Xylem Anatomy Analysis
To characterize self-supporting and lianescent xylem anatomy, we sampled the longest stems from the five unpruned plants (Fig. 2b after two years of cultivation, in May 2018. Due to differences in sampled stem lengths, which ranged from 3.6 to 8.4 m, self-supporting and lianescent xylems were characterized at 4/5 of the distance to the base, in the same cross-section. After collection, the samples were immediately fixed in FAA 50 (10% formalin, 5% acetic acid, 50% alcohol). Subsequently, the samples were rehydrated, embedded with polyethylene glycol 1500, and transversal and longitudinal sections from 8 to 15 µm in thickness were made using a sliding microtome, as described by Barbosa et al.(2010). Additionally, portions of self-supporting and lianescent xylem were dissected under a stereomicroscope and macerated in Franklin’s solution (Berlyn & Miksche, 1976).
Photomicrographs were captured using a Leica DML photomicroscope attached to a digital camera DFC 310FX, and measurements were performed using ImageJ v1.52a (Schneider et al. , 2012). Secondary xylem characterization was based on the IAWA List of Microscopic Features for Hardwood Identification (IAWA Committee, 1989) and Scholz et al. (2013). Secondary xylem characters were analyzed with a magnification of 200× or 400×, while the intervessel pit diameter was the only analyzed parameter requiring a higher magnification of 1,000× under oil immersion. Relative areas of fibers, axial parenchyma, and rays were calculated by analyzing six 0.1 mm² images from random portions of the transverse section (Gerolamo & Angyalossy, 2017). The amount of xylem produced by the cambium was measured by calculating the average length of three straight lines from the cambium to the pith. The vessel grouping index was measured by the ratio of total number of vessels to total number of vessel groupings, including solitary and grouped vessels (Scholz et al. 2013). The equivalent vessel diameter (Dv; µm) was calculated by measuring the vessel lumen area (A; µm2) and using the formula:
Dv = (4A\(\pi\)-1)1/2
The hydraulic diameter (Dh), corresponding to the mean diameter that all sampled vessels would have to correspond to the total conductivity of the same number of vessels (n) (Tyree et al. , 1994, Scholz et al. , 2013) was calculated using the formula:
\begin{equation} D_{h}=(\Sigma_{1}^{n}\ D_{v}^{4}\ n^{-1})^{1/4}\nonumber \\ \end{equation}
Potential specific conductivity (Kp; kg m-1 Mpa-1 s-1) was calculated following Poorter et al. (2010) using the formula:
Kp = (\(\pi\) ρw128η-1) VdDv 4
where Dh (m) is the vessel hydraulic diameter, ρw is water density at 20 °C (998.2 kg.m-3), η is the viscosity index of water (1.002 x 10-9 MPa s-1 at 20°C), and Vd is vessel density (m-2). Sample sizes were recommended by Scholz et al. (2013). Data normality and variance homogeneity were tested using Shapiro-Wilk and Levene’s tests, respectively. To compare each self-supporting and lianescent xylem anatomy characteristics, the Student’s t-test for parametric data, and the Mann-Whitney test for nonparametric data were applied in R software v.3.6.1 (R Core Team, 2014).