Shifts in bryophyte carbon isotope ratio across an elevation × soil age matrix on Mauna Loa, Hawaii: do bryophytes behave like vascular plants?

Abstract

The carbon isotope ratio (δ¹³C) of vascular plant leaf tissue is determined by isotope discrimination, primarily mediated by stomatal and mesophyll diffusion resistances and by photosynthetic rate. These effects lead to predictable trends in leaf δ¹³C across natural gradients of elevation, irradiance and nutrient supply. Less is known about shifts in δ¹³C for bryophytes at landscape scale, as bryophytes lack stomata in the dominant gametophyte phase, and thus lack active control over CO₂ diffusion. Twelve bryophyte species were sampled across a matrix of elevation and soil ages on Mauna Loa, Hawaii Island. We tested hypotheses based on previous findings for vascular plants, which tend to have less negative δ¹³C at higher elevations or irradiances, and for leaves with higher leaf mass per area (LMA). Across the matrix, bryophytes spanned the range of δ¹³C values typical of C₃ vascular plants. Bryophytes were remarkably similar to vascular plants in exhibiting less negative δ¹³C with increasing elevation, and with lower overstory cover; additionally δ¹³C was related to bryophyte canopy projected mass per area, a trait analogous to LMA in vascular plants, also correlated negatively with overstory cover. The similarity of responses of δ¹³C in bryophytes and vascular plants to environmental factors, despite differing morphologies and diffusion pathways, points to a strong direct role of photosynthetic rate in determining δ¹³C variation at the landscape scale.

Electronic supplementary material

The online version of this article (doi:10.1007/s00442-010-1903-y) contains supplementary material, which is available to authorized users.

Introduction

Foliar carbon isotope ratio (δ¹³C) can reflect ecology and physiology, especially because in vascular plants δ¹³C reflects changes in function along resource gradients. Less is known about bryophyte δ¹³C responses. We aimed to quantify the degree to which bryophytes resemble vascular plants (tracheophytes) in their landscape scale δ¹³C using the bryophyte flora distributed across Mauna Loa, Hawaii, a “natural experiment” for ecological research (Vitousek 2004).

Differences in the relative abundance of ¹³C and ¹²C in plant tissues relative to the atmosphere is caused by discrimination against the heavier ¹³C during photosynthesis by the carbon fixing enzyme RubisCO and during diffusion through air and liquid water (Farquhar et al. 1989). The δ¹³C is negatively related to discrimination; δ¹³C increases as the chloroplast CO₂ concentration (c _c) declines and carboxylation discriminates less against ¹³C (Farquhar and Sharkey 1982). The c _c can be affected by several factors which influence CO₂ drawdown by photosynthesis relative to its rate of diffusion to the chloroplast. A high photosynthetic assimilation rate (A) draws down c _c relative to the CO₂ concentration of source air (c _a) (Farquhar et al. 1989). Vascular plants control CO₂ diffusion into the leaf with their stomata, which respond to numerous factors, especially opening with irradiance and high leaf water status, and closing under opposite conditions. A lower stomatal conductance (g _s) or a lower mesophyll conductance should reduce c _c/c _a and lead to higher δ¹³C (Loreto et al. 1992; DeLucia et al. 2003; Seibt et al. 2008; Niinemets et al. 2009). Bryophytes differ from vascular plants fundamentally in lacking stomata in the dominant life stage (Schofield 1985), but span a wide range of δ¹³C from −21 to −40 ‰ (but primarily −22 to −32 ‰; Rundel et al. 1979; Proctor et al. 1992; Fletcher et al. 2004), very similar to C₃ tracheophytes, which range from −24 to −32 ‰ (O’Leary 1988).

In vascular plants, δ¹³C tends to correlate with resource gradients for given species and across species sets. Typically, δ¹³C increases with elevation, associated with higher leaf mass per area (LMA) at lower temperatures (see below; Körner et al. 1988, 1991, 2003; Vitousek et al. 1990; Cordell et al. 1999). The δ¹³C also tends to correlate negatively with water availability, due to stomatal closure at low water availability (Farquhar et al. 1989). Further, vascular plant canopies exhibit increasing foliar δ¹³C with height (Medina and Minchin 1980; Garten and Taylor 1992; Ometto et al. 2006) due to effects linked with three resource gradients. According to the “water stress-g _s effect”, higher irradiance with increasing canopy height (CH) may be associated with lower leaf water status, and lower g _s and c _c (Niinemets et al. 2004). According to the “irradiance-A effect”, high irradiance may drive higher A relative to g _s, decreasing c _c (Brooks et al. 1997; Le Roux et al. 2001; Duursma and Marshall 2006). Finally, according to the “source air effect”, δ¹³C may vary across natural irradiances due to a vertical gradient in air δ¹³C caused by soil respiration (Francey et al. 1985; Schleser and Jayasekera 1985; Ehleringer et al. 1986; Farquhar et al. 1989; Medina et al. 1991; Buchmann et al. 1997b; Sternberg et al. 1989, 1997; Schedlbauer and Kavanagh 2008); this effect is dampened by air mixing and typically accounts for only ca. 10–30% of the difference in foliar δ¹³C from understory to top-of-canopy (Broadmeadow et al. 1992; Brooks et al. 1997; Buchmann et al. 1997a, 2002; Sternberg et al. 1997).

