ARTICLE

Growth and secondary compound investments in the epiphytic lichensLobaria pulmonaria and Hypogymnia occidentalis transplanted along analtitudinal gradient in British ColumbiaMassimo Bidussi, Trevor Goward, and Yngvar Gauslaa

Abstract: We investigated altitudinal variation (550–1650 m) in relative growth rates (RGR) and carbon-based secondary com-pounds (CBSC) in the cephalolichen Lobaria pulmonaria (L.) Hoffm. and the chlorolichen Hypogymnia occidentalis L. H. Pike trans-planted for 14 months in a U-shaped valley in inland southern British Columbia. Prior to transplantation, half of the thalli weretreated with phosphorus (P) to examine effects of P on carbon allocation. Growth in L. pulmonariawas substantially higher in thetoe-slope position, corresponding to much higher bark pH. Sixty-four percent of the variation in RGR was accounted for bypositive pH effects and adverse effects of direct light exposure in the best subset multiple regression model. For H. occidentalis,57% of the variation in RGR was accounted for by positive and negative effects of indirect and direct light, respectively. Neitheraltitude nor P had a noticeable effect on RGR, the former possibly reflecting a trade-off between orographic precipitation andinversion-boosted nocturnal dew in valley bottom localities. Neither was there any correlation between altitude and CBSCs,although treatmentwith P did significantly trigger secondarymetabolism in L. pulmonaria, but not inH. occidentalis. No significantintraspecific relationship between growth and CBSC investments was noted.

Key words: relative growth rate, lichen compounds, phosphorus, bark pH, elevation.

Résumé : Les auteurs ont examiné la variation altitudinale (550–1650 m) sur les taux relatifs de croissance (TRC) et les composéssecondaires basés sur le carbone (CSBC) chez le céphalolichen Lobaria pulmonaria (L.) Hoffm. et le chlorolichen Hypogymniaoccidentalis L. H. Pike, transplantés pendant 14 mois dans une vallée en U, dans le sud de l’intérieur de la Colombie canadienne.Avant la transplantation, on a traité la moitié des thalles avec du phosphore (P) pour examiner ses effets sur l’allocation ducarbone. On a observé une croissance substantiellement plus grande chez le L. pulmonaria au pied de la pente, correspondant aun pH beaucoup plus élevé de l’écorce. Soixante-quatre pour cent de la variation du TRC s’explique par les effets positifs du pHet les effets adverses de l’exposition directe a la lumière dans le sous ensemble du modèle de régression. Chez le H. occidentalis,57 % de la variation du TRC s’explique par les effets positifs ou négatifs de la lumière indirecte ou directe, respectivement. Nil’altitude ni le P n’exercent un effet sur le TRC, le premier reflétant possiblement un compromis entre la précipitationorographique et la rosée accentuée par l’inversion dans les localités du fond de la vallée. On ne retrouve pas non plus decorrélation entre l’altitude et les CSBC, bien que le traitement avec le P ait significativement accéléré le métabolisme secondairechez le L. pulmonaria, mais non chez le H. ocidentalis. On n’a observé aucune relation intraspécifique entre la croissance et lesallocations aux CSBC. [Traduit par la Rédaction]

Mots-clés : taux relatif de croissance, composés lichéniques, phosphore, pH de l’écorce, élévation.

IntroductionEpiphytic lichen richness and species composition vary consid-

erably with altitude (e.g., Goward and Ahti 1992; Wolf 1993;Pirintsos et al. 1995; Cornelissen et al. 2001; Austrheim 2002;Werth et al. 2005; Berryman and McCune 2006; Bruun et al. 2006;Grytnes et al. 2006; Baniya et al. 2010). Presumably, this reflectsconcurrent changes in microclimate and habitat, as, for example,type, frequency and duration of hydration, surface temperatures,stand structure (light), nutrient availability, and substrate tex-ture. Compared with plant diversity, lichen species richness oftendeclines more slowly with altitude (e.g., Vittoz et al. 2010), as wellas with latitude (Mattick 1953), although surprisingly little isknown about altitudinal effects on epiphytic lichen growth rates.Generally speaking, ambient diurnal temperature tends to de-crease by approximately 0.7 °C for each 100 m rise in elevation.Quantification of altitudinal variation in growth rate may thus

provide a basis for evaluating effects of predicted climate change.In general, our knowledge on elevation dependency of specificlichen traits is sparse, although investments of defense com-pounds in terms of carbon-based secondary compounds (CBSC)have been found to decrease in lichens with increasing altitude(Swanson et al. 1996; Bjerke et al. 2004; McEvoy et al. 2007; Vatneet al. 2011), despite the fact that some cortical CBSCs have beenshown to be induced by UV-B (as reviewed by Solhaug and Gauslaa2012). However, as altitudinal studies of CBSCs so far have beendone in naturally occurring lichens, it can be questioned if re-ported altitudinal declines are environmentally regulated or ge-netically controlled.

Here we aim to quantify altitudinal (550–1650 m) variation ingrowth rates and CBSC investments in transplants of two epi-phytic lichens, the cephalolichen Lobaria pulmonaria (L.) Hoffm.and the chlorolichenHypogymnia occidentalis L. H. Pike.Hypogymniaoccidentalis is common on acidic bark of the conifers selected for

Received 8 April 2013. Accepted 9 June 2013.

M. Bidussi and Y. Gauslaa. Department of Ecology and Natural Resource Management, Norwegian University of Life Sciences, P.O. Box 5003, N-1432 Ås, Norway.T. Goward. UBC Herbarium, Beaty Museum, University of British Columbia, Vancouver, BC V6T 1Z4, Canada. (Mailing address: Enlichened Consulting Ltd., 5369 Clearwater Valley Road,Upper Clearwater, BC V0E 1N1, Canada.)

