Andrew S Carr

Brian M Chase

Arnoud Boom

Michael E Meadows

Javier Medina Sanchez

Variability in soil and foliar stable carbon and nitrogen isotope compositions in the winter rainfall biomes of South Africa

Keywords: CAM plant, climate, photosynthesis, organic matter, Fynbos, Succulent Karoo Highlights

We present the stable carbon and nitrogen isotope compositions of 204 plants and 99 soils from South Africa's Fynbos and Succulent Karoo Biomes, from which we assess variability in  13 C and  15 N within and between the biomes. Higher foliar  13 C in the Succulent Karoo (-22.4±4.7‰) primarily reflects a greater abundance of CAM photosynthesis relative to Fynbos (-25.7±3.5‰). For C3 plants within the Fynbos Biome, there is no statistically significant relationship between  13 C and climate, suggesting that within the present sampling framework, local-scale variability masks expected climatic controls on C3 plant  13 C.

Soil and foliar  15 N are highly variable, but foliar  15 N is significantly higher in the Succulent Karoo (7.7±3.9 ‰) compared to the Fynbos (4.4±3.3 ‰). Soil and foliar  15 N correlate, with soils 3±1‰ more enriched in 15 N. Both soil and foliar  15 N show stronger correlations with climate than  13 C, and they accord with the previously observed tendency for higher  15 N with greater aridity. The relationship between  15 N and mean annual rainfall is, however, relatively steep (2.4±0.6‰ per 100 mm rainfall), potentially implying additional controls on  15 N variability. These data provide a benchmarking of contemporary isotopic variability, anticipated to support future archaeological, palaeoenvironmental and C and N cycling research.

Introduction

Stable carbon and nitrogen isotope analyses provide key insights into the nature and functioning of terrestrial ecosystems [START_REF] Dawson | Stable isotopes in plant ecology[END_REF][START_REF] Craine | Global patterns of foliar nitrogen isotopes and their relationships with climate, mycorrhizal fungi, foliar nutrient concentrations, and nitrogen availability[END_REF]. Across southern Africa, several studies have examined variability in foliar  13 C and  15 N [START_REF] Vogel | Occurrence of C4 plants in the Central Namib Desert[END_REF][START_REF] Heaton | The 15 N/ 14 N ratios of plants in South Africa and Namibia: relationship to climate and coastal/saline environments[END_REF]Schulz et al. 1996;[START_REF] Swap | Natural abundance of 13 C and 15 N in C3 and C4 vegetation of southern Africa: patterns and implications[END_REF][START_REF] Aranibar | Nitrogen cycling in the soil-plant system along a precipitation gradient in the Kalahari sands[END_REF][START_REF] Wang | Patterns and implications of plant-soil  13 C and  15 N in African savannah ecosystems[END_REF]2013;[START_REF] Codron | Plant stable isotope composition across habitat gradients in a semi-arid savanna: implications for environmental reconstruction[END_REF][START_REF] Herrmann | Sources, transport and deposition of terrestrial organic material: A case study from southwestern Africa[END_REF][START_REF] Carr | Stable isotope analysis of rock hyrax faecal pellets, hyraceum and associated vegetation in southern Africa: implications for dietary ecology and palaeoenvironmental reconstructions[END_REF]. These studies show the varying sensitivities of isotopic values to climatic conditions and biome type, and present opportunities for further investigations into the isotopic and geochemical responses of plants and soils across this region. The increasingly routine use of stable isotope data in palaeoenvironmental and archaeological research in this region also provides an important impetus to benchmark contemporary soil and plant isotopic compositions across the region's biomes [START_REF] Chase | Rock hyrax middens: a palaeoenvironmental archive in southern African drylands[END_REF][START_REF] Sealy | Late Quaternary environmental change in the Southern Cape, South Africa, from stable carbon and oxygen isotopes in faunal tooth enamel from Boomplaas Cave[END_REF], Foliar  13 C varies with plant photosynthetic pathway, viz C3 (generally trees, shrubs and coolseason grasses), C4 (generally grasses adapted to warm/dry conditions) and Crassulacean Acid Metabolism (CAM) plants [START_REF] O'leary | Carbon isotopes in photosynthesis[END_REF]. The distribution of C3 and C4 plants in southern Africa reflects rainfall seasonality and growing season temperature, with a greater abundance of C4 grasses in the summer rainfall (Nama Karoo, Savanna and Grassland) biomes (Vogel and Seely, 1987;[START_REF] Vogel | Isotopic assessment of the dietary habits of ungulates[END_REF]. Variations in soil organic matter  13 C, are also known to respond to the abundance of C4 grasses [START_REF] Bond | Has the Karoo spread? A test for desertification using carbon isotopes from soils[END_REF][START_REF] Herrmann | Sources, transport and deposition of terrestrial organic material: A case study from southwestern Africa[END_REF]).

In the winter rainfall zone of the western and southwest margins of southern Africa, particularly in the Fynbos Biome (Figure S1), where the vegetation is dominated by C3 plants, intrinsic water use efficiency (WUEi) is anticipated to generate variability in carbon isotope discrimination (and foliar   C  [START_REF] Cernusak | Environmental and physiological determinants of carbon isotope discrimination in terrestrial plants[END_REF][START_REF] Farquhar | Carbon isotope discrimination and photosynthesis[END_REF]. Globally, one third to one-half of global-scale variation in C3  is explained by mean annual precipitation [START_REF] Diefendorf | Global patterns in leaf 13 C discrimination and implications for studies of past and future climate[END_REF][START_REF] Kohn | Carbon isotope compositions of terrestrial C3 plants as indicators of (paleo)ecology and (paleo)climate[END_REF]. At the regional scale similar trends are observed along environmental gradients, (e.g. [START_REF] Stewart | 13 C natural abundance in plant communities along a rainfall gradient: a biological integrator of water availability[END_REF][START_REF] Van Der Water | Leaf  13 C variability with elevation, slope aspect and precipitation in the southwest United Sates[END_REF][START_REF] Peri | Carbon (δ 13 C) and nitrogen (δ 15 N) stable isotope composition in plant and soil in Southern Patagonia's native forests[END_REF]. At the local scale, foliar  13 C variability can be high (e.g. [START_REF] Aranibar | Nitrogen cycling in the soil-plant system along a precipitation gradient in the Kalahari sands[END_REF][START_REF] Araya | Variation in δ 13 C among species and sexes in the family Restionaceae along a fine-scale hydrological gradient[END_REF][START_REF] Codron | Plant stable isotope composition across habitat gradients in a semi-arid savanna: implications for environmental reconstruction[END_REF], with other biotic and abiotic factors influencing  (e.g. leaf nutrient content, taxonomic effects, altitude, irradiance, air temperature, atmospheric CO2 content [START_REF] Cernusak | Environmental and physiological determinants of carbon isotope discrimination in terrestrial plants[END_REF]). Local environmental factors, and variability associated with plant morphologies, functional types or life histories can lead to up to 6‰ differences in  between biomes [START_REF] Ehleringer | Correlations between carbon isotope ration and microhabitat in desert plants[END_REF][START_REF] Hartman | Isotopic values of plants in relation to water availability in the Eastern Mediterranean region[END_REF][START_REF] Mosher | Spatial variability in foliar carbon and nitrogen isotope values on Tenerife reflects both climate and soils: Establishing a framework for future work[END_REF]. In this respect, the degree of variability in foliar  13 C and, therefore, carbon isotope discrimination in C3 plants across the botanically diverse and topographic complex Fynbos biome remains poorly defined and beginning to address this knowledge gap is a goal of this paper. Such information will also be of wider utility in the interpretation of faunal and isotopic data from archaeological sites across this biome, where past environmental changes, potentially involving shifts in C3/C4 vegetation abundances, and/or more subtle changes in aridity/micro-habitat and C3 carbon isotope discrimination, might be inferred (e.g. [START_REF] Sealy | Late Quaternary environmental change in the Southern Cape, South Africa, from stable carbon and oxygen isotopes in faunal tooth enamel from Boomplaas Cave[END_REF][START_REF] Roberts | Climate, environment and early human innovation: stable isotope and faunal proxy evidence from archaeological sites (98-59ka) in the southern Cape, South Africa[END_REF] Located north of the Fynbos biome, the Succulent Karoo Biome (Figure S1) -the world's most species rich semi-desert region [START_REF] Rutherford | Biomes and bioregions of southern Africa. The vegetation of South Africa[END_REF]) -is renowned for an abundance of CAM plants.