Bryophytes may follow similar trends in δ¹³C with gradients as tracheophytes. The δ¹³C may correlate positively with elevation, as reported for Sphagnum (Menot-Combes et al. 2004; Skrzypek et al. 2007), potentially arising because of a “temperature effect” if lower temperature at high elevation reduces respiration relative to A, leading to reduced loss of the heavier isotope (cf. Gillon and Griffiths 1997; Barbour and Hanson 2009), thus reducing discrimination (Hamerlynck et al. 2002; Skrzypek et al. 2007), and/or because of lower atmospheric pressure leading to lower c _c (Menot and Burns 2001). A positive correlation of bryophyte δ¹³C with water availability has also been shown due to “liquid film effect” that reduces CO₂ diffusion (Proctor et al. 1992; Rice and Giles 1996; Williams and Flanagan 1996; Meyer et al. 2008). Bryophytes may also show increased δ¹³C with higher irradiance as found for the moss Tortula ruralis (Hamerlynck et al. 2002) and for three liverworts (Fletcher et al. 2006). This pattern could arise from the “irradiance-A effect” and/or from the “source air effect”, if forest floor bryophytes experience elevated CO₂ originating from soil respiration (DeLucia et al. 2003).

The δ¹³C may also relate to leaf structure and composition. Across species of tracheophytes, δ¹³C correlates positively with LMA and nitrogen concentration per area (N _area), themselves related (Hultine and Marshall 2000; Takahashi and Miyajima 2008; Li et al. 2009). A similar pattern was found for the Hawaiian tree Metrosideros polymorpha across elevations: δ¹³C positively correlated with LMA, apparently due to higher carboxylation rate related to the higher N _area (Vitousek et al. 1990; Cordell et al. 1998, 1999). In bryophytes, canopy mass per ground area (CMA) is analogous to tracheophyte LMA (Waite and Sack 2010) and may be related to higher N per ground area, and hence bryophyte δ¹³C might be expected to correlate with CMA.

Previous work has highlighted trends for a few bryophyte species along specific gradients. In a previous study, we found strong variation in bryophyte N and P stoichiometry on Mauna Loa (Waite and Sack 2011). Here, we conducted a landscape-scale analysis of variation in δ¹³C within and across species, and its correlation with climate (temperature and precipitation), forest and soil development, and overstory cover (OC). We also determined the correlation of δ¹³C with other functional traits and nutrient composition. Based on patterns within and across tracheophyte species, we hypothesized bryophyte δ¹³C would increase at (1) higher elevation, (2) higher irradiance, (3) lower precipitation and (4) higher CMA.

Materials and methods

Study site, species and sampling

Sampling took place across an elevational gradient on the windward slope of Mauna Loa, Hawaii Island, a model system for studies of ecosystem development (Vitousek et al. 1995; Raich et al. 1997). Mean annual temperature (MAT) declines with elevation on this slope at a lapse rate of 6.4°C/km (Juvik and Nullet 1994). Bryophytes were sampled at six elevations (287–2,239 m) on young lava flows (126–152 years old) and old lava flows (≥3,400 years old; Lockwood et al. 1988) at ten sites (Table 1; Fig. 1). Bryophytes occupied seven primary substrates (bark, humus, leaf litter, rock, soil, rotted wood, and tree fern trunk), and accounted for 10–80% of surface cover.

Table 1.

Bryophyte taxa and substrates sampled from young and/or old lava flows at seven elevations on Mauna Loa, Island of Hawaii, and the minimum, mean, and maximum values for average carbon isotope composition (δ¹³C) across individuals at the sampled sites, and modeled climates for each site

