Nostoc colonieswithout direct contact with the surrounding medium. The effect-ivediffusion pathinvolves both the diffusive boundary layer sur-rounding the colony and the path through the gel to the sites offixation in the Nostocfilaments, but the internal resistance hasnot been accounted for.

While increasing theresistance to influx of gasand solutes, thegelwillalsosloweffluxesand contribute tothe retention ofnight-time respiratory CO2 and facilitate accumulation of internal DIC

pools. To ameliorate carbon limitation of photosynthesis andprevent excessive photorespiration,Nostoccolonies, like othercyanobacteria (Badger and Price, 1992,2003), must attain rea-sonably high quotientsof CO2 to O2 atthe site of Rubisco activitywithin the cells. To achieve this,Nostoc colonies must be able toextract high proportionsof theDIC pool in thewaterand have lowcompensation points of CO2 and HCO3

(Sultemeyer et al.,1998). The ability to accumulate DIC pools within the colonyabove immediate needs for photosynthesis and to retain respira-tory CO2 from night-time respiration for later use during daytimephotosynthesis could assist the carbon supplyeven further. Suchefficient extraction and accumulation of carbon are known for

TA B L E 4. Photosynthesis light variables of threeNostocspecies at 15 8C

Species Pmax Pmax/dark respiration Icomp(mmol m2 s1) Quantum efficiency

(mmol O2/mol photons)Absorption (%)

(nmol O2cm2 h1) (mg O2mg Chl

1 h1)

N. commune 353376 2.4 2.5 9.5 19 55

N. pruniforme 552654 2.1 10 90N. zetterstedtii 206409 0.7 2.0 5.8 19.3 20 96

Data are means of measurements in several series.Pmax, maximum net photosynthesis at light saturation;Icomp, light compensation point.Sources:N. commune(K. Sand-Jensen, unpubl. data); N. pruniforme(Raunet al., 2009);N. zetterstedtii(Sand-Jensenet al., 2009b).



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free-living cyanobacteria (Kaplan etal., 1980; Sultemeyer et al.,1998) and phototrophs in lichen photosynthesis (Green et al.,1994), and the potential may also exist for Nostoc trichomesburied within the colony gel.

We found a high mean extraction capacity, of between 74 and82 % of the initial DIC pool in the water (0.3 1.1 mM), during

20 h of photosynthesis in closed bottles with N. commune andN. zetterstedtiiwhere DIC was gradually depleted and pH andO2increased over time (Table5). The mean extraction capacitywas lower (58 %), but probably not fully expressed, in experi-ments with N. pruniforme, though it still reflected very activeDIC uptake. Efficient DIC use is also known forN. flagelliforme and edibleNostocGe-Xian-Me (Gao and Zou,2001;Qiu et al., 2004;Ye et al., 2012). Efficient active use ofexternal DIC by N. commune and N. zetterstedtii was alsoreflected in the high final pH in the surrounding water (10.6011.11) and the low final concentrations of HCO3

(675 mM)and CO2(0.37 nM) as measures of HCO3

and CO2compensa-tion points (Table6). Concentrations of HCO3

and CO2at thesites of Rubisco within the cells must be markedly higher and

molar quotients of O2 to HCO3

+ CO2 lower than in the sur-rounding water (860) to ensure net carbon fixation andreduce photorespiration (Kaplanet al., 1980;Priceet al., 2008).

The ability of all three Nostocspecies to accumulate DIC tomuch higher concentrations within the colony than in the exter-nalwater is supportedby the relativelyhigh ratesof O2 release byphotosynthesis for 20 h in water of low DIC (0.1 mM, 0.1mmol cm3) without any appreciable uptake of external DIC.Assuming that O2is produced and inorganic C consumed fromthe colony itself during extended photosynthesis with a molar

quotient of1.0, we determined that a DIC pool of 16.324.5mmol g1 FM was available for photosynthesis in colonies ofN. zetterstedtii and about half this pool size (5.810.2 mmolg1 FM) was available in N. commune and N. pruniforme(Table 7). Direct measurements of the internal DIC pool inN. zetterstedtiiyielded 19.024.7 mmol g1 FM for colonies

incubated in water containing 0.15 mMDIC and these measure-ments fully corresponded with estimates derived from the

O2DIC balance (Sand-Jensenet al., 2009b). Measured DICconcentrations were 150-fold higher within the colonies(average of cells and matrix) than in the external water and Iexpect that DIC concentrations would be even higher withinthe cells, resulting in a higher accumulation quotient than 150between cells and water. Accumulation quotients of 5001000are known for free-living cyanobacteria (Kaplan et al., 1980;Badger and Price, 2003).