CAM photosynthesis, found in ~6% of all vascular plants, is a mechanism for water conservation, whereby night-time CO2 fixation and storage is followed by daytime decarboxylation and CO2 reduction [START_REF] Osmond | Crassulacean acid metabolism: a curiosity in context[END_REF][START_REF] Winter | Crassulacean acid metabolism: a continuous or discrete trait?[END_REF]. In South Africa, CAM plants are especially prominent in the Succulent Karoo (von Willert et al. 1985;[START_REF] Veste | Variability of CAM in leaf-deciduous succulents from the Succulent Karoo (South Africa)[END_REF]Veste and Theide, 2004). Isotopically,  13 C in CAM plant tissue varies between species and individuals, depending on the degree of CAM use [START_REF] Winter | Ecophysiology of constitutive and facultative CAM photosynthesis[END_REF]. In the Succulent Karoo, considerable variability in CAM foliar  13 C has been documented [START_REF] Rundel | Ecology and phylogenetic patterns of carbon isotope discrimination in the winter-rainfall flora of the Richtersveld, South Africa[END_REF], and it is anticipated that the abundance of CAM species in this biome will render it isotopically distinct from the Fynbos Biome.

Stable nitrogen isotope ( 15 N) data provide insights into the functioning of the N cycle (Robinson, 2001;[START_REF] Pardo | Using nitrogen isotope ratios to assess terrestrial ecosystems at regional and global scales[END_REF], but variability in soil and foliar  15 N can be challenging to interpret given the number of the fractionating transformations within the N cycle [START_REF] Craine | Global patterns of foliar nitrogen isotopes and their relationships with climate, mycorrhizal fungi, foliar nutrient concentrations, and nitrogen availability[END_REF]2015a). As such, the roles of specific environmental variables in controlling foliar / soil  15 N are not always clear [START_REF] Peri | Carbon (δ 13 C) and nitrogen (δ 15 N) stable isotope composition in plant and soil in Southern Patagonia's native forests[END_REF][START_REF] Pardo | Using nitrogen isotope ratios to assess terrestrial ecosystems at regional and global scales[END_REF][START_REF] Díaz | Nitrogen cycling in an extreme hyperarid environment inferred from δ 15 N analyses of plants, soils and herbivore diet[END_REF]. At the regional to global scale, foliar  15 N correlates with aridity [START_REF] Austin | Foliar  15 N is negatively correlated with rainfall along the IGBP transect in Australia[END_REF][START_REF] Handley | The 15 N natural abundance (δ 15 N) of ecosystem samples reflects measures of water availability[END_REF][START_REF] Amundson | Global patterns of the isotopic composition of soil and plant nitrogen[END_REF][START_REF] Aranibar | Nitrogen cycling in the soil-plant system along a precipitation gradient in the Kalahari sands[END_REF][START_REF] Murphy | Kangaroo metabolism does not cause the relationship between bone collagen  15 N and water availability[END_REF][START_REF] Craine | Global patterns of foliar nitrogen isotopes and their relationships with climate, mycorrhizal fungi, foliar nutrient concentrations, and nitrogen availability[END_REF]cf. Nel et al. 2018). This is thought to reflect changes in the  15 N of associated soils, and the increased significance of N losses (relative to the size of the soil N pool) in arid environments [START_REF] Austin | Nutrient dynamics on a precipitation gradient in Hawai'i[END_REF][START_REF] Amundson | Global patterns of the isotopic composition of soil and plant nitrogen[END_REF]. Foliar  15 N can also vary with the presence and type of symbiotic mycorrhizae, and due to human impacts [START_REF] Craine | Global patterns of foliar nitrogen isotopes and their relationships with climate, mycorrhizal fungi, foliar nutrient concentrations, and nitrogen availability[END_REF][START_REF] Pardo | Using nitrogen isotope ratios to assess terrestrial ecosystems at regional and global scales[END_REF].

In southern Africa, foliar  15 N has been considered in the context of animal diet reconstructions [START_REF] Sealy | Nitrogen isotopic ecology in southern Africa: implications for environmental and dietary tracing[END_REF][START_REF] Codron | Reliability of δ 13 C and δ 15 N in faeces for reconstructing savanna herbivore diet[END_REF], analyses of nitrogen fixation (e.g. [START_REF] Muofhe | Nitrogen nutrition in nodulated field plants of the shrub tea legume Aspalanthus linearis assessed using 15 N natural abundance[END_REF][START_REF] Morris | Ecophysiological traits of invasive alien Acacia cyclops compared to co-occurring native species in Strandveld vegetation of the Cape Floristic Region[END_REF] and in palaeoclimatic reconstructions from rock hyrax middens [START_REF] Chase | Rock hyrax middens: a palaeoenvironmental archive in southern African drylands[END_REF]. Studies have shown that, in relation to climate, foliar  15 N in southern Africa broadly corresponds with global scale trends. For example, [START_REF] Heaton | The 15 N/ 14 N ratios of plants in South Africa and Namibia: relationship to climate and coastal/saline environments[END_REF] showed a weak negative correlation between foliar  15 N and mean annual precipitation (MAP) for 33 sites across central and eastern South Africa and western Namibia. [START_REF] Swap | Natural abundance of 13 C and 15 N in C3 and C4 vegetation of southern Africa: patterns and implications[END_REF] measured foliar  15 N for C3 vegetation from 21 sites across central and eastern South Africa, Botswana, northwest Namibia and western Zambia, with more than half of the  15 N variation (R 2 =0.54) in C3 plants (n=143) explained by MAP, despite a range of substrates and land uses (see also [START_REF] Wang | Patterns and implications of plant-soil  13 C and  15 N in African savannah ecosystems[END_REF]. Across the uniform substrate of the Kalahari sands, [START_REF] Aranibar | Nitrogen cycling in the soil-plant system along a precipitation gradient in the Kalahari sands[END_REF] demonstrated a negative correlation between foliar  15 N (non-N2 fixing C3 trees, shrubs and forbs) and MAP (range of 978-230 mm per annum), and a negative correlation between non-N2 fixing foliar  15 N and foliar C/N ratio.

Furthermore, the relationship between foliar  15 N and rainfall was steeper for plants sampled during the wet season, highlighting the importance of spatial/temporal variability in water availability for soil and plant  15 N compositions [START_REF] Wang | Ecosystem-scale spatial heterogeneity of stable isotopes of soil nitrogen in African savannas[END_REF]. [START_REF] Carr | Stable isotope analysis of rock hyrax faecal pellets, hyraceum and associated vegetation in southern Africa: implications for dietary ecology and palaeoenvironmental reconstructions[END_REF] presented foliar  15 N data from seven sites across a variety of biomes in South Africa and Namibia. Again, a negative correlation between MAP and foliar  15 N was reported, although the samples were from a limited number of widely spaced sites.

In this paper, we seek to build a more systematic understanding of foliar and soil  13 C and  15 N variability by employing a paired soil and plant sampling strategy across two South African biomes, viz.

the semi-arid to arid winter rainfall Fynbos and Succulent Karoo Biomes. Particularly in the case of  15 N, isotopic variabilities in these biomes have not been explored. We seek to build on existing works to assess foliar  13 C variability in C3 and CAM plants within and between these biomes, and to consider patterns in   C and  15 N in different plant growth forms. The specific objectives are to: 1) Quantify variability in foliar  13 C / 15 N within and between the Fynbos and Succulent Karoo;

2) Assess the contribution of CAM photosynthesis to foliar  13 C variability in the Succulent Karoo Biome;

3) Assess the degree to which variability in foliar  13 C and  15 N is captured in associated soils; 4) Assess the relationships between climatic parameters and soil/foliar  13 C and  15 N.

Study region and sample sites

Across both biomes, geological substrate, topography, rainfall amount and rainfall seasonality exert strong controls on vegetation. The Fynbos Biome is a Mediterranean-type ecosystem characterised by a high diversity of ericaceous, small leaved shrubs, sclerophyllous proteiod shrubs, Cape Reeds (Restionaceae) and geophytes [START_REF] Cowling | Fynbos[END_REF]. This biome is characterised by woody shrubs (C3) and the associated climate is predominantly winter rainfall (> 66% of mean annual rainfall in May-September; [START_REF] Chase | Late Quaternary dynamics of southern Africa's winter rainfall zone[END_REF], which determines any grasses that are present are C3 varieties [START_REF] Vogel | Isotopic assessment of the dietary habits of ungulates[END_REF].

Within the Fynbos Biome, the Montane Fynbos eco-region is associated with the uplands (~200-1600 m above sea level (asl)) of the Cape Fold Belt Mountains and occurs on shallow, low-nutrient sandy soils derived from the quarzitic Table Mountain Group sandstones [START_REF] Campbell | Montane plant environments in the Fynbos biome[END_REF].

