Experiment 3: Is the Pneumatron underestimating embolism resistance compared to the optical method?
The optical method and Pneumatron were simultaneously applied to the same detached leaves for all six species. We were unable to obtain Pneumatron measurements for B. pendula , because the gas volume that could be extracted from the leaves was too small to meet the sensitivity requirements of the tube volume, even if we reduced the volume of the discharge tube to the minimum volume of 1.1 mL.
Vulnerability curves based on the two methods were obtained for five species (Figure 4). The PEP12, PEP50 and PEP88 values of detached leaves obtained with the optical method in experiment 1 (Fig.1, Table S2) did not differ significantly from those obtained in experiment 3 (Table S3). A difference in embolism spreading from cut conduits to intact ones versus spreading between intact conduits would especially be reflected in PAD12 and PEP12 values. Indeed, average PAD12 values based on the Pneumatron were lower than the average PEP12 values based on the optical method for four species, except for P. avium . The difference between PAD12 and PEP12 was more than 0.6 MPa for Q. petraea , F. sylvatica , and C. betulus , although these differences were not statistically significant. Also, there was considerable variation among the samples tested (Fig. 5a), with several samples showing a slightly more negative PAD12 value than PEP12 for P. avium and L. tulipifera .
Despite minor differences between PEP12 and PAD12, the vulnerability curves based on the optical and pneumatic method showed strikingly similar patterns for the five species tested, with a strong correlation between PAD50 and PEP50, and between PAD88 and PEP88 (Fig. 5b, c). When intraspecific differences in embolism resistance were found within a species, both methods matched each other very well (Figure 4, 5, Figure S6). Although curves based on the optical method had a relatively steeper slope compared to the pneumatic curves for F. sylvatica , C. betulus and Q. petraea , this difference was not significant. No significant difference was found between PAD50 and PEP50, and between PAD88 and PEP88. Nevertheless,P. avium showed a 0.67 MPa difference (P = 0.287) between PEP50 and PAD50, and a 0.61MPa difference (P = 0.454) between PEP88 and PAD88 (Table S3).