Leaf anatomy for delimiting Atlantic Forest species of Psidium (Myrtaceae)

Abstract Leaf anatomical features are widely used to better understand angiosperm taxonomy. However, such information is scarce for the family Myrtaceae. Aiming to fill this knowledge gap, we studied anatomical and micromorphological leaf characters of ten species of Psidium: P. cattleyanum, P. cauliflorum, P. corynanthum, P. guajava, P. guineense, P. macahense, P. myrtoides, P. oligospermum, P. ovale and P. sartorianum. Uniseriate epidermis, paracytic stomata, secretory cavities, and adaxial hypodermis are common among the studied species and are typical characteristics of Myrtaceae. The presence of three or more layers of palisade parenchyma and the absence of sclerenchyma are diagnostic characters of P. guajava. The abaxial epidermis of Psidium cattleyanum and P. macahense possess curved walls. Psidium corynanthum and P. macahense are the only species with sinuous anticlinal walls on both sides of the epidermis, with all other species having straight or curved walls. Open bicollateral vascular bundles are present in all the studied species except P. guineense. With the exception of P. cauliflorum, all the studied species possess druses. The absence of collenchyma in the adaxial face is exclusive to P. ovale. The leaf anatomy of P. macahense, P. corynanthum, P. oligospermum and P. ovale are described for the first time here.

Taxonomic revisions of Neotropical Eugenia (Hussin et al. 1992;Cardoso & Sajo 2004), Campomanesia (Oliveira et al. 2011), and Myrcia, Myrceugenia and Plinia (González 2011), briefly assessed leaf anatomical characters and indicated their significance for the systematics and taxonomy of these genera.The characteristics found to be most useful in distinguishing species of Myrtaceae are leaf venation pattern, vascular arch shape, presence and types of trichomes, form and arrangement of epidermal cells, and presence of secretory cavities.
Relatively few leaf anatomical studies have included species of Psidium (see Soares-Silva & Proença 2008;Gomes et al. 2009;Al-Edany et al. 2012;Oliveira et al. 2017).Amongst leaf anatomical characters common within the genus are uniseriate epidermis; hypostomatic leaves (amphistomatic in P. ratterianum; Proença et al. 2010), paracytic stomata and abundant tector trichomes on the abaxial surface and rare on the adaxial surface.Furthermore, the mesophyll is dorsiventral with one to three layers of palisade parenchyma and a compact arrangement of spongy parenchyma; the hypodermis is adaxial and formed by two continuous layers; the vascular bundle of the midvein is bicollateral and protected by lignified fibers; and subepidermal secretory cavities and prismatic crystals are present throughout the mesophyll.
Herein, we describe anatomical and micromorphological leaf characters of ten species of Psidium found in the Atlantic Forest of Brazil to determine whether anatomy can provide useful information for species taxonomy.Based on previous anatomical studies within Myrtaceae, we hypothesized that the arrangement of epidermal cells, vascular arch shape and the presence and type of trichomes to be the most distinctive characters among, but invariable within the studied species.Finally, we submitted the data to multivariate analysis to better determine those features that explain differences among species.

