Eos m10

For histochemical staining, phloroglucinol (Wiesner) staining was performed as described in Yeung (1998 ). Briefly, hand-cut equatorial sections of five developmental stages were left for 20 min in phloroglucinol-HCl staining solution [20 mg/mL phloroglucinol in 20% ethanol and mixed with 12 N HCl (v:v=80:20)], and immediately photographed with a Canon EOS M10 fitted with a macro lens (35 mm, f/2.8 Canon Inc., Tokyo, Japan). The Wiesner reagent (phloroglucinol-HCl) mainly reacts with O-4-linked coniferyl and sinapyl aldehydes in lignifying cell walls (Pomar et al., 2002 (link)) and was used to follow the onset of lignification.
Sections from July and October nuts were stained with Fuchsin-Chrysoidine-Astrablue (FCA) solution [0.1 mg/mL of New Fuchsin, 0.143 mg/mL Chrysoidine, 1.25 mg/mL Astra blue and acetic acid (v:v=1:50)], and then washed step by step with distilled water, ethanol (30, 70%) and isopropanol. The samples were immersed in the FCA-solution for 48 h to guarantee a full penetration of the staining into the dense microstructure of the cell walls. Stained sections were embedded in Euparal and photographed under a Labophot-2 microscope (Nikon Corporation, Tokyo, Japan).

Water caltrops were photographed with a macro camera (EOS M10, Canon; objective EFS 35 mm). For imaging of the spines, a stereomicroscope (SMZ800, Nikon) was used.