Background and aims – Calcium oxalate crystals are the most common biominerals in plants and have a wide variety of forms, such as styloids, druses, raphides, prisms, and crystal sand. The shape, position, and number of crystals in plant tissues can have taxonomic value. For Rubiaceae, one of the most diverse angiosperm families, the presence of crystals may be taxonomically informative. In Mexico, one of the most diverse Rubiaceae genera is Randia, which occurs in arid, tropical, temperate, and coastal dune vegetation. The aim of this study is to explore the taxonomic value of calcium oxalate crystals in Randia. Furthermore, we aim to ascertain whether these crystals respond to the environmental conditions in which the plant grows.

Material and methods – Ten Mexican Randia species were selected, three of which included individuals from different vegetation types. For each individual, three mature leaves obtained from herbarium samples from MEXU and FESC were selected for SEM processing and leaf clearing.

Key results – Druses in the spongy parenchyma and palisade parenchyma were constant in all the species studied. The druses in the palisade parenchyma were always larger than those in the spongy parenchyma. In addition to druses in the intracellular parenchyma, R. tomatillo presented extracellular prisms in the epidermis as well as extracellular prisms and aggregate prisms in the mesophyll, which is a rare characteristic in Rubiaceae.

Conclusion – The constant presence of druses in the mesophyll could serve as a possible characteristic to diagnose the genus Randia, whereas the variable presence of druse crystals in the epidermis and veins could be related to environmental factors. The prisms in R. tomatillo may be related to its occurrence in the harsh environment of coastal dunes.

druses, extracellular prisms, leaves, Randia, palisade parenchyma, spongy parenchyma

In plants, three typical forms of biominerals occur: calcium carbonate, amorphous silica, and calcium oxalate (Franceschi and Horner 1980; Arnott 1982; Franceschi and Nakata 2005; Bauer et al. 2011); the last is the most common (McNair 1932; Franceschi and Nakata 2005). Raphides, styloids, druses, prisms, and crystal sand are part of the great morphological diversity of calcium oxalate crystals (Franceschi and Nakata 2005; Raman et al. 2014). The shape, type, and number of these crystals, as well as their location in the plant body, have taxonomic significance (Franceschi and Horner 1980; Prychid and Rudall 1999; Hartl et al. 2003; Prychid et al. 2003; Leszczuk et al. 2014). Crystal formation can be a product of genes (Franceschi and Nakata 2005; Kausch and Horner 1982), the environment (Faheed et al. 2013), or a combination of both (Franceschi and Nakata 2005).

Calcium oxalate crystals have been identified in approximately 215 plant families, including Rubiaceae (McNair 1932), which is the fourth-largest flowering plant family with more than 14,000 species (Razafimandimbison and Rydin 2024). For Rubiaceae, the systematic value of crystals has been evaluated at different taxonomic levels; for example, the presence of raphides is diagnostic for the Rubioideae subfamily in some classification schemes (Verdcourt 1958; Bremekamp 1966), at the generic level, as in members of the tribe Chiococceae (Aiello 1979), and at the species level, e.g. Simira Aubl. (Moraes et al. 2009) or Cephalanthus L. (Romero et al. 2019).

In Mexico, Rubiaceae is one of the most diverse plant families, with 111 genera and 724 species, among which Randia L. stands out as the second most diverse genus, with 64 species (Torres-Montúfar and Torres-Díaz 2022). It comprises trees, shrubs, and lianas. It occurs in arid regions and grows in deciduous and evergreen vegetation from sea level to 3300 m in elevation (Borhidi 2012). The neotropical genus Randia is a member of the tribe Gardenieae and comprises approximately 90 species (Gustafsson 1998, 2000; Gustafsson and Persson 2002; Judkevich et al. 2020). The morphology of the genus is diverse with respect to habit (trees, shrubs, and lianas), leaf shape, and floral sexuality (mainly dioecious, but with some bisexual species). It is characterized by the presence of brachyblasts bearing clustered stipules and leaves, pollen grains released in tetrads, and fleshy fruits containing numerous discoidal seeds embedded in dark pulp (Gustafsson 2000; Borhidi 2019; Lorence 2012; Judkevich et al. 2015, 2016, 2020). Randia is considered a taxonomically problematic group (Borges et al. 2021). The aim of our study is to identify crystal types in Randia and explore their locations in leaf tissue as well as their associations with different vegetation types.