Corresponding author: Massimo Bidussi (e-mail: massimo.bidussi@umb.no).

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Botany 91: 621–630 (2013) dx.doi.org/10.1139/cjb-2013-0088 Published at www.nrcresearchpress.com/cjb on 9 July 2013.

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transplantation, whereas L. pulmonaria occurs on such trees onlyin somewhat nutrient-rich sites (Gauslaa andHolien 1998; Gowardand Arsenault 2000a; Gauslaa and Goward 2012). As phosphorushas been found to increase the growth of Lobaria (e.g., Benner andVitousek 2007; Benner et al. 2007), we included phosphorus (P)additions on 50% of the transplants of both species. In this way, wehoped to detect possible differences in species-specific responsesat different points along our elevation transect. Furthermore,P addition may influence allocation of carbon (C) in lichens(Palmqvist 2000). Lichens use C both for biomass growth and forproduction of large amounts of carbon-based defense compounds,andwe hypothesize that the addition of Pwould prompt our studylichens to use more C for new biomass growth at the expense ofdefense investments in terms of CBSCs.

Our study was also specifically designed to give special empha-sis to bark chemistry and light availability. Several earlier studieshave underlined the strong stimulating effect of relatively highbark pH on the presence of epiphytic cyano- and cephalo-lichens(Gauslaa 1985, 1995; Goward and Arsenault 2000a), whereas mostchlorolichens can occur on acidic bark. Possible altitudinal varia-tion in bark chemistry was evaluated by measuring bark pH. pHand (or) pH-dependent elemental availability is important for li-chen growth (Gauslaa and Goward 2012) as well as for CBSC invest-ments (e.g., Hauck et al. 2009; Vatne et al. 2011). Light exposure isanother important determinant of epiphytic lichen growth (e.g.,Gauslaa et al. 2006b; Jansson et al. 2009; Boudreault et al. 2013),and for this reason we quantified altitudinal variation in canopyopenness in terms of direct and indirect solar radiation. By mea-suring both bark pH and canopy openness along an elevationalgradient, we hoped to discriminate their effects relative to tem-perature and other environmental factors. Similarly, we hopedthat examining a chlorolichen and a cephalolichen (with N-fixingcyanobacteria as the secondary photobiont) would make it possi-ble to detect possible altitudinal variation in N availability.

Because thallus temperature likely exerts a significant influ-ence on lichen growth (Lange 2002; Bidussi et al. 2013) and CBSCinvestments (as discussed in Vatne et al. 2011 and Gauslaa et al.2013), growth rates and CBSCs in lichen transplants should de-crease with increasing altitude, provided that the temperatureduring hydration periods follows the general environmental lapserate at all altitudes. However, as windward-facing mountainscause rainfall to increase with altitude during the day (orographicprecipitation), and dewfall normally requires strong nocturnalcooling (temperature inversions at lower altitudes), the relation ofactive lichen metabolism and position along an altitudinal gradi-ent may not be linear. Our main objectives are first to identify thetype of altitudinal response and second to discuss lichen growthand secondary metabolism within a U-shaped, intermontane val-ley subject to considerable diurnal temperature fluctuation. Fi-nally, we will search for possible trade-off between growth and(or) CBSC production to test the hypothesis that growth and sec-ondary metabolism compete for declining pools of fixed carbon.

Materials and methods

Lichen materialOne hundred and eighty-eight specimens of L. pulmonaria and

H. occidentaliswere collected between 19 May and 23 May 2010 in theClearwater Valley of southern inland British Columbia. Lobariapulmonariawas gathered from Picea and Abies in a humid site 18 kmnorth of our transect, whileH. occidentaliswas collected on varioustree species in the vicinity of the transect itself. These collectinglocalities were located between 700 and 800 m a.s.l. Thalli ofL. pulmonaria and H. occidentalis had start dry mass (DM) of 117.0 ±3.8 mg (mean ± 1 SE; n = 94) and 156.05 ± 7.1 mg, respectively, withcorresponding thallus area (A) of 14.0 ± 0.4 and 11.4 ± 0.3 cm2.

Shortly after collection, 94 thalli of each species were sub-merged air dry in an aqueous solution of 500 mg·L−1 K2PO4 for

5 min, whereas the 94 remaining thalli were similarly treated indeionized water. In a preliminary experiment, the P treatmentwas found to have no adverse effects on themaximal photosystemII efficiency repeatedly measured over 120 h (data not shown).After submersion, all thalli were kept hydrated onmoist paper fora period of 1 h in low light. They were then left to desiccate atroom temperature and low light for 2 days before dry mass wasmeasured (±0.1 mg). To calculate the oven DM of our transplants,we subjected some additional thalli of each species to the sametreatment. Afterward, these additional thalli were kept 24 h at70 °C and then reweighed. We computed DM by multiplying theair-dried mass with a correction factor made from the oven-driedthalli. The same weighing protocol was repeated after the trans-plants were harvested 14 months later.

Thallus area (A) for each thallus at the start of our transplantexperiment was computed with the imaging tool ImageJ 1.46fversion (Wayne Rasband, National Institutes of Health, USA) usingphotos taken of each separate thallus while fully hydrated (photoswere taken both prior to and after field transplantation, using aNikonD300 camerawith a Nikon AF-SMicro Nikkor 105mm lens).At the end of the experiment, a leaf area meter (LI3100 Licor;Lincoln, Nebr., USA) was used for measuring thallus area. Gauslaaand Goward (2012) showed that these two methods for area deter-mination are closely correlated in the two species examined here(r2adj = 0.982 and 0.985). These regression models were used toadjust the Aend measured by the area meter to the thallus area asmeasured by ImageJ.