Material and Methods
The taxonomic sampling of this study included ten species of Psidium that occur in the Atlantic Forest of Brazil: P. Study material was obtained from herbarium samples (VIES).Analyses were performed with completely expanded leaves of three different individuals per species (except to Psidium cauliflorum, P. oligospermum, P. sartorianum and P. corynanthum).The leaf samples were submitted to the reverse herborization process (Smith & Smith 1942) and boiled in distilled water for (ca.) 5 minutes.After cooling down to room temperature, the materials were distended in a 2% potassium hydroxide (KOH) solution (Smith & Smith 1942) for 2 hours, then washed 3 times in distilled water for 50 minutes, and stored in 70% ethanol (Johansen 1940).
For analysis of the type of epidermis and stomata, the samples underwent diaphanization, for which they were clarified with 50% bleach and stained with alcoholic safranin (Johansen 1940).The slides were mounted with glycerinated gelatin (Kaiser 1880) and sealed with colorless enamels (Kraus & Arduin 1997).
For the mesophyll analysis, transverse freehand sections were cut from the apex, middle, and basal regions of the leaf blade, which were clarified in 50% sodium hypochlorite and stained with astrablue/safranin (9:1%) for 5 seconds.The sections were then mounted on slides using Rodriguésia 74: e00472022.2023 glycerinated gelatin and sealed with colorless nail polish (Kraus & Arduin 1997).Morphoanatomical analysis was performed using Leica DM2500 equipment with Leica MC170 HD image capture attached.The classification of structures followed the terminology of Metcalfe & Chalk (1979).
The analysis was divided into three approaches.(1) Gower's dissimilarity coefficient (Legendre & Legendre 2012) is used for mixed data and was applied here since it does not consider the absence of data as evidence of dissimilarity between taxa.(2) Cluster analysis was performed using the unweighted pair group method with arithmetic  (3) A heat map (Hummel et al. 2017) was generated to represent clusters through the association between variables and species, represented on a numerical scale ranging from 0 (no association) to 1 (association), with stronger associations having darker colors.
The heat map was produced using the distance matrix obtained with the Gower method and the morphological data matrix.All data analyses were developed in the R environment (R Core Team 2020), using the packages Vegan (Oksanen et al. 2020) and Cluster (Maechler et al. 2020).
In adaxial view, the epidermal cells of P. guajava and P. guineense have straight walls on both sides (Fig. 1b); P. cattleyanum has straight to curved walls on the adaxial surface and straight walls on the abaxial surface (Fig. 1c); P. sartorianum and P. ovale have straight walls on the adaxial surface (Fig. 1d-e) and straight slightly sinuous to sinuous walls on the abaxial surface; P. oligospermum and P. myrtoides have straight walls on the adaxial surface, and straight to slightly sinuous walls on the abaxial surface; P. corynanthum has slightly sinuous and sinuous anticlinal walls on both sides of the epidermis; P. cauliflorum has straight sinuous walls on the adaxial surface and slightly sinuous to sinuous walls on the abaxial surface; P. macahense has sinuous walls on the adaxial surface and curved to sinuous walls on the abaxial surface.All the analyzed leaves are hypostomatic and have paracytic stomata (Fig. 1f).