The Lowland Fynbos eco-region (WWF 2020) is located on the coastal plain of the South African west coast, at altitudes below 200 m asl (Figure S1; Table 1). Substrates here comprise less acidic (than Montane Fynbos) free draining sandy soils (Arenosols) [START_REF] Carr | Biome-Scale characterisation and differentiation of semi-arid and arid zone soil organic matter compositions using pyrolysis-GC/MS analysis[END_REF]. The Succulent Karoo Biome is associated with a wider range of substrates [START_REF] Milton | Succulent Karoo[END_REF] and, in contrast to the Fynbos Biome, soils are generally finer grained, higher in nutrient content [START_REF] Cowling | Fynbos[END_REF] and more varied in terms of texture and clay mineralogy (e.g. [START_REF] Carr | Biome-Scale characterisation and differentiation of semi-arid and arid zone soil organic matter compositions using pyrolysis-GC/MS analysis[END_REF]). The vegetation is drought-adapted, with trees and large shrubs rare. Leaf succulents and dwarf shrubs from the Aizoaceae, Crassulaceae and Euphorbiaceae families are especially abundant [START_REF] Cowling | a. Plant biogeography, endemism and diversity[END_REF][START_REF] Milton | Succulent Karoo[END_REF]. The Succulent Karoo Biome is a winter rainfall desert (in contrast to subtropical, summer rainfall deserts), experiencing higher summer temperatures and more elevated moisture stress than the Fynbos Biome. This winter rainfall, although low, contributes to a relatively reliable rainfall regime (Cowling et al., 1999b).

Sample sites

All plant and soil samples were obtained during a campaign of systematic sampling in April-May 2010 (the end of the summer dry-season). Mean annual rainfall across the sampling region ranges between approximately 480 mm a -1 and 160 mm a -1 . Rainfall seasonality varies from ~50% during the austral winter in the northern reaches of the region (SK7) to ~90% winter rain at SV1 in the south; Figures S1 andS2; Table 1). Mean annual temperatures range between 14 and 19 °C; maximum monthly temperatures lie between 27 and 34 °C [START_REF] Hijmans | Very high resolution interpolated climate surfaces for global land areas[END_REF] depending on elevation and latitude. Aridity index values, which are lowest in the Succulent Karoo, are between 0.1 and 0.4 and are generally lower in the Lowland Fynbos than in the Montane Fynbos.

Ten plots were surveyed and sampled in the Fynbos Biome (including the Lowland and Montane eco-regions) and 15 plots in the Succulent Karoo Biome. These sites were previously described with an emphasis on their soil organic matter compositions in [START_REF] Carr | Biome-Scale characterisation and differentiation of semi-arid and arid zone soil organic matter compositions using pyrolysis-GC/MS analysis[END_REF]. At each site, a 10 x 10 m vegetation survey plot was laid out and a small sample of photosynthetic material from each plant was sampled (usually a leaf, but occasionally a small piece of stem material in the case of stem succulents, or a clearly identifiable and recent piece of leaf litter). Plants were identified with reference to the University of Cape Town herbarium collection, although this was not always possible, particularly in cases (e.g. Aizoaceae) where flowers were absent at the time of sampling (see Table S1). In most cases, we sampled one specimen of each identified plant type at each site. Soil samples were obtained from the same plant survey plots at four fixed locations (plot corners). Following removal of surficial leaf litter, each 200-400 g sample was obtained from the upper 10-15 cm of the soil (where possible, as some soils were rather thin). This broadly corresponded with the soil A Horizon, but many soils were lithosols, lacking well-developed soil profiles, particularly in the Succulent Karoo Biome [START_REF] Carr | Biome-Scale characterisation and differentiation of semi-arid and arid zone soil organic matter compositions using pyrolysis-GC/MS analysis[END_REF].

Methods

Freeze-dried plant material was homogenised and encapsulated in tin cups, with each leaf mixture measured in triplicate. Soil samples were sieved at 2 mm, sub-sampled using a riffle box and homogenised in a ball mill. For total organic carbon (TOC) and  13 CTOC determination, soils were treated with dilute hydrochloric acid, rinsed in deionised water and freeze-dried. TN and soil  15 N measurements were made on (freeze dried) untreated soil samples. Plot average TOC and TN values for soils are reported in [START_REF] Carr | Biome-Scale characterisation and differentiation of semi-arid and arid zone soil organic matter compositions using pyrolysis-GC/MS analysis[END_REF]. Here we present results for the individual soil samples and additionally report the  13 CTOC and  15 N obtained in the same analyses. Total carbon (TC), Total nitrogen (TN) and  13 C and  15 N were determined using a SerCon ANCA GSL elemental analyser interfaced to a SerCon Hydra 20-20 continuous-flow isotope ratio mass spectrometer. Measurement precision was better than ±0.1‰ for  13 C and ±0.2‰

for  15 N.

Climatic data, interpolated surfaces based on climate data from individual weather stations, were obtained from WorldClim 1.4 [START_REF] Hijmans | Very high resolution interpolated climate surfaces for global land areas[END_REF], with site aridity indices from [START_REF] Trabucco | Global Aridity Index (Global-aridity) and Global Potential Evapotranspiration (Global-pet) Geospatial Database[END_REF]. Due to the low density of climate observation stations and high topographic heterogeneity in the study region, only generalised definitions of climate gradients are feasible. Statistical analyses were carried out using PAST [START_REF] Hammer | PAST: Paleontological statistics software package for education and data analysis[END_REF]. To explore our data we used non-parametric tests of difference (Mann-Whitney and Kruskall-Wallis) to assess differences in isotopic values between biomes or between plant functional types. To consider isotopic-climatic relationships we used site average foliar and soil data (as will be shown later, inter-sample (plot scale) variability was relatively high) and we report the Pearson correlation coefficients between isotopic values and climatic parameters from the WorldClim data. We also inspected the results of Spearman's Rank Order correlations in these instances, although similar conclusions were drawn from such an analysis. The results of ordinary least squares regression analysis are reported for some analysis (e.g. plot-wise soil vs. foliar isotopic comparisons).

Results

Foliar C and N content

Foliar C content varies from 20 % to 59 %, with means and standard deviations of 45 ± 4 % (n=46) for the Montane Fynbos, 42 ± 4 % (n=24) for the Lowland Fynbos and 39 ± 6 % (n=135) for the Succulent Karoo Biome (Figure 1). The difference between the Succulent Karoo (n=135) and Fynbos Biomes (all Fynbos samples, n= 70) is statistically significant (Mann Whitney U = 2136, p= < 0.001). Foliar N content from the Succulent Karoo Biome (mean 1.8 ± 0.9%, n=135) is higher (and more varied) than in the Fynbos Biome (Figure 1). This difference is statistically significant whether the Fynbos Biome as-a-whole is compared to the Succulent Karoo (Mann Whitney U =1233, p = <0.001), or if it is treated as separate eco-regions. Within the Fynbos Biome, Montane Fynbos Foliar N (mean 0.7 ± 0.2 %, n=41) is significantly lower (Mann Whitney U= 163, p=<0.0001) than the Lowland Fynbos Foliar N (mean 1.2 ± 0.5 %, n=23). Foliar C/N is highly variable, but is highest in the Montane Fynbos (average 76 ± 27, n=41) and lowest in the Succulent Karoo Foliar  15 N spans a total range of 22.7‰ (-2.0 to 20.7‰ Considering the foliar stable isotope dataset in total, there is weak but significant positive correlation between foliar  13 C and foliar  15 N (Pearson's r = 0.21, p = 0.003), but no such correlations when the biomes are considered separately (r = 0.10, p = 0.25 for the Succulent Karoo Biome, and r = 0.13, p=0.30 for the Fynbos) (Figure 2). There is no correlation between foliar  15 N and C/N ratio in the Succulent Karoo Biome. The two variables are correlated in the Fynbos Biome, for both soils and plants (Figure 2). S1). For the purposes of this analysis, no differentiation is made between the Lowland and Montane Fynbos eco-regions. Woody Succulents (n=62) were the dominant functional type sampled in the Succulent Karoo, while Woody Shrubs (n=52) dominated in the Fynbos Biome. In the Fynbos Biome the foliar  13 C values of Woody Shrubs and Herbs are indistinguishable (-26.9 ± 1.6‰ (n=51) and -26.5 ± 1.4‰ (n=7) respectively; Figure S3). Plants in the Succulent (mean = -18.4 ± 5‰ (n=5)) and Woody Succulent (mean = -21.2 ± 3.8‰ (n=5)) classes account for the seven high  13 C values previously noted.

These are all likely to be CAM plants (three are identified as Euphorbia mauritanica or Tylecodon paniculata). In the Succulent Karoo, Woody Succulents and Succulents, exhibit a very wide range of  13 C (Figure S3). The mean foliar  13 C values of Succulents (-19.4 ± 4.3‰ (n=21)) and Woody Succulents (-21.6 ± 4.5‰ (n=62)) are higher than those of the Herbs (-25.7 ± 4.5‰ (n=9)) and Woody Shrubs (-25.1 ± 3.0‰ (n=34)). The mean foliar  13 C of Herbs in the Succulent Karoo is indistinguishable from that of Herbs in the Fynbos Biome. Grasses, which were only sampled in the Succulent Karoo, include both C3 and C4 varieties (-14.7 ± 1.4‰ (n=3) and -27.2 ± 0.7‰ (n=4)).

