Alam Et Al-2015-New Phytologist

Soluble carbohydrates and relative growth rates in chloro-,cyano- and cephalolichens: effects of temperature and nocturnalhydration

Md Azharul Alam, Yngvar Gauslaa and Knut Asbjørn Solhaug

Department of Ecology and Natural Resource Management, Norwegian University of Life Sciences, PO Box 5003, NO-1432�As, Norway

Author for correspondence:Yngvar GauslaaTel: +47 67231784

Email: [email protected]

Received: 11 December 2014Accepted: 5 May 2015

New Phytologist (2015)doi: 10.1111/nph.13484

Key words: hydration regime, Parmelia

sulcata, Peltigera aphthosa, Peltigera canina,photoinhibition, polyols, relative growth rate(RGR), temperature.

Summary

� This growth chamber experiment evaluates how temperature and humidity regimes shape

soluble carbohydrate pools and growth rates in lichens with different photobionts.� We assessed soluble carbohydrates, relative growth rates (RGRs) and relative thallus area

growth rates (RTAGRs) in Parmelia sulcata (chlorolichen), Peltigera canina (cyanolichen) and

Peltigera aphthosa (cephalolichen) cultivated for 14 d (150 lmol m�2 s�1; 12-h photoperiod)

at four day : night temperatures (28 : 23°C, 20 : 15°C, 13 : 8°C, 6 : 1°C) and two hydration

regimes (hydration during the day, dry at night; hydration day : night).� The major carbohydrates were mannitol (cephalolichen), glucose (cyanolichen) and arabitol

(chlorolichen). Mannitol occurred in all species. During cultivation, total carbohydrate pools

decreased in cephalo-/cyanolichens, but increased in the chlorolichen. Carbohydrates varied

less than growth with temperature and humidity. All lichens grew rapidly, particularly at

13 : 8°C. RGRs and RTAGRs were significantly higher in lichens hydrated for 24 h than for

12 h. Strong photoinhibition occurred in cephalo- and cyanolichens kept in cool dry nights,

resulting in positive relationships between RGR and dark-adapted photosystem II (PSII) effi-

ciency (Fv/Fm).� RGR increased significantly with the photobiont-specific carbohydrate pools within all spe-

cies. Average RGR peaked in the chlorolichen lowest in total and photobiont carbohydrates.

Nocturnal hydration improved recovery from photoinhibition and/or enhanced conversion

rates of photosynthates into growth.

Introduction

A lichen is a symbiotic association of a mycobiont and its auto-trophic photobiont(s). The photobiont, an alga and/or a cyano-bacterium, provides its heterotrophic partner with fixed carbon.The mycobiont regulates the light transmission through theupper cortex for its underlying photobionts (Gauslaa & Solhaug,2001), facilitating photosynthesis in hydration periods andreducing photoinhibitory stress, particularly during periods ofdesiccation (Gauslaa & Solhaug, 1996; Solhaug & Gauslaa,2012). As a result of such strategies, lichens can inhabit niches inwhich isolated lichen bionts would not thrive alone. Lichengrowth results from resource gain and subsequent biosynthesis ofcellular compounds minus loss by dispersal, fragmentation orgrazing (Palmqvist, 2000). Their three-dimensional growth(Gauslaa et al., 2009) involves biomass gain through photosyn-thesis, whereas area extension depends on cell division and expan-sion (Palmqvist, 2000). Lichens are nutritionally specializedfungi acquiring carbon from their photobionts (Honegger, 1991;Richardson, 1999) that deliver carbohydrates to their fungal part-ner (Armstrong & Smith, 1994). Green algal photobionts

produce polyols, whereas cyanobacteria produce glucose(Richardson & Smith, 1966, 1968; Hill & Smith, 1972). Thepolyol type depends on the algal partner; Trebouxia, Coccomyxaand Myrmecia export ribitol, Trentepohlia erythritol andHyalococcus sorbitol (Smith et al., 1969; Richardson, 1985). Thetotal pool of polyols in lichens is considered to represent a physi-ological buffer and to protect against various stressors, asreviewed by Farrar (1976c), but relationships between polyolpools and relative growth rates (RGRs) are poorly known.

Studies of growth (Pearson & Benson, 1977; Denison, 1988;Larsson et al., 2009; Bidussi et al., 2013) and metabolism(Solhaug et al., 2003; Solhaug & Gauslaa, 2004) have shown thatlichens are dynamic. As a result of fast lichen responses, short-term growth chamber experiments can be useful tools for study-ing the effects of environmental factors on growth and carbohy-drates. The growth of lichens is coupled to the length of time forwhich thalli are wet and metabolically active during light periods(Palmqvist & Sundberg, 2000). Nevertheless, nocturnal hydra-tion has been shown recently to boost lichen growth (Bidussiet al., 2013), meaning that the regulation of growth is more com-plex than previously thought. Temperature has been considered

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less important than hydration and light (Nash, 1996), althoughit impacts significantly photosynthesis and respiration. High tem-peratures decrease carbon gain as a result of increased respiration,particularly at nights (Lange et al., 1994; Zotz et al., 1998).Although lichen growth depends on carbohydrate transfer fromthe photobiont to the mycobiont and the subsequent formationof new cells, few functional studies have assessed how environ-mental factors impact lichen-soluble carbohydrate pools and pro-ductivity. Carbohydrate is the main substrate for respiration.Almost 50% of the daily carbon gain (Palmqvist, 2000), or morein warm humid habitats (Zotz et al., 1997), can be consumed bylichen respiration. At the same time, respiration delivers theenergy needed for lichen growth, as reviewed by Palmqvist et al.(2008).

Here, we investigate how environmental factors affect solublelichen carbohydrates and growth. More specifically, we report theeffects of moisture and temperature regimes on lichen growthand carbohydrate pools in growth chamber experiments usingthree common and locally dominant foliose lichens: the cephalo-lichen Peltigera aphthosa with both green algal (Coccomyxa) andcyanobacterial photobionts (Nostoc) located in external cephalo-dia; the cyanolichen Peltigera canina with Nostoc as its onlyphotobiont; and the chlorolichen Parmelia sulcata with the greenalga Trebouxia. These functional groups were selected to comparecarbohydrate pools and growth in lichens with different photo-bionts. We study growth as the RGR, relative thallus area growthrate (RTAGR) and change in specific thallus mass (DSTM, a sim-ple proxy for the change in thallus thickness) in growth chamberswith optimal conditions for cyano- and cephalolichen membersof Lobaria (Bidussi et al., 2013). Our main aims are: to quantifythe effects of temperature and humidity on soluble carbohydratesin the three functional groups; to search for and assess the rela-tionships between growth and soluble carbohydrate pools; and touse the results to discuss the physiological buffering hypothesis ofpolyols in lichens (Farrar, 1976c) by comparing two hydrationtreatments: a continuous hydration for 14 d imposing severeenvironmental stress according to Farrar (1976b), and a mildertreatment with alternations between hydration and desiccation.