Mesophyll and midrib region
In transverse section, all the studied species have a uniseriate epidermis (Fig. 2).Below the epidermis on the adaxial face are two to three layers of cells with anticlinal walls that are higher than the other cells in the epidermis (Fig. 2a-d).
The mesophyll is dorsiventral with one to three layers of palisade parenchyma and a compact arrangement of spongy parenchyma (Fig. 2a-d).
All the studied species have secretory cavities in the epidermis (Fig. 2a-b,e,g) and prismatic crystals associated with the vascular bundle.Druses are present in the mesophyll of all species (Fig. 2a-b) except for P. cauliflorum.
The shape of the midrib of all the studied species is flat-convex (Fig. 2e).Some layers of angular collenchyma are found in both faces of all species (Fig. 2f) except for P. ovale, for which they are on the abaxial face.The collenchyma thickness varies among species (Tab.S1, available on supplementary material <https://doi.org/10.6084/m9.figshare.22253401.v1>).Open bicollateral vascular bundle are present in all species (Fig. 2e) except for P. guineense it is closed (Fig. 2f).
A binary data matrix of the qualitative and quantitative leaf characters was constructed and submitted to grouping analysis for the ten species (horizontal grouping) and for the characters (vertical grouping) in the heat map (Fig. 4).The resulting grouping of 31 characters (Fig. 4, vertical) revealed a group of characters conserved in all or almost all species.
The heat map showed identical profiles among the qualitative characters.Thus, no significant differences were found among species (number of layers of epidermis, presence of cuticle, presence of prismatic crystals, presence of secretory cavities, midrib flat-convex, bicollateral vascular bundles, paracytic stomata and hypostomatic leaves), while others have almost identical profiles but with exclusive characteristics in relation to the other species.Psidium guajava, P. cattleyanum and P. macahense are the Psidium cattleyanum, P. guajava and P. guineense have two or more layers of hypodermis on the adaxial face, whereas all other species have a single layer of hypodermis.Psidium cattleyanum and P. macahense have curved walls on the abaxial epidermis (Tab.S1, available on supplementary material <https://doi.org/10.6084/m9.figshare.22253401.v1>).
Psidium oligospermum, P. corynanthum, P. ovale and P. macahense lack non-ramified unicellular tector trichomes in the adaxial epidermis, whereas all other species have them present.Psidium corynanthum and P. macahense are the only species that have slightly sinuous and sinuous anticlinal walls on both sides of the epidermis, with all other species having straight or curved walls.
Hypostomatic leaves (amphistomatic in P. ratterianum; Proença et al. 2010), and paracytic stomata are common within Myrtaceae, having been reported in Eugenia, (Behar 1971;Palhares 2003;Alves et al. 2008;Donato & Morretes 2009), Myrcia (Gomes et al. 2009;Donato & Morretes 2011) and Plinia (Donato & Morretes 2013) and all the species of Psidium studied here.Our results confirm literature records of hypostomatic leaves, thus increasing the number of studied species.This trait was also found in species adapted to the interior of the Atlantic Forest (Barros et al. 1997) and is recognized as a strategy related to luminosity and periods of water scarcity by reducing transpiration rates (Dickison 2000).
Dorsiventral mesophyll is typical of the family Myrtaceae (Keating 1984) and was found in most of the species of Psidium studied here.Duarte & Paula (2005) and Gomes et al. (2009) observed this same pattern of subepidermal cells in P. guajava and other species of Myrtaceae, respectively, and  characterized them as hypodermis.However, the authors cite the need for ontogenetic study of this tissue to know its origin since, unlike epidermal cells, which originate from the protoderm, cells of the hypodermis originate from ground tissue (Esau 1965).
Secretory cavities are abundant in all tissues and have variable dimensions.Volatile oils produced by secretory cavities in Myrtaceae have been identified as flavonoids and terpenoids (Wollenweber et al. 2000;Díaz-de-Cerio et al. 2017).They are important in the interaction between plants and their biotic environment, as they are involved in defense against herbivory (Van Poecke et al. 2001;Matsuki et al. 2011) and pathogens (Levin 1976), in addition to attracting pollinators (Pichersky & Gershenzon 2002).
Druses occur throughout the palisade and spongy parenchyma of the studied species except for P. cauliforum.Druses are widely present in a diverse array of vegetative and reproductive structures in several genera of Myrtaceae, (Donato & Morretes 2007;Alves et al. 2008;Cardoso et al. 2009;Gomes et al. 2009).The presence of small crystals in vascular plants is related to the removal of oxalate from the metabolic system and the storage of calcium (Franceschi & Nakata 2005;Korth et al. 2006).
Multivariate analysis revealed quantitative micromorphological leaf characteristics that were informative in differentiating almost all the studied species.
Group 1 gathered the largest number of species and although they share many characteristics they still have unique features that can be used for identification.Group 2 includes Psidium guajava and P. guineense, which are characterized by the combination of three layers of hypodermis on the adaxial face, tector trichomes on the abaxial face and the absence of an open vascular bundle.In addition, P. guajava is the only species that does not possess sclerenchyma, while the vascular bundles of P. guineense are completely circled by sclerenchyma.
The quantitative anatomical characters studied here varied more among species than the qualitative characters, making them more useful delimiting the genus.Phenotypic plasticity may have contributed to the variation found in tissue thickness, although this cannot be stated with certainty as no studies have evaluated this factor.
Quantitative leaf anatomical characters showed potential to segregate species of Psidium.
Incorporating these morpho-anatomical findings into future studies, particularly phylogenetic analyses, may provide a better understanding of the evolution of the genus.Further studies, including a broader sampling of species, are needed towards this possibility.

Table 1 -
The studied species of Psidium used in the epidermis analyses, and their respective collectors and collection numbers for the herbarium samples from which study material was obtained.