Material and methods

Recollection. We obtained four leaf samples (including petiole and lamina) per individual of Ternstroemia lineata ssp. lineata and T. sylvatica in six localities of Mexican TMCF in 2013 and 2014 (three individuals per locality = 18 individuals = 72 leaves). We selected randomly well-developed trees with healthy foliage. We got these leaf samples from the middle part of the trees. The voucher specimens were deposited in the FCME Herbarium of the Facultad de Ciencias, UNAM (Table 1). We also removed four fully developed and healthy leaves (petiole and lamina) of herbarium specimens from three individuals from each of the populations of the chosen species (Table 1).

Foliar architecture. The leaves from herbaria and collected in the field were treated with 20 % sodium hydroxide (NaOH) at 60 ºC for 24 h, then washed with running water, followed by adding 50 % sodium hypochlorite (commercial chlorine, Clorox^®) for 3 h until a whitish color was obtained. The leaves were then washed and dehydrated with a series of ethanol (50, 70, and 96 %) every 24 h, and placed in a modified BB1/4 solution (^{Martínez-Cabrera et al. 2007}) for 4 to 8 days until the leaves got translucent. Immediately, they were placed in 96 % ethanol for 24 h to eliminate the remains of the clarifying solution and stained with safranin for 1 h, rinsed in ethanol (96 and 100 %) until the desired contrast obtained, and mounted with synthetic resin (^{Martínez-Cabrera et al. 2007}).

The width and length of the leaf, venation pattern, vein branching, and areole development were characterized for each individual. We quantified the number of sclereids per mm², the total number of epidermal cells, and stomata per mm² in 25 different fields. We calculated the stomatal index using the last two parameters (^{Salisbury 1928}). The foliar architecture was based on ^{Hickey (1979)} and ^{Ellis et al. (2009)}.

Foliar anatomy (petiole and lamina). For the anatomical study, we fixed the laminas and petioles from the field and herbaria in formaldehyde-glacial acetic acid-ethyl alcohol (FAA; ^{Ruzin 1999}) for 24 h. Next, we added 50 % ethyl alcohol. Then the samples were dehydrated in ascending concentrations of ter-butanol (TBA, 50 - 100 %) in an automatic tissue processor (Leica, TP1020) for 24 h and embedded in paraplast (melting point 60°C). Transverse and paradermal sections 12 to 16 μm thick were obtained with a rotary microtome (Leica, RM2125), stained with safranin-fast green, and mounted with synthetic resin (^{Johansen 1940}).

For each individual, we described the cuticle, epidermis, mesophyll, vascular tissue of the lamina and the epidermis, collenchyma, and vascular tissue of the midrib. In the case of the petiole, we described the morphology, epidermis, cortex, and vascular tissue. In addition, we made in each individual 25 measurements in the transverse sections of the leaves of wide of lamina, thickness of cuticle, length and wide of epidermal cells of the adaxial and abaxial surfaces, and length of palisade parenchyma. The anatomical descriptions of the foliar lamina followed the terminology of ^{Metcalfe & Chalk (1979)}, for the leaf surface, we followed ^{Wilkinson (1979)} and ^{Koch et al. (2009)}, and for petiole ^{Howard (1979)}.

All observations, microphotographs, and measurements were carried out with an Olympus COVER-018 stereomicroscope and an Olympus BX-51 light microscope adapted to an Image-Pro Plus version 7.1 image analyzer (^{Media Cybernetics 2006}). In addition, we carried out statistical procedures and analyses with the R statistical software (^{R Core Team 2020}).

Foliar micromorphology SEM. Only the material collected in the field was used to study the leaf surface in the abaxial and adaxial surfaces through SEM. The were fixed in the field with absolute ethyl alcohol-glacial acetic acid solution (Farmer technique, ^{Ruzin 1999}). We kept some others in cardboard boxes to avoid epicuticular wax modifications. The fixed material was washed with distilled water and cut into sections of the 4.0-5.0 mm foliar leaf. They were gradually dehydrated with ethyl alcohol (30 - 100 %). All the samples were placed in a critical point drier (Hitachi-S-2460N), and mounted in an aluminum sample holder using carbon-based glue and metalized with gold (Hitachi-S-2460N). Each surface was observed and photographed with the SEM (Hitachi-S-2460N SEM) of the Instituto de Biología, UNAM. We described from each photograph the shape of epidermal cells, the limits of anticlinal walls (relief and curvature), and the sculpture of outer periclinal walls (relief, convexity, and microrelief). We mounted 4.0-5.0 mm of the lamina from the material stored in the cardboard box in an aluminum sample holder to study the epicuticular wax, following the method described above. The description of micromorphology followed ^{Barthlott et al. (1998)} and ^{Koch et al. (2009)}.