Taxa sampled	Family	Substrate, average height above ground (m)	Elevations (m) and soil ages sampled							δ13C (‰)
			300	700	1,100	1,650	1,750	1,760	2,200
Acroporium fusco-flavum	Sematophyllaceae	Leaf litter–bark, 0.4	Y	Y, O	Y, O	O	O			−29.0, −30.4, −32.2
Bazzania cf. trilobata	Lepidoziaceae	Bark–humus, 0.4		Y, O	Y, O	O	O	O		−27.1, −29.5, −31.0
Campylopus exasperatus	Dicranaceae	Rock, 0.4		Y						−25.9
Campylopus hawaiicus	Dicranaceae	Humus–bark, 0.6			O	O	O	O		−27.6, −29.1, −30.4
Campylopus incurvatus	Dicranaceae	Bark, 0.4		O						−31.4
Campylopus schmidii	Dicranaceae	Rock, 0.4					O			−27.5
Dicranum speirophyllum	Dicranaceae	Leaf litter–humus, 0.4			Y	Y, O	O	O	O	−28.7, −29.3, −29.9
Leucobryum gracile	Dicranaceae	Bark–humus, 0.6	Y	O	O	O		O		−30.2, −31.2, −32.4
Leucobryum seemannii	Dicranaceae	Humus, 0.4	Y	O						−32.5, −32.5, −32.6
Macromitrium microstomum	Orthotrichaceae	Bark, 1.8	Y	Y, O	Y, O	Y, O	O	O		−28.2, −29.4, −30.7
Pyrrhobryum spiniforme	Rhizogoniaceae	Bark–humus, 0.4		O	O	O		O		−28.2, −28.7, −29.5
Racomitrium lanuginosum	Grimmiaceae	Rock, 0.4	Y	Y	Y	Y	O	O		−24.7, −26.6, −28.9
Modeled climate
Mean annual temperature (°C)			21.0	18.1	15.4	12.8	12.3	12.2	9.7
Mean annual precipitation (mm)			4,514	5,840	4,320	2,840	2,420	2,360	1,510
Mean annual vapor pressure deficit (kPa)			0.573	0.369	0.318	0.352	0.365	0.366	0.430

Species nomenclature follows Staples et al. (2004); family nomenclature follows Tropicos, Missouri Botanical Garden [http://www.tropicos.org/]. Lava age is designated as ‘young’ (Y) for 1855–1881 flows and ‘old’ (O) for the 3,400-year-old lava flow (and the ca. 400-year-old lava flow at the 1,750 m site). R. lanuginosum was present but not sampled at the 2,200 m site because of difficulties distinguishing live from dead material

Fig. 1.

Topographic map of Hawaii Island with 300-m elevational contour lines and sampling locations with elevations

Eleven moss and one liverwort species were collected at ten sites (Table 1). Species were chosen that were sufficiently common to allow collection of replicates at given sites. The species varied in elevational range; the extreme cases were Campylopus incarvatus, found only at the 700 m old flow site, and Racomitrium lanuginosum, which, like the dominant tree species M. polymorpha, occurred at all sampled elevations. We collected samples of 40–150 cm² in projected area from three colonies per species, where possible, at each site (in 6 of 53 cases only a single colony was found for a given species). The projected area of each sample was traced on clear plastic and scanned (300 dots per inch; Epson 3170 Photo Scanner; Seiko Epson, Nagano, Japan) for area determination (using ImageJ 1.42q software; US National Institutes of Health, Bethesda, MD, USA). The substrate and the estimated height above ground for epiphytic colonies were recorded to the nearest 10 cm; non-epiphytic colonies were collected from rocks, fallen logs, and the ground surface. Samples were brought to the laboratory in plastic bags (Ziploc; SC Johnson, Racine, WI, USA) on ice and refrigerated until processing.

Climate data and overstory cover

We estimated the climate at each sampling site using a Geographic Information System model based on climate station data (Cao et al. 2007) and Geographic Positioning System coordinates (GPSmap 60CSx; Garmin, Olathe, KS, USA). We estimated mean annual temperature (MAT), precipitation (MAP), relative humidity (MARH) and vapor pressure deficit (VPD), calculated from MAT and MARH (Campbell and Norman 1998). VPD is a measure of atmospheric drought, i.e., the driving force for evaporation, and can be calculated as an absolute pressure difference (in kPa of vapor) or after normalizing by atmospheric pressure (mole fraction; i.e., unitless); the mole fraction VPD accounts for the effects of atmospheric pressure and temperature on diffusibility and thus more clearly indicates differences in driving force across elevations. Across sites the mole fraction VPD correlated strongly with absolute VPD (r = 0.92; P < 0.001), and the two estimates correlated similarly with other measured traits; only relationships with absolute VPD are presented. Forest overstory cover (OC, %), the proportion of the sky obscured from view (equivalent to canopy closure in Jennings et al. 1999), was visually assessed, to the nearest 10%. Visual canopy cover estimates involve a level of uncertainty, but correlate with measurements using a densiometer or hemispherical photography (Schott and Pieper 1985; Vora 1988; Bellow and Nair 2003; Korhonen et al. 2006), especially given training as was undertaken previously to this study, to achieve a correspondence of ±5% relative to hemispherical photos (Teti and Pike 2005; Korhonen et al. 2006; Paletto and Tosi 2009).