Materials and Methods

Lichen materials

Lichens were collected in autumn 2013 in south-east Norway,120–150 m above sea level (asl). The Peltigera species were col-lected on shallow soils near Koll�asen, Ski: P. aphthosa (L.) Willd.from a north-west-facing open forest edge on a slope facing apond (59.753°N, 10.939°E) and P. canina (L.) Willd. fromnorth-facing forest edges facing open land along a local road(59.741°N, 10.941°E). Parmelia sulcata Taylor was collectedfrom the bark of many Tilia cordata on open, north-facing farmland at Rustad in�As (59.666°N, 10.817°E).

Lichens were air dried, cleaned from debris and stored at�20°C for 1 month, a recommended storage for lichens for laterphysiological experiments (Honegger, 2003). Before cultivation,95 young and healthy thalli of each species without reproductive

structures were randomly selected. These dried samples were keptin the laboratory at 20°C for 48 h before recording the air-driedmass (DM; � 0.1 mg). Ten thalli of each species were addition-ally oven dried for 24 h at 70°C before measuring oven DM. Theoven-dried mass/air-dried mass ratio for these sacrificed thalli wasused to calculate the oven DM for all experimental (n = 80) andcontrol (n = 5) thalli. The controls were directly analyzed for car-bohydrates (see later) to estimate the original concentration ofcarbohydrates in the field (start values). The 80 experimentalthalli were excessively sprayed with de-ionized water before thethallus area (A) was measured by an LI3100 Li-Cor leaf areameter; Lincoln, NE, USA).

Experimental design

The growth experiment, modified after Bidussi et al. (2013), wasrun in Sanyo MLR-351 growth chambers (Sanyo Electric, Osaka,Japan) for 14 d with four day : night temperature regimes(28 : 23°C, 20 : 15°C, 13 : 8°C, 6 : 1°C) and two hydration treat-ments (�12 h wet (day) + 12 h dry (night) and �12 h wet(day) + 12 h wet (night); in short referred to as 12- and 24-h wettreatments). The photoperiod (photosynthetic photon fluencerate, 150 lmol m�2 s�1) was 12 h. Fluorescent lamps (Mitsubi-shi/Osram FL 40SS W/37) were used as light source. Twentythalli for each species–temperature combination were cultivatedby placing one specimen of the three species on top of 10 layersof filter paper in each of 20 open Petri dishes (diameter, 9 cm).Peltigera aphthosa, P. canina and Parmelia sulcata had DMstart of184.1� 4.7, 175.2� 5.4 and 240.5� 5.3 mg (mean� 1SE;n = 80), respectively, with corresponding A values of 13.3� 0.3,14.4� 0.4 and 10.5� 0.2 cm2. The thallus size before the startdid not differ significantly between the treatments (ANOVA;data not shown). Lichens were hydrated by spraying with de-ion-ized water because, during a previous 3-month growth experi-ment in oligotrophic forests including clearcuts, added nutrientshad just a weak, positive effect on lichen growth compared withthe natural rain and de-ionized water treatments (P = 0.03; Gau-slaa et al., 2006). Preliminary experiments were run to adjustadded water for species and temperature to keep all specieshydrated until nearly the end of the day. In the final experiment,10 Petri dishes with lichens were sprayed at both the start of theday and at night (24 h of hydration); the 10 other dishes weresprayed at the start of the day only (12 h of hydration). Theformer treatment kept lichens hydrated for most of the day andthe entire dark period; the latter treatment kept them moist onlyduring most of the day. To ensure the necessary desiccation inlichens (see Farrar, 1973) at all treatments in the evening, thalliwere transferred to dry filter papers to accelerate drying.

Carbohydrate analyses

The carbohydrates were analyzed following Gordy et al. (1978);100 mg DM of each specimen were ground to a fine powder inan Eppendorf tube with a ball mill (Retsch MM 301, RetschGmbH, Haan, Germany). Soluble carbohydrates were extractedby heating samples in a 45-kHz frequency ultrasonic bath (Model

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USC 200 TH, VWR, Leuven, Belgium) at 60°C for 30 min in80% ethanol with two repeated extractions of ethanol. For eachextraction, extracts were centrifuged at 12 500 g for 3 min. Thesupernatants from the two repeated extractions were combined.The ethanol was completely evaporated from the supernatant at60°C using a vacuum desiccator (Eppendorf AG 22331, Ham-burg, Germany). Afterwards, we added 1.5 ml water to theextract and heated at 60°C for 30 min. The extract was centri-fuged at 12 500 g for 3 min and the supernatant was filteredthrough a 0.45-lm GHP membrane filter (Millipore) beforechromatography.

We ran the extracts on high-performance liquid chromatogra-phy (Agilent 1200 series of HPLC, Agilent Technologies, Wald-bronn, Germany) to separate and identify the carbohydrates.Carbohydrates were separated on the basis of their differentialadsorption characteristics and analyzed by passing the solutionthrough a column (Agilent Hi-Plex Ca USP L19, 4.09 250 nm2,8 lm; p/n PL1570-5810), specialized for separating polyols,detected by a refractive index detector. For the mobile phase, amixture of 30% acetonitrile and 70% water was used as solvent.The flow rate was 0.3 ml min�1 and the column temperature was90°C (Ball & Lloyd, 2013).

Growth, maximum photochemical efficiency ofphotosystem II (PSII) and chlorophylls

We measured RGR = (loge(DMend/DMstart))9 1000/Dt (mg g�1

d�1) and RTAGR = (loge(Aend/Astart))9 100/Dt (mm2 cm�2

d�1). Dt is the 14 d between the start and end at which DM (g)and A (cm2) were measured (Evans, 1972). The specific thallusmass (STM =DM/A) was calculated at the start and end of theexperiment. Percentage changes in STM were calculated asDSTM = 1009 (STMend – STMstart)/STMstart.

