Variation in Plant Functional Traits Across and Within Four Species of Western Australian Banksia (Proteaceae) Along a Natural Climate Gradient

Austral Ecology (2016) 41, 886–896

Variation in plant functional traits across and within four species of Western Australian Banksia (Proteaceae) along a natural climate gradient

ANNE COCHRANE,1,2* GEMMA L. HOYLE,1 COLIN J. YATES,2 TERESA NEEMAN3 AND ADRIENNE B. NICOTRA1 1Evolution, Ecology and Genetics, Research School of Biology, The Australian National University, Canberra Australian Capital Territory, 2Science and Conservation Division, Department of Parks and Wildlife, Locked Bag 104, Bentley Delivery Centre, Western Australia 6983 (Email: [email protected]) and 3Statistical Consulting Unit, The Australian National University, Canberra Australian Capital Territory Australia

Abstract Plant traits are fundamental components of the ecological strategies of plants, relating to how plants acquire and use resources. Their study provides insight into the dynamics of species geographical ranges in changing environments. Here, we assessed the variation in trait values at contrasting points along an environmental gradient to provide insight into the flexibility of species response to environmental heterogeneity. Firstly, we identified how commonly measured functional traits of four congeneric species (Banksia baxteri, B. coccinea, B. media and B. quercifolia)variedalonga longitudinal gradient in the South Western Australian Floristic Region. This regional gradient provides significant variation in moisture, temperature and soil nutrients: soil nitrogen content decreases with declining rainfall and increasing temperature. We hypothesized that (i) the regional pattern in trait–environment associations across the species would match those observed on a global scale and (ii) that the direction and slopes of the within-species relationships would be similar to those across species for each of the measured traits. Along the regional gradient we observed strong shifts in trait values, and cross-species relationships followed the expected trend: specific leaf area was significantly lower, and leaf Narea and seed dry mass significantly higher, at the drier end of the rainfall gradient. However, traits within species were generally not well correlated with habitat factors: we found weak patterns among populations, either due to the small ecological gradient or perhaps because fine-scale structuring among populations (at a micro-evolutionary scale) was low due to high gene flow within species. Understanding how species traits shift as a result of climatic influences, both at the regional (across species) and local (within species) scale, provides insight into plant adaptation to the environment. Such studies have important applications for conservation biology and population management in the face of global change.

Key words: inter-speciﬁc variation, intra-speciﬁc variation, leaf and seed traits, rainfall.

INTRODUCTION Climate and soils are among the most important factors influencing the selection of seed and leaf traits. Plant functional traits, in particular those associated Temperature and water affect the geographical distribu- with leaves and seeds, indicate how plants acquire tion of plant species through their direct impacts on and use resources (Reich et al. 1997; Westoby 1998). reproduction, growth and survival. Their influence can These traits drive the fitness of species by affecting also be indirect. For example, soil nutrient availability reproduction, growth and survival; they integrate the is driven by gradients in precipitation and temperature, ecological and evolutionary histories of a species with nitrogen and phosphorous considered to be two of thereby capturing fundamental trade-offs that deter- the most limiting factors for growth in terrestrial plants mine species ecological roles in the environment (Güsewell 2004). Selection pressures along environ- (Violle et al. 2007). As these traits directly affect plant mental gradients often give rise to predictable variation performance, they are clearly linked to extinction risk in plant functional traits, with strong evidence to show and could therefore be used to predict the impacts of that many plant traits are functionally linked to climate global change on the dynamics of plant performance at local, regional and global scales (Wright et al. 2004, (Chapin 2003). 2005; Moles & Westoby 2006; Moles et al. 2007; Peppe et al. 2011), particularly along nutrient and/or water availability gradients (Martínez-Vilalta et al. 2009). *Corresponding author. Understanding how species traits shift as a result of Accepted for publication March 2016. climatic influences has important applications for