When I accounted forexchange of DIC during photosynthesisand respiration with both the colony matrix and the externalwater, I found mean molar exchange quotients of O2relative toDIC in the light (1.19) and in the dark (0.96) close to 1.0 in

experiments withN. zetterstedtii(Fig. 3). These direct measure-ments confirmed that a high proportion of DIC for photosyn-thesis in the light was consumed from a DIC pool in thecolony, while in the dark a high proportion of respiratory DICaccumulated within the colony (Fig.3). The measured internalDIC pools inN. zetterstedtiicould support maximum photosyn-thesis for 11 and 23 h in 10- and 20-mm-diameter colonies, re-spectively, without uptake of external DIC. WhenN. communewas incubated for 20 h in relatively DIC-rich water containinginitially 1.1 mM, it accumulated 16.718.7 mmol DIC g 1 FMof colony in excess of the immediate requirements for photosyn-thesis, while it consumed 910.2 mmol DIC g1 FM from thecolony to support photosynthesis whenthe initial DICconcentra-tionin the water was only0.2 mM (K. Sand-Jensen, unpubl. data).

Photosynthetic ATP production providesample energy foractivetransport of HCO3

, and at least N. communecan consume DICfor photosynthesis and at the same time accumulate DICwithin the colony in the light when sufficient external DIC isavailable. Thus, the species does not solely rely on accumulationof respiratory DICin thedark butcan also take up externalHCO3

for use in the following light period.The ability ofNostoccolonies to build up substantial internal

DIC pools had been overlooked in the past. This ability meansthat photosynthesis is less dependent on the immediate externalDIC supply. Nonetheless, all three species were limited by the

TA B L E 5. Extraction capacity of the initial DIC pool (%) of threeNostocspecies during photosynthesis for 20 h in closed bottles

SpeciesMean+ s.e.

(%)No. of

measurements Source

N. commune 72+4 16 K. Sand-Jensen(unpubl. data)82+3 12

N. pruniforme * 56+3 5 Raun (2006);Raunet al. (2009)

N. zetterstedtii 74+9 16 Sand-Jensenet al.(2009)

*Full extraction capacity probably not realized.

TA B L E 6. Final pH, final DIC, HCO3 and CO2 and final O2/HCO3 ratio of the fourNostoc species after 20 h of photosynthesis in

closed bottles. Ranges are shown from several experimental series with many replicates

SpeciesInitial DIC(mmol L1) Final pH

Final DIC(mmol L1)

Final HCO3(mmol L1)

Final CO2(nmol L1) O2/HCO3 No. of series

N. commune 106310 10.6811.11 1290 634 1 7 960 5N. pruniforme * 570 10.6 260 110 ,30 5 1N. zetterstedtii 149162 10.9411.06 77174 13 35 3 10 1829 4N. flagelliforme 3300 10.8

*Maximum final pH and minimum final DIC, HCO3and CO2probably not attained.Sources:N. commune(K. Sand-Jensen unpubl. data); N. pruniforme(Raun, 2006;Raunet al., 2009);N. zetterstedtii(Sand-Jensenet al., 2009);N. flagelliforme

(Gao and Zou, 2001).



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external DIC supplyas photosynthetic rates at 0.1 mM DIC exter-nally were 20 % of the maximum rate for N. commune, 11 %for small, 13 % for medium-sized and 25 % for large coloniesofN. pruniforme and 60 % for N. zetterstedtii (Figs4 and 5).As the internal DIC pool inN. zetterstedtiicolonies is graduallyexhausted during 18 h of light incubation in closed bottles, rea-

lized mean rates of O2 production are 1.8-fold lowerin water ini-tially containing 0.29 mM compared with water initially

containing 0.99 mMDIC (Fig.3). InN. pruniforme, which hashigher rates of photosynthesis and DIC requirements persurface area thanN. zetterstedtiiand apparently 2-fold lower in-ternal DIC pools, the dependency of photosynthesis on externalDIC was stronger, showing half-saturation constants of 0.78 mMfor small colonies (0.1 g FM) and 1.24 mM for larger colonies(2.5 g FM) in 2-h experiments under well-stirred conditions(Fig. 4). Large colonies have higher rates of photosynthesis(164 nmol O2cm

2 h1) at near-zero external DIC than smallcolonies (55nmol O2 cm

2 h1), probablybecause of a largerin-ternal DIC pool (being scaled to colony volume) relative tophotosynthetic activity (being scaled to colony surface area).