The elevation gradientOur elevation gradient was placed on the conifer-dominated

western slope of Battle Mountain (51°57=09==N, 119°51=17==W), start-ing with a site near Hemp Creek at 550 m and terminating at1650m, approximately 400m below the alpine timber line. In thisgradient, L. pulmonaria was a locally dominant species on conifersat altitudes from 700 to 950 m, but additionally occurred onspruce in the dripzone of Populus and on trunks of scattered Salixscouleriana up to at least 1300 m (T. Goward, personal observation,2010). Hypogymnia occidentalis increased in abundance with alti-tude up to the highest elevation, where it was one of the dominantlichen species. Bioclimatically, our transect ranged from thelower oroboreal subzone, also classified regionally as the MoistWarm Interior Cedar–Hemlock Zone, upward to the middle oro-boreal subzone, or the Engelmann Spruce – Subalpine Fir Zone(Meidinger and Pojar 1991). Two trees were selected at each of thefollowing altitudes: 550, 700, 800, 920, 1030, 1150, 1280, 1400, 1500,1600, and 1650 m. The lowermost trees along this gradient werelocated on a west-facing slope, while those at 700m represented agentle rolling plateau surface. The trees at 800–920 m were lo-cated in the toe position of the valley wall (Fig. 1d), while those at1030–1400 m were on steep, west-facing slopes, and the threeuppermost sets of trees represented slightly less steep slopes(Fig. 1d). The models of Wang et al. (2012) were used to approxi-mate temperature and precipitation normals for the period 1961–1990 for each position along our elevation gradient (Table 1).

Transplantation designUsing polyester thread, the thalli were sewn in groups of four

onto 45 strips of fine, pale plastic netting trimmed to 24 × 7 cm.Each group of four thalli included (i) L. pulmonaria with P,(ii) L. pulmonaria without P, (iii) H. occidentalis with P, and(iv) H. occidentalis without P. The sequence of thalli was random-ized for each net. Only the basal parts of L. pulmonaria were fas-tened, thereby allowing the younger tips to hang freely. Thispermitted the thalli to curl when dry, a natural response to desic-cation providing photoprotection by self-shading (Barták et al.2006). Hypogymnia occidentalis does not curl in this way and, hence,was more broadly fastened to the nets.

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Netswith lichenswere transplanted to branches of Picea glauca ×engelmannii, and (or) Abies lasiocarpa where Picea was absent; bothtree species commonly host these two lichen species in the gen-eral study area. The thalli were fastened to the branches using fineplastic netting trimmed to 24 × 7 cm (see photo of the transplanttechnique in Gauslaa and Goward 2012). For each tree, twobranches at 1.5–2 m above ground height were selected, and onenet was fixed to each branch. The samples were transplanted overtwo days in early June 2010 andwere left in the field for 14monthsuntil mid-August 2011. Photos of the samples were taken at thestart and end of the experiment; these were later carefully com-pared, revealing that 12 samples of H. occidentalis had lost smallportions of their thalli. Growth measurements were then cor-rected consequently for thallus fragmentation.

Growth measurementsGrowth during the transplant period was quantified as percent-

age increases of DM and A (%DM and % A). Growth was also mea-sured as relative growth rate, RGR = (ln(DMend/DMstart)) × 1000/�t (mg·g−1·day−1) and as relative thallus area growth rate, RTAGR =(ln(Aend/Astart)) × 100/�t (mm2·cm−2·day−1) (Evans 1972), t = 432 to438 days. Specific thallus mass (STM) was calculated at the begin-ning and end of the experiment as STM = DM/A. Changes in STMwere expressed as �STM = 100 × (STMend − STMstart)/STMstart.

Extraction of secondary compoundsThe lichens were ground to powder using a mixer mill (MN 301,

Retsch GmBH). For each sample, 15 mg were taken and put into avial with 500 �L of 100% acetone for the extraction. After 10min ofextraction, they were centrifuged for 3 min at 15 000 r·min−1 andacetone was poured into a 10 mL tube. The extraction procedurewas repeated three times and the liquids were put together. Theextracts were left to evaporate from the glass tube using a Con-centrator plus/Vacufuge plus (Eppendorf, Hamburg, Germany)prior to analysis.

HPLC analysesThe extracts were dissolved in 500 �L of 100% acetone and the

carbon-based secondary compounds (CBCSs) were measured byHPLC on a Hewlett-Packard (Palo Alto, Calif., USA) 110 serieschromatograph (Agilent Technologies, Waldbronn, Germany)utilizing an ODS Hypersil column (50 × 4.6 mm); for the mobilephases at 2 mL·min−1, we used 0.25% orthophosphoric acid and1.5% tetrahydrofuran in Millipore (Millipore, Billerica, Mass., USA)water (solution A) and 100% methanol (solution B), and UV detec-tion at 245 nm. The injection volume was 10 �L. The run startedwith 30% B, increased to 70% after 15 min and to 100% after an-other 15 min. This condition was maintained for 5 min and thensolvent B decreased to 30% in 1 min. After that, a 10 min post-runwith 30% solvent B was performed before the next analysis wasdone. The identification of CBSCs was based on retention time, on-line UV spectra, and co-chromatography of commercial standards.

pH measurementsDefoliated branch segments 6 cm long were removed at the end

of the transplant period from each of the 22 transplant trees. Eachbranch segment was then cleaned of lichens and submerged in avial containing 6 mL 25 mmol KCl·L−1, consistent with the ionexchange method described by Gauslaa and Holien (1998). Thevials were kept 1 h at room temperature and shaken at regularintervals. The branch segments were then removed and the re-maining solution immediately subjected to pH measurement(Orion pH meter model 420A, Boston, Mass., USA; frequently cal-ibrated against standard buffers during measurements).