After their last night during cultivation, all thalli were sprayedwith water and dark adapted for 15 min. Then, the dark-adaptedphotochemical efficiency of photosystem II (Fv/Fm) was mea-sured with a PAM 2000 fluorometer (Walz, Effeltrich, Ger-many). In chloro- and cephalolichens, Fv/Fm is normally > 0.7;cyanolichens have consistently lower values (Jensen & Kricke,2002). In cyanolichens, such as P. canina, slightly higher PSIIefficiency (ɸPSII) than Fv/Fm can be measured under mediumlight level because the non-photochemical quenching in cyano-bacteria is higher in darkness than in medium light (Campbell &€Oquist, 1996; Sundberg et al., 1997). Cyanolichens have maxi-mal ɸPSII in low–medium light, not in darkness. Thereby, themaximal PSII efficiency for cyanolichens is not the dark-adaptedFv/Fm, but ɸPSII in low light. The dark-adapted Fv/Fm is just< 10% lower than the maximal ɸPSII (Solhaug et al., 2014).Because we planned to analyze the results for each species sepa-rately, we decided to use the dark-adapted Fv/Fm values for alllichen species.

Chlorophyll contents were extracted following Palmqvist &Sundberg (2002); 10–12 mg DM from each specimen wasground to a powder in an Eppendorf tube with a ball mill; 1.5 mlof dimethylsulfoxide (DMSO) with MgCO3 was added. Thetubes were incubated at 60°C in a water bath for 40 min and

repeatedly vortexed. Afterwards, extracts were centrifuged at18 000 g for 5 min and the absorbance of the supernatant wasmeasured (Shimadzu UV2101 PC spectrophotometer, Kyoto,Japan) at 665 and 649 nm; the baseline absorbance was at750 nm. Chlorophyll a and b (Chla and Chlb) concentrations(mg g�1) were calculated according to Wellburn (1994).

Statistical analyses

General linear models (GLMs) were run in Minitab 16 (MinitabInc., State College, PA, USA) to study the effects of treatments(temperature and humidity regimes) on the measured parametersin each species separately. In growth analyses (RGR, RTAGR,DSTM), we used STMstart, Chla and total carbohydrate concen-tration as covariates. In carbohydrate analyses (glucose, ribitol,arabitol, mannitol and total carbohydrate), we used STMstart andChla as covariates. Some variables were log-transformed to meetthe requirements for the GLM. Fv/Fm did not follow a normaldistribution or satisfy the equal variance test. Furthermore, as Fv/Fm was strongly coupled to hydration regime, Fv/Fm was notincluded in any GLMs. Means� 1SE are given in the text.

Results

Carbohydrates

Under field conditions, total measured soluble carbohydrate con-centrations in our control samples representing the start valueswere 11.8� 0.8% (P. canina), 12.9� 1.5% (P. aphthosa) and4.2� 0.6% (P. sulcata; Fig. 1; Table 1). The substantially lowercarbohydrate pool in P. sulcata was related to its high STM(Fig. 1o); area-based total carbohydrate pools varied from0.99� 0.03 (P. canina) to 1.19� 0.04 g m�2 (P. sulcata). After14 d of cultivation, the average total concentration acrosstreatments had decreased in P. aphthosa (7.7� 0.2%) andP. canina (7.8� 0.2%), but increased in P. sulcata (5.0� 0.1%;Table 1).

The cyanolichen P. canina contained glucose and mannitol;the cephalolichen P. aphthosa contained glucose, ribitol, arabitoland mannitol, whereas the chlorolichen P. sulcata contained ribi-tol, arabitol and mannitol (Fig. 1a–l). All soluble carbohydratesin P. aphthosa declined during cultivation (Fig. 1a,c,e,g); onlyglucose declined in P. canina (Fig. 1b), whereas they stayed con-stant (Fig. 1d,f) or increased (Fig. 1i) in P. sulcata. Only mannitoloccurred in all species, but in greater amounts in P. canina(3.41� 0.09%) and P. aphthosa (3.03� 0.06%) than inP. sulcata (1.27� 0.07%; n = 80). The major cyanolichen carbo-hydrate was glucose (4.49� 0.13%) vs arabitol (3.15� 0.09%)for the chlorolichen. The glucose concentration was 1.8 timeshigher in the cyanolichen than in the cephalolichen. Moreover,the ribitol concentration in the chlorolichen (0.69� 0.03%) was2.5 times higher than in the cephalolichen, 4.7 times higher whengiven per thallus area. The overall species-wise ratios of myco-(mannitol, arabitol) to photobiont (glucose, ribitol) carbohydratedid not change much from the start to the end of the experiment,but were substantially higher in P. sulcata (6.98� 0.24) than in


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Glu

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Cultivation temperature (day : night °C)6/1 13/8 20/15 28/23Field 6/1 13/8 20/15 28/23Field 6/1 13/8 20/15 28/23Field

Peltigera aphthosa Peltigera canina

Parmelia sulcata

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Fig. 1 Concentration of carbohydrates (glucose, ribitol, arabitol, mannitol, total carbohydrates; a–l) and specific thallus mass (STM; m–o) at the start of theexperiment (white columns) in Peltigera aphthosa, Peltigera canina and Parmelia sulcata, as well as after 14 d of cultivation in four temperature regimes(28 : 23, 20 : 15, 13 : 8 and 6 : 1°C day : night) all with a 12-h daily photoperiod (150 lmol photonsm�2 s�1) and two diurnal hydration treatments: wet for12 h during the daytime (grey columns) and wet for 12 h during the daytime + 12 h at night (black columns). Error bars indicate +1 SE; n = 5 for values atthe start (field level); n = 10 for values after treatments.



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P. aphthosa (1.81� 0.05), and P. canina (0.82� 0.03) in partic-ular (Table 1; experimental thalli).