Measurements of structure, nutrient concentrations and δ¹³C

CH was determined with a ruler to the nearest mm of the green portion of the canopy at three random positions in each sample. The upper, green portions of the stems were cut from the older brown tissue using a razor blade, dried at 80°C for at least 48 h before determination of dry mass (AB204S/FACT Analytical Balance; Mettler Toledo, Columbus, OH, USA). Canopy mass per area (CMA) was determined by dividing dry mass by ground area. Dried samples were analyzed for δ¹³C, nitrogen per mass (N _mass) and carbon per mass using high temperature combustion in an elemental analyzer (Costech ECS 4010; Valencia, CA, USA), with effluent passed into a continuous flow isotope ratio mass spectrometer (ThermoFinnigan Delta V Advantage with a Conflo III interface; ThermoFisher Scientific, Waltham, MA, USA; Fry et al. 1996). Samples were dry ashed in glass vials (Miller 1998), dissolved in 1 N HCL and analyzed for phosphorus per mass (P _mass) using inductively coupled plasma-optical emission spectroscopy (Varian Vista MPX Instrument; Varian, Palo Alto, CA, USA; Porder et al. 2005). Concentrations of nitrogen and phosphorus per ground area (N _area and P _area) were determined as, respectively, N _mass and P _mass multiplied by CMA. N:P was determined by dividing N _mass by P _mass; C:N and C:P by dividing carbon per mass by, respectively, N _mass and P _mass.

Statistical analyses

Statistical procedures were applied using R 2.6.1 (R Development Core Team 2007). We tested for differences in δ¹³C across elevations, soil ages and species using three-way analysis of variance (ANOVA) and across substrate types using one-way ANOVA after log-transformation to improve normality and homoscedasticity (Zar 1999). Multiple pairwise camparisons of δ¹³C among substrate types was calculated with the Tukey honestly significant difference test (HSD; Zar 1999). Subsequent correlation analyses used species mean values for each site. Pearson correlations were determined for linear and power law relationships (i.e., linear relationships after log-transformation) for δ¹³C against environmental variables and bryophyte composition and structure variables. Relationships were tested for species mean values across all sites, and separately across only young and old soil ages independently, and for each of the six taxa that were sampled at six or more sites (five species: A. fuscoflacum, Bazzania cf. trilobata, D. speirophyllum, M. microstomum, R. lanuginosum; and one genus, Campylopus). The δ¹³C relationship with OC was tested across substrate types using raw data rather than species means because a single species could be found on several substrates within a single site.

For relationships that were significant for all species across all sites and also for individual soil ages and/or for more than one individual taxon, we tested for shifts in the relationship. We tested for similarity of the slope and intercept between the soil ages or among taxa, using untransformed or log-transformed data according to which fitted best the relationship for all species across all sites (using SMATR; http://www.bio.mq.edu.au/ecology/SMATR; Warton et al. 2006). Ordinary least squares regression was used for testing relationships between δ¹³C and climate variables or OC, and standard major axes were used in testing relationships between δ¹³C and structure and composition traits, as appropriate given co-dependence of variables (Sokal and Rohlf 1995).

When bryophyte traits were correlated with climate variables that were themselves inter-correlated, we calculated partial correlations to resolve underlying relationships (using the corpcor package in R; Schaefer et al. 2007).

Results

Species varied strongly in δ¹³C; average values for species collected from at least four sites ranged from −26.3‰ ± 0.4 for R. lanuginosum to −31.2‰ ± 0.4 for L. gracile. Additionally, individual taxa varied strongly in δ¹³C across sites; for R. lanuginosum, δ¹³C differed by 4.2‰ from 300 to 1,650 m elevation on young soil, and within the genus Campylopus, δ¹³C differed by 5.5‰ from C. exasperatus at 700 m to C. hawaiicus at 1,100 m (Table 1). Across all sampled colonies, the δ¹³C showed significant differences across taxa, elevations and soil ages (three-way ANOVA; P < 0.001 for each factor), with less negative values at higher elevation and on younger soil (Appendix Table A); on average the bryophytes on young and old soil had δ¹³C of −28.7‰ ± 0.5 and −29.7‰ ± 0.2, respectively. Notably, all the climate variables correlated with elevation (for MAT, r = −0.999; for MAP, r = −0.89; for VPD, r = −0.68; P < 0.001), and MAT correlated with MAP (r = 0.92, P < 0.001) and VPD (r = 0.53, P < 0.001), though MAP was independent of VPD (r = 0.11, P = 0.45; Table 2). Across all the species and sites, δ¹³C correlated positively with elevation and negatively with MAT (|r| = 0.31–0.32, P < 0.05; Fig. 2a) but not significantly with MAP or VPD (r = −0.23 to −0.25; P = 0.07–0.10; Fig. 2b). Across all sampled colonies, the δ¹³C showed significant differences across substrate type (one-way ANOVA; P < 0.001) with colonies on the rock substrate showing δ¹³C values significantly higher than those on bark, humus and tree fern trunk (Tukey HSD; P < 0.001) but no significant differences among other substrates (Tukey HSD; P = 0.08–0.99; Appendix Table B).

Table 2.