There is considerable variability in foliar  15 N within each Fynbos Biome plant functional type (Figure S3), but there are no significant differences in foliar  15 N between groups (Kruskall-Wallis, p = 0.051).  15 N is also highly variable in the Succulent Karoo Biome and, as in the Fynbos Biome, there are no significant differences (Kruskal-Wallis, p =0.053) between plant functional type (Figure S3).

Soils

Summarised (plot average) soil TOC and TN contents were presented previously [START_REF] Carr | Biome-Scale characterisation and differentiation of semi-arid and arid zone soil organic matter compositions using pyrolysis-GC/MS analysis[END_REF]) and these data are here reported on a per sample basis in Figure 3. Soil TOC varies between 0.2 and 5.8 %. At the biome scale, average values are 0.94 ± 0.70 % (n=60) and 1.37 ± 1.32 % (n=39) for the Succulent Karoo Biome and Fynbos Biomes respectively. Lowland Fynbos soil TOC (1.89 ± 1.68 %, n=19) is higher than Montane Fynbos (0.88 ± 0.55 %, n=20). The average TN ranges from 0.01% to 0.30%. Overall mean values are 0.07 ± 0.05 % (n=60) and 0.09 ± 0.11 % (n=39) for the Succulent Karoo and Fynbos Biomes respectively.

These indistinguishable soil N values contrast with the higher foliar N contents in Succulent Karoo plants compared to the Fynbos.

Soil  13 CTOC tracks the foliar data in that, on average, it is highest and most varied in the Succulent Karoo Biome (mean -23.2 ± 1.8‰, n=60) and lowest in the Montane Fynbos (mean -26.2 ± 1.1‰, n=19, compared to -25.2 ± 0.9‰, n=20 for the Lowland Fynbos). The isotopic variability within each biome/ecoregion is markedly reduced in comparison the foliar data (Figures 1 and3). Soil  15 N is highest the Succulent Karoo Biome (mean 10.5 ± 2.7‰, n=60). As observed for the foliar data, Lowland Fynbos soil

 15 N is intermediate (8.1 ± 2.6‰,

Soil -plant comparisons at the plot scale

Comparing plot average soil  13 CTOC and foliar  13 C across the total data set, we observe a significant correlation (r 2 = 0.67, p = < 0.001) between soil and foliar  13 C (Figure S4). The slope of this relationship is less than 1 (0.58 ± 0.08), and this remains the case when the Fynbos and Succulent Karoo Biomes are analysed separately (0.51 ± 0.10 for Succulent Karoo Biome, and 0.30 ± 0.15 for Fynbos (Figure S4)).

Turning to nitrogen, plot average soil  15 N and foliar  15 N are positively correlated (Figure S4; r 2 =0.59, p=<0.0001) with a slope closer to 1 (0.84 ± 0.15) and regression intercept of 3 ± 1‰. Considered as individual biomes (Figure S4), the correlation is markedly higher in the Fynbos Biome (r 2 = 0.80, slope 0.70 ± 0.12 and intercept 2.8 ± 0.7‰) compared to the Succulent Karoo (r 2 = 0.34, and slope = 0.60 ± 0.24 and intercept 5.8 ± 2.0‰).

Discussion

These data provide an opportunity to explore variability in, and controls upon, foliar and soil  13 C and  15 N across these semi-arid to arid winter rainfall biomes. The discussion focuses on variability at the plot and biome scales, the relationship to climatic conditions, and the relationships between the isotopic signatures of plants and associated soils.

Controls on foliar  13 C

The highest foliar  13 C values are associated primarily with Succulent and Woody Succulent growth forms (Figure S3). High  13 C values are particularly associated with the Crassulaceae, Aizoaceae and Asphodelaceae families. Some sampled species (e.g. Tylocodon paniculatus, Euphoriba mauritanica, Aloë dichotoma, Monolaria moniliformis) are CAM plants [START_REF] Mooney | Carbon isotope ratio measurements of succulent plants in southern Africa[END_REF][START_REF] Veste | Variability of CAM in leaf-deciduous succulents from the Succulent Karoo (South Africa)[END_REF], while other specimens are from genera known to contain CAM species [START_REF] Mooney | Carbon isotope ratio measurements of succulent plants in southern Africa[END_REF][START_REF] Rundel | Ecology and phylogenetic patterns of carbon isotope discrimination in the winter-rainfall flora of the Richtersveld, South Africa[END_REF]).

In the Succulent Karoo, the occurrence of CAM plants results in a characteristically broad foliar  13 C distribution, reflecting a mix of C3 and obligate/facultative CAM plants [START_REF] Rundel | Ecology and phylogenetic patterns of carbon isotope discrimination in the winter-rainfall flora of the Richtersveld, South Africa[END_REF][START_REF] Winter | Crassulacean acid metabolism: a continuous or discrete trait?[END_REF]; Figure 4). Isotopic discrimination for Succulent Karoo Biome CAM plants has previously been considered [START_REF] Rundel | Ecology and phylogenetic patterns of carbon isotope discrimination in the winter-rainfall flora of the Richtersveld, South Africa[END_REF], with specimens largely drawn from the Richtersveld to the north of our study region. These results are combined with the data from this study (Figure 4) after converting to isotopic discrimination () as follows:

∆ = 𝛿 13 𝐶 𝑎𝑡𝑚 -𝛿 13 𝐶 𝑙𝑒𝑎𝑓 1 + 𝛿 13 𝐶 𝑙𝑒𝑎𝑓 /1000

Where  13 Catm at the time of sampling ( 2010) was estimated as -8.2‰ (Keeling et al.

http://cdiac.ess-dive.lbl.gov/trends/co2/iso-sio/iso-sio.html. In terms of taxonomy, the lowest measured Δ is observed in the Chenopodiaceae (including Salsola tuberculata), Aizoaceae (Ruschia sp.), Crassulaceae (e.g. Tylocodon sp.), Eurphorbiaceae (e.g.

Euphorbia mauritanica) and Asphodelaceae (Aloë sp.) families (Table S1). While Salsola likely utilises C4 photosynthesis [START_REF] Pyankov | Photosynthesis in Salsola Species (Chenopodiaceae) from Southern Africa Relative to their C4 Syndrome Origin and their African-Asian Arid Zone Migration Pathways[END_REF], the lowest values for the other plants ( of ~5-7‰ = foliar  13 C of ~12-15‰) imply near 100% dark fixation or "strong CAM" [START_REF] Winter | How closely do the  13 Cvalues of crassulacean acid metabolism plants reflect the proportion of CO2 fixed during day and night?[END_REF][START_REF] Winter | Ecophysiology of constitutive and facultative CAM photosynthesis[END_REF].

However, as a whole, the Succulent Karoo Biome is characterised by a bimodal  distribution (Figure 4) comparable to previous CAM plant compilations [START_REF] Cernusak | Environmental and physiological determinants of carbon isotope discrimination in terrestrial plants[END_REF][START_REF] Winter | How closely do the  13 Cvalues of crassulacean acid metabolism plants reflect the proportion of CO2 fixed during day and night?[END_REF][START_REF] Winter | Crassulacean acid metabolism: a continuous or discrete trait?[END_REF]. The distinct reduction in  at 12-14‰ ( 13 C values -20 to -19‰), implying <> 40% dark fixation [START_REF] Winter | How closely do the  13 Cvalues of crassulacean acid metabolism plants reflect the proportion of CO2 fixed during day and night?[END_REF][START_REF] Cernusak | Environmental and physiological determinants of carbon isotope discrimination in terrestrial plants[END_REF], is apparent in the Succulent Karoo Biome.

Variability in across Succulent and Woody Succulent specimens is also clear (see also Figure S3), with the range of values for a single well-sampled family in the Succulent Karoo (Aizoaceae, Figure S5) 4).The highest foliar  13 C measured for an individual (assumed) non-CAM plant Montinia caryophyllacea) is -20.3‰. Thus, distinguishing CAM from C3 on the basis of foliar isotope data alone is difficult [START_REF] Winter | How closely do the  13 Cvalues of crassulacean acid metabolism plants reflect the proportion of CO2 fixed during day and night?[END_REF]. This applies particularly to "weak CAM" [START_REF] Winter | Ecophysiology of constitutive and facultative CAM photosynthesis[END_REF]) and is exemplified here by a specimen of Monolaria moniliformis (a "CAM cycler" or Weak CAM; Veste et al.

demonstrating
2001; Winter 2019, respectively) whose foliar  13 C (-22.5‰) is identical to that of some C3 shrubs (e.g. a specimen of Galenia africana; SK13 plant 2).

