Leaflet Blade Epidermis and Its Taxonomic Significance in 13 Species of Bignonieae (Bignoniaceae) from Pico Do Jabre, Paraíba, Northeast of Brazil

Botany

Leaflet blade epidermis and its taxonomic significance in 13 species of Bignonieae (Bignoniaceae) from Pico do Jabre, Paraíba, Northeast of Brazil

Journal: Botany

Manuscript ID cjb-2020-0051.R4

Manuscript Type: Article

Date Submitted by the 08-Sep-2020 Author:

Complete List of Authors: Lopes-Silva, Rafael ; Universidade Federal da Paraíba, Programa de Pós- Graduação em Biodiversidade Silva, Anauara; Universidade Federal da Paraíba, Programa de Pós- GraduaçãoDraft em Produtos Naturais e Sintéticos Bioativos Santos, Ednalva ; Universidade Federal da Paraíba, Programa de Pós- Graduação em Biodiversidade Agra, Maria de Fátima; Universidade Federal da Paraíba, Programa de Pós-Graduação em Produtos Naturais e Sintéticos Bioativos

epicuticular waxes, idioblasts, leaf anatomy, micromorphology, Keyword: neotropical lianas

Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? :

Leaflet blade epidermis and its taxonomic significance in 13 species of

Bignonieae (Bignoniaceae) from Pico do Jabre, Paraíba, Northeast of Brazil

Rafael Francisco Lopes-Silva1, Anauara Lima e Silva2, Ednalva Alves Vital dos Santos1 and

Maria de Fátima Agra1,2,3

1Programa de Pós-graduação em Biodiversidade, Centro de Ciências Agrárias, Universidade

Federal da Paraíba, Areia, Paraíba, Brazil. E-mail: [email protected] and E-mail:

[email protected]

2Programa de Pós-graduação em Produtos Naturais e Sintéticos Bioativos, Universidade Federal da

Paraíba, João Pessoa, Paraíba, Brazil. E-mail: [email protected] Draft

3Author for correspondence:

Maria de Fátima Agra

UFPB – Universidade Federal da Paraíba, Centro de Biotecnologia, R. Tab. Stanislau Eloy, 41-769

- Castelo Branco, João Pessoa - PB, 58033-455.

+5583988077888

E-mail: [email protected]

1 © The Author(s) or their Institution(s) Botany Page 2 of 42

Abstract: Bignonieae is the largest tribe of Bignoniaceae, with 21 genera and 393 species of lianas and shrubs, 2–3-foliolate with the terminal leaflet modified as tendrils. We examined the micromorphologies of the leaflet blade epidermises of 13 species of Bignonieae belonging to

Amphilophium, Anemopaegma, Bignonia, Cuspidaria, Dolichandra, Fridericia, Pyrostegia,

Tanaecium, and Xylophragma, from Pico do Jabre, Paraíba, Brazil. These are lianas except for

Tanaecium parviflorum (shrubby). We sought to identify epidermal leaflet parameters to support their taxonomy subject to great similarities between their vegetative characters, mainly in species of the same genus and related genera. Analyses were performed using light and scanning electron microscopy, and showed five types of epicuticular waxes, four cuticle types, three epidermal cell anticlinal wall types, and non-glandular and glandular trichomes. Hypostomatic leaves showed ten different types of stomata, with stomatal indices from 6.21% (Bignonia ramentacea) to 23.52% (Tanaecium parviflorum) and stomatal densitiesDraft from 76 stomata/mm² (Pyrostegia venusta) to 752.9 (T. parviflorum). The presence of raphides in Amphilophium crucigerum and styloids in

Fridericia pubescens constitute the first records for these genera. Epidermal micromorphology provided a set of distinctive characters to separate these species, representing an additional tool to support their taxonomies, as well as that of tribe Bignonieae.

Key words: epicuticular waxes, idioblasts, leaf anatomy, micromorphology, Neotropical lianas.

2 © The Author(s) or their Institution(s) Page 3 of 42 Botany

Résumé: Bignonieae est la plus grande tribu de Bignoniaceae avec 21 genres et 393 espèces de lianes

et d’arbustes, 2–3-foliolés avec la foliole terminale modifiée en vrille. Nous avons examiné la

micromorphologie des épidermes de folioles de 13 espèces appartenant à Amphilophium,

Anemopaegma, Bignonia, Cuspidaria, Dolichandra, Fridericia, Pyrostegia, Tanaecium et

Xylophragma, du Pico do Jabre, Paraíba, Brésil. Ces espèces sont toutes des lianes sauf Tanaecium

parviflorum (espèce arbustive). Nous cherchons à identifier les paramètres de l'épiderme foliolaire en

soutien à la taxonomie au regard des grandes similitudes des caractères végétatifs, en particulier entre

espèces du même genre et genres apparentés. Les analyses ont été effectuées en utilisant la

microscopie optique et la microscopie électronique à balayage, et démontrent cinq types de cires

