Leaf anatomy of Rubiaceae species in a semiarid area of Brazil

Considering the importance of anatomical characters for delimiting Rubiaceae subgroups, the difficulties involved in identifying certain taxa and the lack of studies on that group in the semiarid region, this work aimed to characterize the anatomy of the leaves of 15 species belonging to the genera: Borreria (2), Cordiera (1), Eumachia (1), Hexasepalum (4), Mitracarpus (4), Richardia (1), Staelia (1), and Tocoyena (1) collected in the Serra Branca/Raso da Catarina Environmental Protection Area (Jeremoabo-BA, Brazil), and describe useful elements supporting the group’s taxonomy. Variations were found in the shapes and contours of epidermal cells, the presence/absence and types of trichomes, mesophyll type, stomatal type and position, subsidiary cell shapes, vascular system organization, and the occurrence of collector cells and twinned stomata. The results also demonstrated that the anatomical characteristics, when considered together, represent good taxonomic tools for separating the genera and species of Rubiaceae.

According to Metcalfe & Chalk (1979), traditional study methods that use herborized plants and their external morphologies as the primary tools for defining the taxa often create persistent taxonomic problems. Although plant anatomy, cytogenetics, and molecular genetics are not as accessible as external morphologies, new tools have been developed in these areas that can define new characters and parameters useful for solving numerous taxonomic problems (Mayr 1998). Anatomical analyses can provide important additional data (in addition to traditional external morphological characteristics) and can be useful for solving taxonomic problems.
Angiosperm leaves demonstrate wide structural variations among different species having high complexity and either simple or highly subdivided structures (Sinha 1999), which can be used to differentiate between individuals from the same population (Mccauley & Evert 1988).
Although the leaf is the plant organ most exposed to the environment, and therefore the most variable, numerous anatomical characters have systematic value, including the leaf epidermis (Metcalfe & Chalk 1979;Dickinson 2000), which can be used in phylogenetic studies of the Rubiaceae (Andrade et al. 2015), as taxonomists have continually sought additional anatomical characters that can help identify its species (Solereder 1908;Metcalf & Chalk 1950, 1979. The importance of leaf anatomical characters for Rubiaceae was first proposed by Verdcourt (1958) and Bremekamp (1966); Barroso et al. (1978) later separated Rubiaceae subfamilies by their leaf raphides and septate trichomes, with the presence of raphides placing Borreria into Rubioideae (Spermacoceae tribe) and their absence in Chiococca, placing it among the Cinchonoideae (Chiococceae tribe).
The present study aimed to characterize the leaf anatomy of 15 species of Rubiaceae species occurring in the Brazilian semiarid region to identify useful characters that could support the group's taxonomy.
The adult leaves of three individuals (each), located between the third and fourth node (from the apex to the base) of 15 Rubiaceae species were collected: Borreria spinosa ( Botanical collections were undertaken on different days from May/2014 to June/2015, covering both the rainy and hot/dry seasons, totaling six collections. Samples harvested in the field were herborized following the methodology of Fosberg & Sachet (1965) and Mori et al. (1989), and subsequently deposited in the herbarium of the state University of Bahia (HUNEB -Paulo Afonso Collection). Fully expanded sun leaves of the third and fourth nodes were selected for study. The fresh materials were fixed in 70% FAA for 72 hours, according to the methodology described by Johansen (1940). The leaves were then transferred to 70% ethyl alcohol (v/v) for anatomical procedures.

Light microscopy
Cross-and paradermic sections of the leaves were prepared following the methodology described by Kraus & Arduim (1997). The samples were sectioned manually using a razor blade, and stained with astra blue, safranin, and toluidine blue. Semi-permanent slides were prepared using 50% glycerin (v/v) and photographed using a digital camera (AxioCam ERc5s) coupled to a light microscope (Zeiss Primo Star). The anatomical classifications followed Solereder (1908), Metcalfe & Chalk (1950), and Appezzato-da-Glória B & Carmello-Guerreiro SM (2012).

Similarity analysis
Cluster analysis was performed to evaluate the degrees of similarity among the species studied based on the absence (0) or presence (1) of certain characters, using Euclidean distances and the Average method. Analyses with co-phenotype indices greater than 0.7 were considered significant. Cluster analyses were performed using SYSTAT version 13.0 software (SYSTAT Inc., USA).