Indirect measures of light exposureHemispherical digital photos were taken and analyzed to quan-

tify canopy cover (Englund et al. 2000). Digital photos were takenfrom a horizontal position close to each transplantation site, us-ing a Nikon Coolpix 4500 camera with a Nikon Fisheye Converter(FC-E8). Magnetic north was indicated in all photos. Image analy-sis was done by means of HemiView 2.1 (Delta-T Devices, Burwell,Cambridge, UK) to measure direct (DSF) and indirect (ISF) sitefactors (Anderson 1964). DSF is the proportion of direct solar radi-ation reaching a given location, while ISF is a measure of diffusesolar radiation under the canopy as compared with an open site.

Statistical analysesANOVA analyses were made in Minitab 16 (Minitab Inc., State

College, Penn., USA). Three-way ANOVA for growth rates was doneusing a General Linear Model with the following factors: species,phosphorus addition and elevation, with tree nested in elevation,and STM at start as covariate. Regression analyses were run inSigmaPlot 11.0 (Systat Software Inc., Ashburn, Va., USA). When ana-lyzing possible effects of external factors (pH and site factors) ongrowth rates or CBSCs, growth rates were averaged for each tree(n = 44).

ResultsPhosphorus fertilization did not significantly influence mea-

sured growth rates in either of the two species (Table 2). For thisreason, we averaged growth parameters across both P treat-ments in further analyses of growth. The two species had

Fig. 1. Mean environmental conditions for each of the 11 elevationpositions. Canopy openness is given as (a) a direct site factor (DSF)reflecting exposure to direct solar radiation, and as (b) an indirectsite factor (ISF), a measure of diffuse light. Bark pH of trees used fortransplantation (c) and the altitude profile (d) are also given.

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highly significantly different growth rates, with L. pulmonariagrowing twice as fast as H. occidentalis (Fig. 2). Mean RGR for allelevations was 0.86 ± 0.04 mg·g−1·day−1 for L. pulmonaria and0.39 ± 0.02 mg·g−1·day−1 for H. occidentalis, equivalent to percent-age DM gain of 49.0% ± 2.3 and 18.2% ± 1.1%, respectively. RGR ineach species was strongly influenced by elevation, and inL. pulmonaria, it was also influenced by tree-dependent variables(Table 2). In L. pulmonaria, RGRs increased from 0.79 ± 0.08mg·g−1·day−1

at 550 m to 1.37 ± 0.08 mg·g−1·day−1 at 920 m, where this specieshad its greatest abundance, followed by consistently lowergrowth (≈0.7mg·g−1·day−1) at all higher elevations. The lowest RGR(0.61 ± 0.13 mg·g−1·day−1) was at 1400 m (Fig. 2), slightly above theextreme uppermost natural occurrence of this species.

RGRs of H. occidentalis responded differently, exhibiting slightlydecreasing values with increasing elevation (r2adj = 0.104; p = 0.002;Fig. 2), and no significant tree-specific effect (Table 2). But whilethe overall decrease was slight, the extremes were notable.Hypogymnia occidentalis had its highest RGR at 700 m (0.70 ±0.10 mg·g−1·day−1), growing three times faster than its slowestgrowth at 1400 m (0.24 ± 0.04 mg·g−1·day−1; see Fig. 2). Thus, there

Table 1. Temperature and precipitation normals for 1961–1990 from Climate BC modeling, usingWang et al. (2012) for the studied positions in the altitudinal gradient.

Mean air temperature (°C) Precipitation (mm)

Elevation(m) Annual Winter Spring Summer Autumn Annual As snow

550 4.9 −6.1 5.4 15.5 4.8 616 204700 4.1 −6.6 4.5 14.5 4.1 687 250800 3.5 −7.0 3.7 13.3 3.6 783 307920 3.2 −7.1 3.3 13.3 3.5 980 3991030 2.8 −7.4 2.8 12.8 3.1 1060 4521150 2.4 −7.7 2.3 12.3 2.8 1075 4771280 2.0 −7.9 1.8 11.8 2.4 1102 5121400 1.6 −8.2 1.3 11.3 2.0 1141 5541500 1.1 −8.6 0.7 10.7 1.5 1209 6161600 0.6 −8.9 0.1 10.2 1.1 1262 6701650 0.4 −9.0 −0.2 9.9 0.8 1292 703

Table 2. ANOVA table for relative growth rates and change in specificthallus mass (STM) in Lobaria pulmonaria and Hypogymnia occidentalisover a 14-month period, as recorded at the time of harvest.

RGR RTAGR �STM

Source df F p F p F p

Lobaria pulmonariaSpecific thallus

mass1 2.07 0.156 3.85 0.055 19.24 0.000

Phosphorus (P) 1 2.68 0.108 0.24 0.625 7.01 0.011Elevation (E) 10 9.19 0.000 4.71 0.000 2.03 0.050Tree (elevation) 11 3.84 0.000 2.35 0.020 1.16 0.337Error 50Total 83r2adj 0.591 0.397 0.347Hypogymnia occidentalisSpecific thallus

mass1 12.89 0.001 1.69 0.199 2.36 0.131

Phosphorus (P) 1 0.06 0.804 3.79 0.057 3.40 0.071Elevation (E) 10 3.64 0.001 1.01 0.452 2.38 0.022Tree (elevation) 11 1.61 0.124 0.80 0.639 1.05 0.421Error 50Total 83r2adj 0.427 0.000 0.203

Note: Half of the thalli had been treated with phosphorus (P) at two trees ineach at 11 elevations from 550 to 1650 m. Tree variable is nested in elevation.Specific thallus mass at start is given as a covariate. Interaction terms wereinsignificant (p> 0.05) and are therefore not included. RGR, relative growth rate;RTAGR, relative thallus area growth rate; �STM, change in specific thallus mass.