The more favourable SA:Vratio in small colonies causes photo-synthesis to increase more steeply with increasing external DIC(a-CO2,319nmolO2 cm

2 h1 for a 1-mmol L1 increasein ex-ternal DIC) than in large colonies [a-CO2,197nmolO2 cm

2 h1

(mmol DIC L1)1] (Fig.4).The terrestrial and semi-terrestrial N. commune and

N. flagelliformecan use CO2and HCO3 under water and CO2

alone in air. Under water, N. commune experiences limitationof photosynthesis at low DIC as rates rise to at least 1 mM. Thediffusion coefficient of CO2 in air is 2 10

4 times higherthan that of HCO3

in water, and for the same DBL thicknessand with complete DIC depletion at the colony surface the CO2gradient is 30 50 times lower in air (external CO2 0.02 mM)than in water containing 0.61.0 mM HCO3

. Thus, the potential

fluxwouldbesubstantiallyhigher(500-to700-fold)inairthaninwater through a diffusive boundary layer of the same thicknessoverlying the colony surface, but this is followed by the same re-sistance through thecolony to theNostocfilaments in bothterres-trial and aquatic habitats. At high irradiance and twice daily

20

Small colonies

Medium-sized colonies

Large colonies

15

10

05

20

15

Ne

tp

ho

tosyn

thesis

(mg

O2

mgc

hl1

h1)

10

05

20

15

10

05

00 1 2

External DIC (mM)

3

F IG . 4. Mean net photosynthesis (+ s.d. of four replicates) of small (0.18 gFM), medium (0.84 g FM) and large (2.71 g FM) spherical colonies of

N. pruniforme as a function of external DIC concentration (.95% HCO3).

The apparent half-saturation constant for small, medium and large colonieswas 0.78, 0.78 and 1.24 mMHCO3

, the initial linear slope of net photosynthesisat low limiting DIC concentration was 319, 305 and 197 nmol O2cm

2 h1 (1mmol DIC L1)1 and photosynthesis extrapolated to zero DIC was 55, 72 and

164 nmol O2cm2 h1. FromRaun (2006).

800

N. commune

N. pruniforme

N. zetterstedtii600

Ne

tp

ho

tosyn

thes

is(nmo

lO

2cm2

h1)

400

200

00 1000

HCO3concentration (mol L1)

2000 3000

F IG . 5. Mean DIC uptake during photosynthesis in high light as a function ofmean DIC concentration in the water for N. commune, N. pruniforme and

N. zetterstedtii. Sources: N. commune (K. Sand-Jensen, unpubl. data);N. pruniforme (Raunet al., 2009);N. zetterstedtii(Sand-Jensenet al., 2009b).

TA B L E 7. DIC consumed from the colony volume of threeNostocspecies during extended light periods (20 h)

Species

DIC consumed(mmol C g1 fresh

weight)

SourceMean 95 % CL

N. commune 5.8 1.1 K. Sand-Jensen (unpubl. data)6.5 0.59.0 1.7

10.2 2.3N. pruniforme 8.67 1.33 Raun (2006),Raunet al. (2009)

8.57 0.84N. zetterstedtii 16.3 2.9 Sand-Jensenet al. (2009)

24.5 5.6



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watering, growth of N. flagelliforme was doubled inCO2-enriched air (1500 ppm) compared with atmospheric air(350 ppm atthe time; GaoandYu,2000). The realizedphotosyn-thetic rate ofN. communewas the same in stirred air-saturatedwaterof1mM DIC(0.98 mM HCO3

+ 0.02 mM CO2)asinatmos-

pheric air with 0.02 mM CO2 (Sand-Jensen and Jespersen, 2012).

Thus, theoptimal habitat ofN. commune couldbewetsoilswherewater and inorganic carbon and nutrients are supplied to thelower colony surface, while CO2and light are supplied to theupper surface. This is the habitat in 1-cm-deep water-filleddepressions in the limestone pavements where we have observedmassive growth ofN. commune(Sand-Jensenet al., 2010).