In P. aphthosa, temperature significantly influenced the con-centration of all carbohydrates (P < 0.001; Table 2) exceptglucose. They decreased at the highest temperature, particularlyin those hydrated for 24 h (Fig. 1c,e,g,j). Ribitol and arabitolreached their maxima at intermediate temperatures (Fig. 1c,e),yet the concentration of these two carbohydrates did not correlatewith each other (r = 0.109; P = 0.338). The hydration regimeinfluenced mannitol only (P = 0.02; Table 2) by slight reductions

in thalli kept wet at nights. At the two lower temperatures, carbo-hydrates in continuously hydrated P. aphthosa were higher or ashigh as in those experiencing nocturnal desiccation. At highertemperatures, this was reversed with lower concentrations inthalli hydrated for 24 h, evidenced by a significant (P < 0.02)temperature9 humidity interaction for all carbohydrates exceptmannitol (Table 2). Thus, continuous hydration reduced carbo-hydrates at high, but not low, temperatures. The strongestpositive correlation between carbohydrates in the cephalolichenwas between glucose and mannitol (r = 0.586; P < 0.001),

Table 1 Mean species-specific soluble carbohydrate pools as a percentage of dry mass (� 1SE) measured in field controls (n = 5), representing start values,as well as in experimental thalli (n = 80) after 14 d of cultivation

Parameter

Peltigera aphthosa Peltigera canina Parmelia sulcata

Field Experiment Field Experiment Field Experiment

Total carbohydrates 12.85� 0.83 7.68� 0.19 11.80� 0.83 7.94� 0.17 4.20� 0.55 5.11� 0.13Total polyols 8.68� 1.12 5.18� 0.14 3.41� 0.40 3.45� 0.08 4.20� 0.55 5.11� 0.13Mycobiont carbohydrates (M) 8.26� 1.11 4.91� 0.13 3.41� 0.40 3.45� 0.08 3.59� 0.42 4.42� 0.11Photobiont carbohydrates (P) 4.59� 0.46 2.77� 0.07 8.39� 0.64 4.49� 0.13 0.62� 0.13 0.69� 0.03M : P ratio 1.79� 0.12 1.81� 0.05 0.41� 0.05 0.82� 0.03 6.20� 0.56 6.98� 0.24

Mycobiont carbohydrates: arabitol and mannitol; photobiont carbohydrates: ribitol and glucose. Here, the term carbohydrates includes polyols andglucose.

Table 2 General linear model (GLM) for carbohydrates (glucose, ribitol, arabitol, mannitol and total carbohydrates) in Peltigera aphthosa, Peltigera caninaand Parmelia sulcata cultivated for 14 d in four temperature regimes (T) and two hydration treatments (H)

Parameterdf

Glucose

df

Ribitol*

df

Arabitol*

df

Mannitol(*)

df

Total

Source F P F P F P F P F P

Peltigera aphthosaTemperature 3 1.87 0.142 3 8.40 0.000 3 94.27 0.000 3 16.19 0.000 3 27.11 0.000Humidity 1 3.45 0.067 1 0.74 0.391 1 1.51 0.223 1 5.64 0.020 1 0.52 0.475T9H 3 4.20 0.009 3 3.64 0.017 3 7.07 0.000 3 1.42 0.245 3 6.31 0.001STMstart 1 17.86 0.000(+) 1 36.59 0.000(�) 1 4.83 0.031(+) 1 4.83 0.031(+)

Error 71 71 72 71 71Total 79 79 79 79 79r2adj 0.335 0.430 0.791 0.422 0.564Peltigera caninaTemperature 3 9.79 0.000 3 9.87 0.000 3 11.81 0.000Humidity 1 23.08 0.000 1 1.03 0.313 1 9.25 0.003T9H 3 0.64 0.590 3 1.09 0.359 3 0.43 0.733STMstart 1 18.75 0.000(+) 1 7.88 0.006(�) 1 5.70 0.020(+)

Chla* 1 8.63 0.005(+) 1 21.97 0.000(+)

Error 70 69 69Total 78 78 78r2adj 0.439 0.284 0.381Parmelia sulcata

Temperature 3 3.37 0.023 3 13.06 0.000 3 163.12 0.000 3 7.20 0.000Humidity 1 14.47 0.000 1 7.50 0.008 1 6.12 0.016 1 14.41 0.000T9H 3 2.63 0.057 3 2.45 0.071 3 2.91 0.040 3 2.03 0.118STMstart 1 28.21 0.000(�) 1 13.95 0.000(�) 1 14.54 0.000(�)

Chla* 1 31.99 0.000(+) 1 27.02 0.000(+) 1 24.10 0.000(+)

Error 68 68 71 68Total 77 77 78 77r2adj 0.504 0.557 0.864 0.451

Specific thallus mass at start (STMstart) and chlorophyll a (Chla) are included when they contribute significantly.*The GLM was run on log-transformed values. (*)Mannitol was also log-transformed in P. sulcata. From the start, all covariates (specific thallus mass at startSTMstart and/or Chla) were included. The least significant covariate was stepwise eliminated until the remaining covariates, if any, contributed significantly.The included covariates show a positive (+) or negative (�) relationship with the respective carbohydrates.





followed by arabitol and mannitol (r = 0.465; P < 0.001). Glu-cose (P < 0.001) and mannitol (P = 0.031) increased withincreasing STMstart, whereas ribitol (P < 0.001) decreased(Table 2). The Chla content was not a significant covariate forany measured carbohydrates in P. aphthosa.

In P. canina, both temperature and humidity influenced theconcentration of glucose and total carbohydrates, whereas manni-tol was affected by temperature alone (Table 2). Glucose andmannitol, which were weakly positively correlated (r = 0.270;P = 0.016), peaked at 13 : 8°C–20 : 15°C (Fig. 1b,h). The con-centration of glucose was consistently higher (P < 0.001) in thallihydrated for 24 h (4.9� 0.15%) than for 12 h (3.9� 0.16%;Fig. 1b; Table 2). The temperature9 humidity interaction wasnot significant (Table 2). High STMstart was associated withincreased glucose (P < 0.001) and decreasing mannitol(P = 0.006) concentrations; high Chla content was associatedwith high mannitol and total carbohydrate concentrations(P < 0.001; Table 2).

In P. sulcata, ribitol and arabitol responded in similar ways,evidenced by a strong positive correlation between their concen-trations (r = 0.756; P < 0.001). They decreased at 28 : 23°C(Fig. 1d,f), whereas mannitol (Fig. 1i) increased with temperature(Table 2). Continuous hydration lowered the carbohydrates atthe three lowest temperatures (Fig. 1d,f,i), but with no stronginteraction term (Table 2). Chla content was a positive andSTMstart a negative covariate for ribitol, arabitol and total carbo-hydrates (P < 0.001; Table 2).