Correlations of δ ¹³C with other measured traits from all sites, from young sites, old sites, and for given taxa

	All species/all sites (n = 53)	All species/young soil (n = 17)	All species/old soil (n = 33)	Within-species
				CAMP (n = 6)	RALA (n = 6)
Sampling height above ground	−0.08 (n = 51)	−0.03	−0.11 (n = 31)
Climate variables
Elevation	0.31*	0.56*	0.54***	0.07	0.40
Mean annual temperature	−0.32*	−0.55*	−0.54***	−0.08	−0.44
Mean annual precipitation	−0.25	−0.14	−0.55***	−0.04	0.01
Vapor pressure deficit (absolute)	−0.23	−0.55*	0.17	0.21	−0.81
Overstory cover	−0.63***	−0.75***	−0.42*	−0.89*	−0.84*
Canopy morphology and composition
Canopy height	0.15 (n = 49)	−0.09 (n = 13)	0.28	−0.82 (n = 5)	−0.52 (n = 4)
Canopy mass per area	0.37**	0.50*	0.27	0.52	0.01
Canopy density	0.13 (n = 49)	0.32 (n = 13)	0.00	0.65 (n = 5)	0.27 (n = 4)
Nitrogen per mass	−0.20	−0.23	−0.22	0.09	−0.39
Nitrogen per area	0.32*	0.40	0.21	0.61	−0.13
Phosphorus per mass	−0.03	0.16	−0.09	0.75	−0.02
Phosphorus per area	0.27	0.38	0.17	0.67	0.03
Nitrogen:phosphorus ratio	−0.05	−0.19	−0.17	−0.47	0.11
Carbon:nitrogen ratio	0.26	0.19	0.35*	−0.19	0.34
Carbon:phosphorus ratio	0.14	0.06	0.05	−0.77	0.27

Pearson correlation coefficients, with italicized values derived from log-transformed data, providing better fit and stronger significance. Replication is provided in parentheses in the first row, with exceptions denoted in the table

CAMP Campylopus spp., RALA Racomitrium lanuginosum

Significant correlations in bold: * P < 0.05; ** 0.01 ≥ P > 0.001; *** P ≤ 0.001. No within-species correlations were found for Acroporium fuscoflavum (n = 7) Bazzania cf. trilobata (n = 7) Dicranum speirophyllum (n = 6) or Macromitrium microstomum (n = 9)

Fig. 2.

Relationship between bryophyte δ¹³C and a elevation and b mean annual precipitation (MAP) on Mauna Loa, Hawaii. Open and closed symbols represent colonies on young and soil, respectively. Linear regression fitted to data in (a) δ¹³C = −30.6 + 0.00104 × elevation. *P < 0.05

Across sites, δ¹³C correlated negatively with OC (r = −0.63, P < 0.001; Fig. 3a). In turn, OC was independent of elevation, MAT, MAP, and VPD (r = 0.09–0.22, P = 0.12–0.52). Part of the effect may have related to soil age, as OC differed between young and old soil sites from 52 to 62% on average (three-way ANOVA; elevation, P = 0.008; substrate age, P < 0.001; taxon, P < 0.001), and, as described above, δ¹³C was higher on younger soil. However, the linkage of δ¹³C with OC also held on soils of given age (see below). The linkage of δ¹³C with OC also held on most individual substrates (bark, leaf litter, and rock) for which there were more than four samples (r = 0.45–0.68, P < 0.001; Appendix Table B); the exception was on humus (r = 0.29, P = 0.14). The δ¹³C did not correlate with sampling height (r = −0.08, P = 0.58; a typical value of 0.4 m was used for non-epiphytic species on ground, rocks or logs).

Fig. 3.

Relationship between bryophyte δ¹³C and a elevation overstory cover (OC), and b bryophyte canopy mass per area (CMA). Open and closed symbols represent colonies on young and soil, respectively. Linear regression fitted to data in (a) δ¹³C = −25.3 − 0.068 × OC. Standard major axis power law fitted to data in (b) log (−1 × δ¹³C) = 1.70 − 0.110 × log CMA. The correlation in (b) remained significant when the outlier at the right was removed (r = 0.30, P = 0.03). **0.01 ≥ P > 0.001; ***P ≤ 0.001. Notably, the relationship between δ¹³C and CMA appeared due to both variables being associated with OC; see partial correlation analysis in Table 3, and text

Across all species and sites, δ¹³C correlated with CMA and N _area (r = 0.32–0.37, P = 0.01–0.02; Fig. 3b; Table 2). However, δ¹³C was independent of bryophyte CH, canopy density, and other nutrient traits (N _mass, P _area, P _mass, N:P, C:N, and C:P; |r| = 0.03–0.26, P = 0.06–0.81).

The relationships of δ¹³C with elevation, climate, morphology and composition that held across all sites were similar when calculated only for sites on young or old substrates. Thus, on both young and old soil ages considered independently, δ¹³C correlated with elevation (r = −0.53 to −0.54, P < 0.001–0.02), MAT (r = 0.55–0.54, P < 0.001–0.02), and OC (r = 0.42–0.75, P < 0.001–0.01), but not with bryophyte CH, density, or nutrient composition (N _area, N _mass, P _area, P _mass, N:P, C:P, CH, and CD (|r| < 0.01–0.32, P = 0.06–0.81). The slopes of δ¹³C against elevation and MAT were similar for both soil ages (P = 0.65–0.69) with higher intercepts for the young soil (P < 0.001). For δ¹³C against OC, the two soil ages showed the same relationship (i.e., similar slopes and intercepts; P = 0.28–0.36). However, in contrast with the data for all species and sites, on the young but not the old soil sites, δ¹³C correlated negatively with VPD and CMA (r = −0.50 to −0.55, P = 0.02–0.03), and on old but not on young soil, δ¹³C correlated positively with C:N and negatively with MAP (|r| = 0.35–0.55, P < 0.001–0.04).