Fig. 2. Growth rates measured in transplanted thalli of thecephalolichen Lobaria pulmonaria and the chlorolichen Hypogymniaoccidentalis after a 14-month residency at the 11 altitudes shown inFig. 1d. Error bars indicate ±1 SE. RGR, relative growth rate; RTAGR,relative thallus area growth rate; STM, specific thallus mass.

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was an apparent discrepancy between increasing dominance ofH. occidentaliswith elevation in natural vegetation and concurrentdeclining growth rates.

Area gain (RTAGR) averaged for all elevations was again nearlytwice as high for L. pulmonaria (0.084 ± 0.003 mm2·cm−2·day−1) asfor H. occidentalis (0.046 ± 0.002 mm2·cm−2·day−1; Fig. 2), corre-sponding to a percentage area growth of 45.1% ± 1.7% and 22.8% ±1.2%, respectively. The highest RTAGR occurred at 920 m for bothL. pulmonaria (0.120 ± 0.007 mm2·cm−2·day−1) and H. occidentalis(0.057 ± 0.012 mm2·cm−2·day−1; Fig. 2). Only in L. pulmonaria wasthere a clear trend in RTAGR with elevation, this essentiallymirroring the RGR trend (Fig. 2; Table 2). None of the factorsincluded in the ANOVA (Table 2) significantly influenced RTAGRin H. occidentalis.

STMstart wasmuch lower for L. pulmonaria than forH. occidentalis;overall means were 8.43 ± 0.11 and 13.59 ± 0.44 mg·cm−2, respec-tively. These STM means changed only slightly during the exper-iment (0.6% for L. pulmonaria and −3.0% for H. occidentalis). Thenegative value in the latter species shows that its area growthrates often exceededmass growth (Fig. 2). In thalli ofH. occidentalis,having the largest variation in STMstart, RGR highly significantlydeclined with increasing STMstart; no such effects occurred inL. pulmonaria (Table 2). Phosphorus addition increased �STM inL. pulmonaria from −1.82 ± 1.13 to 2.99 ± 1.43 mg·cm−2 (p = 0.011;Table 2). Furthermore, �STM slightly decreased with altitude inH. occidentalis (p = 0.016; Table 2; Fig. 2).

Bark pH varied greatly between the studied trees (3.95–6.02)and showed a distinct altitudinal pattern (Fig. 1c; p < 0.001 in anANOVA; data not shown). On average, bark was approximately10 times less acidic at 800and920mthan in thevalleybottomandonsteeper slopes rising above (Fig. 1d). RTAGR and RGR increasedsignificantly with bark pH in L. pulmonaria; the strongest relation-ship was found for biomass growth (r2adj = 0.395; p < 0.001; Fig. 3).Canopy openness in terms of direct (DSF) and indirect site factors(ISF) varied between elevations, but without clear altitudinaltrends (Figs. 1a–1b). These factors were among the tree variablesgiving the highly significant tree effect on RGR in L. pulmonaria(Table 2). Increasing direct light exposure reduced RGR (Figs. 4a, 4c),particularly in L. pulmonaria, while increasing diffuse light stimu-lated RGR in H. occidentalis (Fig. 4d) and had no effects on thecephalolichen (Fig. 4b). Overall canopy openness (data not shown)varied between 11% and 22%, with higher values only at 700 m(33%). The importance of external factors (bark pH, elevation, DSF,and ISF) on lichen growth was analyzed by searching for the bestsubset multiple linear regression model. As these external vari-ables were measured relative to individual study trees, the aver-

age growth rate for all thalli in each species on one tree wastreated as one observation. The best RGR model for L. pulmonariaaccounted for 64% of the variation (p < 0.001), versus 58% for thebest RTAGR model (p < 0.001; Table 3). Bark pH positively influ-enced RGR and RTAGR, while DSF, a measure of direct sun expo-sure, reduced both growth rates. The best subset multiple linearregression model for RGR for H. occidentalis explained 57% of thevariation (p < 0.001; Table 3). Canopy openness strongly influ-enced RGR in H. occidentalis (Table 3): diffuse light (as estimated byISF) stimulated lichen growth, while direct sun exposure (in termsof DSF) had a negative effect. There was no significant correlationbetween direct and indirect site factors.

Total amount of CBSCs wasmuch higher inH. occidentalis (36.5 ±0.9 mg·g−1 or 5.09 ± 0.18 g·m−2) than in L. pulmonaria (20.4 ±0.9 mg·g−1 or 1.93 ± 0.08 g·m−2; mean ± 1 SE; n = 94). Hypogymniaoccidentalis had only one medullary compound, physodic acid,comprising 80.5% of the total CBSC concentration, and two corti-cal compounds, atranorin and chloroatranorin, present in con-centrations of 5.10 ± 0.18 and 2.02 ± 0.07 mg·g−1, or given ascontent per area: 0.713 ± 0.035 and 0.272 ± 0.012 g·m−2, respec-tively. Lobaria pulmonaria had no extractable cortical compounds.Among its medullary compound, stictic acid accounted for 69% ofthe total CBSC concentration, followed by cryptostictic acid (16%),constictic acid (11%), and three minor compounds (Table 4). Nei-ther the concentration nor the content per area of medullary andcortical CBSCs showed clear altitudinal trends for either of thetwo species (Fig. 5). Lobaria pulmonaria had fairly constant concen-trations of CBSCs for most elevations apart from the somewhathighermean at 920mand the peak value at 1650m,whichwas theonly site where CBSC concentrations in L. pulmonaria matchedthose in H. occidentalis.

