Succulents are popular ornamental plants that have the capability to live in arid environments. Approximately 12,500 species from 70 flowering plant families have been registered as succulents and are related to the families Crassulaceae, Agavaceae, and Asphodelaceae (Males 2017).
Historically, the taxonomy of the Crassulaceae family and its genera has exhibited complexity and reorganization because of the homoplasia of their morphological characteristics. For example, Pachyphytum has been suggested for inclusion in Echeveria (Thiede and Eggli 2007). Oliveranthus and Urbinia genera are sometimes considered part of the Echeveria genus (Walther 1972;Kimnach 2003) although they have been separated from Echeveria in previous studies (Britton and Rose 1903).
Most of the species in this genus are poly- or para-phyletic in which they are clustered with representative species from Graptopetalum, Sedum, Pachysedum, and Thompsonella (Carrillo-Reyes et al. 2008) and have been named the “Echeveria group” (Thiede and Eggli 2007). Despite considerable effort, distinguishing plants within the genera of the Crassulaceae family is still challenging because of morphological similarities such as the differentiation of Echeveria in the “Acre clade,” which is often confused with Pachyphytum (De La Cruz-Lopez et al. 2019), Sedum (Nikulin et al. 2016), and Graptopetalum (Acevedo-Rosas et al. 2004).
Within the Crassulaceae family, the Echeveria genus has a high level of horticultural interest, both for its ecological benefits and economic value because of its rosette appearance (Borys and Leszcyńska-Borys 2013). This genus is native to southern Mexico and comprises approximately 140 species that grow in diverse habitats and have a high level of adaptability. This means that they can be cultivated as both indoor and outdoor plants for aesthetic purposes (Kimnach 2003;Males 2017). Nowadays, succulents, including Echeveria cultivars, have been used extensively for green roofing, urban green walls, natural air purifiers, and interior plant decors (Grace 2019;Wolf 2015). Because of this, research on cultivar development and the identification of taxonomic relationships have progressed in recent years (Cabahug et al. 2020;Cabahug et al. 2022).
Despite their high degree of endemism and wide distribution, natural hybrids are not easily produced because of their different flowering times (De La Cruz-Lopez et al. 2019). Hybridization of Echeveria requires patience and practice but has been a hobby among plant collectors and breeders aiming to develop new cultivars or mutants with unique morphological traits. New cultivars demand higher prices, which has increased the production of new plants with novel traits. Therefore, the number of cultivars has continued to increase.
With the increasing number of Echeveria cultivars, studies on Echeveria related to phenotypic analysis and anatomy to improve ornamental management and identification are highly relevant. There have been relatively few studies, many of which have had contradictory results, and further studies are required to fully understand this genus. Therefore, this study aimed to identify and summarize the available and published research on leaf anatomy that has been conducted on the Echeveria genus.
1. Background of Succulents
1.1 Characterization of succulent plants
The term “succulence” refers to the substantial amount of usable water in living cells in semi-arid climates or in any plant organ that can specialize in water-storing tissues (Rowley 1977). Leaf and stem succulence are the most common genotypes, whereas root, tuber, and bulb genotypes are present in some plants (Eggli and Nyffeler 2009;Griffiths and Males 2017). Plants with swollen morphological traits with water tissues from large mesophyll cells and vacuoles are often associated with crassulacean acid metabolism (CAM) plants (Griffiths and Males 2017;Winter and Smith 1996).
Abdel-Raouf (2012) reported that succulence could be achieved by either all-cell or storage succulence. All-cell succulents possess undifferentiated tissues composed of parenchyma cells that store water and perform photosynthesis (von Willert et al. 1992). In contrast, storage succulence retains water in achlorophyllous hydrenchyma cells and reallocates it to photosynthetic chlorenchyma cells when water is scarce (Ihlenfeldt 1985). The arrangement of hydrenchyma, that is, the water storage cells, and chlorenchyma cells, namely photosynthetic cells, varies among species. In earlier reports by Nobel (1988), succulence was said to primarily comprise the thin parenchymal cell wall, which allows the cells to swell and easily use water for other tissues that are required for metabolic activities.
Often originating in arid environments, succulents are subjected to limited water supplies. Therefore, to ensure survival, succulents need to a) store water temporarily in living tissues, b) ensure that the water is used independently, and c) maintain their metabolic activities to survive when the external water supply is limited (Eggli and Nyffeler 2009;von Willert et al. 1992). Larcher (2001) explained that succulents possibly react in either “attenuation,” which weakens the effects of stress factors, or “tolerance,” which increases the resistance of desiccation tolerance. Therefore, succulence is assumed to be fundamental at the cellular level and is not only present in morphological features.