When the taxa that occurred at six or more sites were analyzed separately, due apparently to the much lower variation in δ¹³C (Table 1), the only significant correlation of δ¹³C and an environmental or compositional trait was with OC for Campylopus and R. lanuginosum (r = −0.84, P < 0.05); the two taxa had similar slopes (P = 0.25) with the higher intercept for R. lanuginosum (P < 0.001). All other species showed non-significant empirical trends for this relationship (r = −0.43 to −0.73; P > 0.05).

Partial correlation analyses were used to resolve the relationships of δ¹³C with OC, MAT, and MAP, keeping other variables constant. The correlations of δ¹³C with OC and MAT could be resolved as independent effects; the correlation with OC remained significant after partialing out the effects of MAT and MAP (r _partial = −0.61; P < 0.001; Table 3), and the correlation with MAT remained significant after partialing out the effect of OC when log-transformed data were analyzed (r _partial = −0.28; P < 0.05; Table 3). Notably, the relationship of δ¹³C with MAT lost significance after partialing out MAP, as did that with MAP after partialing out MAT (r _partial = −0.15 to 0.03; P = 0.29–0.83; Table 3); thus, elevation, MAT and MAP could not be independently resolved as drivers of δ¹³C, given their inter-correlation across the Mauna Loa matrix.

Table 3.

Partial correlation analyses of δ¹³C and environmental variables

	OC	Elevation	MAT	MAP	CMA	N area
1	−0.63***	0.31*
2	−0.62***		−0.28*
3	−0.61***		−0.15	0.03
4			−0.21	0.06
5	−0.57***				0.03	0.03
6	−0.57***				0.12
7	−0.59***					0.12

Each row represents a separate partial correlation analysis of δ¹³C with the factors in the columns, partialling out the effect of all the other factor(s) in that row. For example, the first value in row (1) presents the partial correlation of δ¹³C and OC, controlling for the effect of elevation

Values in italics were derived from log-transformed data (see Materials and methods)

Bold type indicates correlations that remained significant after partialling out the other factor(s)

CMA, canopy mass per area; MAP, mean annual precipitation; MAT, mean annual temperature; N _area, nitrogen per area; OC, overstory cover

Significance: * P < 0.05, ** 0.01 ≥ P > 0.001, *** P ≤ 0.001. The correlations with elevation and MAT remained significant after partialling out the effect of OC for log-transformed data but not untransformed data. Correlations shown without significance were non-significant using log-transformed or untransformed data

Partial correlation analyses were also used to resolve the relationships of δ¹³C with OC, CMA, and N _area, because N _area and CMA were correlated (r = 0.91; P < 0.001), and both were negatively correlated with OC (r = −0.46 to −0.37; P < 0.001–0.006). The relationship of δ¹³C with OC remained significant after partialing out CMA and N _area (r _partial = −0.57; P < 0.001; Table 3). However, the relationship of δ¹³C with CMA or N _area lost significance after the other was partialed out, or after OC was partialed out (r _partial = 0.03–0.12; P = 0.40–0.83; Table 3).

Discussion

Across the Mauna Loa matrix landscape, the bryophytes varied greatly in δ¹³C due to species differences and their being located across sites ranging markedly in elevation, climate and soil age. Across the landscape, the bryophytes exhibited similar correlations of δ¹³C with elevation and habitat irradiance as previously reported for tracheophytes. This finding indicates a potential for parallel underlying processes, despite the lack of stomata in the dominant bryophyte gametophyte life stage, and provides insights into functional relationships. These trends in bryophytes have previously been reported within a few individual species, primarily in the genus Sphagnum (Menot and Burns 2001; Fletcher et al. 2006; Skrzypek et al. 2007). Our study extends these findings across 12 species within eight genera, across natural gradients at a landscape scale. Notably, the bryophytes on Mauna Loa showed only a weak, non-significant negative correlation of δ¹³C with rainfall (MAP), despite its nearly three-fold variation, thus suggesting no important “liquid water film effect” integrated over long-term growth across sites. This finding contrasts with positive trends of δ¹³C with water availability previously reported in studies of Sphagnum species across varying water table heights and moisture contents (Proctor et al. 1992; Rice and Giles 1996; Williams and Flanagan 1996; Loisel et al. 2009) and precipitation (Menot and Burns 2001; Zhu et al. 2009). Notably, Sphagnum species are unusual among mosses in having photosynthetic cells that are surrounded by dead, hollow cells that hold water and would contribute to greater CO₂ diffusion resistance. Without such modified water-holding cells, the leaves of typical bryophyte species may quickly drain to an optimal water content less restrictive to CO₂ diffusion (Dilks and Proctor 1979). Of the study taxa, only Leucobryum has a cellular morphology approaching that of Sphagnum.