© 2016 Ecological Society of Australia doi:10.1111/aec.12381 VARIATION IN PLANT FUNCTIONAL TRAITS 887 conservation biology and population management in the considerable trait differentiation. However, our under- face of global change. standing of what drives variation between species is still Leaves directly support plant growth by converting limited and conflicting (Cornwell & Ackerly 2009; light and carbon dioxide into chemical energy via photo- Kichenin et al. 2013; Read et al. 2013; Richardson synthesis. High temperature and drought tend to select et al. 2013). To compound this uncertainty, variation for smaller leaves that reduce evapotranspiration in functional traits also occurs at the intra-specific level, (Cornelissen et al. 2003), but in turn reduce carbon as- often at small spatial scales and can reflect phenotypic similation (Flexas et al. 2013). Specificleafarea(SLA; plasticity in response to the environment, selection for the ratio of leaf area to dry mass) is closely correlated genetic differentiation among populations in trait means with photosynthetic capacity and leaf nitrogen content and/or random genetic drift (Vergeer & Kunin 2011). and is important for growth: species with high SLA are Despite the accepted presence of intra-specific variation, more efficient at capturing light and therefore have high the study of trait variation along environmental gradients photosynthetic capacity (Reich et al. 1997; Wright et al. usually focuses on species mean values (Wright et al. 2004). Specific leaf area typically is lower on dry, nutri- 2001). Within-species variation may be of less impor- ent poor sites and is associated with water and nutrient tance in explaining trait–environment relationships. conservation (Wright et al. 2001; Lamont et al. 2002). However, documenting where such variation exists, in- This leaf trait is also assumed to be correlated with tem- cluding mapping the environmental variables that might perature, though weakly (Wright et al. 2004; Ordoñez explain that variation, may provide insight into how et al. 2009; De Frenne et al. 2013). Low SLA is generally species will perform in future climates (Diaz & Cabido considered to confer enhanced ability to withstand 1997; Dalgleish et al. 2010) and is also interesting in its wilting (Cunningham et al. 1999; Reich et al. 1999). Leaf own right. nitrogen content (on an area basis, Narea)correspondsto Here, we examined the influence of the abiotic envi- the amount of protein and other secondary compounds ronment on five commonly measured plant functional present in the leaf, compounds necessary for photosyn- traits in four endemic Banksia R.Br. (Proteaceae) spe- thesis and growth. Leaf Narea tends to increase as rainfall cies (Banksia subgenus Banksia sensu George; George declines (Wright et al. 2004) but scales negatively with 1981) along a longitudinal gradient of temperature, SLA. On the other hand, N content on a mass basis moisture and soil nutrients within the Mediterranean- (Nmass) decreases with declining rainfall (Cunningham climate ecosystem of the South Western Australian et al. 1999; Jones et al. 2013) and soil nutrient availability Floristic Region (SWARF). The strong seasonal varia- (Wright et al. 2001). tion between wet winter and dry summer conditions in Seeds provide the main opportunity for species to this region plays a major role in determining vegetation disperse and colonize new habitats. Seed mass, an dynamics. The life history attributes of Banksia (long- important regenerative trait, links germination and lived, perennials with long juvenile periods) make them early seedling growth and survival (Leishman et al. particularly sensitive to abiotic factors in the landscape 2000; Westoby et al. 2002; Moles & Westoby 2004; and vulnerable to the predicted effects of climate change Pichancourt & van Klinken 2012) and is a measure of (Fitzpatrick et al. 2008; Yates et al. 2010), with cur- the resources allocated by maternal parent plants to their rent climate conditions at the warmer, drier eastern individual offspring. Seed mass has been shown to end of the gradient projected to resemble those at increase along an aridity gradient (Murray et al. 2003), the cooler, wetter western end in the future. Many with large seeds having a larger food reserve to draw on species in this region exhibit functional traits that con- during harsh times (McWilliams et al. 1968; Keeley fer drought tolerance (e.g. sclerophylly); however, the 1991; Milberg et al. 1998; Leishman et al. 2000). Larger relationship between drought and other environmental seeds commonly confer an advantage over smaller seeds variables and plant traits has not been well examined during early seedling establishment (Coomes & Grubb in Banksia and, to our knowledge, rarely at the popu- 2003; Rodríguez-Pérez & Traveset 2007; Metz et al. lation level. 2010), enabling seedlings to better succeed where com- We sought to characterize the mean functional trait petition is intense and resources are scarce (Leishman values for the four Banksia species and determine et al. 2000). whether these traits change in a linear fashion along an The change in species presence across the landscape environmental gradient. Our aim was to test for consis- plays a dominant role in the underlying trait– tent association between environmental conditions and environment relationships along environmental gradi- plant traits along the entirety of the regional gradient ents; however, within a plant community, there can (i.e. cross-species variation), as well as quantifying the also be huge variation in functional traits, even if magnitude of intra-specific variation attributable to dif- the mean trait values shift predictably along gradients ferences in the environment at the local scale (i.e. among (Ackerly & Cornwell 2007). For this reason, we populations within each species). We hypothesized that may expect to see co-occurring species exhibiting (i) the regional pattern in trait–environment associations