T OL E RANCE OF E NVI RONM E NT AL E XT RE M E S

FreshwaterNostocspecies do not experience extreme tempera-tures like those experienced by terrestrial species. Active growthof N. commune, N. pruniforme and N. zetterstedtii is, nonethe-less, confined to the same temperature range (0358C).

Temperature and desiccation tolerance

The terrestrial and semiterrestrial species N. commune andN. flagelliformeare exceptional in their tolerance of alternatingfreezing and thawing and of desiccation and rehydration atvarying temperatures in their open habitats, located from theArctic to tropical regions (Li, 1991;Sand-Jensen and Jespersen,2012). In contrast, the freshwater species N. pruniforme andN. zetterstedtii are not exposed to freezing and drying or toextreme temperatures in their temperate and sub-Arctic environ-ments. Nonetheless, it appears that active growth of all fourspecies is confined to the same temperature interval between 0and 35 8C and that the fastest growth takes place around 25 8C(Mlleret al., 2014).

BothN. communeandN. flagelliformeare widely distributedspecies in arid and semiarid bare land throughout the world ( Li,1991; Dodds et al., 1995). Nostoc commune survives annualtemperature ranges of60 to 258C under Arctic conditionsand 30 to 508C under temperate conditions (Davey, 1989;Sand-Jensen and Jespersen, 2012). Surface temperatures in theopen habitats of N. flagelliforme can reach 788C in summerand 408C in winter (Li, 1991). Both species can survivemonths or years of frost and drought as inactive desiccatedcrusts, and within minutes to hours can reactivate ion exchange,respiration, photosynthesis and N fixation when water and suit-able temperatures again become available (Scherer et al.,1984;Satohet al., 2002).

Experiments with the temperate species N. commune con-

firmed that photosynthesis and respiration were maintainedafter 36 h of desiccation of wet or already dry specimens at tem-peratures from 269 to 708C, while temperatures above 708Cled todeath(Sand-Jensen and Jespersen, 2012). During repeateddaily cyclesof dryingfor 18 h at 18, 20and 408C and rewettingfor6hat208C,N. commune retained its photosynthetic capacityat 18 and 20 8C but died at 408C, presumably because of thegreater costs of repair of macromolecules at this higher tempera-ture, which would generate stronger thermal damage than lowertemperatures (Sand-Jensen and Jespersen, 2012). In 24-day-longexperiments under permanently submerged conditions,N. commune also died at 458C but survived at 35 8C; growth

peaked at 25 8C and declined at the lower temperatures of 15and 58C(Mlleret al., 2014). Annual carbon fixation of rehy-drated populations ofN. communefrom an Antarctic dry valleywas also a strong positive function of increasing temperaturesabove zero and up to 208C, the highest temperature tested(Novis etal., 2007). Thus, there was no obvious sign of tempera-

ture adaptation or acclimation across geographical ranges.Nostoc flagelliforme, like other terrestrial Nostoc species(Davey, 1989;Dodds et al., 1995;Sand-Jensen and Jespersen,2012), showed great heat resistance when dry, while it wasmore susceptible when wet or immersed (Mei and Cheng,1990). The regular protein pigment structure of photosystem Icomplexes was destroyed at 70 and 80 8C (Hu et al., 2005).Pretreatment of wetN. flagelliformeat65 8Cledtodeathandtem-peratures above 45 8C stopped photosynthesis (Mei and Cheng,1990). The exact temperature effect on survival ofN. communeandN. flagelliformeapparently depends on the duration and fre-quency of heat exposure and on the length of time available forrepair of cellular damage between heat exposures (Sand-Jensenand Jespersen, 2012). Likewise, a longer time was required for

maximal photosynthesis and respiration to recover after rehydra-tion of specimens subjected to extended periods of desiccation(Scherer et al., 1984;Potts, 1994; 2000;Qiu and Gao, 1999).The recovery of rehydratedN. flagelliforme was light-dependent(Gaoet al., 1998) and required potassium (Qiu and Gao, 1999),stressing that a suite of energy-demanding metabolic processesinvolving expensive proteinand lipid synthesis and various cellu-lar repair systems restores cellular structure and catalytic capacity(Angeloni and Potts, 1986;Tarantoet al., 1993).