Results

Quantitative characters are shown with their standard deviations in Table 2 and Table 3 and summarize the most distinctive characteristics of the studied species.

Foliar architecture. Leaves simple, alternate, petiolate, symmetrical, coriaceous, 75.02 mm long, 19.63 mm wide in Ternstroemia lineata ssp. lineata; and slightly coriaceous in T. sylvatica with 58.13 mm long and 19.45 mm wide. Oblanceolate to obovate in T. lineata ssp. lineata (Figure 1A) and narrow-elliptical to obovate shape in T. sylvatica (Figure 1B). Microphyllous in both species. Apex straight to round shape, with an acute angle (less than 90°) and a retuse ending in T. lineata ssp. lineata, while in T. sylvatica apex slightly acuminate to straight with an acute angle, and the termination non-retuse. Base symmetrical and decurrent with an acute angle in both species. The leaf with diminutively serrated margin in T. lineata ssp. lineata and entire margin in T. sylvatica. The petiole slightly winged, more evident near the leaf base due to the extension of the lamina and attached to the leaf margin in both species.

Figure 1 Venation pattern of Ternstroemia lineata subsp. lineata and T. sylvatica, cleared leaves. A, B. Festooned broquidodromous venation pattern, A. T. lineata subsp. lineata, B. T. sylvatica. C. Detail of the midrib, T. sylvatica. D. Areoles moderately developed and third, fourth, and fifth-order veins, T. lineata subsp. lineata. E, F. Veinlets and sclereids, E. T. lineata subsp. lineata, F. T. sylvatica. G, H. Marginal ultimate venation, G. T. lineata subsp. lineata, H. T. sylvatica. I, J. Detail of gland in margin, T. lineata subsp. lineata. Vm = midrib vein; Vs = secondary veins; Vi = intersecondary vein; Vc = fourth-order veins; Vq = fifth-order veins; Vt = tertiary veins. Es = sclereids; Ve = veinlets. Arrows = glandular scar. Scale bar is 1 cm in A, B; 300 µm in C-E, H; 100 µm in F, G, J; 50 µm in I. 

The primary venation pinnate, with a basal and primary vein following a straight course, moderately thick, 502.78 μm wide in both Ternstroemia species (Figure 1A-C). The secondary venation brochidodromous festooned, more developed in T. lineata ssp. lineata (Figure 1A, B). The secondary veins moderately thick; space between them irregular and decurrent, with an acute divergence angle. Intersecondary veins weak to robust, with approximately one per intercostal area (Figure 1A, B). Random reticulate pattern in tertiary veins, with a slightly sinuous course and obtuse angle to the primary vein (Figure 1D). The fourth-order venation irregular, reticulate, with freely branching fifth-order venation and areolae moderately developed, e.g., irregular in shape and more or less variable in size, predominating four to seven-sided areolae (Figure 1D, E). Most veinlets have two or more unequal branches (Figure 1H). Astrosclereids highly branched, usually large, evenly distributed in the mesophyll (Figure 1E, F), but well organized and continuous throughout the margin (Figure 1G, H). Sclereids/mm² 27.77 in T. lineata ssp. lineata and 20.68 sclereids/mm² in T. sylvatica, with significant differences between both species (Table 2). The ultimate marginal venation incomplete in both species (Figure 1G, H), and marginal, glandular, deciduous serrate glands with frequent scars in T. lineata ssp. lineata and infrequent in T. sylvatica (Figure 1H-J). The mucilage all over the leaf lamina of both species visible as brown spots.