at the cross-species level would match those observed on sympatrically, in deep white sands on plains and dunes in a global scale and (ii) that the direction and slopes of the tall shrubland. Their longitudinal ranges are similar (about within-species trait–environment relationships would be 300 km and 360 km, respectively). Banksia media (ser. similar to those across species for each of the measured Cyrtostylis) tends to grow in sand, loam and clay, some- traits (due to plasticity and genetic variation). In accor- times over limestone or granite, in heath, shrubland or open dance with ecological theory (Reich et al. 1999; Wright woodland with a more easterly distribution (range of et al. 2004; Byars et al. 2007; Moles et al. 2007; Peppe ~600 km). With the smallest geographical distribution et al. 2011; Konarzewski et al. 2012), we predicted that (~200 km), Banksia quercifolia (ser. Quercinae) is found in plants inhabiting drier environments would have larger sand, often peaty, in depressions and on swamp margins, seed mass, smaller leaf area, lower SLA and higher leaf in shrubland-sedge formations, and sometimes in low wood- nitrogen (on an area basis), relative to those at more land, at the high rainfall end of its range, where nutrients are mesic sites. generally higher. Banksia media and B. quercifolia flower in autumn or winter, whereas B. coccinea flowers in winter, MATERIALS AND METHODS spring and summer and B. baxteri predominantly in summer. Seeds of all four species are stored in the plant canopy in Study species woody fruits (known as serotiny), with seed release generally confined to the post-fire environment. However, B. coccinea The Banksia species investigated are locally common where will recruit between fire intervals as its fruits are only weakly they occur but have a restricted longitudinal distribution serotinous (Witkowski et al. 1991). Seed set tends to be low along the southern coastal areas of Western Australia in all species (George 1984), and recruitment commonly (Fig. 1). They grow in different but partially overlapping occurs in autumn-winter with the onset of rain and cool habitats and are dominant in many vegetation communities temperatures. and therefore have potentially important cascading effects on ecosystem processes. All four species are long-lived, Data collection woody, evergreen, sclerophyllous shrubs that lack lignotubers and are killed by fire (Lamont et al. 2007), relying entirely Plant traits were measured in six populations of each of the on seed for regeneration. The closely related Banksia baxteri four Banksia species; these geographically distinct localities and B. coccinea (ser. Banksia subser. Cratistylis) grow, often represented the core and range margins of each species

Fig. 1. Seed source sites in the South Western Australia Floristic Region for four study species: Banksia quercifolia (♦), B. coccinea (○), B. baxteri (▲)and B. media (●). Rainfall isohyets demonstrate the longitudinal aridity gradient present.