Considering plant functional type, while on average isotopic discrimination is consistently lowest in the Succulent PFT, low discrimination/high  13 C values are observed in all functional types except herbs.

In the Succulent Karoo Biome,  never exceeds 21‰, regardless of PFT. [START_REF] Rundel | Ecology and phylogenetic patterns of carbon isotope discrimination in the winter-rainfall flora of the Richtersveld, South Africa[END_REF] reported a  mode of 17‰ for C3 plants in their study region, with a range of 8‰ and no correlation with phenology or morphology. This is comparable to our findings (e.g. C3 plant mean of  19 ± 2‰ for the Fynbos Biome (excluding CAM)). For a single species, [START_REF] Rundel | Ecology and phylogenetic patterns of carbon isotope discrimination in the winter-rainfall flora of the Richtersveld, South Africa[END_REF] reported up to 4‰ variability ( of 14-18‰).

Here we observe up to 6‰ variation for the cosmopolitan species Galenia africana (Kraalbos), which was sampled across six sites (Lowland Fynbos and Succulent Karoo Biome) spanning mean annual rainfall range of 158 mm a -1 .

Relationship to environmental variables - 13 C

Table 2: Pearson's correlation coefficients between site-averaged soil and foliar  13 C and climatic data extracted from WorldClim [START_REF] Hijmans | Very high resolution interpolated climate surfaces for global land areas[END_REF]. MAP = Mean annual precipitation MAT = Mean annual temperature. Aridity index taken from [START_REF] Trabucco | Global Aridity Index (Global-aridity) and Global Potential Evapotranspiration (Global-pet) Geospatial Database[END_REF]. Values in bold are significant at p <0.05. The stronger correlations with climatic parameters for plot average soil  13 C most likely reflect averaging of individual plant responses and time averaging (see also [START_REF] Carr | Stable isotope analysis of rock hyrax faecal pellets, hyraceum and associated vegetation in southern Africa: implications for dietary ecology and palaeoenvironmental reconstructions[END_REF]. 2). There are some limitations in this analysis of climatic relationships, notably the relatively limited climate space (particularly when biomes are analysed separately) and the constraints of the interpolated WorldClim database [START_REF] Hijmans | Very high resolution interpolated climate surfaces for global land areas[END_REF]) (e.g. in the topographically complex Cape Fold Belt Mountains)

MAP MAT Aridity

Index

Considering all sites,  13 C from soils and plants is most strongly (negatively) correlated with precipitation of the wettest month (Table 2). Given the range of foliar  13 C measured, the primary cause of this correlation between  13 C and rainfall is most likely photosynthetic pathway, indicating the greater occurrence of CAM plants, and occasional C4 grasses (e.g. SK2, SK9) in the more arid Succulent Karoo.

There are no significant correlations when only samples from the Succulent Karoo Biome are considered.

This most likely reflects the substantial individual plant / soil  13 C variability, driven by the relative abundance of CAM and C3 specimens within the sample plots, and variability in the degree of night-time CAM fixation within individual specimens (at and prior to sampling; [START_REF] Cernusak | Environmental and physiological determinants of carbon isotope discrimination in terrestrial plants[END_REF]. Similarly, for the Fynbos Biome alone there are no significant correlations with climate. Excluding the seven outlier specimens (almost certainly CAM -see above) (Table 2) the strongest (negative) correlation is between foliar  13 C and precipitation of the wettest quarter (PWetQ; r = -0.49, p= 0.15). This is not statistically significant and the absence of correlations between folair  13 C and climate parameters in this biome likely reflects a combination of sample numbers, plot scale variability, topographic variability and the relatively narrow climatic envelope (Figure 5; see also [START_REF] Codron | Plant stable isotope composition across habitat gradients in a semi-arid savanna: implications for environmental reconstruction[END_REF]). These likely mask the expected WUEi signal anticipated in this C3 plant dominated biome.

Soil-plant  13 C relationship

The correlation between soil and foliar  13 C indicates that variability in foliar  13 C is captured within soil organic matter (SOM) (Figures S4). This is not unexpected, as plant-derived organic matter will be the main C input to soils [START_REF] Ehleringer | Carbon isotope ratios in belowground carbon cycle processes[END_REF][START_REF] Wang | Patterns and implications of plant-soil  13 C and  15 N in African savannah ecosystems[END_REF][START_REF] Nel | Correspondence between  13 C and  15 N in soils suggests coordinated fractionation processes for soil C and N[END_REF]. Indeed, in the Succulent Karoo Biome, plot average soil  13 CTOC correlates strongly (r = 0.88 p = < 0.001) with the proportion of sampled plants with foliar  13 C ≥ 20 (excluding C4 grass samples). While only a crude estimate of the likely contribution of CAM plant-derived OM to the soils (i.e. it does not take into account biomass or cover differences), this is markedly stronger than the correlations observed between soil  13 CTOC and climatic parameters.

Comparing these results more explicitly with vegetation in the study region, Figure 6 compares the plot average soil  13 CTOC values with the percentage of plant species in CAM-rich families (Aizoaceae-Mesembryanthemaceae, Crassulaceae, Euphorbiaceae), as obtained from the PRECIS database (SANBI, 2003). The highest soil  13 C values broadly accord with this independent measure of CAM plant abundance, implying contributions to the wider soil and sedimentary organic matter pool in this region cannot be assumed to be negligible. Comparison of figures 1 and 3 suggests that time-space averaging in soils reduces plant-scale isotopic variability and that sedimentary or soil  13 CTOC values > -23 ‰ are potentially indicative of a CAM presence (Figure 3). The importance of considering CAM as well as C3:C4 plant abundance within any interpretative (palaeo) framework is clearly implied. The increased presence of CAM plants in the sampled (winter rainfall) environments is largely driven by aridity (Table 2). For a palaeoenvironmental record, applying such an interpretation would assume inputs from C4 grasses could be excluded (perhaps requiring other lines of evidence). This would be especially important at the margins of the study region in the winter and year-round rainfall zone transition, where C4 grasses are demonstrably present. On average soil  13 CTOC is lower than foliar  13 C and this offset increases at higher foliar  13 C values (Figure S6). In this instance, the variable offset may reflect a single sampling season -the end of the dry season -meaning that the  13 C of sampled plant tissue, which may integrate the most recent conditions, became increasingly higher than the more time-averaged soil organic matter, particularly in the most arid (more CAM, higher foliar  13 C) Succulent Karoo plots. However, 13 C depleted SOM relative to foliar OM has been associated with selective preservation of 13 C-depleted fractions of relatively recalcitrant plant organic matter, such as lignin and lipids (though see [START_REF] Nel | Correspondence between  13 C and  15 N in soils suggests coordinated fractionation processes for soil C and N[END_REF]. In this instance it is possible to compare our results with SOM compositions, obtained via pyrolysis-GC/MS analyses of the same soil samples [START_REF] Carr | Biome-Scale characterisation and differentiation of semi-arid and arid zone soil organic matter compositions using pyrolysis-GC/MS analysis[END_REF]. Here we observe no significant correlations between the soil-foliar  13 C offset and summary variables for this SOM composition analysis. More specifically axes 1 and 2 from a Principal

Component Analysis of the pyrolysis-GC/MS-derived SOM compositions [START_REF] Carr | Biome-Scale characterisation and differentiation of semi-arid and arid zone soil organic matter compositions using pyrolysis-GC/MS analysis[END_REF] were associated with relative abundances of lignin and leaf cuticle-derived organic matter (Axis 1) or a high (low) abundances of short to mid-chain length aliphatics (Axis 2). Both however are uncorrelated with the soilfoliar  13 C offset measured here.

 15 N variability and controls

There is considerable variation in soil and foliar  15 N across the study region. Site averages for soil and foliar  15 N decrease as Succulent Karoo Biome > Lowland Fynbos > Montane Fynbos (Figures 1 and3).

Although there is significant inter-specimen variation in foliar  15 N, even within the same plot, there are no significant differences in foliar  15 N between PFTs (Figure S3). Furthermore, there is no evidence for any differences in foliar  15 N with photosynthetic pathway. (Succulent Karoo Biome foliar  13 C < > -20‰

produces identical mean foliar  15 N values ( 15 N = 7.7‰; < -20 mean vs. > -20‰  15 N = 7.7‰; Mann Whitney U = 1895, p = 0.982; Figure S7)).