Our analysis of CBSC in each species by two-way ANOVAs withelevation and P treatment as factors (data not shown) revealedelevation as marginally significant (p = 0.045) for the total medul-lary compound concentration in L. pulmonaria. No significant ele-vation response in CBSCs was found for H. occidentalis. In thisspecies there was a highly significant, positive regression betweencortical pigments and medullary physodic acid given as contentsper thallus area (r2adj = 0.337; p < 0.001). Phosphorus additionsignificantly increased the concentration of CBSCs in L. pulmonariaby 26% (p = 0.006; Table 4), while the secondary chemistry inH. occidentalis was not affected (Table 4). In L. pulmonaria, sticticacid (p = 0.013), constictic acid (p = 0.037), and methyl norsticticacid (p = 0.031) increased in concentration after the P treatment(Table 4). Other external factors (bark pH, ISF, and DSF) did notsignificantly influence CBSC investments in the two species.

There were no significant relationships between investments inCBSCs and growth within either of the two species (data notshown). However, H. occidentalis, with approximately twice theCBSC investment of L. pulmonaria, grew only half as fast as thatspecies (Figs. 2–5).

DiscussionLichen growth rates were not much influenced by altitude per

se in either of the two species (Fig. 2), despite a vertical distance of1100 m between the lowermost and the uppermost sites. Thisvertical distance equates to a computed temperature difference of5.6 °C in spring and summer (Table 1), slightly lower than theaverage adiabatic lapse rate. Such temperature contrasts shouldin principle exert a profound difference on thallus growth(Bidussi et al. 2013). The situation, however, is complicated by thenightly formation of temperature inversions, which tend to befrequent in mountainous regions such as the study area (e.g.,Geiger 1950; Mbogga et al. 2009). Above the inversion layer, more-over, there often develops a thermal belt with warmer nighttimetemperatures (Geiger 1950), here between about 1000 and 1300 m(T. Goward, personal observation). Inversion layers are especially

Fig. 3. The relationship between relative growth rate (RGR) andbark pH measured at the end of the experiment in Lobariapulmonaria and Hypogymnia occidentalis. Regression line was fitted forL. pulmonaria (RGR = −0.568 + 0.313 pH; r2adj = 0.395; p < 0.001); therewas no significant regression for H. occidentalis. ns, not significant.

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prevalent during winter, but regularly form during clear, calmweather throughout the year. For these reasons, the relation oftemperature to elevation is unlikely to be linear along our verticaltransect — a phenomenon of considerable significance, consider-ing the central importance of dewfall for the photosynthetic acti-vation of many lichens (Lange 2003). What is crucial to lichenestablishment and growth is therefore not temperature duringperiods of thallus desiccation, but rather during periods of activegrowth, which in the present study area are likely to be rathersimilar across our elevational gradient (see Fig. 1.2 in Reifsnyder1980).

A similar conclusion regarding the importance of temperaturecan be arrived at from other lines of evidence. For example, it hasrecently been shown by Sancho et al. (2011) that temperature may

not be the most crucial factor for lichen growth; for the poikilo-hydric lichens, humidity may be more important than tempera-ture. For example, as many as 50 of the oceanic Norwegian lichenspecies have their main distribution area in tropical rain forests(Jørgensen 1996), including some that extend north of the ArcticCircle.

With respect to humidity factors, rainfall and cloud formationtend to increase with altitude (Roe 2005; Ballantyne 2012; Table 1).Rainy eventsmay occur any time of day or night. At the same time,the diurnal temperature range decreases with increasing altitude,influenced by cold-air ponding at valley elevations (Geiger 1950;Oke 2002). A drop of 15 °C from the afternoon to the earlymorningis common in the Clearwater Valley on clear summer nights(T. Goward, personal observation). For example, one of us ob-served full hydration of lichens by dew each morning during thefinal two weeks of the experiment period (Y. Gauslaa, personalobservation, 2010). The diurnal timing of hydration events allow-ing growth may thus differ along the gradient. Hydration likelyoccurs more frequently during cold mornings in the lower eleva-tions versus more frequent hydration by rain and (or) clouds dur-ing (cool) daylight hours at higher elevations. Thus it seemspossible that the absence of any significant difference in lichengrowth rates over a vertical distance of 1100 m may reflect theexistence of separate hydration windows during which tempera-tures are roughly similar.

Three highly significant factors for growth in L. pulmonaria vari-ables pertained to bark pH, canopy DSF, and canopy ISF (Fig. 1;Table 2). Of these, the most important factor was bark pH (Fig. 3;Table 3). The importance of bark pH for epiphytic cephalo- andcyano-lichens has been well documented (Gauslaa 1985, 1995;Gauslaa and Holien 1998; Goward and Arsenault 2000a; Gauslaaand Goward 2012). In the present study, it is remarkable that barkpH was more important than elevation for a lichen species thatdoes not naturally occur in the upper portions of our transect. Itcan also be observed that the two points along our elevationalgradient that yielded the highest bark pH values are each in thetoe slope position (as defined by Luttmerding et al. 1990). Suchsites, which are typically characterized by enriched soils, are well

Fig. 4. The relationship between relative growth rate in Lobaria pulmonaria and Hypogymnia occidentalis versus diffuse (indirect site factor) anddirect site factor solar radiation exposure in their sites in the altitudinal gradient used for transplantation. Regression lines are shown. ns, notsignificant.

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Table 3. Best subset multiple regression models of relative growthrate and relative thallus area growth rate in Lobaria pulmonaria andHypogymnia occidentalis.