This study was conducted on 10 taxa of Randia from different vegetation types. For some species, several samples from different vegetation types were collected. Three mature leaves per individual were removed from herbarium samples deposited at FESC and MEXU (acronyms follow Thiers 2025). A list of the species studied and voucher information is given in Table 1.

The results detailed below are summarized in Table 2.

Crystal types and distribution

All the sampled species of Randia had intracellular druse crystals in the leaf mesophyll, notably in the spongy parenchyma and palisade parenchyma (Fig. 1A, E). Randia tomatillo also presented solitary bipyramidal prisms extracellularly (over or across the cell wall) and aggregated bipyramidal prisms in the epidermis and mesophyll (Fig. 1B–D). The size of these solitary bipyramidal prisms was 5 to 10 µm.

Figure 1. 

Presence of crystals in Randia. A. Intracellular druse in Randia capitata (artificially coloured in yellow, from R. Medina 5703, MEXU). B–E. Randia tomatillo (from L. Gonzalez-Quintero 4534, MEXU). B. Bipyramidal prisms (artificially coloured in purple) and extracellular prism (arrow). C. Prisms in the adaxial epidermis (artificially coloured in purple). D. Bipyramidal aggregate prisms (curly brackets) and bipyramidal prism (arrow). E. Druse in palisade parenchyma. e = epidermis, pp = palisade parenchyma.

Druses in the mesophyll

In all the samples studied for all vegetation types, druses were observed in the palisade and spongy parenchyma. In general, the druses present in palisade parenchyma were larger than those present in spongy parenchyma. In the spongy parenchyma, druses were scarce and varied in size among individuals from different localities and vegetation types (Fig. 2C–E).

Figure 2. 

Crystals in the mesophyll and midveins of leaves. A. Druse in the midvein of Randia hidalgensis (artificially coloured in yellow, from M. Aguilar 9, MEXU). B. Druse in the midvein of R. capitata (arrow, from R. Medina 5703, MEXU). C. Druse in mesophyll of R. capitata (artificially coloured in yellow, from A. Bonfil 275, FESC). D. Druse in mesophyll of R. xalapensis (arrows, from A. Torres-Montufar 664, MEXU). E. Druses in mesophyll of R. thurberi (arrows, from L. Albarran 7, FESC). e = epidermis, p = parenchyma, pp = palisade parenchyma, sp = spongy parenchyma, x = xylem.

Surface distribution patterns

Three types of crystal distribution were recorded: (1) druses in the mesophyll but not in the midvein or the lateral veins (R. thurberi, R. tomatillo) (Fig. 3A), (2) druses in the mesophyll and lateral veins but absent in the midvein (R. capitata) (Fig. 3B), and (3) druses in the mesophyll, the midvein, and lateral veins (R. hidalgensis) (Fig. 3C).

Figure 3. 

Crystal distribution patterns in leaves of Randia. A. Druses in the mesophyll in R. thurberi (from L. Albarran 7, FESC). B. Druses in the mesophyll and the lateral veins in R. capitata (from R. Medina 5703, MEXU). C. Druses in the mesophyll, the midvein, and the lateral veins in R. hidalgensis (from M. Aguilar 9, MEXU). mv = midvein, lv = lateral vein, * = druses. Scale bars = 200 µm.

Druses in Randia leaves have already been described in earlier general observations (Metcalfe and Chalk 1950). The presence of druses in the spongy parenchyma was consistent across all species of Randia studied here, regardless of the vegetation type from which the individuals were collected. This aligns with what has been reported by Martínez-Cabrera et al. (2009) for R. aculeata and R. tetracantha (Cav.) DC., and by Judkevich et al. (2015, 2020) for R. brevituba Judkevich & R.M.Salas and R. heteromera Judkevich & R.M.Salas from South America, where druses were observed in the same tissue. Given the significance of crystals at various taxonomic levels in Rubiaceae (e.g. Bremekamp 1966; Aiello 1979; Moraes et al. 2009), this result could be a taxonomically important characteristic for the genus Randia, pending further studies in related genera.