Foliar lamina. In the surface view, the typical epidermal cells of the adaxial and abaxial epidermis tetragonal and polygonal in shape with irregular sides in T. lineata ssp. lineata and tetragonal in T. sylvatica (Figure 2A). These cells have straight or slightly curved anticlinal walls, rounded ends in both species. Anomocytic stomata surrounded by four to five ordinary epidermal cells (Figure 2A), randomly distributed on both surfaces (amphistomatic leaf). Both, length of guard cells (20.71 μm in T. lineata ssp. lineata; 18.58 μm in T. sylvatica) and stomata/mm² (10.76 in T. lineata ssp. lineata; 5.68 in T. sylvatica) with significant difference between both species (Table 2).

Figure 2 Leaf anatomy of Ternstroemia lineata subsp. lineata and T. sylvatica. A. Tetragonal epidermal cells and stomata, PS, T. lineata subsp. lineata. B. adaxial and C. abaxial epidermal cells, TS, T. lineata subsp. lineata. D. Adaxial epidermal cells and sclereids, TS, T. sylvatica. E. Bifacial mesophyll, TS, T. lineata subsp. lineata. F. Double layered palisade parenchyma and sclereid, TS, T. lineata subsp. lineata. G. Margin, TS, T. lineata subsp. lineata. H. Collateral vascular bundle in secondary vein, TS, T. sylvatica. I. Collateral vascular bundle in tertiary vein, TS, T. sylvatica. J. Midrib diagram; black = sclerenchyma, dots = phloem, parallel lines = xylem, cross lines = collenchyma, white = parenchyma. K. Midrib, TS, T. lineata subsp. lineata. L. Midvein abaxial epidermis, TS, T. sylvatica. Co = collenchyma; Ep = epidermis; Pe = palisade parenchyma; Pes = spongy parenchyma; Es = sclereids; S = stoma; X = xylem; F = phloem; fp = Perivascular fibers; PS = paradermal section; CS = transverse section. Scale bar is 20 µm in A-D, F, H, I; 50 µm in L; 100 µm in E, G; 300 µm in J, K. 

In the transverse-section, the lamina wide 281.80 μm in T. lineata ssp. lineata and 233.86 μm in T. sylvatica, with significant differences between both species (Table 2). On both surfaces, the cuticle smooth and thin, with 1.89 μm thickness in T. lineata ssp. lineata and 1.75 μm in T sylvatica on its adaxial surface, and no significant differences (Table 2).

The epidermis simple on both surfaces (Figure 2B-G). In T. lineata ssp. lineata, the typical cells rectangular with concave and thickened periclinal walls, 18.20 μm length, 6.63 μm wide on the adaxial surface. Epidermal cells on the abaxial surface identical but smaller in size (Figure 2C), with 10.04 μm length and 7.06 μm wide. In T. sylvatica, the adaxial epidermal cells rectangular, round-ended with concave-convex outer periclinal walls due to a more frequent huge cellulose deposit at their adaxial end and thin anticlinal walls (Figure 2D), 18.33 μm length, 9.60 μm wide, and with abaxial epidermal cells identical to those of T. lineata subsp. lineata with 12.49 μm length, 5.74 μm wide (Table 2).

The palisade parenchyma of elongated, tubular cells in T. lineata ssp. lineata with 57.81 μm length when a single stratum and 105.90 μm length in some individuals with two strata (Figure 2F) and of one stratum in T. sylvatica with 40.04 μm length, with differences in the palisade parenchyma length between species (Table 2). The spongy parenchyma is open, having wide, well-developed intercellular spaces, occupying more than two-thirds of the lamina thickness (Table 2, Figure 2C-E, G). Abundant astrosclereids (Figure 2D, E) in the mesophyll, generally long and irregularly distributed. The tannins occluding lumina of the palisade and spongy parenchyma. In addition, suberized cells (cork cells) in the margin corresponding to deciduous glands (Figure 2G). Vascular bundles collateral closed, surrounded by perivascular fibers thick-walled (Figure 2H, I).