Leaf anatomy
The leaves of Rubiaceae plants have a thick cuticular layer covering the uniseriate epidermis. The adaxial epidermal surfaces have papillae and anticlinal walls.
The numbers of palisade parenchyma layers varied greatly, with Borreria verticillata, Hexasepalum gardineri, Hexasepalum radulum, and Eumachia being unistratified (Fig. 2b), Cordiera and Tocoyena being multistratified ( Fig.  2c), while the other species were bistratified. A vascular midrib system of the collateral type in an open arc ( Fig. 2d) was seen in all of the species studied. Tocoyena differed from the others, however, by having a continuous principal vascular bundle in a closed loop (Fig. 1e), with two subjacent accessory bundles; the vascular systems of Cordiera and Eumachia had half-moon outlines (Fig. 1f).
Most species showed angular collenchyma on the abaxial surface of the midrib (Fig. 1g); only Mitracarpus salzmannianus, Richardia, and Staelia did not exhibit that characteristic. Additionally, angular collenchyma were observed on the adaxial faces of Hexasepalum (except Hexasepalum gardineri), Tocoyena, Mitracarpus baturitensis, and Mitracarpus robustus. The taxa Borreria verticillata, Hexasepalum gardineri, Eumachia, and Staelia differed by having collenchyma on the adaxial face of the central cylinder; Cordiera rigida demonstrated both of those characters on the adaxial face.
The vascular bundles of all of those species were composed of sclerenchyma fibers as well as primary phloem and xylem that partially or completely surrounded the main vascular bundles. Cordiera also showed abundant fibers in its secondary bundles.
Tocoyena formosa, which was the only petiolate species in this study, presented a nonfistulous cross section and planar petiole with semi-depressed margins, angular collenchyma with 4-5 cell layers, oval cortical parenchyma cells, and sclerenchyma fibers surrounding the vascular system (which consists of a primary vascular bundle in a closed, continuous arc with accessory strands (Fig. 2h) in the medulla and along the petiole edges), and medulla with irregular cells.
Long, non-glandular trichomes were observed in all species, being mostly erect or curved. Hexasepalum gardineri differed by having hook-shaped trichomes, and Tocoyena had abundant and tangled trichomes throughout the leaf. Borreria, Staelia, Mitracarpus baturitensis, and Mitracarpus salzmannianus demonstrated short, thick trichomes, and Hexasepalum teres also bore unicellular glandular trichomes on the blade borders.

Similarity analyses
Similarity analyses performed based on presence/absence matrices (Tab. 1) distinguished three different groups among the Rubiaceae (Fig.  3a) species studied here. The first group comprised the Psychotrieae tribe (Fig. 3b) (represented by Eumachia depauperata), and the second group the Spermacoceae tribe (Fig. 3c)    The characteristics that distinguished Psychotrieae from Spermacoceae were the absence of trichomes, the presence of normal and abnormal subsidiaries cells in the same individual, half-moon shaped vascular bundles, and isodiametric and bulky epidermal cells on both leaf faces.
The most shared characteristics in the Spermacoceae tribe were: amphistomatic leaves, stomata level with the adaxial face, anomalous subsidiary cells, mesophyll with collector cells, arcshaped vascular bundles, angular collenchyma in the midrib, abaxial face, epidermal cells and adaxial papillose, and the absence of druses.
The characters that grouped the Gardenieae tribe were: cell walls with anticlinal curves on their abaxial faces, the absence of papillae on the adaxial epidermis, hypostomatic leaves, only paracytic stomata, multistrata palisade parenchyma, the presence of angular collenchyma in the midrib and adaxial face, and the presence of druses.

