**2. Material and Methods**

Two adult and healthy plants of five species classified within the Cacteae tribe were collected in their natural habitats (Table 1). For each species, a portion of the plant was prepared as a voucher, which was deposited in the National Herbarium (MEXU). In one of the two plants per species, spines were removed. Then, using a dissecting microscope at different magnifications, the stem of each sample was dissected into the epidermis plus hypodermis (EH), cortex (C), vascular cylinder (VC) and pith (P) (Figure 1A,B). Each tissue was blended with distilled water and filtered with a mesh of approximately 300 μm pore diameter to separate biominerals from cell debris. The biominerals were precipitated, washed with distilled water several times until no residue was observed under a stereomicroscope, and finally, dried at room temperature. Small samples from pith to epidermis were prepared for FE-SEM-EDS (see 2.3, JEOL Ltd., Akishima, Tokyo, Japan).

**Table 1.** Species analyzed and the state of Mexico where they were collected. Vouchers deposited at MEXU.


**Figure 1.** Stem and macro and microscopic tissues studied. (**A**) *Echinocactus texensis* (TT1021), showing the globose depressed stem. (**B**) *Echinocactus texensis*, transverse section illustrating the stem tissues. (**C**) *Mammillaria sphaerica* (TT1051), microscopic transverse section showing the tissues. A = apical, M = medium, B= basal, EH = epidermis-hypodermis, C = cortex, VC = vascular cylinder, P = pith. Bar is 300 μm in C.

The second individual of each species was divided into three parts (apical, median and basal) no larger than 1.5 cm length that included epidermal, cortical, vascular and pith tissues (Figure 1A,C). Each part was immediately fixed in a formalin-acetic acid-ethanol solution (10:5:85) [26] and processed for paraffin embedding according to Loza-Cornejo and Terrazas [27]. Transverse and tangential sections 14 μm thick were made with a rotary microtome, stained with Safranin-fast green, and mounted with synthetic resin.