along the longitudinal gradient (Fig. 1). Canopy-held fruits negative relationship between total soil N% and longitude were collected from 10 maternal plants at each of the 24 (P = 0.008), with N declining along the west–east gradient sites (4 species × 6 populations) to give a representative as soils became more nutrient poor. Phosphorous levels sample of the local genetic diversity (Table S1 in the showed no predictable linear pattern along the longitudinal Supporting Information). Seeds were extracted by burning gradient, however, the ratio of phosphorous to nitrogen woody fruits with a gas torch, soaking fruits in water for increased as precipitation declined. 4–6 h then placing them in a drying room (15 °C and 15% relative humidity) until seeds naturally released (Cochrane et al. 2015b). Twenty seeds from each individual Data analysis plant were weighed to four decimal places (Mettler Toledo AB54) and averaged to provide individual plant seed Mean trait values weights (mg ± standard error). Five leaves from each of the 10 maternal plants were sampled We calculated mean trait values for each species and used ’ fi at each site from the outer north facing canopy where incident analysis of variance and Fisher sleastsigni cant difference test < radiation is greatest in the southern hemisphere. Leaves were at P 0.05 to determine whether trait values differed across selected if healthy, fully expanded and of comparable age, hav- species and among populations within species. ing emerged at the most recent leaf flush. Each leaf was scanned fl fresh on a atbed scanner or photographed and subsequently Trait–environment responses along the gradient dried at 60 °C for 72 h, then weighed. Leaf images were – analysed using ImageJ (Rasband 1997 2012) to obtain the We used a restricted maximum likelihood model to identify 2 fi one-sided projected surface area of each leaf (mm ). Speci c trait–environment associations along the entirety of the gradient leaf area (SLA) was calculated as the ratio of leaf area to dry taking into account all measured trait values for species and 2 À1 weight (mm mg ). Chemical analysis of leaf nitrogen concen- populations (n = 240; d.f. = 222). Traits (i.e. leaf area, SLA, tration was assessed by Kjeldahl acid digestion followed by seed mass and leaf nitrogen content on both mass and area ba- fl colorimetric ( ow injection) analysis, which allowed the sis) were used as the y variate with each environmental variable À1 calculation of leaf nitrogen per unit mass (Nmass,mggm ) fitted as a fixed effect with ‘population’ as a random effect to ac- À2 and per unit area (Narea,gmcm ). count for the representativeness of the population samples. We As moisture and temperature have a strong impact on re- ran the analysis separately against each of the traits with each of production, growth and survival of plant species, particularly the environmental variables (i.e. MAP, MAT, precipitation in in winter wet, summer dry Mediterranean climates the wettest quarter, mean soil temperature, mean soil moisture (Mooney & Dunn 1970), we extrapolated mean annual À total soil N%, total soil P and the ratio of soil N/P), including precipitation (MAP, mm year 1), precipitation in the wettest À longitude. quarter (mm year 1) and mean annual temperature (MAT, °C) for each site from bio-climatic data from WorldClim, a set of global climate layers with a spatial resolution of ap- Trait–environment relationships among and within species proximately 1 km2 (Hijmans et al. 2005) to provide regional- scale climate data. We also gathered local environmental in- We then tested the trait response to longitude both across and formation along the gradient by measuring soil temperature within species using a mixed model analysis with terms that and moisture at the source sites. These recordings were separated out the cross-species patterns from the within-species taken at 2–5 cm below the soil surface at all sites using patterns. We did this by including the mean longitude for each Hobo data loggers (H21-002 micro weather stations with species as a covariate (cross species, which also takes into S-SMC-M005 moisture sensors and S-TMB-M006 temper- account the average phenotype of each species) as well as the ature sensors). Soil temperature and moisture were recorded deviation from the species mean for each population (within- every 30 min for more than 12 months at each site, and species, which takes into account the longitude of each popula- these data were analysed to provide mean daily readings. tion and the average phenotype of that population at that We collected three representative soil samples from the longitude). The model therefore allowed us to determine top 5 cm of the soil profile, from each site, for analytical whether the relationships between longitude and traits within assessment of nitrogen and phosphorous (ChemCentre, species were the same as these relationships across the species. Perth). Concentrations of nitrogen and phosphorous in the We repeated the same analysis using MAP and MAT instead leaves of plants are largely driven by uptake of these nutri- of longitude as the covariate. ents from the soil. All leaf and seed trait values and MAP were natural log The majority of measured abiotic parameters varied transformed prior to the analyses to stabilize the variance predictably, and significantly, with longitude (Table S2). and meet assumptions of normality. Differences were consid- Temperature increased (r = 0.73) from west to east at the ered significant at P < 0.05 unless otherwise stated. All same time that rainfall (r = À0.68) and soil moisture availabil- analyses were performed using GenStat 16th edition (VSN ity (r = À0.65) declined. In addition, there was a significant International).