Midrib. In the transverse-section, the midrib on the adaxial surface slightly concave and convex on the abaxial surface (Figure 2J, K). The cuticle very thin and smooth on both surfaces. Epidermis similar to the lamina, but on the abaxial surface, the typical cells rectangular and convex due to cellulose deposits on their outer periclinal wall. In Ternstroemia lineata ssp. lineata typical cells 20.37 μm length, and 12.21 μm wide and 16.21 μm length and 12.16 μm wide in T. sylvatica, with significant differences in the length of the abaxial epidermal cell of the midrib (Table 2). Palisade parenchyma in the adaxial surface similar to that of lamina but decreasing in length as approaching the central region, interrupted by three to four layers of angular collenchyma (Figure 2J, K). Towards the abaxial surface, eight layers of angular to lacunar collenchyma (Figure 2L). Astrosclereids towards the abaxial surface. Prominent collateral closed vascular bundle, arched in both species and surrounded by perivascular fibers (Figure 2K). In the xylem, vessels arranged in radial rows. Sieve tube elements, companion cells, and parenchyma in the phloem.

Petiole. In transverse-section, the petiole broadly obovate (Figure 3A-C) and slightly winged, evident near the base of the lamina in both species, 7.08 mm length and 1.16 mm wide in T. lineata ssp. lineata with an area of 8.21 mm² and 7.66 mm length and 0.96 mm wide in T. sylvatica, with an area of 7.35 mm² (Table 2). The cuticle and epidermis similar to the midrib (Figure 3E). Subepidermally, three to five layers of angular collenchyma in T. lineata ssp. lineata and one to three continuous layers of angular to lacunar collenchyma in T. sylvatica. Both species have more than 10 layers of isodiametric parenchyma cells, with wide intercellular spaces occupying two-thirds of the thickness of the petiole and more reduced intercellular spaces towards the vascular tissue (Figure 3D). Astrosclereids (Figure 3E) and mucilaginous cells common in both species.

Figure 3 Petiole anatomy of Ternstroemia lineata subsp. lineata and T. sylvatica, transverse sections. A. Petiole at the middle region, T. lineata subsp. lineata. B. Petiole diagram of the middle region; black = sclerenchyma, dots = phloem, parallel lines = xylem, crossed lines = collenchyma, white = parenchyma. C. Petiole at the distal region, T. sylvatica. D. Detail of the open parenchyma, with large intercellular spaces, T. sylvatica. E. Detail of the epidermal cells and sclereids, T. sylvatica. F. Details of the perivascular fibers, T. lineata subsp. lineata. G. Detail of vascular tissue in the distal region, T. sylvatica C = cortex; Ea = intercellular space; Ep = epidermis; Es = sclereids; F = phloem; fp = perivascular fibres; x = xylem. Bar is 300 µm in A-C; 100 µm in D; 20 µm in E, G; 50 µm F. 

The vascular tissue of the distal end is represented by closed, arch-shaped collateral vascular bundle (Figure 3C). The middle region is represented by three vascular bundles surrounded by perivascular fibers with the same arrangement, but the central bundle is arc-shaped and larger than the other two, which are circular and lateral (Figure 3A, B). Near the foliar lamina (proximal end), the petiole is slightly winged and with the same vascular arrangement as the middle region. The xylem and phloem show the same characteristics as the midrib (Figure 3F, G).

Micromorphology of the foliar surface. The epicuticular wax follows a scale-like deposition pattern in T. lineata ssp. lineata (Figure 4A) and a granular-like pattern in T. sylvatica (Figure 4B), in both cases uniformly distributed, being scarce on the abaxial surface of both species. The cuticle with reticulated, striated pattern (Figure 4C). Anomocytic stomata surrounded by grooves arranged in the radial direction of the guard cells, forming extended bands in the larger ones, being more pronounced in T. lineata ssp. lineata (Figure 4D, E). Dome-shaped scars of the deciduous glands in the margin, with a large depression in the center (Figure 4F, G). Protuberances in the petiole unique in T. lineata ssp. lineata (Figure 4H, I).

Figure 4 Epidermal micromorphology of T. lineata subsp. lineata and T. sylvatica leaves, SEM. A, B. Epicuticular wax pattern in the abaxial surface, A. T. lineata subsp. lineata, B. T. sylvatica. C. Detail of cuticular striation, T. sylvatica. D, E. Anomocytic stomata in the abaxial surface, D. T. lineata subsp. lineata, E. T. sylvatica. F, G. Detail of the scar left by the deciduous gland, F. T. lineata subsp. lineata, G. T. sylvatica. H. View of the striate cuticle of the petiole, T. lineata subsp. lineata. I. Detail of the protrusion of the petiole, T. lineata subsp. lineata. Bar is 50 µm in A, B; 100 µm in D, E, G, I; 300 µm in C; 400 µm in F, 500 µm in H. 

