Leaf architecture of Rubiaceae Juss . from caatinga vegetation in Brazil

The study and characterization of leaf venation plays a key role in the recognition of taxonomic groups that have been identified mainly based on reproductive traits. This study aimed at characterizing the pattern of leaf venation of Rubiaceae, recognizing individual characters among the taxa of this group and testing the use of leaf architecture as a taxonomic tool capable of providing vegetative and diagnostically characteristics between species. Leaves of 14 species distributed in eight genera from Rubiaceae were diaphanized and classified. The study area is located in Área de Proteção Ambiental Serra Branca/Raso da Catarina, Bahia State, Brazil. The characterization of secondary veins, including type, spacing and number of pairs, third veining patterns, fourth and fifth order, and the conformation of the areola were useful to delimit taxa when used together, providing subsidies to more comprehensive studies.


Introducion
Rubiaceae Juss. is the fourth largest family in number of species of angiosperms (Robbrecht 1988).In Brazil, the family is represented by 126 genera and 1,412 species, of these 84 genera and 406 species occur in the Northeast, with the greatest diversity in the state of Bahia, where it was recorded 78 genera and 364 species (Barbosa et al. 2018).
Rubiaceae is a monophyletic group of easy circumscription.However, it still faces problems regarding intrafamily classification (Verdcourt 1958, Bremekamp 1966, Bremer & Jansen 1991).The use of vegetative characteristics as diagnostic elements for plant specimens have been considered important in the identification process, in particular those related to the shape of the constituent parts of the plant.
Although the shape is accepted as an important element in the taxonomic study, its application has not been explored to its full potential due to the inherent difficulty of description and comparison (Niklas 1994).
Acording to Stace (1989) states that leaf morphology has always played an important role in plant systematics as a whole, particularly to characterize and identify taxa where variation in floral structures is not informative.The leaves present characteristics that can also be used in evolutionary studies, being considered as useful as the floral characteristics, pollen morphology and most of the anatomical characteristics traditionally used in the systematics (Cabral et al. 2016, Ellis et al. 2009, Judd et al. 2009, Hickey & Taylor 1991).
The study of foliar architecture, which was created to assist in the identification of fossils (Hickey 1973), has developed considerably 1. Borreria spinosa (L.) Cham.& Schltdl.
Symmetrical slide, elliptic to lanceolate, attenuate base, acute apex, entire margin.Pinnate venation, eucamptodromous-brochidodromous.Secondary vein decreasing to the base four-five pairs/side.Intersecondary moderately developed veins, strongly printed.Tertiary veining with alternating percorrent, branched (Figure 1A).Fourth and fifth order reticulated veining in regular polygons.Areola moderately developed, forming three-five sides.Final veins forming one-two branches with no extended vascular endings.Terminal venation of the margin without teeth.
Symmetrical and elliptical slide, truncated base, acute apex, serrated margin.Pinnate venation, eucamptodromous.Secondary veins growing to base with three-four pairs/side.Absent intersecondary veins.Randomly reticulated tertiary veins, sinuous, moderately branched.Dichotomized veins of fourth and fifth order (Figure 1B).Undeveloped areolas with three-five sides (Figure 1C).Free final veins forming a branch with enlarged vascular endings (Figure 1D).Terminal venation of the margin forming incomplete ties without teeth.
Symmetrical slide, elliptical, convex base, acute apex, entire and revolute margin.Pinnate venation, weak brochidodromous.Irregular secondary veins, with 10 pairs/side.Well developed intersecondaries veins, strongly printed.Tertiary veins randomly reticulated, sinuous, branching.Fourth and fifth order reticulated vein in regular polygons.Areola moderately developed (Figure 1H), with three-five sides.Free final veins forming one-two branches (Figure 1I) with no extended vascular endings.Venation margin of terminal forming ties incomplete without teeth.
Symmetrical slide oblong to lanceolate, acute apex, attenuate base, entire margin.Pinnate venation, eucamptodromous-brochidodromous.Secondary veins decreasing to the base with three pairs/side.Absent intersecondaries veins.Tertiary percorrent alternating veins, winding to ramified.Quaternary reticulated in regular polygons veins.Dichotomized fifth order veins.Undeveloped areolas with four-five sides.Free final veins forming one branch with no extended vascular endings (Figure 1J).Venation margin of terminal forming ties incomplete without teeth.over the years, constituting an important tool for classifying and differentiating problematic groups (Ellis et. al. 2009, Judd et al. 2009, Leaf Architecture 1999).
According to Hickey (1973) the term "leaf architecture" indicates the position and shape of the elements that constitute the external expression of the leave structure including venation pattern, marginal configuration, leaf shape and glandular position, this term is appropriate because the elements of the leaves are arranged in certain defined structural standards that can be described.
Therefore, this study aimed at characterizing the pattern of Rubiaceae leaf venation in the Área de Proteção Ambiental Serra Branca/Raso da Catarina, located in Jeremoabo, Bahia State, recognizing individual characters among the taxa of this group and testing the use of leaf architecture as taxonomic tool capable of providing vegetative diagnostic characteristics among species.