Variable Coefficient ± SE t P VIF

RGR for L. pulmonariaConstant −0.0639±0.316 −0.203 0.842 0.000pH 0.257±0.0644 3.990 <0.001 1.054DSF −1.277±0.331 −3.852 0.001 1.054RTAGR for L. pulmonariaConstant 0.0351±0.0231 1.520 0.145 0.000pH 0.0148±0.00472 3.135 0.005 1.054DSF −0.0920±0.0243 −3.791 0.001 1.054RGR for H. occidentalisConstant 0.137±0.0956 1.438 0.167 0.000ISF 1.452±0.320 4.539 <0.001 1.001DSF −0.604±0.213 −2.830 0.011 1.001

Note: Subset multiple regression models are based on four environmentalvariables — elevation, bark pH, direct site factor (DSF), indirect site factor(ISF) — and one internal lichen variable: specific thallus mass at start (n = 11). Nomultiple models could account for more than a small portion of the variation inRTAGR for H. occidentalis. For L. pulmonaria RGR, r2adj = 0.642 and p < 0.001; forL. pulmonaria RTAGR, r2adj = 0.581 and p< 0.001; forH. occidentalis, r2adj = 0.567 andp < 0.001. RGR, relative growth rate; RTAGR, relative thallus area growth rate;VIF, variance inflation factor.

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known to be rich in epiphytic cephalo- and cyano-lichens (e.g.,Radies et al. 2009). Given the data presented in Table 3 and Figs. 2and 4, it seems reasonable to infer that the rich soils in the toeposition enrich the normally acidic conifer bark and result insufficiently high pH to support fast growth and dominance ofL. pulmonaria (see also Goward and Arsenault 2000b). A similar linkbetween rich soils, high bark pH, and the presence of a well-developed Lobarion community was earlier demonstrated for theoak forests of southern Norway (Gauslaa 1985). More recent stud-ies in inland British Columbia (e.g., Radies et al. 2009) have em-phasized the importance of humidity in the establishment of highcyano- and cephalo-lichen diversity and biomass in toe positionforests. Our data suggest that bark pH may be no less important,and indeed, perhaps more so, at least in our study area. Thisquestion is in need of further research. Light is another importantfactor for lichen growth; Coxson and Stevenson (2007), working inthe same region, found that rates of growth in L. pulmonaria in-creasedwith increasing stand spacing up to 30% canopy cover; i.e.,the highest value recorded by us. Stand spacing leading to directlight (Table 3; Figs. 4a, 4c) had a negative impact, presumablybecause it causes rapid desiccation and lasting photoinhibition(e.g., Gauslaa and Solhaug 1996) and thus inactivates metabolicactivity and may result in chlorophyll bleaching (Gauslaa andSolhaug 2000). By contrast, indirect light (Table 3; Figs. 4b, 4d) hasa positive influence on growth, as it is associated with open-shadehabitats sensu Stoutjesdijk (1974) in which nighttime dew, andthus lichen hydration, can last for hours.

The slower RGR of H. occidentalis at high elevations (Fig. 2),where it occurs in the highest abundance, presents an interestinga paradox and suggests that rates of growth and rates of establish-ment in this species are uncoupled. Growth of H. occidentalis wasstrongly boosted by diffuse light (ISF; Table 3; Figs. 4b, 4d), whichwasmost prevalent at lower elevations in our study area (Fig. 1). Atthe same time, H. occidentalis is less adversely affected thanL. pulmonaria by direct solar radiation (Table 3; Fig. 4). All told,the growth responses of these species to DSF, ISF, and pH sup-port the view that (i) H. occidentalis is less shade adapted thanL. pulmonaria and (ii) bark pH and light strongly shape epiphyticlichen communities.

Production of medullary and cortical compounds did not varysignificantly along our elevational transect, in marked contrast tothe altitudinal decline in CBSCs reported by McEvoy et al. (2007)and Vatne et al. (2011). We emphasize, however, that our studydesign is not strictly comparable to those of the earlier studies,which measured growth response on hilltops of differing height

Table 4. Carbon-based secondary compound concentrations and contents in Lobaria pulmonaria and Hypogymnia occidentalis with and withoutadded phosphorus.

−P +PANOVA onconcentrations

ANOVA oncontents

Lichen compoundsConcentration(mg·g−1)

Contents(g·m−2)

Concentration(mg·g−1)

Content(g·m−2) r2adj p r2adj p

L. pulmonariaConstictic acida 1.90±0.24 0.174±0.022 2.61±0.23 0.243±0.023 0.039 0.037 0.042 0.034Peristictic acida 0.46±0.13 0.043±0.012 0.68±0.10 0.063±0.010 n.s. n.s. n.s. n.s.Stictic acida 12.25±1.03 1.220±0.114 15.78±0.94 1.470±0.093 0.059 0.013 n.s. n.s.Cryptostictic acida 3.7±1.11 0.358±0.111 2.85±0.60 0.275±0.062 n.s. n.s. n.s. n.s.Norstictic acida 0.064±0.025 0.006±0.002 0.031±0.01 0.007±0.004 n.s. n.s. n.s. n.s.Methyl norstictic acida 0.20±0.02 0.020±0.002 0.26±0.01 0.024±0.001 0.042 0.031 n.s. n.s.Total medullary comp. 17.72±1.32 1.822±0.141 22.25±0.90 2.083±0.093 0.075 0.006 n.s. n.s.H. occidentalisPysodic acida 28.33±1.18 3.969±0.248 30.11±1.04 4.238±0.193 n.s. n.s. n.s. n.s.Atranorinb 5.11±0.27 0.708±0.044 5.28±0.28 0.754±0.059 n.s. n.s. n.s. n.s.Chloroatranorinb 1.95±0.10 0.260±0.016 2.09±0.10 0.289±0.018 n.s. n.s. n.s. n.s.

Note: Means ± 1 SE (n = 44) are given across all branches, trees, and altitudes. Highest values are shown in boldface. n.s., not significant; P, phosphorus.aMedullary.bCortical compounds.