An additional source of foliar  15 N variability is the presence of N-fixing plants and plants with mycorrhizal associations [START_REF] Craine | Global patterns of foliar nitrogen isotopes and their relationships with climate, mycorrhizal fungi, foliar nutrient concentrations, and nitrogen availability[END_REF]. The latter are associated with several plant families in the Fynbos Biome, but less so with succulent-rich families like Aizoaceae [START_REF] Spriggs | Influence of mycorrhizal associations on foliar  15 N values of legume and non-legume shrubs and trees in the Fynbos of South Africa: Implications for estimating N2 fixation using the 15 N natural abundance method[END_REF]. Mycorrhizal associations are associated with foliar  15 N values up to 6‰ lower than non-mycorrhizal plants [START_REF] Craine | Global patterns of foliar nitrogen isotopes and their relationships with climate, mycorrhizal fungi, foliar nutrient concentrations, and nitrogen availability[END_REF]2015a), while N-fixers typically generate foliar  15 N values close to zero (Craine et al. 2015a and references therein). Although the presence of the former has not been assessed here, our foliar  15 N values are mostly higher than those reported for Fynbos plants with known mycorrhizal associations [START_REF] Spriggs | Influence of mycorrhizal associations on foliar  15 N values of legume and non-legume shrubs and trees in the Fynbos of South Africa: Implications for estimating N2 fixation using the 15 N natural abundance method[END_REF], with only four  15 N measurements in the present study at or below 0‰. Of these, two are from the Fabaceae family (FB5-P4 Aspalanthus sp. and FB5-P19 Rafnia sp.) and indeed their foliar  15 N values are commensurately lower (measured  15 N of 0 and 0.1‰) than their (FB5) plot-mean foliar  15 N (1.4‰). However, one of the lowest measured  15 N values was from a Restionaceae specimen, a

family not thought to be mycorrhizal [START_REF] Allsopp | Mycorrhizal status of plants growing in the Cape Floristic Region, South Africa[END_REF]. Conversely, the  15 N of a sample of Erica sp. (the Ericaceae family is associated with ericoid mycorrhiza, with reported  15 N values < 0‰; [START_REF] Allsopp | Mycorrhizal status of plants growing in the Cape Floristic Region, South Africa[END_REF][START_REF] Spriggs | Influence of mycorrhizal associations on foliar  15 N values of legume and non-legume shrubs and trees in the Fynbos of South Africa: Implications for estimating N2 fixation using the 15 N natural abundance method[END_REF]) had a foliar  15 N close to the plot average (2.6‰ compared to the FB1 plot mean of 3.0‰). The impact of these effects is therefore difficult to evaluate with the present dataset.

Considering  15 N in in terms of climate (e.g. [START_REF] Handley | The 15 N natural abundance (δ 15 N) of ecosystem samples reflects measures of water availability[END_REF]Admunson et al. 2003), noting the previous caveats regarding gridded climate data,  15 N is most strongly correlated with precipitation of the wettest quarter and mean annual precipitation (Table 3; Figure 5). Correlations with temperature-related parameters are largely non-significant, except for the positive correlation between mean annual temperature and soil  15 N, and the negative correlation between aridity and soil  15 N. Correlations are weaker when the Succulent Karoo Biome is considered alone, but the narrower climatic envelope and reduced sample numbers likely account for this. WorldClim [START_REF] Hijmans | Very high resolution interpolated climate surfaces for global land areas[END_REF]. Values in bold are significant at p <0.05. MAP = Mean annual precipitation MAT = Mean annual temperature. Aridity index taken from [START_REF] Trabucco | Global Aridity Index (Global-aridity) and Global Potential Evapotranspiration (Global-pet) Geospatial Database[END_REF]. This level of explanation is comparable to several studies (e.g. [START_REF] Cheng | Climate and ecosystem 15 N natural abundance along a transect of Inner Mongolia grasslands: contrasting regional patterns and global patterns[END_REF][START_REF] Craine | Global patterns of foliar nitrogen isotopes and their relationships with climate, mycorrhizal fungi, foliar nutrient concentrations, and nitrogen availability[END_REF]Perri et al., 2012;[START_REF] Díaz | Nitrogen cycling in an extreme hyperarid environment inferred from δ 15 N analyses of plants, soils and herbivore diet[END_REF][START_REF] Mosher | Spatial variability in foliar carbon and nitrogen isotope values on Tenerife reflects both climate and soils: Establishing a framework for future work[END_REF], although other regional-scale studies have noted both an absence of a relationship between climate and  15 N (e.g. [START_REF] Zhao | Climatic, edaphic and biotic controls over soil δ 13 C and δ 15 N in temperate grasslands[END_REF] or a non-linear relationship [START_REF] Wang | Aridity threshold in controlling ecosystem nitrogen cycling in arid and semi-arid grasslands[END_REF]. [START_REF] Campbell | Montane plant environments in the Fynbos biome[END_REF][START_REF] Muofhe | Nitrogen nutrition in nodulated field plants of the shrub tea legume Aspalanthus linearis assessed using 15 N natural abundance[END_REF] may account for the relatively steep MAP- 15 N gradient observed across the whole dataset. In this respect it is noteworthy that foliar N and  15 N are significantly correlated within the Fynbos Biome (r = 0.43, p < 0.0001), but are uncorrelated in the Succulent Karoo Biome (r= -0.01, p = 0.9), which would be predicted for a more N-limited Fynbos biome. A fuller assessment of this requires further study.

MAP MAT Aridity

Index

Non-climatic factors may also be associated with soil and foliar  15 N variability [START_REF] Craine | Global patterns of foliar nitrogen isotopes and their relationships with climate, mycorrhizal fungi, foliar nutrient concentrations, and nitrogen availability[END_REF][START_REF] Craine | Convergence of soil nitrogen isotopes across global climate gradients[END_REF][START_REF] Nel | Correspondence between  13 C and  15 N in soils suggests coordinated fractionation processes for soil C and N[END_REF]. A correlation matrix for soil and foliar  15 N with associated soil properties is shown in Table 4. There are substantial differences in soil texture, pH, TOC and TN between the Fynbos and the Succulent Karoo Biomes (Figure 3; Figure S2). Apparent correlations between  15 N and TN or TOC may conflate climatic effects (i.e. these are also correlated with climate, via biomass; Table 4; Figure S8). Considering this and accounting for the TOC-MAP (particularly) and TN-MAP relationships across all sites, we note the overall MAP- 15 N relationship remains significant (Figure S8). Considering the biomes separately, while the Fynbos shows no correlation with non-climatic factors, the Succulent Karoo data imply that soil  15 N (but not foliar  15 N) is negatively correlated with TN and TOC (soil) and % fines (Figure S8), but uncorrelated with rainfall-related factors (Table 2). The latter observations are however difficult to assess further given the limited climate space in the Succulent Karoo. S4), consistent with observations elsewhere [START_REF] Craine | Global patterns of foliar nitrogen isotopes and their relationships with climate, mycorrhizal fungi, foliar nutrient concentrations, and nitrogen availability[END_REF][START_REF] Pardo | Using nitrogen isotope ratios to assess terrestrial ecosystems at regional and global scales[END_REF]. As the slope of the relationship between the soil and foliar  15 N is close to 1 (0.84 ± 0.15), these data also imply that the "enrichment factor" (the difference between foliar and soil  15 N i.e.

 15 N) is no greater at higher foliar  15 N sites (cf. [START_REF] Craine | Global patterns of foliar nitrogen isotopes and their relationships with climate, mycorrhizal fungi, foliar nutrient concentrations, and nitrogen availability[END_REF]). This is of potential relevance as  15 N can provide insights into N cycling and plant-available N by removing the effects of variations in the baseline (soil) signature (Craine et al. 2015a). For the total dataset here, there is no correlation between  15 N and any climatic factors (cf. [START_REF] Cheng | Climate and ecosystem 15 N natural abundance along a transect of Inner Mongolia grasslands: contrasting regional patterns and global patterns[END_REF][START_REF] Mosher | Spatial variability in foliar carbon and nitrogen isotope values on Tenerife reflects both climate and soils: Establishing a framework for future work[END_REF], but interestingly it is correlated with % fines for the Succulent Karoo, an observation worth further investigation, particularly given current uncertainties surrounding the drivers of the enrichment factor (Craine et al. 2015a).

Conclusions

Soil and foliar  15 N and  13 C data collected from across the Fynbos and Succulent Karoo Biomes facilitated consideration of a range of environmental variables impacting plant and soil isotopic compositions across these semi-arid to arid winter rainfall biomes. Soil and foliar  13 C for the Fynbos are consistent with a dominantly C3 biome, but these new data provide a more robust indication of the degree of variability This most likely reflects: 1) a limited range of WUEi-induced variability relative to non-WUEi factors, 2) the narrow climatic envelope of the study area, and 3) the limited number of samples.

We observe substantial  15 N variability at the plot scale, but overall, soil and foliar  15 N values conform to the hot/dry (higher  15 N) versus cool/wet (lower  15 N) paradigm outlined in previous works (e.g. [START_REF] Craine | Global patterns of foliar nitrogen isotopes and their relationships with climate, mycorrhizal fungi, foliar nutrient concentrations, and nitrogen availability[END_REF]). Both soil and foliar  15 N are correlated with climate (rainfall and temperature).