épicuticulaires, quatre types de cuticules, trois formes de parois anticlinales de cellules épidermiques

et des trichomes non glandulaires et glandulaires. Les feuilles hypostomatiques présentent dix types de stomates, et des indices stomatiques variantDraft de 6,21% chez Bignonia ramentacea à 23,52% chez Tanaecium parviflorum et des densités stomatiques variaient de 76 stomates/mm² chez Pyrostegia

venusta à 753 chez T. parviflorum. La présence de raphides chez Amphilophium crucigerum et de

styloïdes chez Fridericia pubescens font l’objet de premières descriptions de ces types d'idioblastes

pour ces genres. La micromorphologie épidermique a fourni un ensemble de caractères distinctifs

pour séparer ces taxons et offre un outil additionnel pour soutenir la taxonomie des espèces et de la

tribu Bignonieae.

Mots-clés: anatomie foliaire, cires épicuticulaires, idioblastes, lianes Néotropicales,

micromorphologie.

3 © The Author(s) or their Institution(s) Botany Page 4 of 42

Introduction

Bignoniaceae Juss. is divided into eight main clades, of which Bignonieae is the largest

Neotropical clade of the family, comprising about 21 genera and 393 species (Lohmann and Taylor

2014), of which 327 occur in Brazil, mainly in the Amazon, Atlantic Forest, and dry forest domains

(BFG 2015). The Bignonieae clade constitutes the most diverse and abundant clade of lianas in the

Neotropics (Lohmann 2006; Lohmann and Taylor 2014) and, according to Gentry (1980), those

Brazilian domains are the centers of diversity of the family.

The Bignonieae clade is formed predominantly of lianas and shrubs species, with the greatest morphological diversity among neotropical lianas (Lohmann 2006). Their compound leaves are usually 2–3-foliolate with the terminal leaflets modified as simple, bifid, or multifid tendrils (Sousa-

Baena et al. 2014). The flowers are showy and are visited by a broad range of floral visitors and pollinators (Alcantara and Lohmann 2010).Draft The monophyly of the group is supported by molecular, morphological, and anatomical characters. Morphological and anatomical synapomorphies can be observed in the modification of terminal leaflets as tendrils, the fruits are septicidal capsules with the septum parallel to the fruit valves (Olmstead et al. 2009), and irregular stem growth (which results in the formation of four to thirty-two phloem wedges that interrupt the xylem) (Santos 1995; Lohmann and Taylor 2014).

Anatomical characters constitute an important tool for the taxonomy of flowering plants

(Metcalfe 1946; Metcalfe and Chalk 1950, 1979; Dickison 1975). Leaf epidermal anatomy has revealed important characters in the taxonomy of distinct groups of Angiosperms with diagnostic values at various levels. Carlquist (1961) viewed the leaf as the most variable organ among angiosperms, those anatomical variations are often closely related to the generic and specific (and sometimes family) levels.

The leaf epidermis has revealed several important anatomical structures whose characters have been found to be useful in the taxonomy of various groups of vascular plants, with diagnostic value at different taxonomic levels (Dilcher 1974; Metcalfe and Chalk 1979). According to

4 © The Author(s) or their Institution(s) Page 5 of 42 Botany

Dickison (1975), anatomical characters of the leaf epidermis hold systematic value and

phylogenetic potential, which allow the identification of plants and the resolution of taxonomic

problems in different groups of vascular plants. Among the characters of the leaf epidermis that

most stand out are the morphology of epidermal cuticle and epicuticular waxes (Barthlott 1998), the

presence of inorganic idioblasts with different types of crystals (raphides, druses, and crystal sands)

and lithocysts, as well as epidermal appendages, including different types of stomata and trichomes.

The value of epidermal characters in supporting the taxonomy of Bignoniaceae has been

demonstrated for some African species (Ogundipe and Wujek 2004; Ugbabe and Ayodele 2008) as

well as for Brazilian species of Tabebuia Gomes ex DC. and Handroanthus Mattos (Silva et al.

2009). Additionally, the wood anatomy of Bignoniaceae has corroborated and supported

relationships established within the family (Gerolamo and Angialossy 2017; Santos 2017), and within the tribe Bignonieae, revealing theDraft importance of anatomical studies for the current classification of Bignoniaceae tribes and for understanding their evolution (Pace et al. 2009; Pace et

al. 2015). Additionally, other studies have focused on the quality control of medicinal species

(Mauro et al. 2007; Souza et al. 2007), epidermal structures (Gonzalez 2013), the ontogeny of

stomata (Paliwal 1970), and their taxonomic implications (Nogueira et al. 2013; Firetti-Leggieri et

al. 2014; Sousa-Baena et al. 2014).