1.2 Controversy in the phylogenetic relationship of Echeveria species
In 1828, New World species that possessed a lateral inflorescence were separated from the Cotyledon genus and moved into the Echeveria genus. Since then, several changes have been made. Echeveria belongs to the Echeverioideceae subfamily of the Crassulaceae (Berger 1930) and has been subdivided into 17 infrageneric series. Plants in this genus have rosette formations, flowers with expanded erect sepals, and brightly colored succulent petals which are connate-shaped at the base (Kimnach 2003;Walther 1972).
With the increasing number of cultivars and the discovery of new plants, only using morphological characteristics to determine the relationships in this genus has proven challenging. For example, it is still being debated whether E. heterosepala Rose should be placed under Pachyphytum or in Echeveria (Walther 1931). Similarly, some genera including Oliveranthus and Urbinia have been separated from Echeveria (Britton and Rose 1903), while Thompsonella was included in a section of Echeveria and was then placed in a separate genus (Walther 1972). Some challenges have arisen in distinguishing this genus from other look-a-like species, where Echeveria species have been misidentified as Crassula (Jones 2011), Pachyphytum (Vázquez-Cotero et al. 2017), Sedum (Nikulin et al. 2016), GraptopetalumAcevedo-Rosas et al. 2004), and Aeonium (Griffiths and Males 2017).
Modern technological tools have been developed to address these issues. Several DNA barcoding analyses have been conducted to investigate the phylogenetic relationship of Echeveria within the Acre clade including Echeveria, Graptopetalum, and Thompsonella (van Ham and ‘t Hart 1998). This has shown that some controversial members from the Acre clade are monophyletic in Pachyphytum and Sedum sect. Pachysedum. Based on this report, these species should be in a separate clade, despite being initially within the Echeveria group (Carrillo-Reyes et al. 2008). In contrast, De La Cruz-Lopez et al. (2019) reported that Echeveria is non-monophyletic.
Phylogenetic relationships have repeatedly changed and can remain uncertain, predominantly because of the lack of definitive molecular information and the limited collection of samples. In addition to these barriers, many other samples from Asia and other countries need to be further examined, which will help improve the phylogenetic framework and understanding of the relationships among its species, varieties, and cultivars (De La Cruz-López et al. 2019). The use of traditional taxonomical tools including plant morphology, anatomical evaluation, and growth characteristics combined with modern genetic and cytogenetic methods can provide sufficient information to resolve disputes around these phylogenetic issues.
2. Leaf Anatomical Analysis
To provide the vital information needed to further examine the taxonomy of Echeveria, morphological evaluation studies, together with the anatomical analysis of cells, tissues, and other plant organ structures, provide systematic characteristics for phylogenetic analyses (Simpson 2019). Leaves of plants within the family Crassulaceae are characterized by a thick cuticle and unique anatomical characteristics. To date, standard histological techniques have not been applied, which has limited studies on these species (Rost 1969). However, new techniques and solutions have been developed in recent years.
Qualitative and quantitative anatomical studies on leaves and stems have been successfully performed in Kalanchoe (Moreira et al. 2012), Crassula (Jones 2011), loe (Silva et al. 2014), and Sedum (Ardelean et al. 2009) which has provided information for comparing and differentiating between individual plants within the same genus or species or those thriving in different environments. The results of these anatomical analyses have also helped in understanding the response of plants to the environment by determining the internal structure of the leaf and their physiological adaptation when exposed to xerophytic habitats (Ardelean et al. 2009;Chernetskyy and Weryszko-Chmielewska 2008;Jones 2011;Karwowska et al. 2015). Leaf anatomical analyses usually highlight the characteristics of fundamental tissues, namely the epidermis and hypodermis, stomata, vascular bundles, and other phenolic compounds.
2.1 Fundamental tissues
Plants in arid habitats are commonly reported to have trichomes, papillae, and epicuticular waxes, which are considered xeromorphic traits, as part of their adaptation strategies for harsh environments (Eggli and Nyffeler 2009;Griffiths and Males 2017). Except for trichomes, these xeromorphic features were found in all E. aff. gigantea accessions with thick amphiprotic leaves in a study by Sandoval-Zapotitla et al. (2019). They also reported thickening of the periclinal wall on epidermal cells, slightly sunken stomata, a multilayered hypodermis with thickened cell walls, aquifer mesophyll, and abundant tannin distribution, corresponding to 35% robustness in all plant accessions. These traits and fundamental tissues are the main contributors to the adaptation of ornamental succulents to various light stresses (Mott and Michaelson 1991).