Desiccation is not an issue for freshwater species ofNostoc(except when they are washed ashore) and temperature variabil-ity is also much lower in freshwater than in terrestrial habitats. InDanish lakes, for example, the annualtemperaturerange is typic-ally0258Cinshallowwaterand2158Cindeepwater(Mller

et al., 2014). Nonetheless, the temperature dependence of long-term growth ofN. pruniforme andN. zetterstedtii resembled thatof hydratedN. communein that all three species grew fastest at258C and many times slower at 58C, while 458C led to die-off(Fig. 6 in Mller et al., 2014). So while terrestrial Nostocspecies possess extraordinary desiccation tolerance there is nomajor difference in the relationship of active growth with tem-perature in terrestrial and aquatic specimens from north-temperate localities, though we cannot rule out that differentrelationships of growth with temperature have evolved in speci-mens livingunder Arcticor tropical climates.Because, under tem-perate conditions,temperature ranges between winterand summerare also profound it would notbe a greatsurprise,however,if theserelationships remained constant across geographical ranges. High

temperature requirements (20 8C) of maximum photosynthesisofN. commune from an Antarctic valley with very short frost-freeperiods support the latter suggestion.

pH and salt tolerance

Being photosynthetic organisms capable of using both freeCO2and HCO3

at high efficiency, all four Nostocspecies canpush pH in the external water above 10.5 and in some caseseven above 11 (Table6). Exposure of rehydrated N. communeto a pH gradient for 36 h also showed that it tolerated pHranging from 3 to 10, while it died at pH 2 (Sand-Jensen and



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Jespersen, 2012). Terrestrial Nostoc species should be able tosurvive exposure to rainwater of low pH (typically 3.84.8)and only very acidic habitats receiving sulphuric acid from theoxidation of metal sulphides are likely to be below pH3. Therefore, whenit comesto direct pH tolerancemost terrestrialhabitats should be open to colonization by Nostoc. Although

N. communecan grow between garden tiles and on open sandsoils fed by rainwater, it appears to prefer clay soils and calcar-eous rocks of high pH, perhaps because available HCO3

cansupport photosynthesis during prolonged illumination. The re-quirement of HCO3

for photosynthesis of aquaticNostocis ac-companied by a preference for a pH above 7 in the waterbecause of the buffering and alkalinization effect of HCO3

.Thus, at air saturation, freshwaters with pHs of 6.5, 7.5 and 8.5have HCO3

concentrations of 0.05, 0.5 and 5 mM, respectively(Stumm and Morgan, 1981).

BecauseN. communeandN. flagelliformeoften grow in aridhabitats with salt accumulation they must be tolerant. Indeed,N. communeretained its photosynthetic capacity upon exposureto alkaline freshwater enriched with NaCl to 20 g kg 1, but not

30 g kg1

as in oceanic water (Sand-Jensen and Jespersen,2012). Theclosely relatedN. flagelliformewas also salt-resistant,but photosynthesis, respiration and photosystem II activitydeclined upon exposure to .12 g NaCl kg 1 (Ye and Gao,2004; Yong-Hong et al., 2005). Salt resistance is probablybased on the formation of sucrose and trehalose, both of whichaccumulate under desiccation and exposure to low salt concen-trations (Sakamotoet al., 2009) and therefore play a dual role.Tolerance of freezing and desiccation is extraordinary, whilesalt tolerance is modest. Other cyanobacteria are more salt-tolerant, because of the synthesis of glucosylglycerol inspecies of moderate tolerance and glycine betaine and glutamatebetaine in species showing high tolerance (Mackay etal., 1984).

ECOLOGICAL ADAPTATIONS AND STRATEGIES

Colonial Nostoc species live in resource-poor environments andsuccumb in competition with tall macroalgae and plants underricher conditions.

All four colonial Nostoc species discussed here live inresource-poor environments with a limited supply of water andinorganic nutrients on land and a limited supply of nutrientsand DIC under water. Phosphorus could be a limiting nutrientfor growth but experimental tests are lacking. All speciescan fix elemental N2, but this is a costly process that requires arare co-factor (molybdenum). In suspension culture with

N. flagelliforme, the variable availability of nitrogen and phos-phorus influences the production of new cells and extracellularpolysaccharides (Feiet al., 2012).