Chlorophyll fluorescence

After cultivation, the dark-adapted photosystem II activity (Fv/Fm) showed strong species-specific responses (Fig. 2o–q). Aver-aged across treatments, the chlorolichen had the highest values(0.647� 0.012), followed by the cephalo- (0.506� 0.018) andcyanolichen (0.176� 0.017; n = 80). Temperature and/or hydra-tion regimes influenced Fv/Fm in all species (Fig. 2o–q). InPeltigera, Fv/Fm increased with temperature (Fig. 2o,p), whereasthe chlorolichen (Fig. 2q) had similar values at all treatmentsapart from the strong depression after nocturnal desiccation at28 : 23°C. Moreover, all species had substantially higher Fv/Fmfor thalli hydrated for 24 h than for those hydrated for 12 h.Thalli kept wet for 24 h reached 0.572� 0.021, 0.256� 0.025and 0.677� 0.009 (n = 40) for P. aphthosa, P. canina andP. sulcata, respectively, whereas those hydrated once a day hadthe respective means of 0.439� 0.025, 0.088� 0.013 and0.617� 0.022. Peltigera canina had almost three times higher Fv/Fm values when hydrated for 24 h compared with 12 h (Fig. 2p).In both Peltigera species, photoinhibition was substantial at thelowest temperature and in thalli kept dry at night, whereasP. sulcata was only photoinhibited after dry nights at the highesttemperature (Fig. 2o–q).

Chlorophylls

The average Chla concentration across treatments (n = 80) washighest in P. sulcata (1.44� 0.05 mg g�1), followed by

P. aphthosa (1.10� 0.04 mg g�1) and P. canina (0.76�0.04 mg g�1). Converted to contents per thallus area (Fig. 2j–l),P. sulcata (0.335 g m�2) had 2.3 times more Chla thanP. aphthosa and 3.4 times more Chla than P. canina. The con-trasts between species were strong (Fig. 3). Treatments did nothave large effects, but nocturnal desiccation reduced Chla slightlyin the chlorolichen (Fig. 2j,l; P = 0.035; two-way ANOVA notshown) with a similar tendency in the cephalolichen. Further-more, temperature affected Chla slightly in the cyanolichen(Fig. 2k; P = 0.016; two-way ANOVA not shown). The Chl a/bratio (Fig. 2m,n) was higher (P < 0.001) in P. sulcata (2.94�0.05) than in P. aphthosa (2.67� 0.03).

Relative growth rate (RGR)

Average RGR across treatments (n = 78–80) was 3.96�0.46 mg g�1 d�1 for P. canina, 4.58� 0.29 mg g�1 d�1 forP. aphthosa and 5.92� 0.35 mg g�1 d�1 for P. sulcata. Convertedto percentage DM gain in 14 d, the rates were 6.69� 0.42,6.36� 0.76 and 8.73� 0.52%, respectively. Maximal individualRGR values ranked from 10 (P. aphthosa) to 15 mg g�1 d�1

(P. canina), equivalent to a percentage DM gain of 15% and23%.

Temperature had a strong impact on RGR in all species(P < 0.001; Table 3). For the 24-h hydration treatment, RGRpeaked at 13 : 8°C in all species (Fig. 2a–c). At this treatment,RGR was highest in P. canina (9.43� 0.88 mg g�1 d�1; Fig. 2b),the species showing the highest variation in growth between treat-ments. For 12 h of hydration, RGR was fairly constant and highat the three highest temperatures in both Peltigera species(Fig. 2a,b), and negative or close to zero at the 6 : 1°C regime.The highest mean RGR for P. sulcata occurred at 13 : 8°C inboth hydration treatments with high growth also at 6 : 1°C, butRGR declined strongly at 28 : 23°C (Fig. 1c). The hydrationregime significantly affected RGR in both Peltigera species, butnot in P. sulcata (Table 3). The highest RGRs for P. aphthosa andP. canina (averaged for each humidity; n = 40) consistentlyoccurred at 24 h of hydration (5.34� 0.34 and 5.39�0.67 mg g�1 d�1; respectively) and the lowest RGRs in thosehydrated for 12 h (3.81� 0.43 and 2.56� 0.58 mg g�1 d�1).

Species-wise mean RGRs increased with log-transformed meanChla contents (Fig. 3). In species-specific GLMs (Table 3), Chlacontent was a significant positive covariate for RGR only inP. aphthosa (P = 0.006). STMstart was a highly significant covari-ate in P. sulcata; thick thalli had low RGR (P < 0.001, Table 3).Furthermore, RGRs within all three species increased with theirrespective total carbohydrate concentration when used as a covar-iate (P < 0.001; Table 3). In linear regression analyses, RGRhighly significantly increased with at least one of the photobiontcarbohydrates (glucose and/or ribitol); by contrast, the mycobi-ont-specific mannitol did not (Peltigera species), or negatively(P. sulcata), correlated with RGR (data not shown). Thereby,RGR declined with the myco-/photobiont carbohydrate ratio,particularly in the chloro- and cephalolichen (P ≤ 0.001), but alsoin the cyanolichen (P = 0.012). However, this contrasts thestrong positive interspecific relationship between the mean RGRs



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RG

R (m

g g–

1 d–

1 )

–2

0

2

4

6

8

10

Wet 12 hWet 24 h

RT A

GR

(mm

2 cm

–2 d

–1)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Chl

orop

hyll

a (g

m–2

)

0.0

0.1

0.2

0.3

0.4

Chl

orop

hyll

a/b-

ratio

0

1

2

3

6/1 13/8 20/15 28/23 6/1 13/8 20/15 28/23

Peltigera aphthosa Peltigera canina Parmelia sulcata

Cha

nge

in S

TM (m

g cm

–2)

–1.0

–0.5

0.0

0.5

1.0

1.5

2.0

F v/F

m

0.0

0.2

0.4

0.6

Cultivation temperature (day : night °C)6/1 13/8 20/15 28/23

(a)

(d)

K

(e)

(b) (c)

(n)(m)

(l)(k)(j)

(i)(h)(g)

(f)

(q)(p)(o)

Fig. 2 Growth rate (RGR, relative growth rate; RTAGR, relative thallus area growth rate), change in specific thallus mass during cultivation (DSTM),Chla and dark-adapted photosystem II activity (Fv/Fm) in Peltigera aphthosa, Peltigera canina and Parmelia sulcata cultivated for 14 d in four temperatureregimes (28 : 23, 20 : 15, 13 : 8 and 6 : 1°C day : night temperature) all with a 12-h daily photoperiod (150 lmol photonsm�2 s�1) and two hydrationtreatments (grey columns, wet for 12 h during the daytime; black columns, wet for 12 h during the daytime + 12 h at night). Error bars indicate + 1SE;n = 10.





(given in Fig. 3) and mean myco-/photobiont carbohydrate ratios(Table 1). Finally, RGR also declined with increasing photoinhi-bition, evidenced by decreasing Fv/Fm within all species (Fig. 4;P < 0.001; r2adj = 0.152–0.444).

