Materials and methods

Three adult individuals per species were collected in different locations (Table 1). The Coryphantha clavata individuals were simple globose with 8-15 cm long and 10 cm in the wider diameter of the stem with a thick root; the Coryphantha cornifera individuals had stems that were up to 16 cm long and up to 8.6 cm in total diameter and the Coryphantha radians individuals had four branches with thick stems 5 to 8 cm in diameter and 8 cm long. The three species were in the same phenological stage (without flowering).

Sections with areas of 3 × 3 cm corresponding to the basal, middle and apical parts of the stem of each species were cut and fixed with FAA (formaldehyde, 37 % glacial acetic acid, 95 % ethanol 95 % and distilled water; ^{Ruzin 1999}) for 48 hours. Then, they were washed with running water, sectioned into smaller portions up to the vascular cylinder and dehydrated with alcohol (70 % for one month). The alcohol solution was replaced every two weeks to reduce the mucilage content. The samples were cut with a double-edged knife to obtain transverse planes of the tubercle and stem as well as tangential and radial planes for each section of the stem. Subsequently, they were dehydrated in 96 and 100 % xylene and included in Paraplast^®. The anatomical sections were 14 and 16 µm thick and were obtained using a rotary microtome (Leica RM2125RT, Leica, Wetzlar, Germany). They were stained with safranin and fast green and mounted in synthetic resin according to methods previously reported by ^{Loza-Cornejo & Terrazas (1996)}. Descriptions were made of the three regions (apical, middle, basal) of the epidermal, fundamental and vascular tissues. The terminology used for the description of the secondary xylem is in accordance with the International Association of Wood Anatomists (^{IAWA Committee 1989)}. For the epidermal characteristics, the terminology of ^{Metcalfe & Chalk (1980}, ¹⁹⁸³⁾ was used. The mean and standard error were obtained from 30 measurements per attribute per individual per species. Anatomical characters were quantified with a Zen Lite 3.0 image analyzer.

Results

Epidermis. In the transverse section, the cuticle was smooth for all three species, the thicknesses were as follows: 2.49 ± 0.30 µm for C. clavata, 2.75 ± 0.16 µm for C. cornifera and 3.0 ± 0.13 µm for C. radians. The epidermis was simple and rectangular, and the largest in C. radians (42. 05 ± 1.88 µm), followed by C. cornifera (35.24 ± 4.27 µm) and C. clavata (20.97 ± 2.2 µm), with the outer periclinal wall being more variable (Figure 1).

Figure 1 Epidermis and hypodermis of three species of Coryphantha, transverse sections. A, C. radians, B, C, E. C. clavata, D, F. C. cornifera, e = epidermis, h = hypodermis (square bracket), stoma, (white arrow), crystals = *. Bar: B-D, F = 20 µm, A, E = 50 µm. 

The outer periclinal wall was flat for all three species. The periclinal wall thickness was thicker for C. radians (11.84 ± 0.48 µm) than for C. cornifera (5.55 ± 0.21 µm) and C. clavata (4.68 ± 0.13 µm). For all three species, the stomata were superficial, while in C. radians, the stomata appeared to be sunken only at the base of the stem, and all three species had substomatal cavities throughout the hypodermis (Figure 1E).

Hypodermis. The number of hypodermal layers was variable from two layers in C. cornifera and C. radians to up to three layers in C. clavata (Figure 1B). The hypodermis of C. cornifera possessed parenchymatous cells (Figure 1D), while those of C. radians and C. clavata showed collenchymatous cells (Figure 1A-B). The hypodermal cells of C. cornifera and C. radians had crystal shapes of druses and druse clusters (Figure 1A, C), and only those of C. clavata had rounded and prismatic crystals (Figure 1B, C).

Cortex. All the taxa studied had palisade and storage parenchyma; in the storage parenchyma, collateral cortical bundles were found (Figure 2D). The cortical bundles may have secondary growth. In the xylem, we found few tracheary elements. The phloem was scarce and had sieve tube elements with a simple horizontal sieve plate and one companion cell per element. Mucilage cavities were observed for C. clavata, but they were absent for C. cornifera and C. radians.

Figure 2 Secondary xylem, secondary phloem and periderm in three species of Coryphantha, transverse and tangential sections. A-C. secondary xylem; D. cortical bundle; E, F. secondary phloem; G-I. Periderm. A, E, H. C. radians; B, D, I. C. cornifera; C, F, G. C. clavata. co = cortex, cp = conductive phloem, e = epidermis, h = hypodermis, ncp = nonconductive phloem, pe = periderm, ph = phloem, tpl = thick phellem cells, r = ray, white circle = vessel, wbt = wide band tracheids, x = xylem, yellow arrows = sieve tube element, red arrows = companion cell. Bar: A-C, G-I = 100 µm, F = 50 µm, D, E=20 µm. 