The terrestrial N. commune and N. flagelliforme grow onnutrient-poor, sparsely vegetated or bare soils and rock surfacesthat alternate between being wet and dry. From April toSeptember on the open limestone alvar on Oland, Sweden, mea-surements of humidity and temperature suggested that popula-tions ofN. communewere rehydrated and active at least 26 %of the time (Sand-Jensen and Jespersen, 2012). From Octoberto March, populations were probably active during larger partsof the frost-free time.

Maximum recorded growth rates ofN. commune floating onwater and exposed to atmospheric air under laboratory conditionswere 0.0500.056 d1 at 1525 8C and markedly lower, at0.0200.025 d1, at 5 and 35 8C (Mller et al., 2014). Thesegrowth rates correspond to doubling times of dry mass between12 and 35 d. Growth rates are intermediate when compared with

the extremely low rates (0.0008 d

1

) of oligotrophicN. zetterstedtii and high rates (0.20.4 d1) of nutrient-demanding thin macroalgae (e.g. Cladophora spp. andEnteromorphaspp.;Sand-Jensen and Borum, 1991;Nielsen andSand-Jensen, 1991). According toGrimes (1979)classificationof plant growth strategies in relation to resource richness and dis-turbance, the stress strategy (S) of N. zetterstedtii is linked toresource-poor, stabile habitats, the ruderal strategy (R) of thingreen macroalgae is linked to resource richness and high disturb-ance, and the competitive strategy (C) of thick macroalgae islinked to resource richness and low disturbance. Resource-poor,highly disturbed habitats do not, in Grimes concept, support aviable strategy becausethe growth rate is very low when resourcesare limitingand disturbanceleadsto frequent loss of biomass, thus

preventing long-term survival. However, N. commune (andN. flagelliforme) can survive disturbance by desiccation, freezingand extreme temperatures with restricted biomass loss in a quies-centstage andpossessesa viablestrategy in theresource-poor, dis-turbed terrestrial habitats that Grime considered unoccupied, atleast by higher plants. A suite of drought-resistant lichens,mosses and a few flowering plants are capable of surviving inthe same resource-poor, disturbed habitats as N. commune andN. flagelliforme (Sand-Jensen etal., 2010) and could be classifiedas belonging to a mixed SD strategy, where S represents toler-ance of limiting resources and D represents resistance to disturb-ance. Their biomass survives harsh physical conditions that aredetrimental to most other phototrophs. Likewise,in themarine en-vironment, crust-forming coralline red algae are adapted to an ex-

tremely low supply of light or nutrients, tolerate high disturbanceby wave action and avoid intensive grazing, and thus have a viableSD strategy with extremely slow growth and biomass turnover(Littler and Littler, 1980;Littleret al., 1986).

The freshwaterN. zetterstedtii growsin soft-water, oligotroph-ic lakes and faces the strongest constraints on nutrients and DICsupply.Nostoc pruniformegrows in mesotrophic, alkaline lakesof higher DIC concentrations, where nutrients remain low fromlate spring to early autumn due to nutrient uptake by phytoplank-ton and other benthic phototrophs (Sand-Jensen et al., 2009b).Because bothspecies areperennial, we cannot dismiss the untest-ed possibility that they can take up nutrients in excess during thericher winter season for later use during summer when organicproduction by photosynthesis is high. This mechanism has been

shown for perennial marine macroalgae (Chapman and Cragie,1977;Lundberget al., 1989;Lobban and Harrison, 1997). Thepersistence ofNostoccolonies maythereforeoffer an opportunityto withdraw external nutrients and produce photosynthates whenconditions are favourable and store them within the colony untilthey are needed for growth and survival. The large size and lowSA:Vof the colonies can promote internal circulation of valuablesubstances and reduce their diffuse loss to the environment, aspreviously discussed (see section Functional consequences ofthe use of light and inorganic carbon).