All the species studied here and in other Randia species occurring in Argentina presented druses in the palisade parenchyma, except in Randia micracantha (Lillo) Bacigalupo (Judkevich et al. 2015). It should be noted, though, that we observed in this study that crystals were not maintained in leaf tissue embedded in paraffin or prepared for SEM observations. However, the crystals remain present in the cleared cross sections of the leaf blades. This phenomenon has been reported in the study by He and Kirilak (2014) in which the technique caused crystal dislodging or scattering.

Larger druses in the palisade parenchyma and smaller druses in the spongy parenchyma were also documented by Judkevich et al. (2015, 2020) in Randia calycina Cham., Randia ferox (Cham. & Schltdl.) DC., and Randia heteromera. Larger druses in photosynthetic parenchyma have been linked to light-reflective properties, which may help disperse light to surrounding chloroplasts (Kuo-Huang et al. 2007; Horner 2012). Additionally, they can channel light toward the parenchyma, enhancing photosynthetic efficiency (Pierantoni et al. 2017).

Druses in the epidermis (R. capitata) and vascular bundles (R. aculeata, R. capitata, R. hidalgensis, R. retroflexa, and R. thurberi) were not a consistent finding. They may or may not be present in individuals from the same type of vegetation but different localities (R. thurberi with druses in dry forest LA7, but not in dry forest FM1940). This difference could be due to calcium availability in the soil (Webb 1999; Faheed et al. 2013). The calcium that forms the crystals is obtained from the environment (Franceschi and Nakata 2005). The synthesis of oxalic acid and the formation of crystals takes place in the cell. Alternatively, the presence of druses may be a response to herbivory (Molano-Flores 2001).

The size of the crystals can vary depending on the type of cell in which they form and the amount of calcium available in the environment (Franceschi and Nakata 2005). In this study, no correlation was observed between the type of vegetation from which individuals originated and the size and number of crystals. However, in controlled greenhouse studies, a positive correlation was recorded between higher concentrations of calcium in the substrate and an increase in the number and size of crystals (Faheed et al. 2013).

Certain studies report a possible correlation between druse abundance and ambient conditions. For Schinus terebinthifolia Raddi (Anacardiaceae), De Souza et al. (2022) reported a higher number of druses in leaves of plants from restinga forest (a coastal ecosystem in Brazil) in contrast with leaves of plants growing in savannas. The only study that has performed a correlation analysis is that of Gómez-Espinoza et al. (2021); for Colobanthus quitensis (Kunth) Bartl. (Caryophyllaceae), they found that crystals in leaves were more abundant and larger in plants growing in mountainous environments with low temperatures and abundant rainfall than in plants inhabiting coastal areas. In contrast, some studies suggest the absence of a correlation between ambient conditions and crystals. Palchetti et al. (2014) observed the presence of leaf crystals in 13 species of Capsicum L. (Solanaceae) growing in both humid and dry regions of South America; only two species from humid regions lacked leaf crystals.

Extracellular prismatic crystals, a rare type of crystal among most angiosperms (Hartl et al. 2003, 2007), were observed in R. tomatillo. However, prior to this study, no records of extracellular crystals had been reported for Rubiaceae. These crystals may be present because R. tomatillo grows in coastal dunes. Due to high concentrations of windborne salt, wind speed, erosion, drought, salt spray, and nutrient scarcity (Alcaraz Ariza and Garre Belmonte 1985; Martínez and Moreno-Casasola 1996), plants that grow in these regions must exhibit physiological adaptations common to coastal halophytes, such as an increase in the size of water-storage tissues, cell wall elasticity, and a reduction in number of stomata (Alcaraz Ariza and Garre Belmonte 1985). Small crystals have been described in the epidermis of some species from various plant families distributed in coastal dunes; however, the presence of these crystals has not been associated with this type of vegetation and further studies are needed (Pérez-Cuadra and Cambi 2016).

This study highlights the importance and potential use of crystals in Rubiaceae taxonomy. The prismatic crystals observed in R. tomatillo have not previously been reported for Rubiaceae. The location and size of the druses is variable and, therefore, may be dependent on environmental factors such as the soil. This study can be a starting point for future research to investigate correlations of druses in Randia.

The authors thank Berenit Mendoza Garfias (IBUNAM) for her assistance with MEB photographs at IBUNAM. Special thanks to the herbarium curators and associate personal from MEXU and FESC. This research was supported by the Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica-UNAM: PAPIIT IA205224 and IA202622. We are grateful to the anonymous reviewers.