Potential sources of the δ¹³C—elevation correlation

The landscape-scale increase of δ¹³C with elevation in bryophytes on Mauna Loa was similar to that of M. polymorpha trees at these sites (Vitousek et al. 1990; Cordell et al. 1998, 1999) and that observed generally for vascular plants along elevational gradients worldwide (Körner et al. 1988, 1991, 2003; Morecroft et al. 1992; Hikosaka et al. 2002). This finding extends the elevational trends reported for Sphagnum (Menot and Burns 2001; Skrzypek et al. 2007) to a broader set of genera. In vascular plants, the trend was attributed to reduced CO₂ partial pressure, associated with the lower atmospheric pressure at higher elevations, which would reduce c_c and increase δ¹³C (Körner et al. 1991), but later work concluded that such an effect would be too small to explain observed variation (Terashima et al. 1995; Körner 2003, 2007). The trend in vascular plants has also been attributed to increased leaf mass per area (LMA) with elevation (Körner 1989, 2003; Vitousek et al. 1990; Cordell et al. 1998; Hikosaka et al. 2002; Takahashi and Miyajima 2008; Li et al. 2009). A higher LMA in vascular plants may lead to lower mesophyll conductance, lower ratio of chloroplast to atmospheric CO₂ partial pressure (c _c/c _a) and higher δ¹³C (Terashima et al. 1995) because a greater leaf thickness increases the diffusion path length from the stomata to the mesophyll cell walls (Parkhurst 1994), and/or because thicker mesophyll cell walls reduce CO₂ diffusion from intercellular air space to the chloroplasts (Niinemets 1999; Niinemets et al. 2009). We found that the δ¹³C decreased with decreasing bryophyte canopy mass per area (CMA), analogously to the decrease of δ¹³C with decreasing LMA in M. polymorpha (Vitousek et al. 1988), but whereas in M. polymorpha and in many other vascular plants LMA increases with elevation (Vitousek et al. 1988; Cordell et al. 1998; Hikosaka et al. 2002; Körner 2003), bryophyte CMA did not correlate with elevation but rather with OC (as discussed further below). These results indicate that the altitudinal trend in δ¹³C was likely due to lower CO₂ pressure and/or the “temperature effect” as shown for three Sphagnum species (Menot and Burns 2001; Skrzypek et al. 2007). Such an effect contrasts with tracheophytes, for which there is no evidence for a general direct effect of temperature on δ¹³C (Troughton and Card 1975; Christeller et al. 1976). There is also the possibility that the declining cloud cover and thus increased irradiance, lower MAP and higher VPD at higher elevation might have contributed to the increase of δ¹³C. However, we found no correlation of δ¹³C with MAP or VPD, and thus such an effect was apparently not important at the landscape scale.

Potential sources of the δ¹³C-soil age and δ¹³C–OC correlations

We observed a tendency for lower bryophyte δ¹³C on older soils on Mauna Loa. This finding is similar to that reported for leaves of M. polymorpha which have lower LMA and lower δ¹³C on the young flows (Vitousek et al. 1990). However, for the bryophytes, the effect was weak and apparently linked with the lower OC on young flows.

The strong correlation of bryophyte δ¹³C with OC across the landscape would likely have arisen from several factors. A first candidate is the “source air effect” if incomplete mixing resulted in air enriched in ¹²C due to soil-respired CO₂ at bryophyte-level at sites with higher OC; such an effect would be stronger when canopies are dense, hindering airflow (Schleser and Jayasekera 1985; Broadmeadow et al. 1992). However, the bryophytes in this study were typically collected <0.5 m above ground, and up to only 5.5 m above soil, and tissue δ¹³C did not correlate with vertical height as expected if soil respiration were a major driver of the δ¹³C–OC relationship. The δ¹³C was significantly higher in colonies growing on rock than on organic substrates, as would be expected if substrate respiration had an important effect. However, colonies growing on humus, where substrate decomposition presumably leads to high CO₂ production, did not differ significantly from those growing on bark substrates, and, most importantly, the δ¹³C–OC relationship held on individual substrates, including on rock. Thus, a vertical gradient in source air composition would likely contribute as a subtle effect, rather than as key driver of the relationship. The strong gradient in irradiance was likely the primary driver of the relationship, as reported for leaves of tracheophytes within forests (Buchmann et al. 1997b; Sternberg et al. 1997) and within the canopies of single trees (Le Roux et al. 2001). The δ¹³C–OC relationship may arise from the “irradiance-A effect” effect, i.e. a higher δ¹³C due to increased photosynthetic rate. The relationship might also arise from an indirect effect of water stress in more exposed, drier microhabitats leading to the development of thicker cell walls with greater water storage capacity, which during periods of high water availability would reduce CO₂ diffusion (Dilks and Proctor 1979; Waite and Sack 2010). Such an effect may hold for individual species, but the lack of a significant relationship of δ¹³C with MAP or with VPD despite their varying by two- to three-fold across sites did not support the importance of such an effect at the landscape scale. We note that variation across taxa in cell size might also be expected to play a role; across three species of microalgae, larger cell size was related to reduced CO₂ conductance and higher δ¹³C (Popp et al. 1998). That scenario is unlikely to be important across bryophyte species, in which larger cells tend to be associated with deeper shade (Waite and Sack 2010) while higher δ¹³C was related to lower OC and hence greater irradiance.