In terms of physiological function, the epidermis is the outermost wall that protects plants from exogenous pathogens and controls gas exchange, water, and nutrients (Simpson 2019) (Fig. 1). In all seven E. aff. gigantea accessions in the study by Sandoval-Zapotitla et al. (2019), the cross-sections of the epidermis had a uniseriate layer, which is a typical feature of CAM plants.
The epidermal layer increases the potential for cell size expansion and water-use efficiency (Chernetskyy and Weryszko-Chmielewska 2008;Jones 2011). In the same study, the differences in cell size in the middle and basal regions were similar. The size of the epidermal cells can be interpreted as the evapotranspiration area opening to reduce and control the temperature on both surfaces.
Leaf sections from seven Echeveria aff. gigantea accessions have shown that the habitat influenced the size of epidermal cells (Sandoval-Zapotitla et al. 2019). The epidermis data from accessions collected from areas with high annual temperatures (21.5 °C) and low rainfall (450 mm) had larger epidermal cells. This enlargement of the epidermal cells is attributed to the cooling of the inner leaf temperature between 10 to 15 °C lower compared to the outside air temperature (Taiz and Zeiger 2010).
Underneath the epidermis, the hypodermis forms a cuticle that stores water (Evert 2006). The hypodermis is interpreted as a layer of large cells aligned below a single epidermal layer (as shown in Fig. 1), which mainly originates from the ground meristem (Martins et al. 2012). Chernetskyy and Weryszko-Chmielewska (2008) found that in Kalanchoe pumila, subepidermal tissues located under the epidermis layer have a row of slightly smaller cells and multiple epidermises, with one or two layers of aligned cells developed from the protoderm. This type of formation has been characterized as storage water cells in Peperomia leaves (Kaul 1977). The hypodermis commonly occurs in plants with xeromorphic traits, including Echeveria (Sandoval-Zapotitla et al. 2019) and Myrtaceae (Retamales and Scharaschkin 2015). The presence of multiple epidermis or hypodermis layers is an arid environmental adaptation strategy for xerophyte plants, which helps prevent water loss from excessive evapotranspiration and reduces the absorption of solar radiation by the laminae (Evert 2006). However, Martins et al. (2012) suggested that it is necessary to observe ontogenetic development to avoid misinterpretation between multiple epidermal and hypodermal layers. The presence of hypodermis in plants has also been an indicator of the phylogenetic relationship as was the case with Nothomyrcia fernandeziana wherein the presence of hypodermis cells was a factor in positioning it close to the ‘Pimeta group’ (Cardoso et al. 2009).
2.2 Stomatal characteristics
2.2.1 Stomatal pattern and type
Succulent plants typically display a scotoactive stomatal type. This type can fix CO2 assimilates at night to produce malic acid by the phosphor-enol-pyruvate carboxylase enzyme, and then store them in mesophyll cells to be re-fixed the following day (Winter and Smith 1996). The stomata regulate water use and gas exchange by adjusting the guard cells, in which the stomatal pore aperture opens widely, to enhance rates of CO2 and water loss (Kollist et al. 2014). To improve water-use efficiency, the shape of the guard cells and the presence or absence of subsidiary cells influence stomatal behavior (Franks and Farquhar 2007).
The stomata of members of the Crassulaceae family, such as Crassula, Kalanchoe, and Echeveria are anisocytic (Fig. 2a), meaning that they comprise three subsidiary cells (Karwowska et al. 2015;Moreira et al. 2012;Sandoval-Zapotitla et al. 2019). However, it has also been reported that there is an anisocytic stomatal complex, similar to that in Kalanchoe spp. with anisocytes and heliocytes in a single leaf (Gray et al. 2020), and in aquatic Crassula inanis with the presence of two subsidiary cells (Moteetee and Nagendran 1997). According to Wilkinson (1979), these species are rarely found in the family Crassulaceae. Stomatal complexes are a series of asymmetric cell divisions in which subsidiary cells are created through the division of meristemoid or meristemoid mother cells (Metcalfe and Chalke 1957). Similarly, anisocytic stomata are generated after amplifying division, and the heliocytic stomatal complex is generated by additional division (Rudall et al. 2018). The number, morphology, and function of subsidiary cells vary considerably and provide ions to guard cells and facilitate stomatal movement. This was shown by the findings of a study by Franks and Farquhar (2007) on Triticum aestivum and T. virginiana, whose subsidiary cells assisted stomatal pores to open wide, resulting in high rates of carbon fixation.