Discussion
The leaf epidermis proved to be useful for the systematics of the Rubiaceae, providing important distinguishing features such as presence of papillae, different epidermal cell sizes, wall shapes and thicknesses, stomatal diversity, and trichome morphologies and distributions (Metcalf & Chalk 1979;Dickinson 2000;Araújo 2008).
All of the species have leaf blades with uniseriate epidermises, usually with epidermal cells of different sizes on both faces; isodiametric epidermal cells were also commonly found on the adaxial face. Similar results were reported by Mantovani et al. (1995) for Rudgea species. The adaxial faces of the leaves were bulky in most species. Vitarelli (2008) reported the same trait when studying Psychotria carthagenensis. Light and water availability can modulate cell growth and influence cell division processes, epidermal development, and cell volumes (Taiz & Zeiger 2004;Rizzini 1997;Lambers et al. 1998;Larcher 2000;Zini et al. 2016). The greater volumes of the epidermal cells on the adaxial faces of xeromorphic individuals likely reflects increases in vacuolization to ensure greater water reserves when water restrictions exist due to the high solar irradiation exposure, as in the APASB.
Most of the species studied showed adaxial epidermal papillary cells, with conservative characteristics in Borreria, Hexasepalum, Mitracarpus (except M. robustus), and Staelia that could be used to solve delimitation problems in Spermacoceae, as noted by Mattos (2011) for Borreria. Kay et al. (1981) and Zini et al. (2016) suggested that a papillose epidermis could help reflect sunlight, thus reducing water losses and stabilizing the physiological balances of the plant.
The contours of the leaf epidermal cells corresponded to the patterns described by Metcalfe & Chalk (1950), with most species demonstrating sinuous cells on the abaxial face, but straight and polygonal cells on the adaxial surface. Isanogle (1944), Combes (1946, Hughes (1959), Gusmão et al. (1992), Dickinson (2000), and Alquini et al. (2012) reported that the sinuous anticlinal walls of epidermal cells are directly related to light effects, and the greater shading the greater the sinuosity. In this study, however, Mitracarpus, Staelia, and B. spinosa demonstrated twisted adaxial epidermal cells even in nonshaded sites; although growing in shaded sites, Hexasepalum teres and Eumachia depauperata had straight, polygonal abaxial epidermal cells. Light, therefore, is not influencing that character in those Rubiaceae species, which enhances its taxonomic usefulness -corroborating Mantovani et al. (1995) and Mattos (2011). According to Medri & Lleras (1980), the low sinuosity of the cell walls reflects an adaptive strategy against water losses, while Haberlandt (1928) observed that wall sinuosity increased cell stiffness, thus preventing cell collapse under conditions of water stress.  Rodriguésia 70: e01562018. 2020 The amphistomatic stomatal positions observed in the present study differed from the hypostomatic pattern described for most Rubiaceae species by Solereder (1908), Metcalfe & Chalk (1950), Robbrecht (1988), andMattos (2011). Only Cordiera, Eumachia, Tocoyena, and Hexasepalum radulum demonstrated hypostomatic leaves. That character therefore demonstrated taxonomic value that could be used in genera circumscriptions, as well as differentiating H. radulum from other species in its genus. That stomatal type also differed from the traditional paracytic stomatal pattern associated with Rubiaceae (the 'Rubiaceous type') (Solereder 1908;Accorsi 1947;Metcalf & Chalk 1950;Bahadur et al. 1971). Here, we identified anisocytic stomata in Hexasepalum (except H. radulum), Eumachia, and Mitracarpus baturitensis in addition to the paracitic stomata type. Those characteristics are essential for differentiating H. radulum and M. baturitensis from the other species of the genus. Variations of stomatal type in Rubiaceae were also observed by Mantovani et al. (1995), Da Cunha & Vieira (1997), Vitarelli (2008), and Mattos (2011), who all reported paralelocytic stomata. Accorsi (1947) and Pant & Mehra (1965) observed that the stomatal subsidiaries cells in Rubiaceae species showed anomalous morphological defects due to incomplete and uneven development. Although the ontogeny of those peculiar stomata in the species studied here was not examined, they were considered anomalous because of their similarities to those described by the aforementioned authors.
Some species demonstrated grouped stomata (described as twinned stomata by Accorsi [1947]) or malformed subsidiary cells; those finding agree with the studies of Mantovani et al. (1995) and Gavilanes et al. 2016. Accorsi (1947 reported the presence of twinned stomata in 39.45% of a total of 601 Rubiaceae species. Most of the species studied here showed anomalous stomatal cells, and only the genera Cordiera and Staelia, and H. teres genera showed normal cells. The genus Eumachia demonstrated both normal and abnormal stomata cells; twinned stomata were recorded in the genera Cordiera, Hexasepalum, Mitracarpus (except M. robustus), and Tocoyena.
