Disentangling the relative importance of species occurrence , abundance and intraspecific variability in community assembly : a trait-based approach at the whole-plant level in Mediterranean forests

Understanding which factors and rules govern the process of assembly in communities constitutes one of the main challenges of plant community ecology. The presence of certain functional strategies along broad environmental gradients can help to understand the patterns observed in community assembly and the filtering mechanisms that take place. We used a trait-based approach, quantifying variations in aboveground (leaf and stem) and belowground (root) functional traits along environmental gradients in Mediterranean forest communities (south Spain). We proposed a new practical method to quantify the relative importance of species turnover (distinguishing between species occurrence and abundance) versus intraspecific variation, which allowed us to better understand the assemblage rules of these plant communities along environmental gradients. Our results showed that the functional structure of the studied plant communities was highly determined by soil environment. Results from our modelling approach based on maximum likelihood estimators showed a predominant influence of soil water storage on most of the community functional traits. We found that changes in community functional structure along environmental gradients were mainly promoted by species turnover rather than by intraspecific variability. Specifically, our new method of variance decomposition demonstrated that between-site trait variation was the result of changes in species occurrence rather than in the abundance of certain dominant species. In conclusion, this study showed that water availability promoted the predominance of specific trait values (both in above and belowground fractions) associated to a resource acquisition or conservation strategy. In addition, we provided evidence that changes on community functional structure along the environmental gradient were mainly promoted by a process of species replacement, which represent a crucial step towards a more general understanding of the relative importance of intraspecific versus interspecific trait variation in these woody Mediterranean communities.


Measurements of plant functional traits
Nine above-ground and two below-ground functional traits related to morphology, physiology and chemical composition were measured. These traits, at leaf, stem, root and whole plant level (Table   1) are related to the ability to acquire, transport and fulfill plant water and nutrient requirements.
Plant height and cover were measured in ten individuals, per species and site, with a tape except for the taller species, whose height was estimated with the ´Christen height` meter based on trigonometric principles (Klein 2007). Plant cover area was estimated by ellipse area equation (major and minor diameter of the canopy projection).
For leaf and stem measurements, six individuals per species and site were chosen. A few branches with young, fully expanded leaves and a portion of stem of the previous year were collected from each individual plant. These branches were stored in plastic bags to prevent water loss and further transported to the laboratory, where they were maintained with the basal portion of the stem submerged in water at 10 ºC for 24h in darkness to allow a complete re-hydration.
A subsample of leaves was removed from the stem, the petiole was excised and the leaves were fresh-weighted and scanned. The leaf area was calculated using image analysis software (Image-Pro 4.5). Leaves were oven-dried for at least 48 h at 60ºC, and further weighed with a precision of 0.001 g. Specific leaf area (SLA, m 2 kg -1 ) was calculated as the ratio between the leaf lamina area and its dry mass. Leaf dry matter content (LDMC, mg g -1 ) was calculated as the ratio between dry and saturated fresh mass of the leaf lamina.
Leaves were ground with a stainless steel mill for nitrogen and δ 13 C content analysis. The nitrogen concentration was measured using an elemental analyser. The isotopic analysis of C (δ 13 C) was carried out at the Laboratorio de Isótopos Estables of the Estación Biológica de Doñana (LIE-EBD, Spain). All samples were combusted at 1020ºC using a continuous flow isotope-ratio mass spectrometry system by means of an elemental analyzer coupled to an isotope ratio mass spectrometer . Replicate assays of laboratory standards routinely inserted within the sampling sequence, and previously calibrated with international standards, indicated analytical measurement errors of ±0.1‰. For stem traits, we selected young stems from the last growing season with an approximate length of 10 cm. Stems were oven-dried for at least 48 h at 60ºC and weighed to obtain stem dry mass. Stem dry matter content (SDMC, mg g -1 ) was obtained as the ratio between dry and saturated fresh mass.
To better understand the plant-soil interactions, we measured two functional traits from fine roots (< 2 mm in diameter), which are related to water and nutrient uptake (Jackson 1997). We collected the root samples in the first 20-30 cm of soil digging close to the plant basal stem and we collected only those fine roots emerging from these primary roots. Sampling roots were stored in plastic bags to be transported to the laboratory and washed there with distilled water to remove soil residuals. Cleaned roots were maintained in water at 4ºC for 24 h in darkness for a complete rehydration. Root measurements were obtained from fine roots (< 2 mm in diameter). Roots were weighed for saturated mass and scanned. Images were analyzed with WinRHIZO 2009 for root length. Root dry mass was obtained after oven-drying them at 60ºC for 48h. Specific root length Details of the method used to disentangle the relative importance of species occurrence, abundance and intraspecific variability on changes in community functional structure.
First, we calculated the three types of CWM parameters proposed by Lepš et al. (2011): 1) 'specific' average traits, using trait values of each species within each site, whose variation can be caused by both species turnover and intraspecific trait variability: where p i is the abundance of the i-th species in a given community, S is the number of species in this community, and x i _habitat is the specific mean trait value of the i-th species, which is valid just for a given habitat sampled.
2) 'fixed' trait values, using mean trait values of each species along the whole environmental gradient (i.e. site-independent trait values), whose variation is only due to changes in species turnover: Where x i is the fixed mean trait value of the i-th species for all communities where the species is found.
3) ´intraspecific variabilitity` trait values, which are calculated from the differences between 'specific' and 'fixed' average traits and permit an estimation of the pure effects of the intraspecific variability: intraspecific variability parameter = specific parameter -fixed parameter Second, we computed two new community parameters with the aim of disentangling the effects of the two components of species turnover (species occurrence and species abundance): 1) 'unweighted' trait values (UWM), which were calculated similarly to the above-mentioned 'fixed' trait values but without weighting them by their relative species abundances: 2) 'species-abundance' trait values, calculated from differences between 'fixed` and 'unweighted' trait values. Thus, variation in the 'unweighted' trait values is solely affected by changes in species occurrence (presence/absence of species) whereas variation in 'species-abundance' trait values allows us to estimate the pure effects of changes in species abundance as follows: species abundance parameter = fixed parameter (´species turnover`) -unweighted parameter (´species occurrence`) Thus, the complete formula can be defined as: Finally, we explored 'CWM traits -environment' linkages for the two new types of community parameters ('unweighted' and 'species-abundance' trait values) as well as for that used to estimate the pure effects of the intraspecific variability ('intraspecific variability' parameter). To quantify how much variability is accounted for by each individual component (species occurrence, abundance and intraspecific variability), we used the method based on the sum of squares (SS) decomposition from Lepš et al. 2011, using the best likelihood models previously calculated. The SS can be decomposed into the amount of variability explained by individual terms of the model and the unexplained variability (error). Since the effects of the above-explained community parameters do not always vary independently, we also considered the effect of their covariation. In turn, covariation was partitioned into two different components, as specified in the equations: the covariation between species turnover and intraspecific variation (covSSI), and the covariation between species occurrence and abundance (covSSII), as specified in the following equations: covSSI = SS specific -SS fixed -SS intraspecific variability covSSII = SS fixed -SS species occurrence -SS species abundance In summary, the maximum variability included in 'specific' average traits (i.e. that due to changes in species occurrence, abundance and intraspecific trait variability) can be defined as: SSspecific = SS species occurrence + SS species abundance + SS intraspecific variability + covSSI + covSSII

Example
To illustrate this method we developed the results for the case of specific leaf area (SLA).   Table A7. Results of a one-way ANOVA between zones and between slopes for non-correlated abiotic variables.