Discussion

A combination of diacritic morpho-anatomical characters was found that allow the distinction of the two species of Ternstroemia here studied. The diacritic characters are leaf shape including margin, shape of epidermal cells and anticlinals in surface view, abundance of deciduous glands, length of palisade parenchyma, number of stomata/sq. mm; type and number of collenchyma layers in petiole and petiolar protuberances.

Foliar architecture. Although all the specimens examined were collected in tropical montane cloud forests, the lamina is variable in shape between the individuals of the same population, regardless of the locality where they are found. For this reason, an oblanceolate to obovate lamina was described in T. lineata subsp. lineata and narrow-elliptical to obovate in T. sylvatica. According to ^{Weitzman et al. (2004)}, the most common secondary venation pattern of the Pentaphylacaceae is brochidodromous and sometimes reticulodromous. In T. lineata subsp. lineata and T. sylvatica, a festooned brochidodromous venation pattern was observed as described by ^{Hickey & Wolfe (1975)} for the leaves of Ternstroemia tepezapote Schltdl. & Cham. from Belize.

It was observed that the leaf texture depends on the number of sclereids/mm², not on the thickness of the cuticle and that the cuticle does not present significant differences between species. The leaves of T. lineata subsp. lineata presented a coriaceous texture and 27.77 sclereids/mm², while the leaves of T. sylvatica, had a slightly coriaceous texture and 20.68 sclereids/mm², showing significant statistical differences in the abundance of sclereids/mm²

^{Weitzman et al. (2004)} and ^{Stevens et al. (2004)} point out that the presence of a "theoid" glandular tooth is characteristic of Pentaphylacaceae and Theaceae; the glands are deciduous in the first and permanent in the second. However, in this study, the presence of a tooth was not observed in any species, as no vascular relationship was found with the projections of the leaf margin. ^{Royer et al. (2005)} and ^{Ellis et al. (2009)} point out that the teeth are projections of the leaf margin and have some association with the vascular system. Therefore, we described them as deciduous marginal glands, leaving frequent marks in T. lineata subsp. lineata and rare in T. sylvatica; however developmental studies are required to understand why these deciduous glands appear not show vascular tissue.

Anatomy of the lamina.^{Dickison (2000)} pointed out that the leaves of plants that develop in low-light conditions (as Ternstroemia species) show mesomorphic anatomical characters, e.g., thin cuticle, mesophyll with scarce palisade parenchyma, and abundant intercellular spaces in the spongy parenchyma. These traits agree with the characteristics observed in the two species of Ternstroemia studied inhabiting TMCF. In addition, the occurrence of sclereids in the mesophyll contributes to light transfer (^{Karabourniotis et al. 2021}).

A very thin cuticle was observed on both leaf surfaces, impossible to stain due to its physicochemical properties, contrary to the thick cuticle described by ^{Herat & Theobald (1977)} for the adaxial surface of T. emarginata (Gardner) Choisy and T. japonica (Thunb.) Thunb. by ^{Boeger & Wisniewski (2003)} and on both surfaces of the T. brasiliensis Cambess. leaf. The thickness of the cuticle is not remarkably different in both species studied. The stomata in both species studied are anomocytic, as cited by various authors (^{Keng 1962}, ^{Herat & Theobald 1977}, ^{Weitzman et al. 2004}, ^{Zhang & Zhuang 2004}). Some authors pointed out that in Ternstroemia, stomata are confined to one of the surfaces, generally to the abaxial surface. Our study showed stomata on both leaf surfaces (amphistomatic leaf) surrounded by four to five ordinary epidermal cells, as ^{Herat & Theobald (1977)} reported for some Ternstroemia species.