RESULTS nitrogen) with the exception of soil phosphorous along this regional gradient (Table S2). From west to east, Mean species values for measured functional traits moisture and soil nitrogen values declined, and at the same time, temperature increased. The mixed model There was clear evidence of differences among the analyses highlighted a number of significant associations species on the basis of the measured traits (Fig. 2), and between the measured traits and environmental parame- this was confirmed by ANOVA. The greatest difference ters along the gradient when population was taken into among the species was observed in seed mass, with B. account (Table S3). There were some strong linear baxteri having the largest seeds and B. quercifolia the patterns, although not all environmental variables were smallest. Banksia baxteri and B. media shared similar equally good predictors of shifts in trait values along SLAs and leaf nitrogen on an area basis, but not the the longitudinal gradient. Mean annual precipitation same leaf area: leaf area in B. baxteri was more than three and precipitation in the wettest quarter were significantly times that of B. media, and overall, the leaves of B. media associated with more traits than any of the other environ- were significantly smaller than those of the other three mental drivers. These variables, in conjunction with the species. Banksia coccinea and B. quercifolia shared similar ratio of N/P in the soil, had a small but significant impact leaf area, leaf nitrogen per unit area, but not SLA. Spe- on leaf area along the gradient: leaf area increased with cificleafareawassignificantly higher for B. quercifolia. increasing precipitation and a higher N/P ratio. All envi- À1 Values of leaf Nmass (mg gm ) were similar among B. ronmental variables with the exception of soil P and the baxteri, B. media and B. quercifolia but significantly lower N/P ratio affected SLA with increasing MAP and precip- for B. coccinea. itation in the wettest quarter most dominant in their effects on higher average SLA. Precipitation (as either Changes in functional traits along the MAP or precipitation in the wettest quarter) was the best environmental gradient predictor of variation in seed mass, with seed mass increasing as conditions became drier. There was no Longitude was highly correlated with all environmental significant relationship between leaf nitrogen on a mass measures (MAT, MAP, precipitation in the wettest basis and measures of temperature or moisture along quarter, soil moisture, soil temperature and soil the gradient; however, soil nutrients, in particular total

Fig. 2. Species mean values (untransformed) ± standard error for each measured trait (n = 60 samples per species). Fisher’s protected LSD test at P < 0.05 used to determine differences between species. Species that share the same letters for each trait are not signiﬁcantly different from each other. doi:10.1111/aec.12381 © 2016 Ecological Society of Australia VARIATION IN PLANT FUNCTIONAL TRAITS 891

