Xylem traits
At the final harvest, three to four seedlings per species were chosen
randomly for xylem traits measurements. For each individual, one fully
expanded leaf as well as the stem was pickled, and the middle part of
each leaf (including the middle of the midvein) as well as the middle of
each stem were cut transversely. The materials were embedded in 5% agar
and progressively dehydrated in 50, 70 and 95% ethanol (2h per
solution), after which the small blocks of agar were infiltrated for 15
days with resin JB 4 Polysciences (Polysciences Inc., Warington, Pa.,
USA). After polymerisation of the resin, 2 µm thick cross-sections were
obtained with a glass ultra-microtome, then sections were stained with
5% toluidine blue and permanently mounted onto slides with DPX (dibutyl
phthalate in xylene). The cross-sections of leaves and stems were
studied with a light microscope (Zeiss Axioskop; Carl Zeiss, Jena,
Germany) on a computer screen with image analysis software (Aequitas IA
v. 1.25) (Castro-Díez, Puyravaud & Cornelissen, 2000, Castro-Díez,
Puyravaud, Cornelissen & Villar-Salvador, 1998).
For stems, stem xylem area and stem xylem conductance area (stem xylem
area minus cell wall area) were circled and measured. The proportion of
cell wall area relative to xylem area in transverse section was measured
in three to four microscopic fields per slide using Aequita tools
(Castro-Díez et al. , 1998). For leaves, leaf midvein xylem area
and minor vessel area were circled in light microscope images and
measured, and the minor vessel area was calculated as the average area
of the ten smallest vessels of the cross-section of leaves, which were
defined as the distal conduits. The plant minor vessel number
(N vessel) was theoretically approximated as:N vessel = A/(
π*R s*R m), where A is stem
xylem conductance area, R s is the radius of the
biggest vessel in stem medium, and R m is the
radius of the minor vessel in leaves. This calculation was based on the
pipe model, which states that the sum of all vessel inner diameters at
each vein order is equal (Shinozaki et al. , 1964). We used the
stem (rather than leaf) xylem conductance area to calculateN vessel because it is difficult to gain the leaf
xylem conductance area in a representative way from entire leaf
cross-sections, especially for species that have big leaves.