The most common type of vascular midrib seen here was in the form of an arc, corroborating the studies of Holm (1907), Solereder (1908, and Metcalfe & Chalk (1950); a half-moon type variation was seen in the genera Cordiera and Eumachia. The genus Tocoyena could be distinguished by the arrangement of its vascular system, with a continuous main bundle in a closed arc, with two subjacent accessory bundles on the adaxial face; Coelho et al. (2006) reported that same morphology.
Sclerenchyma fibers were associated with the phloem in most species, completely or partially surrounding the vascular bundles. Sclerenchyma fibers were abundant in the principal and secondary veins in the genus Cordiera genus, but those fibers were scarce and ungrouped in Tocoyena and collateral (except in T. formosa), and were usually surrounded by an endoderm layer; a vascular sheath was seen in the species B. verticillata, H. apiculatum, and H. teres, as well as in Richardia and Staelia. The abundances of those fibers are related to greater tissue stability in xerophytic plants, and they help avoid cell collapse during dry periods as well as mechanical stress due to strong winds (Esau 1977;Krahl et al. 2013;Krahl & Krahl 2017).
A dorsiventral mesophyll was observed in the majority of the species studied here, corroborating with the family descriptions of Solereder (1908) and Metcalfe & Chalk (1950). Hexasepalum apiculatum and Staelia galioides, however, showed an isobilateral mesophyll, which is an important character for their differentiation. Collector cells were observed in Cordiera, Borreria, Hexasepalum, Staelia, Mitracarpus baturitensis, and M. longicalyx. Collector cells are formed by the connection of spongy parenchyma cells to palisade parenchyma cells (thus differing from other foam cells). Those cells consistently occur in Rubiaceae species, and have taxonomical value (Lersten 1974;Scatena & Scremin-Dias 2012).
According to Mattos (2011) and Teixeira et al. (2016), trichomes have taxonomic value in the Rubiaceae. Their importance may vary according to the hierarchical level analyzed, with Kocsis et al. (2004) demonstrating that trichome types, especially abaxial leaf trichomes, can be used to distinguish Rodeletia species. Martínez-Cabrera et al. (2009) observed that the types, sizes, and distributions of trichomes are important characteristics for separating genera in the Hamelieae tribe. Trichomes have an indirect influence on water conservation in plants by reflecting back solar radiation that strikes the leaves (Salatino et al. 1986;Larcher 2000) and, at high densities, they are responsible for maintaining a saturated water vapor atmosphere (Fahn & Cutler 1990;Larcher 2000). Trichomes were found here to be important in distinguishing species such as Hexasepalum apiculatum, which has dense, elongated multicellular trichomes, but H. teres does not; H. teres, on the other hand, has glandular marginal trichomes but H. apiculatum does not. The presence of glandular trichomes in H. teres was also reported by Mussury et al. (2012). Those species are commonly confused in taxonomic studies, resulting in misidentifications. The anatomical characters observed here point to identifiable differences between the species.
Hexasepalum apiculatum is described here for the first time or the APASB.
Similarity analyses showed that the presence of papillae on the adaxial epidermis, the presence or absence of trichomes, stomatal type, variations of palisade layer morphologies, stomatal classification and position, subsidiary cell shape changes during vascular system formation, epidermal cell sizes, the morphologies of the anticlinal walls, the presence of collector cells, the types of crystals, and the angular dispositions of the midrib collenchyma are useful characteristics for the taxonomy of Rubiaceae species. According to Vasconcelos et al. (2017), crystal types represent important diagnostic features for the genera and species of Rubiaceae.
Some of these characteristics have been used in anatomical studies applied to the taxonomy of the Rubiaceae (Tavares &Vieira 1994;Nascimento et al. 1996;Kocsis et al. 2004;Moraes et al. 2009;Martínez-Cabrera et al. 2009;Moraes et al. 2011;Mattos 2011), so that the results reported here can contribute to a better understanding of the phylogenetic relationships among the taxa of that family.
The similarity analyses corroborated the current circumscription of Rubiaceae proposed by Bremer & Eriksson (2009).
These results confirm the importance of leaf anatomical characters as additional tools that can support taxonomic studies in Rubiaceae and increase our anatomical, ecological, and physiological knowledge of the subfamily, tribe, genera, and species, and indicate promising characters for future taxonomic and phylogenetic approaches.