N%, had a role to play in changes in this trait along the B. media at the dry end of the gradient demonstrated gradient: leaf Nmass showed a small but significant consistently lower leaf area and SLA, and consistently decline as soil nutrients became more limited along the higher seed mass, compared with trait values in popula- gradient (Table S3). Precipitation, again as either total tions of B. quercifolia at the wet end of the gradient, and annual rainfall or precipitation in the wettest quarter, this tended to drive the patterns of association at the re- provided the driving force for changes in leaf Narea along gional scale. Low SLA is a good indicator of water and the gradient, although total % soil N made a significant nutrient conservation (Wright et al. 2001; Lamont et al. contribution to the observed variation. These associa- 2002) and has been previously reported for Western tions were all negative: the drier and more nutrient-poor Australian Banksia species growing in dry, nutrient-poor the site, the greater the leaf N on an area basis. Mean soils (Witkowski et al. 1992). values for each trait and population were plotted against Direct comparison of the slopes of the trait– longitude, MAP and MAT to demonstrate the effect of environment associations in our study to global patterns the gradient on shifts in trait values (Fig. 3a–e). Along is problematic due to the difference in the number of the gradient from west to east, leaf area and SLA samples investigated. However, if we do compare the declined and seed mass and leaf Narea increased in the association between SLA (or its inverse, leaf mass area) transition from the wetter, nutrient-rich habitats of B. and precipitation, we see a much smaller slope in our quercifolia to the drier, more nutrient-poor sites regional gradient than that seen at the global scale for supporting B. media. Precipitation had a larger impact evergreen species (Wright et al. 2005) or species from on trait values than did MAT. California (Ackerly & Cornwell 2007). One of our key expectations was to see intra-specific Patterns of within-species trait variation variation mirroring the overall pattern across the gradient. However, the observed within-species trait– Despite some strong shifts in trait values observed along environment associations were generally absent, much the gradient, the within-species trait–environment asso- weaker or in the opposite direction to that at the regional ciations (i.e. within each species geographical distribu- scale. Hence, a comparison of the strength and direction tion) were mostly missing or weaker than that at the of the slopes at the two hierarchies of scale is again diffi- larger regional scale (Table 1; Fig. 3). The trends in trait cult when the slopes did not go in the same direction and shifts were mostly due to a change in species along the were generally not significant. However, where there is gradient, and the within-species variation in traits was some comparison of the direction of slopes, the within- relatively small and mostly non-linear. species slopes were generally much smaller than that across the species. Trait–environment associations DISCUSSION among populations within species have often been interpreted as local adaptation, but in this study, trait As predicted, we observed some significant trait– values were rarely associated with position on the local environment associations as functional trait values climate gradients of the species. We had expected that shifted in response to changes in the environment along if the environment and plant traits were correlated at this the longitudinal gradient. At the regional level, and local scale, we could use the gradient of sites within each across all species, the trait–environment associations species geographical distribution to infer shifts in the we observed in this Mediterranean-climate ecosystem future (i.e. temporal trends), similar to ‘space-for-time’ of the SWARF matched our expectations of those seen analyses. However, the lack of strong geographical pat- on a global scale (i.e. SLA was significantly lower and terning in the local gradient of each species precludes leaf Narea and seed dry mass significantly higher at the our ability to do this. These weak within-species trait– drier end of the climate gradient where plants are under environment associations and the resulting non-linear greater water stress and nutrients are more limited) responses to environmental variables have been ob- (Wright et al. 2001; Ordoñez et al. 2009; Kattge et al. served previously in European temperate forest herbs 2011). Overall, our results suggest that precipitation from a latitudinal gradient (De Frenne et al. 2011), in has perhaps the strongest influence on changes in plant populations of Eucalyptus salubris in Western Australia functional traits along this regional longitudinal gradi- (Steane et al. 2015) and in calcareous alpine grasslands ent. MAT had a lesser influence on plant traits, with (Rosbakh et al. 2015). Lack of local clinal patterns in the exception of SLA and leaf Narea, diverging from the the Banksia species may have been due to the small observations of Moles et al. (2014) that MAT was more number of populations sampled and the comparatively strongly correlated with plant traits than MAP at the short, longitudinal gradient selected, and it may well be global scale. that the variation observed here reflects smaller-scale The observed divergence in trait means for the two microsite or developmental differences, but more con- species inhabiting opposite ends of the gradient was sideration is needed to understand the relative impor- expected from their location along a climate gradient: tance of drift versus selection on within-species trait–

Fig. 3. Patterns of trait variation in log transformed species mean values for (a) leaf area, (b) SLA, (c) seed mass, (d) leaf Nmass and (e) leaf Narea. Banksia baxteri (●), B. coccinea (○), B. media (▲)andB. quercifolia (Δ)inrelationtolongitude,meanannualprecipitation (MAP) and mean annual temperature MAT). The solid black lines represent the regression coefficient for significant cross-species relationships between trait values and longitude, MAP and MAT along the gradient (n = 24). See Table S3 for slope and significance of trait-longitude, MAP and MAT relationships. environment relationships (Steane et al. 2015). Although gradients were ecologically small and fine-scale structur- the local gradients generally covered the geographical ing among populations (at a micro-evolutionary scale) range of each of the four species, the within-species may be limited by high gene flow within species. Banksia

Table 1. The slope of the linear relationship between log transformed trait values and longitude, log MAP and MAT at the different ecological scales

