Ds 5m

Cross-sections (thickness, 10 μm) were cut from frozen samples of 14 days OL plantaris muscle and stained with hematoxylin & eosin (Sakura Finetek) according to standard procedures. Stained cross-sections were observed under a bright field microscope (Eclipse TE300; Nikon, Tokyo, Japan) and captured with a digital camera (DS-5M; Nikon, Tokyo, Japan). From photomicrographs of each muscle, cross-sectional area (CSA) of ≥1000 fibers was measured using ImageJ v. 1.46r (National Institutes of Health, Bethesda, MD, USA). The plantaris muscle has slow fibers in its deep region and fast fibers with different fiber size in its middle to surface region [22 (link)]. To avoid such a region-dependent bias, the measurement of CSA were made evenly for fibers in all captured images. As with muscle weight, fiber CSA is thought to be affected by body weight, so that it was expressed relative to body weight, too (CSA/body weight).

A freshly prepared silicon sample was bonded to a Peltier stage (Deben MK3 Coolstage) with thermal paste and placed under a top-down optical microscope (Nikon Eclipse LV150). The ambient temperature was T_∞ = 21.3 ± 0.3 °C and the relative humidity was H = 40 ± 3%, corresponding to a dew point of T_DP = 7.2 ± 1.3 °C. The substrate was initially kept dry at 10 °C and was then cooled beneath the dew point to 5 °C to allow condensation to form in order to make the patterns visible. The desired pattern type (circles, triangles, or stripes) was centered and focused under a 5× objective lens, such that the 2P, 4P and 8P arrays were all visible within the microscope’s field-of-view.
Once the field-of-view was set, the sample was immediately heated to 40 °C to dry the surface. The wafer was then cooled back down to 10 °C, followed by further cooling to either 5 °C or −10 °C to observe the resulting condensation with a digital camera (Nikon DS-5M) attached to the microscope. The transient of the Peltier stage was 5 ± 1 s to cool from 10 °C to 5 °C and 30 ± 1 s to cool from 10 °C to −10 °C. The forming condensation was imaged for 10 min, and the surface was then heated back to 40 °C to dry out the surface for the next trial. Three trials were obtained for both condensation temperatures and a new chip was used for each pattern type to minimize surface contamination.

To analyze root length, five-day-old seedlings grown on agar (for details see Plant Material) were transferred on agar plates containing 1/8 MS, pH 4 supplemented with 200 μM AlCl₃. After 9 day incubation, plates were scanned (Canon CanoScan 8800F) and the root growth was measured using the JMicroVision 1.2.7 software.
To measure pollen tubes length, tobacco pollen was transiently transformed with AtNPC4:YFP by particle bombardment. After 6 h of germination in dark, pollen tubes were incubated in liquid simple sucrose medium (pH 5) with or without 50 μM AlCl₃ for additional 2 h. Mean growth rate of pollen tubes expressing AtNPC4:YFP and vector-only control was evaluated using the fluorescence microscope Olympus BX-51.
To determine survival rate, 7-day-old seedlings grown on agar (for details see Plant Material) were transferred to 6-well plates with liquid 1/8 Hoagland’s (Kocourková et al., 2011 (link)) solution (pH 4) with 100 μM AlCl₃ for 22 days. Plates pictures were taken by Nikon SMZ 1500 zoom stereoscopic microscope coupled to a Nikon DS-5M digital camera. The survival rate was calculated as a number of viable true leaves.

Morphological assessments were based on herbarium specimens and our field collections. Further, protologues of all published names in the genus Tinnea from Africa and all relevant taxonomic literature [1 (link)–3 , 15 , 16 , 20 ] were consulted and reviewed. To delimit the potential new taxa from closely similar species, morphological variations were compared with herbarium (FHI, GSUH, K, S; acronyms follow [21 ]) specimens including type materials on JSTOR [22 ] and POWO [2 ]. A dissecting microscope (Leica GZ4) or stereomicroscope (Leica S9i) fitted with digital camera (Nikon DS-5M) and eyepiece micrometer were used to measure and photograph stems, leaves, inflorescences, flowers and fruits.