Thus, the results best support a direct effect of irradiance on photosynthetic rate as a key driver of the bryophyte δ¹³C–OC relationship. Such an effect was found experimentally for two liverwort species grown at four irradiance levels; δ¹³C correlated positively with growth irradiance (Fletcher et al. 2006). Our findings extend this pattern across multiple species and show its potential importance at the landscape scale, across sites varying in forest successional stages. The effect is quantitatively strong; when the range of OC was divided into five bins, the average δ¹³C shifted from −27.1 to −30.6‰. The effect was also strong on bark, leaf litter and rock substrates individually; the lack of the correlation on humus may be due to the lower range of OC values compared to the other substrate types. Notably, the correlation of OC and δ¹³C observed across all species and sites was also found within two of the six tested taxa, i.e., for Campylopus and in R. lanuginosum, which had the greatest range in OC values. Whereas these within-species relationships emerged from their wide distribution across OC levels, the across-species relationship reflected the assembly of diverse species along a light gradient (cf. Ackerly and Cornwell 2007).

We found that bryophyte δ¹³C correlated with the morphological variable CMA, analogously to LMA for tracheophyte leaves. This finding points to integrated adjustment of canopy form and physiology with irradiance for bryophytes across forest successional stages. However, by contrast with tracheophytes, in which the correlation of δ¹³C and LMA may arise in part from their relationship with mesophyll conductance, our partial correlation analysis indicated that in bryophytes, the relationship of δ¹³C with CMA arose indirectly due to relationships with OC. The high scatter in the correlation of δ¹³C with CMA, and their joint relationship with OC indicates that additional data would be needed to support a direct control of δ¹³C by CMA for any given species.

Implications and future work

The bryophytes of this study exhibited great similarities with vascular plants in landscape-scale patterns, showing independent and strong trends for lower carbon isotope fractionation with younger soil age, greater canopy openness and increasing elevation. The similarity with tracheophytes is especially remarkable given the centrality of stomata in explaining carbon isotope fractionation in tracheophytes and the lack of these structures in bryophytes. Our findings point to direct environmental impacts of light and temperature on gas exchange rates as primary mechanisms for these landscape-level trends. We inferred the importance of given potential effects from correlation analyses at landscape scale; individual species may differ in the factors that determine δ¹³C. These findings highlight a necessity for additional mechanistic studies to clarify the underlying basis for these landscape-level trends. Indeed, for bryophytes, we lack a physiological predictor of δ¹³C signals, in the way that stomatal water use efficiency can be used for tracheophytes (reviewed in Seibt et al. 2008). This study, showing strong δ¹³C signals across the landscape scale, points to the importance of elucidating the determinants of carbon isotope discrimination in bryophyte structure and biochemistry (see Meyer et al. 2008). Such studies will need to determine isotope fractionation in real time for given species under ranges of controlled conditions to deepen the understanding of the landscape scale patterns demonstrated here.

We found the variation in δ¹³C in bryophytes at the landscape scale—including diverse taxa across diverse systems—to be information-rich, reflecting the signal of multiple environmental factors. This finding indicates the possibility of extending the usefulness of preserved bryophyte tissue to estimate past environmental conditions. Previous work emphasized that the lack of stomata can link bryophyte δ¹³C more closely with atmospheric CO₂ concentration than for vascular plants (White et al. 1994; Fletcher et al. 2005, 2006, 2008), and pointed to the δ¹³C of fossilized Sphagnum as a proxy for moisture conditions, given that the “liquid film effect” would lead to higher δ¹³C in wetter climates (Loisel et al. 2009; Zhu et al. 2009). However, the lack of a relationship in our study between δ¹³C and MAP in the Mauna Loa bryophytes suggests that not all species would be good proxies for moisture conditions, though they may be proxies for other environmental factors. An avenue for future work is to determine how to model the combined effects of elevation, irradiance, soil age and temperature on given bryophyte floras. Alternatively, it may be possible to tease apart specific environmental signals, perhaps by using additional isotopes to carbon. Thus, bryophytes, by occupying, and responding to, a very wide range of environments could provide in their tissues recoverable information not only of physiological responses across gradients but also information of their climates and microhabitats.

Electronic supplementary material

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