Pith. The pith had only parenchyma cells and medullary bundles were present; druses occurred for all three species as in the cortex but were more abundant for C. cornifera.

Secondary tissues. Secondary xylem.- For all three species, vessels were embedded in a matrix of wide-band tracheids (Figure 2A-C). The vessels were mostly solitary and narrow, with radial and tangential diameters of 33.42 ± 1.14 µm and 31.68 ± 1.43 µm for C. cornifera, 31.73 ± 0.97 µm and 31.25 ± 0.89 µm for C. clavata and 27.88 ± 1.81 µm and 29.26 ± 3.22 µm for C. radians, respectively. Vessel elements had simple perforation plates and helical or annular secondary wall thickenings (Figure 2D). The wide-band tracheids had the same type of secondary walls as the vessel elements with radial and tangential diameters of 40.35 ± 0.96 µm and 38.41 ± 1.23 µm for C. cornifera, 24.34 ± 0.72 µm and 23.42 ± 0.49 µm for C. clavata and 27.65 ± 0.86 µm and 26.06 ± 0.85 µm for C. radians. For C. radians, the primary and secondary rays are distinctive (Figure 2A). The rays were unlignified (2-4-seriate for C. radians, 2-6-seriate for C. clavata and 2-3- seriate for C. cornifera) and composed almost entirely of erect cells. Druses were observed in some ray cells for all three species.

Secondary phloem.- The phloem of all three studied species had sieve tube elements with companion cells (STE-CCs) and axial and radial parenchyma. In the conductive phloem, the STE-CCs were arranged in groups among the diffuse axial parenchyma cells; the CC occurred on one side of the STE (Figure 2E, F). The STEs had simple sieve plates, with small sieve areas on the sidewalls. In the nonconductive phloem, the STE-CCs collapsed and formed a furrow region between the dilate rays (Figure 2E, F).

Periderm.- For all three species, the periderm was unidirectional. The phellogen was epidermal in origin. The phellem showed thin-walled suberized cells alternating with one to two strata of very thick-walled lignified cells for C. clavata and C. radians (Figure 2G, H), while for C. cornifera, the lignified and suberized cells maintained thin walls, and the outer cells collapsed (Figure 2I).

Discussion

The cuticle thicknesses showed a variation of less than one µm among the three species studied here, and this variation was similar to that exhibited by Coryphantha pallida and Coryphantha retusa (^{Loza-Cornejo & Terrazas 2003}). However, this cuticle thickness was greater (2.49 - 3.00 µm) than the cuticle thickness of 1.8 µm for Coryphantha elephantidens (^{Gasson 1981}). Variations in cuticle thicknesses do not determine survival in arid and semiarid species (^{Gibson & Horak 1978}). The cuticle was smooth for all three species, as for C. elephantidens (^{Gasson 1981}), and did not present ornamentations, as for other Cactaceae species (^{Loza-Cornejo & Terrazas 2003}). The presence or absence of cuticle ornamentations and papillose epidermis may allow recognition between genera. Such is the case for Peniocereus, genus where its seven species have a papillose epidermis and smooth cuticle (^{Loza-Cornejo & Terrazas 2003}, ^{Franco-Estrada et al. 2021}). Whereas the former subgenus Pseudocanthocereus (now in Acanthocereus, ^{Arias et al. 2005}) has two species that have cuticles with micropapillae (^{Sánchez-Mejorada 1974}), one of them Peniocereus fosterianus (=Acanthocereus) (^{Loza-Cornejo & Terrazas 2003}). Acanthocereus needs further studies since A. chiapensis, A. horridus and A. tetragonus have a smooth cuticle (^{Loza-Cornejo & Terrazas 2003}, ^{Martínez-Quezada et al. 2020}). The simple epidermises of the three species had greater sizes (height 20.97 µm, 42.05 µm width) than those reported for other species of Coryphantha (^{Gasson 1981}, ^{Loza-Cornejo & Terrazas 2003}). The outer periclinal wall exhibited greater thickening than the rest of the walls that made up the epidermal cells. This thickening is common for various species of the Cactaceae family and requires future studies to determine the nature of the primary wall thickening and confirm whether it is collenchymatous with the ability to provide flexibility and storage of water (^{Leroux 2012}). The superficial positions of the stomata are maintained along the stem for C. cornifera and C. clavata, which is similar to that recorded by ^{Eggli (1984)}, while C. radians exhibited sunken stomata similar to Coryphantha elephantidens (^{Gasson 1981}). If the stomata are sunken throughout the stem and this is a characteristic of the species, it should be studied in a greater number of species of the same genus to evaluate whether the sunken stomata are associated with species from drier habitats and could be an adaptation to reduce water loss through transpiration (^{Gibson 1983}, ^{Garcia et al. 2012}).