Relative thallus area growth rate (RTAGR) and change inspecific thallus mass (ΔSTM)

Average RTAGR across all treatments was 0.72�0.05 mm2 cm�2 d�1 for P. aphthosa, 0.24� 0.04 mm2 cm�2 d�1

for P. canina and 0.48� 0.03 mm2 cm�2 d�1 for P. sulcata(n = 80). Temperature and humidity significantly influencedRTAGR in all species (Table 3), but with no significant interac-tion. All species had highest RTAGR at 13 : 8°C and 24 h ofhydration (Fig. 2d–f). RTAGR increased strongly with STMstart

in P. aphthosa (P < 0.001) and with the total carbohydrate con-centration in P. sulcata (P = 0.022; Table 3).

The ΔSTM value facilitates comparisons of biomass and areagains, because constant STM necessarily implies balanced A andDM gains. However, each species showed a distinct ΔSTM

Mean chlorophyll a content (mg m–2) 80 90 200 300100

Mea

n re

lativ

e gr

owth

rate

(mg

g–1

d–1 )

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

Peltigera canina

Peltigera aphthosa

Parmelia sulcata

Fig. 3 Species-specific mean values of relative growth rate (RGR) andchlorophyll a content across treatments for a 14-d growth chamberexperiment for Peltigera canina, Peltigera aphthosa and Parmelia sulcata.Vertical and horizontal bars show �1SE (n = 80). The regression line isshown.

Table 3 General linear model (GLM) for growth rates (RGR, RTAGR, DSTM) in Peltigera aphthosa, Peltigera canina and Parmelia sulcata cultivated for 14 din four temperature regimes (T) and two hydration treatments (H)

Parameterdf

RGR

df

RTAGR

df

DSTM

Source F P F P F P

Peltigera aphthosaTemperature 3 39.59 0.000 3 6.61 0.001 3 2.99 0.037Humidity 1 10.96 0.006 1 6.14 0.016 1 0.52 0.473T9H 3 2.16 0.100 3 0.31 0.819 3 1.74 0.166STMstart 1 24.98 0.000 1 25.42 0.000Chla* 1 8.17 0.006Carbohydrates 1 23.18 0.000Error 70 71 71Total 79 79 79r2adj 0.691 0.359 0.257Peltigera canina

Temperature 3 27.98 0.000 3 10. 60 0.000 3 7.58 0.000Humidity 1 11.92 0.001 1 5.95 0.017 1 1.01 0.318T9H 3 1.06 0.371 3 0.36 0.782 3 0.42 0.740Chla* 1 10.01 0.002Carbohydrates 1 14.83 0.000 1 7.25 0.009Error 68 72 69Total 76 79 78r2adj 0.615 0.287 0.415Parmelia sulcataTemperature 3 23.77 0.000 3 6.77 0.000 3 7.00 0.000Humidity 1 0.14 0.706 1 11.91 0.001 1 13.68 0.000T9H 3 0.33 0.801 3 0.93 0.431 3 1.18 0.325STMstart 1 17.36 0.000 1 11.61 0.001Chla* 1 6.22 0.015Carbohydrates 1 25.22 0.000 1 5.51 0.022Error 69 70 69Total 78 78 78r2adj 0.540 0.240 0.351

Significant covariates were included: specific thallus mass at start (STMstart), Chla and/or total carbohydrate concentration.*The GLM was run on log-transformed values. From the start, all covariates were included. The least significant covariate was stepwise eliminated until allremaining covariates contributed significantly.RGR, relative growth rate; RTAGR, relative thallus area growth rate; DSTM, change in specific thallus mass.



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pattern (Fig. 2g–i). Peltigera aphthosa consistently reduced STM(Fig. 2g) from its field level (see Fig. 1m–o), with only weakeffects of temperature (P < 0.037; Table 3), meaning that Aincreased more than DM. In P. canina, ΔSTM increased withtemperature at both hydration treatments (P < 0.001; Table 3;Fig. 2h), implying increasingly higher DM than A gain withincreasing temperature. High Chla content and total carbohy-drate concentration were associated with high ΔSTM (Table 3).A more complex pattern occurred in P. sulcata with decliningΔSTM at increasing temperature and with 12 h of hydration(P < 0.001), and an increase from 24- to 12-h humidity regimes(P < 0.001; Fig. 2i), but no significant interaction (Table 3). TheChla content in P. canina and P. sulcata was a significant covari-ate that increased with ΔSTM (Table 3).

Discussion

Carbohydrates

The reported carbohydrates correspond to those known inlichens with similar photobionts (Lewis & Smith, 1967; Richard-son & Smith, 1968; Armstrong & Smith, 1994). Our green algalphotobionts (Coccomyxa and Trebouxia) provide their myco-bionts with ribitol; the cyanobacteria provide glucose (Lewis &Smith, 1967; Richardson, 2002). However, glucose concentra-tions as high as those in P. aphthosa (Fig. 1a) have not beenrecorded previously in cephalolichens. The glucose was probablyproduced by Nostoc in the cephalodia. Secondary Nostoc photo-bionts have been thought to contribute fixed nitrogen rather thancarbon, because nitrogen fixation is energy demanding (Marsch-ner, 1993). It is not clear whether the reported glucose passes onto the mycobiont, or accumulates in the photobiont in, for

example, extracellular sheaths around cyanobacterial cells(Honegger, 1991). The strong correlation between RGR and glu-cose in P. aphthosa and P. canina suggests that glucose is a sourceof biomass growth in both species, and that glucose inP. aphthosa is not just retained in cyanobacteria.

The species-specific average total polyol concentrations(excluding glucose) in our field-harvested lichens (3.4–8.7%:Table 1) are within the normal range of 2–10%, as reviewed byPalmqvist (2000). By including glucose, the average value of totalsoluble carbohydrates in Peltigera before the start of the experi-ment was 12–13% (Table 1). Honegger et al. (1993) reported≤1% polyols in 11 cultured mycobionts, showing that high poly-ol pools characterize the symbiotic state. High concentrations ofthe fungal polyols, arabitol and mannitol, in our chloro- andcephalolichens suggest that the largest soluble carbohydrate poolsare located in their mycobiont, and that much assimilated CO2

in the photobiont eventually is released to its heterotrophic part-ner (Fahselt, 1994). By contrast, our cyanolichen had high poolsof photobiont-derived glucose, resulting in a myco-/photobiontratio of soluble carbohydrates as low as 0.41� 0.05 in field-harvested thalli (Table 1).

