3.4 Leaf thickness
Leaf thickness (Lth, µm or mm) is one of the key components of SLA (see Sections 3.1 and 3.3), because
SLA 

1/(tissue density × Lth) (where density = dry mass/volume 

LDMC; see Section 3.3). Lth also plays a
key role in determining the physical strength of leaves (see Section 3.7). For example, leaf ‘work to shear’ is
(by definition) the product of Lth and tissue toughness. Optimisation theory, balancing photosynthetic benefits
against C costs of respiration and transpiration, predicts that Lth should be higher in sunnier, drier and less
fertile habitats, as well as in longer-lived leaves. These patterns are indeed often observed, at least in
interspecific studies. Within individuals, many studies have shown that outer-canopy ‘sun’ leaves tend to be
thicker than those from more-shaded parts of the canopy. Both within and among species, the strongest
anatomical driver of variation in Lth is the number and thickness of mesophyll layers. Consequently, Lth is a
strong driver of leaf N per area. Although higher Lth should lead to faster photosynthetic rates per unit LA
(via a higher N : area ratio), this relationship is often weak in interspecific studies, for a combination of
reasons. First, because of covariance of SLA and %N, thicker leaves often have lower %N and longer leaflifespan (which are associated with lower photosynthetic rate per unit leaf mass). Second, thicker-leaved
species may have slower CO2 diffusion (lower mesophyll conductance) via longer diffusion pathways,
greater internal self-shading of chloroplasts, or higher optical reflectivity in combination with lower internal
transmittance. Thick leaves are also a feature of succulents.
What and how to collect?
Follow similar procedures as for Section 3.1. In many cases, the same leaves will be used for the
determination of SLA, Lth and LDMC (and perhaps Section 3.7). For recommended sample size, see
Appendix 1.
Storing and processing
Similarly as for SLA. Lth is strongly affected by LWC; hence, some form of rehydration should be
seriously considered, as described for SLA, particularly if using a digital micrometer, where any slight loss
of turgor results in an underestimation.
Measuring
Thickness tends to vary over the surface of the leaf, generally being thickest at the midrib, primary veins,
margins and leaf base. Depending on the research question, you may be interested in the average thickness
across the leaf, or the thickness at special locations or of special tissues. Often one measurement per leaf, at a
position as standard as possible within the lamina (e.g. at an intermediate position between the border and the
midrib, and between the tip and the base of the leaf, avoiding important secondary veins) is acceptable for
broad interspecific comparisons. When more precision is needed, the average of thickness measurements at
several points in the lamina will be more appropriate. Another way to estimate the average thickness over the
entire leaf surface is to back-calculate it from the leaf volume divided by LA; however, it is laborious to
accurately measure leaf volume, e.g. with a pycnometer. A relatively fast approximation of whole-leaf
average Lth can be obtained by dividing leaf fresh mass by LA (which is the same as calculating 1/SLA ×
LDMC), i.e. by assuming that leaf fresh mass and volume are tightly related. This approach does not take
into account the higher density of dry material in the leaf, or the lower density as a result of intercellular
spaces; however, as an approximation it works well.
Other approaches are needed if one wants to distinguish between thickness of midrib, margin and
intercostal regions of the leaf, or to compare replicates at a given point on the leaf, e.g. half-way between the
leaf base and the tip, as is commonly done. One method is to measure these quantities directly from leaf
cross-sections (hand-sections), or to use image analysis (see Section 3.1 for free software) to calculate
average Lth across the section, by dividing the total cross-sectional area by the section width. On the positive
side, this method enables reasonably accurate measurements to be made. On the down side, soft tissue may
distort when hand-sectioned, and the method is relatively slow (e.g. 15 min per measurement).
Probably the fastest approach is to measure Lth using a dial-gauge or a digital micrometer (or even a linear
variable displacement transducer; LVDT). Multiple measurements can be made within quick succession and
averaged to give an indicative value of Lth for the feature in question (such as e.g. midrib or lamina between
the main veins) or region of interest (e.g. near midpoint of leaf). If necessary, we recommend replacing the
original contact points on the micrometer with contacts 2–3 mm in diameter; i.e. narrow enough to fit
between major veins, but sufficiently broad so as not to dent the leaf surface when making measurements.
However, for soft-leaved species such as Arabidopsis, permanent deformation is difficult to avoid.
Special cases or extras
(i) Needle leaves. For needle leaves that are circular in cross-section, average Lth can be quickly estimated
as Diameter × π/4 (equivalent to cross-sectional area divided by cross-section width). Still, because
needle leaves typically taper towards the leaf tip, several measurements would normally need to be
made.
References on theory, significance and large datasets: Clements (1905); Givnish (1979); Parkhurst (1994);
Enríquez et al. (1996); Knapp and Carter (1998); Smith et al. (1998); Wilson et al. (1999); Green and Kruger
(2001); Niinemets (2001); Díaz et al. (2004).
More on methods: Witkowski and Lamont (1991); Garnier and Laurent (1994); Shipley (1995); Wright
and Westoby (2002); Vile et al. (2005); Poorter et al. (2009); Hodgson et al. (2011).