The relationship with climate, particularly with MAP is, however, relatively steep compared to previous compilations. This is not a result of systematic differences in foliar  15 N for succulent growth forms or CAM plants, but mycorrhizal associations, and contrasting soil /nutrient properties between the Fynbos and Succulent Karoo are potential contributors to within and between biome variability. Soil and foliar  15 N are correlated (more strongly than the equivalent analysis of  13 C) and replicate the ~3‰ offset between soil and foliar  15 N reported across a range of ecosystems.

Overall, these data provide a starting point for future studies of N and C cycling in these biomes, as well as important baseline isotope ranges for palaeoecological, palaeoclimatic and archaeological

research. An understanding of spatial variability in isotopic compositions, and particularly variability within any biome or climatic zone, is clearly necessary for robust interpretations of changing (palaeo) climates, animal diets and human diets [START_REF] Sealy | Nitrogen isotopic ecology in southern Africa: implications for environmental and dietary tracing[END_REF][START_REF] Szpak | Carbon and nitrogen isotopic survey of northern Peruvian plants: baselines for paleodietary and paleoecological studies[END_REF]. A challenge in this region, particularly near to the winter rainfall-year round rainfall transition zone, will be to disentangle the roles of CAM (aridity) and C4 (rainfall seasonality) plants within stable isotopic records. , 2003), along with the mean soil  13 CTOC from the study sites (as circles). To allow comparison between these two datasets their colour ramps are matched. The size of the circles has been varied to allow easier differentiation of soil  13 CTOC values between sites, but does not convey additional information

Figure Captions:

  Biome (29 ± 23, n=135). The difference in foliar C/N between the Succulent Karoo Biome (n=135) and the Fynbos Biome (all samples, n = 64) is significant (Mann Whitney U = 1004, p= < 0.001). The difference between the Lowland (42 ± 19, n= 23) and Montane Fynbos (average 76 ± 27, n=41) is also significant (Mann Whitney U = 131, p = <0.001).

Figure 1 :

 1 Figure 1: Violin plots showing variability in foliar total carbon (top left) and total nitrogen (top right), and variability in foliar  13 C (bottom left) and  15 N (bottom right) for the sampled biomes/eco-regions. SK = Succulent Karoo, LF = Lowland Fynbos and MF = Montane Fynbos.

Figure 2 :

 2 Figure 2: Scatter plots of foliar (upper left) and soil (upper right)  13 C and  15 N split by biome. The correlation between soil  13 C and  15 N is statistically significant for the Fynbos Biome (Pearson's r = 0.51, p < 0.0001). Bottom: Scatter plots of foliar (left -note log scale x axis) and soil (right) C/N and  15 N split by biome. The correlation between foliar C/N and  15 N for the Fynbos Biome is statistically significant (Pearson's r = -0.52, p < 0.0001), as is the correlation for soil C/N and  15 N for the Fynbos Biome (Pearson's r = 0.44, p < 0.001). The variables are uncorrelated in the Succulent Karoo Biome.

  n=20) between the Succulent Karoo and Montane Fynbos (4.8 ± 1.9‰, n=19). Succulent Karoo soil  15 N is significantly higher than Montane Fynbos (Mann-Whitney U = 61, p = <0.001). The difference between Montane and Lowland Fynbos is also significant (Mann-Whitney U = 55, p = < 0.001), as is the difference between the Succulent Karoo and Lowland Fynbos (Mann-Whitney U = 302, p= < 0.001). As observed in the foliar data, for all samples, across both biomes,  13 CTOC and  15 N are weakly, but significantly correlated (Pearson's r = 0.51, p<0.001, n=98) (Figure2). When biomes or ecoregions are considered individually there is a correlation for the Fynbos Biome (Pearson's r = 0.51, p<0.001, n=39; Figure2), but no correlation within the Succulent Karoo Biome (Pearson's r = 0.11, p=0.4, n=60).

Figure 3 :

 3 Figure 3: Violin plots showing variation in soil total nitrogen (TN; top left), soil total organic carbon (TOC; top right) contents, soil  13 CTOC (bottom left) and soil  15 N (total N; bottom right). SK = Succulent Karoo, LF = Lowland Fynbos and MF = Montane Fynbos.

  The range in Δ across the Succulent Karoo Biome, spanning the Richtersveld in the north to the Fynbos Biome in the south, is ~19‰ (Figure4). Compared to[START_REF] Rundel | Ecology and phylogenetic patterns of carbon isotope discrimination in the winter-rainfall flora of the Richtersveld, South Africa[END_REF], we observed a larger proportion of specimens with higher discrimination, probably reflecting reduced CAM use under less arid conditions (mean annual precipitation in the[START_REF] Rundel | Ecology and phylogenetic patterns of carbon isotope discrimination in the winter-rainfall flora of the Richtersveld, South Africa[END_REF] study sites was ~40-100 mm). The distributions are otherwise quite similar.

Figure 4 :

 4 Figure 4: Histograms of isotopic discrimination for this study (Fynbos and Succulent Karoo Biome), and those of Rundel et al. (1999) from the Richtersveld region of the Succulent Karoo Biome -in each case C4 grasses have not been plotted

Figure 5 :

 5 Figure 5: Scatter plots considering  13 C in relation to climatic parameters extracted from the WorldClim data. The upper two plots show soil (solid fill) and foliar (open symbol)  3 C in relation to mean annual temperature and precipitation of the wettest quarter (see table 2). The lower two plots show soil (solid fill) and foliar (open symbol)  5 N in relation to mean annual temperature and precipitation of the wettest quarter (see table 2).

Figure 6 :

 6 Figure 6: Map showing regional-scale variation percentage of plant species in the vegetation from CAM-dominant families (Aizoaceae-Mesembryanthemaceae, Crassulaceae, Euphorbiaceae), as obtained from the PRECIS database (SANBI, 2003), along with the mean soil  13 CTOC from the study sites (as circles). To allow comparison between these two datasets their colour ramps are matched. The size of the circles has been varied to allow easier differentiation of soil  13 CTOC values between sites, but does not convey additional information

Figure 1 :

 1 Figure 1: Violin plots showing variability in foliar total carbon (top left) and total nitrogen (top right), and variability in foliar  13 C (bottom left) and  15 N (bottom right) for the sampled biomes/eco-regions. SK = Succulent Karoo, LF = Lowland Fynbos and MF = Montane Fynbos.

Figure 2 :

 2 Figure2: Scatter plots of foliar (upper left) and soil (upper right)  13 C and  15 N split by biome. The correlation between soil  13 C and  15 N is statistically significant for the Fynbos Biome (Pearson's r = 0.51, p < 0.0001). Bottom: Scatter plots of foliar (left -note log scale x axis) and soil (right) C/N and  15 N split by biome. The correlation between foliar C/N and  15 N for the Fynbos Biome is statistically significant (Pearson's r = -0.52, p < 0.0001), as is the correlation for soil C/N and  15 N for the Fynbos Biome (Pearson's r = 0.44, p < 0.001). The variables are uncorrelated in the Succulent Karoo Biome.

Figure 3 :

 3 Figure 3: Violin plots showing variation in soil total nitrogen (TN; top left), soil total organic carbon (TOC; top right) contents, soil  13 CTOC (bottom left) and soil  15 N (total N; bottom right). SK = Succulent Karoo, LF = Lowland Fynbos and MF = Montane Fynbos.

Figure 4 :Figure 5 :

 45 Figure 4: Histograms of isotopic discrimination for this study (Fynbos and Succulent Karoo Biome), and those of Rundel et al. (1999) from the Richtersveld region of the Succulent Karoo Biome -in each case C4 grasses have not been plottedFigure 5: Scatter plots considering  13 C in relation to climatic parameters extracted from the WorldClim data. The upper two plots show soil (solid fill) and foliar (open symbol)  13 C in relation to mean annual temperature and precipitation of the wettest quarter (seetable 2). The lower two plots show soil (solid fill) and foliar (open symbol)  15 N in relation to mean annual temperature and precipitation of the wettest quarter (see table 2).

Figure 6 :

 6 Figure6: Map showing regional-scale variation percentage of plant species in the vegetation from CAMdominant families (Aizoaceae-Mesembryanthemaceae, Crassulaceae, Euphorbiaceae), as obtained from the PRECIS database(SANBI, 2003), along with the mean soil  13 CTOC from the study sites (as circles). To allow comparison between these two datasets their colour ramps are matched. The size of the circles has been varied to allow easier differentiation of soil  13 CTOC values between sites, but does not convey additional information

Figure S2 :Figure S3 :

 S2S3 Figure S2: Soil vs plant  13 C and  15 N split by biome (Succulent Karoo in closed circles and Fynbos in open squares). Linear regression results for the individual biomes are as follows:  15 N Fynbos y = 0.70x + 2.8 r 2 =0.80;  15 N Succulent Karoo: y = 0.60x + 5.8 r 2 =0.33;  13 C Fynbos y = 0.30x -18.1 r 2 =0.32;  13 C Succulent Karoo y = 0.51x -11.7 r 2 =0.66

Figure S4 :

 S4 Figure S4: The magnitude of soil-foliar  13 C offset ( 13 C) (obtained from plot average data) is negatively correlated with foliar  13 C.