In the case of the epidermis, ^{Keng (1962)} pointed out that the size and shape of the epidermal cells and the nature of their anticlinal walls (straight or wavy) can be of value for the identification of genera and species of the family. In Ternstroemia lineata subsp. lineata and T. sylvatica an unilayered epidermis, was observed on both surfaces, as reported by other genus species (^{Keng 1962}, ^{Herat & Theobald 1977}, ^{Boeger & Wisniewski 2003}, ^{Zhang & Zhuang 2004}). Differences were recorded between the epidermal cells of the two Ternstroemia species in both surface and transverse-section views. We did not find statistically significant differences in the epidermal, adaxial and abaxial cells of both Ternstroemia species studied. Other studies in Ternstroemia reported some similarities and differences regarding the characteristics described for the epidermis. For example, ^{Boeger & Wisniewski (2003)} described adaxial epidermal cells of greater size than the abaxial surface, but polygonal in shape, thickened walls, anticlinals wavy, and the outer periclinal convex in T. brasiliensis. ^{Herat & Theobald (1977)} described in T. emarginata square adaxial cells with straight walls, and for T. japonica smaller epidermal cells also with straight walls. ^{Weitzman et al. (2004)} reported the occurrence of hypodermal layers, however they were nor observed in the two species studied.

In Ternstroemia lineata subsp. lineata and T. sylvatica, we found a bifacial mesophyll, differentiated into palisade and spongy parenchyma, similar to other species of the genus (^{Herat & Theobald 1977}, ^{Cao 2000}, ^{Boeger & Wisniewski 2003}). ^{Weitzman et al. (2004)} pointed out that the palisade parenchyma consists of a variable number of layers in the members of the family. On the other hand, ^{Metcalfe & Chalk (1972)} mentioned that the number of layers of palisade parenchyma is of some value for the identification of genera and species of the family. ^{Herat & Theobald (1977)} considered that the number of layers of palisade parenchyma helps differentiate genera, reporting one to four layers in different species of Ternstroemia. ^{Cao (2000)} reported a palisade parenchyma stratum in T. aneura Miq. and ^{Boeger & Wisniewski (2003)} two strata in T. brasiliensis as do occur in T. lineata subsp. lineata. Spongy parenchyma is open, with abundant large intercellular spaces, occupying more than two-thirds of the thickness of the lamina, as described by ^{Herat & Theobald (1977)} for T. emarginata and T. japonica.

We found, as earlier authors (^{Keng 1962}, ^{Metcalfe & Chalk 1972}, ^{Weitzman et al. 2004}, ^{Stevens 2021}), the occurrence of astrosclereids. In addition, we found that sclereids are concentrated towards the margin or scattered through the lamina but not densely grouped around the veins, as ^{Stevens (2021)} mentioned for members of the Ternstroemieae subfamily. As pointed out by ^{Karabourniotis et al. (2021)}, the sclereids may contribute to improving photosynthetic performance and defense protection. ^{Herat & Theobald (1977)} observed, as in the two studied Mexican Ternstroemia, that the size of the sclereids varies within the same leaf. ^{Keng (1962)} suggested that sclereids formed during the early leaf development are small, unlike those formed later; he pointed out that sclereids are of value for delimiting taxa at the generic level. We found particular similarities in T. lineata subsp. lineata and T. sylvatica with other Ternstroemia species worldwide. Other authors such as ^{Rao (1951a}, ^b) described the sclereids in T. japonica, ^{Herat & Theobald (1977)} in T. emarginata and T. japonica, ^{Cao (2000)} in T. aneura Miq., and ^{Boeger & Wisniewski (2003)} in T. brasiliensis. Our observations agree with ^{De Roon (1966)} in the presence of highly branched sclereids (astrosclereids) with pointed ends.

^{Azcárraga-Rossette (2010)} mentioned the usual presence in the adaxial epidermis and the mesophyll of pigments that help capture light. We observed the presence of tannins in both species studied, occluding the lumen of the palisade and spongy parenchyma cells, less frequent in the epidermal cells. Tannins protect the protoplast against desiccation and predators; they are essential in the starch metabolism, formation and transport of sugars, as antioxidants, and as colloids maintaining the homogeneity of the cytoplasm (^{Bonfil-Campos 2010}). The presence of tannins was reported in different species of Ternstroemia (^{Metcalfe & Chalk 1972}, ^{Herat & Theobald 1977}, ^{Boeger & Wisniewski 2003}, ^{Stevens 2021}). ^{Metcalfe & Chalk (1972)} reported cork cells and tannins on the abaxial surface in Ternstroemia. In both species, cork cells were observed, corresponding to the deciduous glands leaving frequent scars in the margin of T. lineata subsp. lineata and rare in T. sylvatica.