Fig. 5. Carbon-based secondary compounds (CBSC) concentrationsand contents in the cephalolichen Lobaria pulmonaria and in the chlo-rolichen Hypogymnia occidentalis measured at 11 positions along theelevation gradient. Total cortical and total medullary CBSC are givenseparately for H. occidentalis, though, only medullary CBSCs arereported for L. pulmonaria, which lacks extractable cortical pigments.Error bars indicate ±1 SE.

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and over a much greater horizontal distance. Moreover, becausealtitudinal trends in temperature are likely often absent or mini-mal in the present study area during periods of active thallusmetabolism (see above), our CBSC data can have little bearing onthe hypothesis that medullary defense compounds increase withtemperature. This hypothesis has been supported by other altitu-dinal studies (Swanson et al. 1996; Bjerke et al. 2004; McEvoy et al.2007; Vatne et al. 2011), latitudinal studies (Huovinen 1985), sea-sonal studies (Gauslaa et al. 2013), and by laboratory experimentsin which lichens are exposed to increasing temperature (Hamada1982; Bjerke et al. 2003).

Phosphorus is important for lichens insofar as growth is ad-versely affected when this element is in low concentration (Hoganet al. 2010a, 2010b; Johansson et al. 2011). Nevertheless, the avail-able information on its effects on lichen growth is not entirelyconsistent. While some studies show a positive effect on cya-nolichen growth in rainy, presumably P-limited habitats (e.g.,Benner and Vitousek 2007; Benner et al. 2007; McCune andCaldwell 2009), others (e.g., Campbell et al. 2010; Gauslaa andGoward 2012) have failed to document any such effect, as in thepresent case (Table 2). Even so, P addition was the only factor ormeasured parameter in our study that significantly influenced theproduction of CBSCs (see L. pulmonaria in Table 4), consistent withSolhaug and Gauslaa (2004) and McEvoy et al. (2006), who haveshown that experimentally added carbohydrates can boost thesynthesis of some CBSCs in lichens. Presumably this results froma three-part process involving (i) an increase in N-fixation by thelichen cyanobiont (Benner et al. 2007); (ii) movement of the fixedNinto the green algal photobiont, which raises photosynthetic ca-pacity; and (iii) enhanced carbohydrate production, which stimu-lates CBSC synthesis. At the same time, it is also possible thatP addition triggers bacterial communities (growth of bacteria isgenerally increased by P because bacteria are richer in DNA andRNA than eukaryotes) and may in turn cause the lichen to dedi-cate more carbon toward production of defense compounds.Recent studies have pointed to the physiologic importance of bac-teria living in symbiosis with lichens, raising the possibility thatthese organisms are not merely a symbiosis between a myco- anda photo-biont, but include other potentially harmful organisms(e.g., Liba et al. 2006; Grube and Berg 2009; Grube et al. 2009; Bateset al. 2011; Schneider et al. 2011; Cardinale et al. 2012).

The concentrations and contents of CBSCs in our study areawere substantially lower than the levels found in Scandinavia forL. pulmonaria (see, for example, Nybakken et al. 2007; McEvoy et al.2007; Asplund et al. 2009; Vatne et al. 2011). AlsoH. occidentalis, notpresent in Europe, had much lower defense compound invest-ments than was recently documented in European populations ofthe circumpolar lichen H. physodes (Solhaug et al. 2009; Asplundet al. 2012; Hauck et al. 2013). An important role of medullaryCBSCs in these species is to deter herbivores (see, for example,Gauslaa 2005; Asplund et al. 2010; Solhaug and Gauslaa 2012).Whereas climbing gastropod populations and grazing marks onepiphytic lichens are abundant andwidespread inwestern Europe(Gauslaa et al. 2006a; Vatne et al. 2010), these were not observed inthe study area. The low medullary CBSC concentration in ourlichens in habitats poor in lichen feeders is consistent with theherbivore defense hypothesis.

The substantially higher growth rates in L. pulmonaria than inH. occidentalis across all elevations is likely influenced by the ability ofthe former species to fix nitrogen, as well as by its dependence onricher soils and bark providing better access to mineral nutrients.In suitable habitats, L. pulmonaria is thus a stronger competitorthan the acidophilic H. occidentalis, which probably uses a stress-tolerant strategy (sensu Grime 1977), hence allocating more of itscarbon to compounds thatmay, for example, help to deter grazers(Fig. 5) (Gauslaa 2005) and (or) achieve metal homeostasis in acidsites with toxic ions (Hauck 2008; Hauck et al. 2013).

In conclusion, altitude per se apparently has a negligible influ-ence on CBSCs and carbon allocation to lichen growth across our1100 m elevational transect. The fact that RGR and CBSCs weresimilar within each species at all elevations may point to a trade-off between increasing orographic precipitation at higher eleva-tions and inversion-induced nocturnal dew events in the valleybottom. In this view, lichen metabolic activity at low and highaltitudes likely begins under similar temperatures, albeit at dif-ferent periods of the day. By contrast, the elevated RGRs observedin L. pulmonaria in our toe-slope position sites is likely attributableto a corresponding spike in bark pH in these nutrient-enrichedlocalities.

AcknowledgementsWe thank senior lecturer Nancy J. Flood for providing access to

a scale in the laboratory of the Department of Biological Sciences,Thompson Rivers University, and professors Mikaell OttossonLöfvenius (Swedish University of Agricultural Sciences) andKristin Palmqvist (Umeå University) for helpful discussion. We alsothank Jason Hollinger for kind assistance with our elevation profile(Fig. 1d), and Professor Darwyn Coxson (University of Northern Brit-ish Columbia) for providing facilities and laboratory for bark pHmeasurements.

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