Vein networks constrain the transport of water, C and nutrients within the leaf. Vein density, the length of minor veins per unit LA (mm mm–2) can characterise the structure of these networks. Vein density is a structural determinant of hydraulic conductance and photosynthetic rate. Depending on the species set considered, the vein density may correlate with other leaf traits, such as Lth, stomatal density and maximum gas-exchange rates. This trait shows plasticity across environments and is highly variable among species, showing both broad phylogenetic trends and potential adaptation to resource gradients.
Fresh or dried leaves may be used. For the leaf collecting protocol, see Section 3.1. Sampling intensity should account for known trait variation between sun and shade leaves and across the lamina of given leaves. If possible, measurements should be made on sections of lamina containing no large veins. For smaller leaves, use the entire leaf; for larger leaves, a 1-cm2 section is sufficient.
Soak the leaf in 5% w/v NaOH–H2O for 24–72 h, until it becomes transparent. If the NaOH solution turns an opaque brown colour during soaking, replace the soaking solution. Then rinse the leaf in H2O and transfer to a 2% w/v NaOCl–H2O solution for 5–30 min, until it becomes bleached.
Rinse the leaf in H2O and transfer via dehydration series to pure ethanol (e.g. 30%, 50%, 70%, 100% ethanol, or 50%, 100% ethanol, each step lasting 30 s, for tender leaves, and 5 min for tougher leaves). Stain the leaf for 15 min in 1% w/v safranin O in ethanol and/or other lignin stains. Destain in ethanol. The leaf can be mounted in water or glycerol on plastic transparency film, or permanently, after transfer to 100% toluene, in immersion oil or Permount (allow several days after mounting for toluene to fully evaporate). Veins will appear red. Take particular care to ensure that all veins in the network are visible. The most numerous and functionally important minor veins can be very hard to see, and often require the epidermis to be removed for accurate visualisation. Vein counts should be made using microscope objectives of ×4 for ferns and up to ×40 for tropical angiosperms. Photograph the venation network under a light microscope, ensuring a large enough field of view (e.g. 1–10 mm2). Then measure the total length of veins in the image and divide this number by the image area to obtain the vein density, using image analysis software (see Section 3.1). This image analysis process may be semi-automated with freely available software (see More on methods, below in the present Section), but the accuracy should be tested.
Special cases or extras
(1) Leaf handling. Leaves become delicate during processing and should be moved carefully between solutions or solution should be vacuumed out of the dish, so that leaves will not puncture or tear. For best results in clearing leaves of given species, the details of the protocol can be modified. Warm NaOH may be used (although not boiling), and longer clearing times, or more concentrated NaOCl–H2O solution for a shorter time period. Small or thin leaves may require less soaking time in all solutions. Conversely, very thick or dense leaves may require several days in the NaOH solution before they become transparent. Hairy leaves may require removal of epidermis.
(2) Sclereids. In some species, leaves may have sclereids that can be mistaken for veins, and also have an important hydraulic function. Additional venation traits can be measured for more detailed investigations of leaf structure and function (see references below in the present Section).
References on theory and significance: Uhl and Mosbrugger (1999); Roth-Nebelsick et al. (2001); Sack and Frole (2006); Sack and Holbrook (2006); Brodribb et al. (2007, 2010); Boyce et al. (2009).
More on methods: Dilcher (1974); Gardner (1975); Brodribb and Feild (2010); Price et al. (2011).