Mucilage-bearing idioblasts were found among the mesophyll cells of both species; the presence of mucilage in the family was reported earlier by various authors (^{Keng 1962}, ^{Metcalfe & Chalk 1972}, ^{Herat & Theobald 1977}, ^{Weitzman et al. 2004}, ^{Stevens 2021}). However, we did not find solitary or grouped crystals in the mesophyll despite using polarized light, as mentioned by ^{Metcalfe & Chalk (1972)} for the family. The vascular tissue of T. lineata subsp. lineata and T. sylvatica coincide with the description of ^{Metcalfe & Chalk (1972)} and ^{Herat & Theobald (1977)} for some species of Ternstroemia. In both species, perivascular fibers and astrosclereids are clearly related with a “coriaceous” leaf texture.

There are few studies concerning the midrib of Ternstroemia subsp., so this study is the first to detail the tissues of this region of the lamina. ^{Herat & Theobald (1977)} reported collateral vascular bundles in the midrib of T. emarginata and T. japonica with a sclerenchyma sheath. These authors pointed out that the shape of the vascular bundles varies within the genus, but they are consistent within the species. They found in T. emarginata a U-shaped vascular bundle, while in T. japonica, it has a sunken U-shaped one. In T. lineata subsp. lineata and T. sylvatica, the vascular tissue of the midrib, is represented by a prominent closed, arch-shaped bundle in both species, as in T. emarginata and T. japonica, surrounded by perivascular fibers. Additionally, Herat & Theobald (1977) observed that xylem vessels are interrupted by rays with cells containing tannins. In addition, they recorded scattered tanniferous parenchyma cells in the phloem of some species. We did not observe tannins either in the xylem and phloem of the studied species.

Petiole anatomy. There are few studies on the petiole of Ternstroemia subsp. ^{Herat & Theobald (1977)} reported that in T. emarginata and T. japonica, a relatively thick cuticle and small epidermal cells in the petiole compared to the foliar lamina. In the case of T. lineata subsp. lineata and T. sylvatica, the cuticle and epidermis are similar to the midrib. ^{Esau (1985)} pointed out that the collenchyma or sclerenchyma are the supporting tissues of the petiole, arranged similarly to that of the stem. ^{Herat & Theobald (1977)} observed parenchyma exclusively in the cortex of the petiole in Ternstroemia. In our study, we found angular, 3-5-layered collenchyma in T. lineata subsp. lineata and angular to lacunar 1-3-layered collenchyma in T. sylvatica. ^{Rao (1951a)} reported that in other Ternstroemia species, there are wide intercellular spaces and astrosclereid in the petiole, as do occur in the species here studied. We did not find crystals in the petiole, as ^{Metcalfe & Chalk (1972)} recorded for some family species.

^{Howard (1979)} suggested that the anatomy of the petiole may be helpful as a taxonomic character. In this study, we found differences in the vascular system between both species at different petiole levels (distal end, middle region, and proximal end). ^{Metcalfe & Chalk (1972)} commented that “in transverse-section, the petiole of several genera of the Pentaphylacaceae family is characterized by a vascular bundle, but in some cases, small lateral bundles are present”. Our study observed that closer to the lamina (proximal end), the petiole is slightly winged and has three vascular bundles. There is a prominent vascular bundle, with the same arrangement as the midrib, surrounded by perivascular fibers and two circular lateral bundles. This arrangement was also recorded in the middle region of the petiole. At the distal end of the petiole, we only observed one vascular bundle. The findings of ^{Keng (1962)}, ^{Metcalfe & Chalk (1972)}, and ^{Weitzman et al. (2004)} coincide with our study, arguing that the distal end of the petiole of some members of Pentaphylacaceae exhibits an arch, U, or V vascular bundle surrounded by fibers. ^{Weitzman et al. (2004)} pointed out that the lateral bundles of the leaf diverge from the end of the trace, and there are from one to five lateral bundles in the petiole of Pentaphylacaceae. ^{Herat & Theobald (1977)} pointed out that in Ternstroemia emarginata and T. japonica, there is a U-shaped closed collateral vascular bundle surrounded by perivascular fibers.