2.2.2. Stomatal size and density
The stomata of ornamental succulents are undisputed in terms of their water-use efficiency. Under water stress, they tend to reduce stomatal density (Griffiths and Males 2017;Bertolino et al. 2019) and possess a lower stomatal density than C3 and C4 species to decrease evapotranspiration (Silva et al. 2001). The stomatal density and index are affected by the amount of solar radiation (Pandey et al. 2007). Stomatal density on the adaxial surface (topside) is higher than that on the abaxial (underside) area of succulent plants (Chernetskyy and Weryszko-Chmielewska 2008;Moreira et al. 2012) (Fig. 2b and 2c).
In the case of E. aff. gigantea, accessions grown in shaded environments with high rainfall and low temperature had shorter occlusive cells and lower stomatal frequency in the adaxial epidermis (Sandoval-Zapotitla et al. 2019). The ability to reduce the number of stomata on the adaxial surface prevents water loss and allows for adaptation to various stress conditions. Despite having xeromorphic features, stomatal density is higher on the abaxial area of Haworthia species because their leaf form is directed upward and outward and can be exposed to the sun from all sides (von Willert et al. 1992). Caballero-Ruano and Jiménez-Parrondo (1978) have found that the distribution of the number of stomata on both sides is similar despite having the same anisocytic stomata type in some other taxa including Aeonium, Monanthes, and Greenvia.
2.3 Vascular tissue arrangement
Vascular patterns are typically composed of the phloem and xylem, in which the phloem is the route for dissolving carbohydrates and the xylem transports water and mineral nutrients from the roots to the upper parts (Taiz and Zeiger 2010). These organs are involved in plant responses to stressful conditions through water and nutrient regulation. E. aff. gigantea accessions in which collateral-type arrangements with xylem cells were forwarded to the upper side and phloem cells were oriented on the opposite side (Melo-de-Pinna et al. 2016). They showed poor overall plant development, similar to that reported for Kalanchoe and Sedum species (Ardelean et al. 2009;Chernetskyy and Weryszko-Chmielewska 2008;Sandoval-Zapotitla et al. 2019).
According to Ogburn and Edwards (2013), the vascularization pattern of succulent plants was found to have a 3D arrangement in which the main vascular bundles were in the middle of the mesophyll and peripheral vascular bundles which was also observed with those of Echeveria ‘Chubby Bunny’ (Fig. 3a). However, for some accessions that displayed a scattered vascular bundle arrangement in the mesophyll, this type of vascular tissue arrangement would likely have a broad vascular distribution that could allow faster and more efficient transport of water to all the mesophyll cells (Caballero-Ruano and Jiménez-Par-rondo 1978). This arrangement of vascular bundles was shown to have contributed to their adaptation strategy for various habitats, whether under conditions of low rainfall or high temperatures.
While collateral vascular bundles (Fig. 3b) are commonly found in succulent plants (Melo-de-Pinna et al. 2016), the amphicribal vascular bundles having phloem surrounding the xylem present in the middle region of the leaf were discovered in two accessions of E. aff. gigantea which is an uncommon vascular formation in angiosperm leaves (De Bary 1884). However, gain-of-function in KAN genes (MYB-like GARP transcription factors) has resulted in an amphicribal vascular bundle in Arabidopsis (Kerstetter et al. 2001). This is consistent with studies that have shown that some plants belonging to the Crassulaceae family, namely Aeonium canariense, A. lindleyi, A. haworthii, have collateral vascular bundles in the center of the leaf and amphicribal vascular bundles in the basal region. This has suggested that this is a common dimorphic vascular feature in this family (Caballero-Ruano and Jiménez-Par-rondo 1978).
Succulent plants are known to have a combination of morphological and anatomical features that have evolved to use water and adapt to temporarily arid conditions. These have also been described in the leaf morphological structure and specific organs and tissues in Echeveria species. The increasing demand for Echeveria species in horticultural markets, owing to their aesthetic beauty, a wide range of colors, and unique morphological leaf structures, has led to the improvement of the visual quality of plants and numerous hybrids and cultivars. However, it has also resulted in many challenges in identifying and classifying them because of their homoplastic morphologic features. The study of the anatomical features of plants of this genus is still limited owing to their thick cuticles, the presence of tannins, and high mesophyll cells. These require a standard method to enhance our understanding of their relationship issues and/or physiological aspects. Aside from anatomical leaf feature studies, there is still an opportunity to expand the knowledge encompassing Echeveria species, including a combination of various fields such as cytogenetic tools and DNA markers, which can provide reliable data to deepen our knowledge of Echeveria species and/or other related species and genera.