Leaf area SLA mm Seed Leaf Nmass Leaf Narea À À (mm2) (mg 1) mass (mg) mg (mm 1) gm (cm2)

Cross-species Longitude 0.1153 À0.2395 0.8018 0.0346 0.2741 MAP À0.4040 0.8395 À0.2811 À0.1210 À0.9610 MAT 0.3400 À0.7063 0.2365 0.1020 0.8080 Within-species Longitude B. baxteri 0.2089 0.0050 À0.1723 0.0079 0.0029 B. coccinea 0.3079 À0.1321 À0.0843 À0.2969 À0.1651 B. media 0.0937 0.0052 À0.0043 0.0217 0.0165 B. quercifolia À0.1831 À0.0217 À0.0760 0.1298 0.1515 Within-species MAP B. baxteri À0.381 À0.071 0.443 À0.298 À0.227 B. coccinea À0.028 0.251 0.492 0.234 À0.017 B. media 0.595 0.275 0.143 0.100 À0.176 B. quercifolia 0.294 À0.028 0.180 À0.289 À0.261 Within-species MAT B. baxteri 0.350 0.0145 À0.2929 À0.0024 À0.0169 B. coccinea 0.260 À0.0714 À0.0366 À0.2404 À0.1689 B. media À0.036 À0.1079 À0.0019 0.0466 0.1541 B. quercifolia 0.831 0.0145 À0.4559 0.1716 0.1571

Cross-species slopes estimate the trait association based on species averages and represent the larger regional scale. Trait values in bold denote significance at P < 0.05 (Table S3). Within-species slopes estimate the trait association within each species based on population averages and represent the smaller local gradient defined by each species geographical distribution. have been hypothesized to have low levels of genetic ecology, they have not previously been empirically structure at the population level (Coates 2000), and demonstrated for these Banksia species. Furthermore, these mainly bird-pollinated species are likely to have quantification of trait responses across various ecological extensive long-distance gene flow between populations scales is frequently lacking (Lavorel & Garnier 2002) which can reduce the scope for local adaptation, thereby and so what is new is that we have compared the relation- reducing trait–environment correlations. ship between traits and some of the forces that influence We had anticipated some similarity in trait values in trait variation both among and within-species in one the closely related and frequently co-occurring species, framework. B. baxteri and B. coccinea, as edaphic specialization is ex- In this current study, the majority of variability in seed tremely high in the SWARF (Hopper 1992). However, and leaf traits along the regional gradient of our Banksia mean species trait values for these two species often dif- was a result of variation among species; however, varia- fered significantly, suggesting that these traits may have tion within species was not entirely absent. These results developed independently due to unique evolutionary generally concur with other studies in trait ecology that histories (Prunier et al. 2012) or the species were show that variation among species is generally consid- responding in different ways to the different attributes ered to be greater than variation within species (Kitchen of the environment as a mechanism of co-existence 2001; Wright et al. 2001; Roche et al. 2004; Kattge et al. (Chesson 2000). These two species have already demon- 2011; Kazakou et al. 2014), although instances of strated divergence in their responses to simulated comparable variation in foliar traits have been reported drought and heat stress (see Holloway-Phillips et al. across ecological scales (Messier et al. 2010). Seed mass, (2015) for water use strategies and Cochrane et al. in particular, is considered a highly conserved trait (2015a) for leaf and allocational traits). Although re- across environmental gradients on global (Harper lated, these species are ecologically distinct and employ 1977; Moles & Westoby 2003; Kazakou et al. 2014) different strategies for survival such as strong versus weak and regional scales (Murray et al. 2004; Konarzewski serotiny, large versus small leaves and seeds, fire- et al. 2012) and this we did see. dependent versus inter-fire recruitment (George 1981), High ambient temperatures and low water availability perhaps in part as a result of selection for differentiation already pose a series of limitations on plant growth and in niche (Richardson et al. 1995). survival in Mediterranean-climate ecosystems (Sala Although the broader regional trait–environment as- et al. 2000; Yates et al. 2010). These limitations are sociations we observed as a result of changes in species expected to compound in the future as local climate presence along the gradient are not a new concept in patterns change as a consequence of global warming

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