Few studies (Armstrong, 1975; Armstrong & Smith, 1993;Armstrong & Smith, 1994) have emphasized the relationshipsbetween environmental conditions and carbohydrate allocationpatterns. The photobiont provides carbohydrates used for lichengrowth and respiration. For Peltigera, the lower carbohydratepools (Table 1) in fast-growing experimental thalli than in slowergrowing field specimens suggest that these lichens use their solu-ble carbohydrates with a faster turnover for growth and mainte-nance in growth chambers than they do in nature. In P. aphthosa,reduced ribitol at extreme temperatures (Fig. 1c) can be explainedby strong photoinhibition at low temperature (Fig. 2o) and highrespiration at high temperatures. The higher ribitol pool inP. sulcata than in P. aphthosa (Fig. 1) is consistent with Richard-son (2002), emphasizing that Trebouxia lichens have four timesmore ribitol than lichens with other green algae. In P. sulcata,higher Chla contents per thallus area may have contributed tosubstantial ribitol production. Less glucose in P. canina hydratedfor 12 h than for 24 h (Fig. 1b) is consistent with the strong pho-toinhibition after dry nights (Fig. 2o) that probably results inlower photosynthesis at 12 h of hydration. The high myco-/photobiont ratio of soluble carbohydrates in both P. sulcata(Table 1) and Platismatia glauca (Palmqvist & Dahlman, 2006)suggests that chlorolichen mycobionts represent a strong sink ofsoluble carbohydrates. In both P. aphthosa and P. sulcata, thearabitol pool decreased at the maximum temperature (Fig. 1),whereas mannitol increased strongly in P. sulcata, supporting theview that mannitol is synthesized from arabitol. Under stress,arabitol decreases and mannitol increases (Farrar, 1973). Arabitolvaried with external conditions (Fig. 1; Table 2), consistent withthe hypothesis that arabitol is a short-term flexible reserve (Lewis& Smith, 1967; Armstrong & Smith, 1993). According to Rich-ardson (2002), large pools of mannitol can support several daysof respiration; mannitol functions as a low-molecular-weightstorage compound (Sturgeon, 1985) with rapid turnover (Farrar,1988).

Fv/Fm

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

Rel

ativ

e gr

owth

rate

(mg

g–1

d–1 )

–5

0

5

10

15 Peltigera aphthosaPeltigera caninaParmelia sulcata

Fig. 4 The relationships between relative growth rate (RGR) and Fv/Fmmeasured at the end of the experiment in the thalli of Peltigera aphthosa,Peltigera canina and Parmelia sulcata. All species showed highlysignificant linear regressions (P < 0.001) between RGR and Fv/Fm:cephalolichen RGR =�0.54 + 10.119 (Fv/Fm); r

2adj = 0.444; cyanolichen

RGR = 0.87 + 18.229 (Fv/Fm); r2adj = 0.395; chlorolichen

RGR =�1.18 + 10.979 (Fv/Fm); r2adj = 0.152.





Temperature had a much lower impact on carbohydrate con-centrations in lichens than in plants, probably because tempera-ture and photoperiod are important signals in plants, regulatingthe allocation of photoassimilates to storage or growth as anadaptation to seasonal growth and dormancy (Wardlaw, 1990and references therein). The low variation in the lichen carbohy-drate pool is probably related to active growth in all seasons withno dormancy (Larsson et al., 2012).

Photoinhibition

Fv/Fm is a commonly used viability measure in photosyntheticorganisms. In the absence of other harmful agents, depression ofFv/Fm indicates photoinhibition. Although P. sulcata had normalFv/Fm, the two Peltigera species were photoinhibited (consistentwith Demmig-Adams et al., 1990b; Manrique et al., 1993; Gau-slaa & Solhaug, 1996), particularly at low temperature. Thestrong photoinhibition in P. canina is consistent with Demmig-Adams et al. (1990a,b) reporting stronger high-light susceptibilityin hydrated cyanolichens than in chloro-/cephalolichens. Cyan-olichens lack the high-light-protecting zeaxanthin–violaxanthincycle (Demmig-Adams et al., 1990a,b) and their PSII reactioncenter protein, D1, has an inherently lower resistance to photoin-hibition (Clarke et al., 1993). During rehydration, antioxidantsdecrease and lichens produce reactive oxygen species (ROS)(Weissman et al., 2005), which may lower Fv/Fm. The strongphotoinhibition in P. aphthosa is consistent with the reportedhigh-light susceptibility in other cephalolichens (Gauslaa &Solhaug, 1996). However, while desiccated, cephalolichens aremore susceptible to high light than cyanolichens (Gauslaa et al.,2012), suggesting that photoinhibition in P. aphthosa occurredduring drying at the end of the day.

The chlorolichen was hardly photoinhibited, regardless ofhydration regime (Fig. 2q). This may explain why its carbohy-drate concentration did not drop during cultivation (Table 1).The photoinhibition in the chlorolichen at the highest tempera-ture for thalli hydrated for 24 h (Fig. 2q) was accompanied by adrop in Chla (Fig. 2l) and visible browning, which did not occurat lower temperatures. These symptoms probably indicatesubstantial irreversible damage.

Growth rates

By comparing field (Dahlman & Palmqvist, 2003; Gauslaa &Goward, 2012; Larsson et al., 2012) and growth chamber (Bid-ussi et al., 2013) measurements of RGR and/or RTAGR, lichenscan grow much more rapidly in a growth chamber than they doin nature. Our maximal mean RGR obtained was recorded inP. canina for the 24-h hydration treatment at 13 : 8°C. Thismean matched the growth rate recorded in cut discs from the cy-anolichen P. praetextata during a 14-wk cultivation with addedK, P, Mg, Ca, Na, Fe and S (Scott, 1956). Assuming continuousexponential growth over time, the treatment with the highestmean RGR would have doubled the DM after 90 d inP. aphthosa, 73 d in P. canina (both at 13°C : 8°C, 24-h wet) and87 d in P. sulcata (13°C : 8°C, 12-h wet), corresponding to

annual RGRs of 53.2, 58.5 and 53.8 g g�1 y�1, respectively. Inthe field, lichens often become active at suboptimal temperaturesand light (Green et al., 2008), for example, in cool mornings withdew or in dark rainy periods (Lange & Green, 2005). However,kept hydrated for most of the day at 150 lmol m�2 s�1, theygrow rapidly (Fig. 2). With such high RGRs, the effects of envi-ronmental factors can rapidly be detected. Because growthparameters (RGR and/or RTAGR) integrate responses affectingviability, reproduction and fitness, they are important tools forunderstanding lichen functioning.