Material and Methods
The Área de Proteção Ambiental Serra Branca/Raso da Catarina (APASB) is located in the municipality of Jeremoabo, northeast of Bahia, between the coordinates 09º53'15.5''to 09º44'34.6''Sand 38º49'36,1'' to 38º52'20,4"W.The area have 67,237 hectares, bordering to the north with the Estação Ecológica Raso da Catarina (ESEC) and to the south with the Vaza-Barris river, which is the main affluent of the São Francisco river in the region.The area is flat with sandstone formations (Szabo et al., 2007).
There were a total of six collection of the botanical material in different days, from May/2014 to June/2015, covering both periods: rainy and hot/dry.The samples obtained in the field were herborized, according to the methodology of Fosberg & Sachet (1965) and Mori et al. (1989), and are deposited in the herbarium of the Universidade do Estado da Bahia (HUNEB -Paulo Afonso Collection).Fully expanded sun leaves of the third and fourth knot were selected for the study.The fresh materials were fixed in 70% FAA according to the methodology described by Johansen (1940), remaining for 72 hours in the solution.Thereafter, the leaves were transferred to 70% ethanol solution (v/v), where they remained preserved until the technical diaphanization procedure.
For each species, 3-4 leaves were selected.The leaves were mostly intact and uninjured.The leaves were diaphanizated in order to not occur any distortion of venation or leaf shape as a whole, resulting from the process of pieces of assembly.
The diaphanization technique used in this work was a modification of the method proposed by Shobe & Lersten (1967).Posteriorly, semipermanent slides with 50% glycerin solution (v/v), and slides with Canada balsam were prepared, and were photographed using a camera (AxioCam ERc5s) attached to the microscope (Zeiss Primo Star).Hickey (1973), Leaf Architecture (1999) and Ellis et al. (2009) were used as references for the description and classification of the veining patterns.

Results
There were a total of 14 species distributed in eight different genera of Rubiaceae of APASB, and described below.1K).Areola moderately developed with four-five sides.Final ending veins forming an enlarged vascular termination branch (Figure 1L).Venation margin of terminal forming ties incomplete without teeth.