Micromorphology of the foliar surface. We undertook micromorphology employing scanning electron microscopy (SEM). By this method, we found in T. sylvatica and T. lineata subsp. lineata characters not observed through conventional microscopy. For the first time, we observed anomocytic stomata on the foliar lamina of T. lineata subsp. lineata and T. sylvatica, with a clear pattern of stomatal dimorphism in the same lamina. Stomata have striae arranged radially to the guard cells, forming extended bands in the larger stomata, more visible in T. lineata subsp. lineata, similar to those described by ^{Wilkinson (1979)} for Nyssa sylvatica Marshall (Nyssaceae). We observed in T. lineata subsp. lineata and T. sylvatica, a cuticle with a reticulated-striated pattern with thin striae and open valleys. In the case of the petiole, the cuticle had a reticulated pattern of thick striations and very closed valleys.

Epicuticular wax is essential for the functionality and structuring of the cuticle (^{Koch et al. 2009}). Therefore, wax morphology is mainly used as an additional diagnostic character sensu^{Wilkinson (1979)} for taxonomists. The epicuticular wax sensu^{Barthlott et al. (1998)} has a short-tubular deposition pattern in T. lineata subsp. lineata and a granular-like in T. sylvatica, uniform and sparsely distributed on the abaxial surface of the lamina of both species. In some parts of the leaf margin, we observed dome-shaped scars of the deciduous glands, with a large depression in the center; these depressions correspond to cork regions in the transversal sections. In the case of the petiole, we found sparse protrusions only in T. lineata subsp. lineata. These structures are not visible in the petiole cross-sections.

Few studies about the anatomy and foliar architecture of Ternstroemia exist worldwide. Our studies on the foliar architecture and anatomy on the leaves of T. lineata subsp. lineata and T sylvatica reveal similarities and differences between them and other species of Ternstroemia described elsewhere, which may contribute to the separation of species. Furthermore, the complete micromorphology of the Ternstroemia leaf is described for the first time and can contribute to the resolution of taxonomic problems.

We found in both species a glandular margin composed of deciduous glands, a stomatal dimorphism pattern in the size of the guard cells on both sides of the lamina, and adaxial epidermal cells with concave-convex periclinal walls due to a large cellulose deposit, characteristics observed for the first time in these taxa.

Several conclusions are rejected for the genus because we found stomata on both leaf surfaces (amphistomatic leaves). We did not find crystals and hypodermis in the mesophyll, nor was the presence of tannins in the xylem or mucilage in the epidermis. We conclude that the leaf texture (described as leathery for most species) depends on the number of sclereids/mm² and the abundance of perivascular fibers. These plants have been described as having thick cuticles, but the reality is that they have very thin cuticles.

Ternstroemia sylvatica and T. lineata subsp. lineata preferably inhabits the Mexican tropical montane cloud forest. The genus is highly heterogeneous, e.g., the morphology of the leaf varies between individuals of the same population regardless of the locality where it is found. The foliar anatomy of the studied species shares some characteristics with other plants from humid environments, such as thin cuticle, mesophyll with sparse parenchyma palisade and abundant intercellular spaces in the spongy parenchyma, and pigments (tannins) for light capture and protection. Several Ternstroemia species developed specific mechanisms to adapt to humidity and shadow-fog conditions. These features are shared with other TMCF diagnostic/characteristic tree species, such as certain species of Clusiaceae Lindl. (Clusia L.), Staphyleaceae Martinov (Turpinia Vent.), some members of Ericaceae Juss. (Vaccinium L.) and Magnoliaceae Juss. (Magnolia L.), Styracaceae DC. & Spreng. (Styrax L.), Symplocaceae Desf. (Symplocos Jacq.), other Pentaphylacaceae (Cleyera Adans., Freziera Sw. ex Willd., Symplococarpon Airy Shaw), allowing them to flourish in these peculiar environments (^{Schadel & Dickison 1979}, ^{Rodríguez-Ramírez et al. 2021}).

This study represents the first attempt at the anatomy, foliar architecture, and micromorphology of Pentaphylacaceae in Mexico. It intends to contribute basic information on Mexican tropical montane cloud forest diagnostic taxa. This information is also helpful for taxonomic, ecological, and identity purposes.