Nostoc zetterstedtiiis exceptional among aquatic phototrophicorganisms due to its exceedingly low rates of growth, respiration



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and mortalityas a consequence of the large spherical colonysizeand high CMA (Sand-Jensen and Mller, 2011). Because directselection for low growth rate is not a viable evolutionarystrategy(Lambers etal.,2008), lowgrowthrateshould, as emphasized, beregarded as an indirect consequence of high CMA and invest-ment in a large arsenal of organic compounds as protection

against environmental hazards, grazers and pathogens. Themaximum growth rate ofN. zetterstedtii in laboratory experi-ments was only 0.78 103 d1, corresponding to a mean doub-ling time of colony biomass of 2.4 years (Mlleret al., 2014).This value resembled the annual mean doubling time of 2.63.3 years in field experiments (Sand-Jensen and Mller, 2011).With such low growth rates, the largest colonies, measuring7 cm in diameter, are 2431 years old. We found no senescenceand mortality in the examined population of colonies in the fieldduring13 608incubationdays. This findingimpliesthat themor-tality rate was,7.3 105 d1 and longevity.24 years, in ac-cordance with the high age of the largest colonies. MaceratedN. zetterstedtiicolonies diluted 10-fold in water and added to asample of lake bacteria immediately stopped all cell division

(Sand-Jensen et al., 2009a). Likewise, no grazing by smallmacroinvertebrates has been observed, and large potentialgrazers, such as crayfish, fish and swans, are probably unableto consume the large, firm colonies. Recent experimentsshowed that colonies survived for 14 months in complete dark-ness at 58C with integrity of photosynthesis and fresh mass ofthe colonies and extremely low daily respiration rates of0.21 mmol C (mol organic carbon) 1 corresponding to a half-time of the colony carbon biomass of 9 years (K. Sand-Jensen,unpubl. data). I anticipate that Nostoccells can utilize organiccompounds from the colony matrix for basal respiration andlong-term survival.

The ecological strategy of N. zetterstedtii is therefore anextreme example of a stress-selected species living in a very

resource-poor environment, growing extremely slowly, beinghighly persistent thanks to efficient physical and chemical pro-tection against pathogens and grazers, and having efficient recir-culation of inorganic carbon (Fig.3) (Sand-Jensen etal., 2009b).Measured growth rate in dry mass of N. zetterstedtii was10-fold lower thanrates predictedfrom general allometricrela-tionships of growth rate with CMA of aquatic macroalgae(Markager and Sand-Jensen, 1996) and, likewise, of predictedgrowth rate with the thickness of photosynthetic tissue for abroad range of terrestrial and aquatic plants (Nielsen et al.,1996). This finding suggests that there are extra constraints onthe growth ofN. zetterstedtiilinked to limited DIC supply and/or production of the colony matrix and the protective com-pounds. The proposed reduced loss of organic solutes from

large colonies to the surrounding water has not been tested asyet, though it is plausible given the ability ofN. zetterstedtiitomaintain colony integrity and fresh mass during 14 months indarkness (K. Sand-Jensen, unpubl. data).

Nostoc pruniforme grows in oligo-mesotrophic lakes richer inDIC than the main habitats ofN. zetterstedtii, and its growth,mortality and ecological strategy are also markedly different.Maximum growth rate of N. pruniforme in laboratory experi-ments was 75-fold higher than that of N. zetterstedtii, corre-sponding to a biomass doubling time of 12 d (Mller et al.,2014). Growth rates in a clear-water, DIC-rich lake duringearly summer resembled these laboratory rates (Mller and

Sand-Jensen, unpubl. data 2013). A high DIC (2.0 mM) in thelake water was required to sustain a biomass turnover of 12 d,while incubation in low-DIC water (0.2 mM) reduced net photo-synthesis 4-fold and prolonged biomass turnover (Mlleret al.,2014). Field studies confirmed thatN. pruniformeperiodicallyundergoes mass mortality, presumably by attack of microbial

pathogens. In terms of the ecological strategy, N. pruniformehas species traits belonging to a mixture of ruderal-, stress- andstress-disturbance-selected strategies. The expression of thesetraits is plastic depending on whether the species lives in cold,oligotrophic freshwaters and forms very large and old coloniesor colonies that live for shorter periods in warmer, mesotrophiclakes and reach smaller sizes.

All fourNostocspecies have one important trait in common.They live directly on the ground surface and are unable tosurvivein competition withtall,dense terrestrial or aquatic vege-tation. This is a main reason why they are primarily confined toresource-poor habitats where tall, dense vegetation cannotdevelop. TheNostoc species therefore face the risk of disappear-ing when their habitats are subjected to nutrient enrichment. The

freshwater Nostoc organisms in Scandinavian lakes have beenfurther threatened by large-scale leaching of dissolved humicsubstances from low-buffered, acidic soils.