Discussion
The most used vegetative morphological characters for taxa delimitation are the forms, especially the leaves, because it is the growing body of better access.In this study, these elements were not relevant since there was no significant variation between species, characterized primarily by elliptical type, which may be related to genetic issues.This statement agrees with Parkhurst & Loucks (1972) who state that the size and shape of the leaves are controlled by heredity, as demonstrated in their studies, by high variation of types that occur between different species coexisting in a particular environment.Still, it should be noted that the origin of leaf form occurs for a brief morphogenesis initial period, being more influenced by the formation of secondary veins, since the veins of smaller caliber arise only during expansion of the sheet (Dengler & Kang 2001).
The primary standards of leaf architecture of the studied species correspond to the pinned type, where a main vein of larger caliber diverges from the base to the tip of the slide.In general, the primary and secondary veins are the backbones of the leaf, and tertiary veins are on the smaller-caliber veins, which form a kind of mesh or lattice.The primary vein has the highest caliber and generally extends along the leaf, starting from the base or near the base of the leaf and moving toward the margin or apex (Obermüller et al. 2011).
The secondary veins ranged from broquidodromous to eucamptodromous, although this first type is a common standard for Rubiaceae (Mattos 2011, Mantovani et al. 1995, Da Cunha & Vieira 1997), the latter was well represented in this study, constituting 50% of the species studied, Varjão et al. (2013) observed the same parameters.The species Borreria spinosa, Cordiera rigida, Hexasepalum teres e Mitracarpus salzmannianus showed a variation between these two types, described as eucamptodromous-brochidodromous, where secondary veins are strongly curved, joining lightly to each other.In addition, Eumachia depauperata showed the standard semibroquidodromous, where secondary veins grow towards the margins and unite forming slightly undeveloped arches.The secondary veins are the next set to be measured after the primaries, they often also have a long haul, and that usually goes from the base of the leaf or a primary vein toward the margin or following in arcs towards the apex (Obermüller et al. 2011).
The spacing between the secondary veins varied between species, allowing the delimitation of taxa.Borreria spinosa presented secondary veins decreasing to base with four-five pairs/side, whereas in B. verticillata they were found growing to the base with three-four pairs/ side.The species of the genus Hexasepalum showed decreasing spacing to the base with three pairs/side in H. apiculatum and four-five pairs/side H. radulum.On the other hand, H. teres secondary veins were found to grow for four base pairs.All species of the genus Mitracarpus presented spacing of secondary veins decreasing to the base, with four-five pairs/ side in M. baturitensis, five-six pairs/side in M. longicalyx, five-seven pairs/side in M. robustus and five pairs/side in M. salzmannianus.
The other genera, which are represented in this study by one species each, also presented different spacings.In Cordiera rigida, secondary veins were falling to the base with five-seven pairs, in Eumachia depauperata, the spacing is irregular with ten pairs.Richardia grandiflora and Staelia galioides showed growing spaces for the base, with five pairs in the representative of the genus Richardia and fourfive pairs for the kind of Staelia.Tocoyena presented secondary veins with uniform spacing.
Tertiary veins and smaller orders veins are less visible and keep a similar caliber, forming reticles.Tertiary veins generally have a narrower size than the set of secondary veins and often connect with the primary and secondary veins throughout the sheet (Obermüller et al. 2011).The importance of the tertiary venation pattern is emphasized by Hickey (1973) and Dilcher (1974).The pattern is a dense, well-structured network that supports webs of smaller calibers.The percorrent alternate type was the most representative of the species, which agress to Saha et al. (2014).Tertiary patterns found in this study were essential to distinguish species.
The highest standards of crosslinking are formed until the fifth order of veins for most species herein studied.Only Hexasepalum radulum presented veins just until the fourth order.This ratio of the number of cores orders in one species is not linked to the size of the leaf, since relatively large Mitracarpus leaf has the same number of cores orders compared to the Borreria species, which have tiny slides.These observations are supported by the work of Saha et al. (2014) for Rubiaceae in India.Most species were fourth and fifth reticulated order veins in regular polygons.However, H. apiculatum, H. teres, M. robustus e M. baturitensis differed with dichotomized fifth order veins, and B. verticillata and Richardia grandiflora with both dichotomized orders.It is noteworthy that the higher the density of the veins, more channels per unit area are available for driving, thus helping transpiration and therefore the maintenance of the leaves for water supply (Fonsêca et al. 2006), and sustention (Souza et al. 2013).
Smaller areas formed by tertiary veins and lower orders of veins originate the so-called areolas, which can provide development, arrangement and different form and may or may not present free

Figure 1 .
Figure 1.Diaphanized leaves of Rubiaceae of Área de Proteção Ambiental Serra Branca/Raso da Catarina, Bahia.(A) Borreria spinosa, detail of branched tertiary veins; (B-D) Borreria verticillata, B-with the fourth and fifth order dichotomized veins, C-undeveloped areolas and D-extended vascular endings; (E-G) Cordiera rigida, E-showing secondary veins decreasing to the base, F-intersecondary veins and G-developed areolas.(H) Hexasepalum apiculatum, detail of the final free vein with extensive vascular endings; (I,J) Eumachia depauperata, I-detail of moderately developed areolas and J-free end with two branches; (K,L) Mitracarpus salzmannianus, K-detail of the fourth and fifth order reticulated veins in regular polygons and L-free end veins with one branch.