Dry mass gain results from net photosynthesis during lightperiods minus dark respiration during nights. Thereby, DM gainis constrained by environmental factors limiting photosynthesis(Palmqvist, 2000; Dahlman & Palmqvist, 2003). The averageDM gain was > 30% higher in P. sulcata than in cephalo-/cyan-olichens. Much higher Chla content per thallus area may havecontributed to this. The photosynthetic capacity of lichens isstrongly coupled to their Chla content (Tretiach & Pecchiari,1995; Valladares et al., 1996; Palmqvist et al., 2002), and lightuse efficiency increases with increasing Chla (Dahlman & Palmq-vist, 2003). Strong photoinhibition reduces RGR (Fig. 4). InP. canina, the strong depression of RGR in the 12-h relative to24-h hydration regime (Fig. 2b) was probably related to thesubstantial photoinhibition that did not relax without hydrationat night. Trebouxia lichens can induce photosynthetic electrontransport and CO2 fixation more rapidly after hydration(≤ 10 min) than can lichens with Coccomyxa and Nostoc (Palmq-vist, 2000). This may also contribute to the high RGR inP. sulcata. Although thalli kept dry at nights avoided dark respira-tion loss, their RGR was reduced significantly (Fig. 2; Table 2)compared with thalli hydrated for 24 h. Dark respiration inP. aphthosa is c. 4–10 nmol CO2 g

�1 DM s�1 (Kershaw & Mac-Farlane, 1980; Sundberg et al., 1999). In addition, respirationgradually, but substantially, declines with time after hydration(Sundberg et al., 1999). A net CO2 exchange during the daytimeof 3 nmol CO2 g

�1 DM s�1 is necessary for a total mean RGR of5.92 mg g�1 d�1 (Fig. 2) for thalli with no respiration during thenight. A net fixation of CO2 during the daytime of 7–13 nmol CO2 g

�1 DM s�1 is necessary to compensate for a darkrespiration of 4–10 nmol CO2 g

�1 DM s�1 for the wet nighttreatment. Therefore, although thalli hydrated during the nightmay lose > 50% of net fixed CO2 during the daytime, their fastrecovery from photoinhibition (Fig. 2) and/or the positive effectsof respiration more than compensate for nocturnal respiratoryCO2 loss. Nocturnal hydration also boosted RGR in cephalo-/cy-anolichen members of Lobaria (Bidussi et al., 2013). Preliminarymeasurements did not show diurnal variation of soluble carbohy-drates in P. aphthosa and P. canina under the growth chamberregimes used (data not shown). If all fixed CO2 is stored as solu-ble carbohydrates during the 12-h light period or if soluble car-bohydrates only are respired during the dark period, a reductionin soluble carbohydrates of 10% is expected, which is probablyless than that needed to be significant at P = 0.05. Another reasonfor the lack of diurnal changes could be that other compounds,such as sugar phosphates, are involved in short-term carbonexchange.



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The change in STM (Fig. 2) showed that A growth exceededDM growth in P. aphthosa, and vice versa in the two other spe-cies, with some deviations at extreme temperatures. This supportsthe view that A and DM growth are different processes (Gauslaaet al., 2006, 2009). Photobionts contribute to mass growth bytheir carbon gain, whereas mycobionts contribute to area growth.According to Jahns (1988), thallus area expansion results frommarginal hyphal growth in growing hyphal tips. Cell expansionin plants depends on cell wall properties and turgor pressure(Nobel, 1999). With no turgor pressure, area growth hardlyoccurs. Water availability drives turgor pressure and fungalhyphal expansion (Wessels, 1993; Lew, 2011) and RTAGR (Gau-slaa et al., 2009). Higher long-lasting turgor pressure under con-tinuous hydration, boosted by high soluble carbohydrate pools,may explain the higher RTAGR in cephalo-/cyanolichenshydrated for 24 h (Fig. 2). Area-expanding effects of moisturewere found by Gauslaa et al. (2009), showing that high rainfallsupported wider and thinner lobes than a drier climate. Further-more, increased DSTM in P. canina experiencing nocturnalhydration indicates that DM increased despite dark respiration.It is not known why DSTM of the chloro- and cephalolichensresponded differently.

The physiological buffering hypothesis proposed by Farrar(1976c, 1988) implies that the pool of soluble carbohydrates, asa source of protection and as a substrate with high turnover forrespiration, stabilizes lichen metabolism in a fluctuating envi-ronment. It predicts that reduced polyol pools imply increasedrisk for damage or adverse effects of, for example, long-termextensive hydration periods (Farrar, 1976a,b). In our experi-ment, the average soluble carbohydrate concentration across alltreatments was reduced to 60–67% of the starting values inPeltigera species (Table 1). Nevertheless, lichens grew rapidly,particularly under the 24-h hydration regime (Fig. 2a,b), whichwas considered to be the most damaging treatment (see Farrar,1976b). Parmelia sulcata, the least buffered lichen with lowpolyol pools, had the highest average RGR and experienced a21% increase in soluble carbohydrates during cultivation(Table 1). It seems that conversion rates from soluble carbohy-drates to new lichen tissue also matters, and that nocturnal res-piration during wet nights boosts lichen growth. Conditionsmay occur when growth is more limited than photosynthesis,resulting in the accumulation of polyols. This probably happensin the field because optimal net photosynthesis is often encoun-tered at intermediate water contents (Lange et al., 2001) withinsufficient turgor pressure for good hyphal expansion. Suchconditions may lead to a partitioning in time, with active pho-tosynthesis during daylight periods and conversion of photosyn-thates into growth of hyphae at humid nights during dewformation and thus high turgor pressure at maximum watercontent. Because ≤ 10% of soluble carbohydrates are consumedby nocturnal respiration, such pools can sustain thallus areagrowth through a number of successive nights even withoutintermittent hydration periods during daylight. In this perspec-tive, high polyol pools and high nocturnal respiration can be auseful combination buffering lichen functions across diurnalcycles and environmental variation.

In conclusion, there was a shift from low growth and highcarbohydrate pools in the field to high growth and lower carbo-hydrate pools in the growth chamber for the two Peltigera species,consistent with the hypothesis that turnover rate rather than theconcentration of carbohydrates matters for growth. However,the concentration of the specific carbohydrate delivered by therespective primary lichen photobiont boosted growth in all threefunctional groups.

Acknowledgements

Thanks to Annie Aasen for help with carbohydrate extractionand HPLC analyses.

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