Materials and methods
The five vessel-bearing angiosperm species used in this study wereIlex verticillata (L.) A.Gray [Aquifoliaceae], Rhododendron hirsutum L. [Ericaceae], Ficus religiosa L. [Moraceae], Tilia cordata Mill. [Malvaceae] andLindera benzoin L. [Lauraceae], the vessel-less angiosperm was Drimys winteri J.R. Forst & G.Forst. [Winteraceae], and three conifer species were Agathis robusta (C.Moore ex. F.Muell) Bailey [Araucariaceae], Tsuga canadensis (L.) Carrière [Pinaceae] and Torreya californica Torr. [Taxaceae]. Plants of Ti. cordata , and Ts. canadensis were grown outside in the grounds of the Botanical Gardens of Ulm University, Ulm (Germany) (48° 25’ N, 9° 57’ E), F. religiosa , A. robusta , and D. winteri in the glasshouses of the Botanical Gardens of Ulm University, I. verticillata , L. benzoin and To. californica were grown in the glasshouses of Purdue University, West Lafayette (Indiana, USA) (40° 25’ N, 86° 54’ W, elevation: 187 m).R. hirsutum was collected in the Allgäu Alps near Oberstdorf (Germany) (47° 25’ N, 10° 17’ E, elevation: 2600 m). Care was taken to ensure no pre-existing embolism was present in the stems prior to experiments, with all plants grown under well-watered conditions for the duration of the growing season prior to harvesting. All stems were collected between May and June 2019 prior to dawn and were more hydrated than -0.4 MPa. Furthermore, air-filled xylem conduits appear as a blank area in the xylem when image processing is performed using the optical method. In all experiments no blank areas were detected in the observed areas of xylem at the completion of the experiment, providing support for no substantial, pre-existing embolism being present in any sample measured prior to the experiment.
Terminal branches ranging from 0.3 to 2.0 m long (all exceeding the length of the longest vessel determined by air injection for the five angiosperm species) were collected prior to dawn, cut under water, and bagged for approximately 1 h, so that all experiments started with a stem water potential (Ψstem) after equilibration of at least -0. 4 MPa. Stems were measured in all species, except L. benzoin in which experiments were conducted on leaves. Stems and leaves were fixed under a stereo microscope (SZMT2, optika, Italy) or in a Raspberry Pi clamp (opensourceOV.org). Vulnerability curves for stems were conducted on a region of the stem in which the bark was gently removed by hand, without touching the xylem beneath, and an adhesive gel (Tensive) immediately applied to the surface of the stem xylem to avoid dehydration and provide greater optics. A glass cover slip was placed on top of the adhesive gel to aid imaging. A stem psychrometer (ICT International, version 4.4) was then attached to the stem beyond the length of the longest vessel from the area imaged and Ψstem measurements were collected every 10 min. Branches were allowed to naturally dry while images were captured every 3 min.
All branches were rehydrated across a range of dehydrated Ψstem to build a data set spanning a range of variation in the percentage of embolism at the point of rehydration across the species selected. At the point of rehydration, the end of the branch was excised under water (removing c. 1 cm from the cut end) until Ψstem had fully recovered. Rehydration with water potentials approaching 0 MPa generally occurred in less than 60 mins in all species. Branches were allowed to remain at a relaxed, high water potential for a variable amount of time ranging from 30 mins to 5 h. The cut end of the stem was removed from water once rehydrated, and the branch was then allowed to bench dry a second time until all conduits had visibly embolized. Stem or leaf images were analysed using ImageJ (version 1.52h, NIH, USA) to quantify the accumulation of embolized xylem area through time. Optical vulnerability curves were constructed as described by Brodribb et al. (2016b).
Based on the dehydration-rehydration-dehydration curves, we were able to determine the area of xylem that was embolised on the second cycle of dehydration at a more hydrated Ψ than when rehydration occurred (E pr) as follows:
\begin{equation} \ E_{\text{pr}}=\left(\frac{E_{r2}-E_{r1}\ }{100-E_{r1}\ }\right)\ X\ 100\nonumber \\ \end{equation}
Where \(E_{r1}\) is the percentage of accumulated embolized xylem area at the moment of rehydration and \(E_{r2}\) was the percentage of accumulated embolized xylem area that occurred prior to when plants reached the same Ψ at which rehydration occurred.
Based on the dehydration-rehydration-dehydration curves, we also determined the P 50 (calculated by considering all events of embolism obtained using the dehydration-rehydration-dehydration curves), and an apparentP 50 (P 50r) that was obtained based only on the embolism events that occurred on the second cycle of dehydration. From these values we were able to calculate the percentage change in P 50 due to pre-existing embolism using the follow equation:\(\text{Percent\ change\ in\ apparent\ }P_{50\ }=\left(\frac{P_{50}-P_{50r}\ }{P_{50}\ }\right)\ X\ 100\)
We conducted the experiment at least once in all species, three times inI. verticillata and R. hirsutum and twice in L. benzoin and F. religiosa . For To. californica , three rehydration curves were performed at the same \(E_{r1}\) and the results are presented as a mean of these three experiments (Supplementary Figure S1).