Figure S5 :

 S5 Figure S5: Box and whisker plot of foliar  15 N in the Succulent Karoo Biome, split for plants  13 C < > -20 ‰

Table 1 :

 1 Climatic envelopes (means an standard deviations) for the sampling sites within the considered biomes/ecoregions (from WorldClim 1.4;[START_REF] Hijmans | Very high resolution interpolated climate surfaces for global land areas[END_REF]

Biome Number of sites Altitude range (m) Mean annual Precipitation (mm) Mean annual temperature (°C) Aridity Index Precip. WetQ (mm) Temperature of the driest month (°C)

	Lowland Fynbos	5	7-119	297 ± 105	17.3 ± 0.6	0.21 ± 0.11	142 ± 44	21.1 ± 0.8
	Montane Fynbos	5	227-947	305 ± 45	17.2 ± 1.6	0.20 ± 0.04	150 ± 20	22.4 ± 1.5
	Succulent Karoo Biome	15	93-1072	201 ± 51	17.5 ± 1.7	0.13 ± 0.03	85 ± 22	21.4 ± 3.6

  ). It is highest and most varied in the

	Succulent Karoo Biome (mean 7.7 ± 3.9‰ n = 135) and lowest in the Montane Fynbos eco-region (mean
	3.2 ± 2.0‰ n =41). At the plot scale, variation in foliar  15 N is substantial, with a maximum (10x10 m)
	sample plot range of 15.4‰ (SK7; n=11). The difference between Succulent Karoo and Fynbos Biomes
	(Fynbos sample mean = 4.6 ± 3.3‰ n=64) is significant (Mann Whitney U = 2286, p = < 0.001), as is the
	difference between the Montane and Lowland (7.1 ± 3.6‰ n =23) Fynbos eco-regions (Mann Whitney U

= 152, p< 0.0001). There is no significant difference between foliar  15 N in the Lowland Fynbos and Succulent Karoo (Mann Whitney U = 1470, p=0.69). The highest measured value (20.7‰ from a woody succulent of the Aizoaceae family) approaches the highest foliar  15 N values reported globally (21.4‰;

Craine et al., 2015a)

.

  significant variability in CAM use. Climate is seemingly one factor generating this variability; the correlation between  and aridity for this plant family is significant (Aizoaceace vs Aridity

	index; Pearson's r =0.36, p = 0.01, n= 48).
	For Lowland and Montane Fynbos, the  13 C data indicate the expected dominance of C3 plants,
	although CAM species are present.  13 C from C3 plants within arid regions can span a range of -20 to -31‰
	(e.g. Ehleringer, 1989 Elheringer et al. 1998). Considering only the most likely C3 plants (i.e. the Fynbos
	Biome samples with the seven aforementioned outliers removed), the  13 C range is approximately -23 to
	-31‰. (Figure

Table 3 :

 3 Pearson's correlations between site-averaged soil and foliar  15 N and climatic data extracted from

Temp of the wettest Quarter Temp of the driest Quarter Precipitation of the Wettest Quarter Precipitation of the Driest

  It is notable, given the range of potentially fractionating processes in the nitrogen cycle, that  15 N shows stronger correlations with climate (precipitation, temperature and aridity; Table4) than  13 C. Additionally, in contrast to  13 C,  15 N shows also shows statistically significant correlations with temperature-related parameters. Multiple regression (Table4) with temperature and precipitation of the wettest quarter (these variables are uncorrelated) produces highest combined R 2 (0.48 and p < 0.001) for total dataset.

	 15 N
	FB Foliar  15 N	-0.70 0.57	-0.56	0.74	0.17	-0.75	-0.65
	FB soil  15 N	-0.43 0.48	-0.26	0.80	-0.06	-0.50	-0.38
							Quarter
	All sites foliar  15 N	-0.57 0.38	-0.52	0.18	0.20	-0.62	-0.19
	All sites soil  15 N	-0.60 0.52	-0.55	0.15	0.23	-0.56	-0.35
	SK foliar  15 N	-0.11 0.32	-0.04	-0.19	0.31	-0.15	-0.07
	SK soil	-0.28 0.68	-0.33	-0.29	0.46	0.15	-0.54

Table 5 :

 5 Results of the multiple regression analyses for  15 N of all soils and plants with MAP and MAT 15 N per 100 mm change in MAP and a 0.72 ± 0.35‰ per °C change in mean annual temperature. The slope for the foliar data is comparable (2.1 ± 0.6‰ per 100 mm). For MAP these slope values are higher than several previous studies (e.g.Perri et al., 2012;[START_REF] Swap | Natural abundance of 13 C and 15 N in C3 and C4 vegetation of southern Africa: patterns and implications[END_REF], substantially so when compared to global scale analyses of  15 N vs. MAP, which incorporate wider ranges of MAP (e.g.Hadley et al. 1999;

		Intercept	MAP slope	MAT slope	R 2	N	p
	Soil  15 N	1.06 ± 6.77	-0.02 ± 0.01	0.72 ± 0.35	0.48	25	0.0007
	Foliar  15 N	4.82 ± 6.75	-0.02 ± 0.01	0.37 ± 0.35	0.38	25	0.0052
	Considering just the relationship between  15 N and precipitation (using MAP for comparison with
	published works; soil n = 25, r 2 = 0.38 p = 0.001) the linear regression slope indicates a 2.4 ± 0.6‰ change
	in 

Table 4 :

 4 Correlation coefficients for all site soil and foliar  15 N. % fines and soil pH data taken from[START_REF] Carr | Biome-Scale characterisation and differentiation of semi-arid and arid zone soil organic matter compositions using pyrolysis-GC/MS analysis[END_REF]. Values in bold are significant at p < 0.05.

					Fynbos	Fynbos		Succulent	Succulent
		All site soil  15 N	All site foliar  15 N	All site  15 N	site average soil	Site average foliar	FB  15 N	Karoo Biome site average	Karoo Biome Site average	SK  15 N
					 15 N	 15 N		soil  15 N	foliar  15 N
	Soil
	total N	-0.35	-0.20	0.26	-0.10	-0.07	0.02	-0.70	-0.34	0.41
	(%)
	Soil TOC (%)	-0.49	-0.43	0.15	-0.29	-0.26	-0.06	-0.66	-0.53	0.17
	Soil pH	0.37	0.42	0.03	0.28	0.01	-0.42	0.05	0.44	0.41
	fines (%
	< 63	0.19	0.34	0.19	-0.29	-0.11	0.26	-0.55	0.08	0.70
	µm)
	Plot average foliar  15 N and soil  15 N are correlated across all sites, with soil 3 ± 1 ‰ more enriched
	in 15 N than the plants (Figure

  (the Fynbos  13 C standard deviation, excluding CAM plants, is 1.6‰) within this biome. Additionally, CAM plants with elevated  13 C, while rare, are present in the Fynbos Biome and contribute to foliar isotopic variability (Fynbos foliar  13 C standard deviation including CAM plants is 3.6‰). Indeed, across the total dataset and the Succulent Karoo in particular, CAM is associated with the greatest carbon isotope variability ( 13 C range be more than 10‰).  13 C is negatively correlated with mean annual or wet season rainfall, a relationship driven largely by the turnover of vegetation from Fynbos (more humid, C3, rare CAM) to Succulent Karoo (more arid, common CAM). The presence of CAM is apparent in soil  13 CTOC, and the data available at present suggest soil  13 CTOC values > -23 ‰ are potentially indicative of CAM presence in the landscape. There are no statistically significant correlations between foliar  13 C (Δ) and climate within the primarily C3 Fynbos sites, despite expectations that WUEi would generate such a relationship.

Acknowledgements

We thank the Leverhulme Trust (F/00 212/AF) for funding the original field sampling campaign and the majority of the laboratory analyses. Matthew Britton, Zoe Roberts and Pippin Anderson are thanked for plant identification, laboratory and field assistance respectively. We thank Alex Cumming and Hannah Grimes for laboratory assistance. BMC's contributions and some laboratory analyses were supported by the European Research Council (ERC) under the European Union's Seventh Framework Programme (FP7/2007-2013) grant agreement no. 258657. We thank the reviewers and editor for their constructive comments on the original version of this paper.

Supplementary figures for Carr et al -Variability in soil and

foliar stable carbon and nitrogen isotope compositions in the winter rainfall biomes of South Africa