The number of strata in the hypodermis was variable for C. clavata (three strata) with respect to the other two species; this variation was recorded for C. elephantidens with one stratum and two strata for C. pallida and C. retusa (^{Boke 1952}, ^{Gasson 1981}, ^{Loza-Cornejo & Terrazas 2003}). The variations in the number of strata and thicknesses of the hypodermis are induced by the environment (^{Nyffeler & Eggli 1997}). In addition, the hypodermis acts as a protective, supportive tissue that provides elasticity (^{Mauseth 1989}, ^{Vázquez-Sánchez et al. 2005}) and is another defense mechanism against herbivory attack and excessive radiation, especially when associated with the accumulation of crystals in hypodermal cells (^{Pierantoni et al. 2017}, ^{De la Rosa-Tilapa et al. 2020}). ^{Loza-Cornejo & Terrazas (2003)} recorded druses in the hypodermis of C. radians and C. retusa, which was also confirmed for C. radians in this work and for C. cornifera. In C. clavata prisms and rounded crystals were detected. Crystals in the hypodermis are of high diagnostic value (^{Terrazas & Arias 2002}, ^{De la Rosa-Tilapa et al.2020}), so the study of the other species of Coryphantha will contribute to support the subclades within the genus according to the recent phylogeny proposed (^{Sánchez et al. 2022}).

It is worth mentioning that C. clavata possessed abundant mucilage cells in the cortex, in agreement with the clade named mucilaginous by ^{Sánchez et al. (2022)}. Coryphantha cornifera belongs to a species with a characteristic watery cortex. Our observations confirmed this: the cortices of the C. cornifera and C. radians individuals in this study lacked mucilage cells.

The tracheary elements of the three species showed the same anatomical characteristics already described for other Coryphantha species: for example, the vessel elements have simple perforation plates and helical thickening (^{Terrazas et al. 2016}); the vessels were narrow and solitary, characteristic of species with small growth forms (^{Gibson 1973}); the diameter of the vessels (27.88-33.42 µm radial, 29.26-31.68 µm tangential) in C. clavata was consistent with that reported by other authors (^{Gibson 1973}, ^{Terrazas et al. 2016}). Wide-band tracheids showed wall thickening similar to that of the vessel elements. However, the diameter was greater than the diameter of the vessel elements, as was also reported for six species of Coryphantha by ^{Terrazas et al. (2016)}. The authors mentioned that wide-band tracheids have the ability to contract and expand depending on the availability of water (^{Gibson 1973}, ^{Mauseth 2004}) and the vessel elements will have the same ability since both have the same characteristics in the primary and secondary walls. The rays were not lignified and were arranged in series of 3 to 7, similar to the results of ^{Terrazas et al. (2016)}, where they observed rays in series of 2 to 5 for the six species of Coryphantha studied; the rays were mainly composed of erect cells.

The divisions of the epidermis originate the periderm; this had already been reported for other cacti species (^{Gibson & Nobel 1986}, ^{Terrazas et al. 2005}, ^{Evans & Dombrovskiy 2020}) and seems to be a conserved characteristic throughout the family relating to late development in the stems, especially of the Cactoideae subfamily (^{Terrazas & Arias 2002}). ^{Evans et al. (1994}, ²⁰⁰¹⁾ suggested that early development of the periderm is associated with the accumulation of epicuticular waxes in the epidermis, which stimulates cell division and in turn the production of phellogen. It is interesting that, of the three species studied here, only one of them does not have thick-walled lignified cells, C. cornifera, which could be a diagnostic characteristic. To support this assertion, further studies should analyze the periderm of a greater number of species of Coryphantha.

In conclusion, we recognized only a few unique characteristics in each analyzed species to discriminate species. The differences found here between C. cornifera and C. radians may contribute to recognize them as distinct entities. For C. cornifera, the hypodermis is parenchymatous and no thick-walled phellem cells occur in the periderm, whereas C. radians has collenchymatous hypodermis and C. clavata possesses distinctive prisms and rounded crystals in the collenchymatous hypodermis and abundant mucilage cells in the cortex. The epidermis and hypodermis were the tissues that presented the greatest variation between species. The type of crystals and their location could be of value and may contribute to the systematics of the genus. The xylem and phloem seem to be highly conserved since the three species showed the same characteristics already described for other members of Coryphantha, but this is not so for the periderm, considering that the phellem cells can have very thick and lignified walls.