OUT L OOK

Most studies on the ecophysiology of phototrophs have focusedon relatively fast-growing species from resource-rich habitats(Larcher, 2003; Lambers et al. 2008). With this review onlarge gelatinousNostoccoloniesI call attention to thesecompeti-tively inferior species from nutrient-poor habitats. I introduceterrestrial and semiterrestrial Nostoc species possessing anunprecedented ability to enter quiescent, desiccated or frozenstages and rapidly resume metabolism when rehydrated. The

molecular, biochemical and physiological sequences leadingto quiescence and reactivation need to be fully explored andthe metabolic costs of frequent desiccation, freezing and rehy-dration and substantial production of extracellular polysacchar-ides need quantification in the future. There is strongmolecular,biomedical and ecologicalinterest in theseprocesses.

The rare freshwater speciesN. zetterstedtii has unprecedentedlow rates of metabolism, growth and mortality in its DIC- andnutrient-poor lake habitats. Its ability to take up external DICand recirculate DIC within the colony is profound. It is challen-ging to determine whether the large colony size also createsadvantages for effective circulation of nutrients within thecolony and strong antibiotic effects for relatively low productioncosts. Future studies should also unravel fluid dynamics and nu-

trient and DIC conditions in the neglected near-bed habitats ofN. pruniforme and N. zetterstedtii on lake sediments. Thiseffort is needed in order to put the presented diffusion modelsforuptake andloss of solutes from sphericalcoloniesof differentsize into proper physical and chemical perspectives in nature.

As free-living organisms and as symbionts in lichens,Nostocspecies are both pioneers and permanent members of the vegeta-tion of deserts, semideserts, dry grasslands and rock surfacesranging in geographical distribution from polar to tropicalregions. Their N input to these biomes can be of utmost import-ance (Dodds etal., 1995; Holst et al., 2009; Caputa et al., 2013).In a carefully mapped Low-Arctic tundra landscape, N2fixation



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by cyanobacteria (0.68kgha1) wastwice the annualwet depos-ition of nitrogen (Stewart et al., 2011). It remains unexploredhow the high water-absorbing capacity and N2 fixation ofNostoccan facilitate the colonization of bare or newly exposedmineral surfaces by mosses and higher plants, thereby formingmore stable vegetation and moreorganic soils. Withthe exposure

of new mineral surfaces behind retreating glaciers on a warmingEarth, this ecosystem service ofNostoc deserves future attention.

ACKNOWL E DGE M E NT S

This work was supported by grants from the Willum KannRasmussen Foundation to the Centre of Excellence for LakeRestoration Research (CLEAR) and from the CarlsbergFoundation. I thank Mikkel ReneAndersen, Michael Kuhl andClaus Lindskov Mller for help and the referees for constructivesuggestions.

L I T E RAT URE CI T E D

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Agusti S, Enriquez S, Frost-Christensen H, Sand-Jensen K, Duarte CM.

1994. Light harvesting among photosynthetic organisms. FunctionalEcology8: 273279.

Al-Najjar MAA, de Beer D, Kuhl M, Polerecky L. 2012.Light utilizationefficiency in photosynthetic microbial mats. Environmental Microbiology14: 982992.

Angeloni SV, PottsM. 1986. Polysome turnover in immobilized cells ofNostoccommune (Cyanobacteria) exposed to water stress.Journal of Bacteriology168: 1036 1039.

Arima H, Horiguchi N, Tkaichi S, et al. 2011.Molecular, genetic and chemo-taxonomic characterization of the terrestrial cyanobacterium Nostoccommune and its neighboring species. FEMS Microbiology Ecology 79:3445.

Badger MR, Price GD. 1992.The CO2concentrating mechanism in cyanobac-teria and microalgae.Physiologia Plantarum84: 606615.

BadgerMR, Price GD.2003. CO2 concentratingmechanisms in cyanobacteria:molecular components, their diversity and evolution. Journal ofExperimental Botany54: 609622.

Bender J, Rodriguez-Eaton S, Ekanemesang UM, Phillips P. 1994.Characterization of metal-binding bio-flocculants produced by the cyano-bacterial component of mixed microbial mats. Applied andEnvironmental Microbiology60: 2311 2315.

Bengtsson R. 1986.The macroalgaNostoc zetterstedtii.Distribution and envir-onmental requirements.Fauna och Flora81: 201212 (in Swedish).

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