One may ask which tissues will explain the variation in LVA between deciduous and evergreen species, mentioned earlier. In most anatomical literature, results of leaf cross-section analysis have been presented as volumetric fractions of different tissues. However, in order to break down LMA into its anatomical building blocks, one needs to calculate the volume of each tissue per unit leaf area (see Section I.2). Analysis of the deciduous–evergreen contrast showed that evergreen species had considerably greater mesophyll tissue volume per unit leaf area (R. Villar, unpublished), which is intriguing because these species did not have higher rates of photosynthesis. Evergreens do have thicker cell walls (Terashima et al., 2006) and possibly they compensate their lower cell wall conductance for CO2 with a larger volume of mesophyll. The larger volume of mesophyll resulted predominantly from evergreens having larger cells, and far less because of an increased number of cells (Castro-Díez et al., 2000; R. Villar, unpublished). Within each group (deciduous or evergreen), high-LMA species had a similar total LVA compared with low-LMA species, but a relatively lower volume of epidermal tissue. Leaves with a higher volume of epidermis had a lower LD. Within the grasses, high-LMA species showed a higher volume of sclerenchymatic tissue and vascular bundles.
A low leaf density could be caused by the presence of a large volume of air spaces. This enhances conductivity within the leaf, which may facilitate photosynthesis (Niinemets, 1999). A high density can be caused by a large fraction of mesophyll, or a high proportion of lignified tissue, that may be important for leaf toughness, and thereby leaf and plant survival (Alvarez-Clare & Kitajima, 2007). To make our investigations in Eqn 3 complete, one not only needs to know the volumes taken up by the various tissues, but also their density. Unfortunately, we have little insight into the density of different tissues, let alone variation between species. Determination of densities of individual leaf fractions is not straightforward, as it is difficult to separate the various tissues from each other. Winter et al. (1993) reported that the volume of epidermal cells in barley consisted of 99% vacuole. In mesophyll cells, vacuoles formed 73% of the volume, with chloroplasts and mitochondria occupying 20%. As these plastids are much denser than the vacuole, one could expect mesophyll to have a higher density than the epidermis. Alternatively, one could reason that the density of epidermal cells is higher, as their cell walls are thicker (U. Niinemets, unpublished). A few studies have determined simultaneously the volume per area (VA) of the different tissues as well as LD and LMA (Van Arendonk & Poorter, 1994; Castro-Díez et al., 2000; R. Villar & H. Poorter, unpublished). Using a multiple regression, we regressed the VA of epidermis, mesophyll and vascular tissue against LMA. In principle, the regression coefficients should then give an estimate of the average density of these tissues over all species. Averaged over all groups of species epidermal tissue was shown to be low in density, mesophyll densities were closest to that of the overall leaf and vascular tissue plus sclerenchyma showed the highest values (Table 2). These are rather rough estimates, owing partly to the fact that these cross-sections only represent part of the leaf and partly to the fact that the densities of the different tissues vary with species. They are, nonetheless, in accord with the observation that the area around the main vein has a higher LMA value than the rest of the leaf (Section V.1). By publishing these first approximations, we hope to stimulate the scientific community to come up with experimentally derived estimates.