The identification of Bignonieae taxa is quite complex without considering reproductive traits

(Gentry 1992), due to their great diversity and numerous patterns of morphological variation, which

often requires analyses of additional diagnostic characters (Lohmann 2006). The importance of

many species of Bignonieae species for ethnobotanical uses, especially in folk medicine (Gentry

1992), reinforces the need for a better understanding of their leaf anatomy, especially because the

vegetative parts of the plants are the most widely used as medicine.

We examined the micromorphology of blade leaflet epidermises of 13 species of Bignonieae

belonging to nine genera: Amphilophium crucigerum (L.) L.G.Lohmann, Amphilophium

paniculatum (L.) Kunth, Anemopaegma citrinum Mart. ex DC., Bignonia ramentacea (Mart. ex

5 © The Author(s) or their Institution(s) Botany Page 6 of 42

DC.) L.G.Lohmann, Bignonia sciuripabulum (Hovel.) L.G. Lohmann, Cuspidaria lateriflora

(Mart.) DC., Dolichandra unguis-cati (L.) L.G.Lohmann, Fridericia pubescens (L.) L.G.Lohmann,

Pyrostegia venusta (Ker Gawl.) Miers, Tanaecium cyrtanthum (Mart. ex DC.) Bureau & K.Schum.,

T. dichotomum (Jacq.) Kaehler & L.G.Lohmann, T. parviflorum (Mart. ex DC.) Kaehler &

L.G.Lohmann, and Xylophragma heterocalyx (Bureau & K.Schum.) A.H.Gentry. All species are lianas, except the shrubby T. parviflorum. They have compound leaves, 1-3-foliolate; of which, six genera have leaves 2-3-foliolate: Amphilophium Kunth, Anemopaegma Mart. ex Meisn., Fridericia

Mart., Pyrostegia C. Presl., Tanaecium Sw., and Xylophragma Sprague; Bignonia L. has 1-2 leaflets; Dolichandra Cham. has exclusively 2-foliolate leaves; and Cuspidaria DC. has 3-foliolate leaves.

Most of these species have distinctive characters when they flower, however, they show great vegetative similarities, which makes theirDraft taxonomic identification quite difficult, especially when congeneric species and related genera share the same habitat, type of habit, and leaf morphology.

This study was carried out in order to identify epidermal leaflet characters and define parameters for

13 species of Bignonieae as an additional tool to support their taxonomy, and that of tribe

Bignonieae.

Materials and methods

Plant material

Leaf material from 13 species of Bignonieae (Table 1) was collected at Pico do Jabre, in the municipality of Maturéia. This area contains the highest elevation areas of Paraíba State and the northern region of northeastern Brazil, at an elevation of up to 1,197 m a.s.l. The vegetation of the area is largely composed of elements of dry forests of Atlantic Forest (Rocha and Agra 2002; Agra et al. 2004; Cunha and Silva-Junior 2014).

Part of the material collected was pressed and dried, according to the usual techniques for herbarium collections (Bridson and Forman 1992), and subsequently deposited in the Prof. Jayme

6 © The Author(s) or their Institution(s) Page 7 of 42 Botany

Coelho de Morais Herbarium (EAN) at the Federal University of Paraíba (UFPB). Duplicates were

deposited in the JPB, MO, RB and SPF herbaria (acronyms according to Thiers 2019). All species

had compound leaves 2-3 foliolate, except for some young branches of B. ramentacea and B.

sciuripabulum with unifoliolate leaves. Leaf samples were collected from adult plants of each

species, from the third to fifth nodes, from three individuals of each species, when the specimens

were available (see Table 1). The leaves were subsequently fixed in FAA (50%) for 48h, according

to Johansen (1940), and then preserved in ethanol 70%.

Leaf epidermis

Samples of fresh and dried leaflets blades were analyzed from 13 species of Bignonieae (Table 1), a

total of 15 samples were analyzed from each species. Samples from three species were exclusively from fresh material (Tanaecium cyrtanthumDraft, Tanaecium dichotomum and Xylophragma heterocalyx), and also samples from two species (Amphilophium paniculatum and Cuspidaria

lateriflora), exclusively from exsiccates of the Herbarium Prof. Lauro Pires Xavier (JPB). The

remaining eight species had both fresh and dry material (see Table 1). The dried material was

rehydrated according to Smith and Smith (1942).

Sections of leaves with two or three leaflets were prepared using standard transverse and

paradermal sections (on the adaxial and abaxial surfaces), subsequently clarified using 2% sodium

hypochlorite, then washed in distilled water, and neutralized with acetic acid 1%. The paradermal

sections were stained with Safranin 0.25%, which appears red in lignified, suberized, and cutinized

cell walls, following Johansen (1940). The cuticle is a non-cellular layer composed of several inert

polymers, especially cutin, that covers the entire leaf surface and most other aerial plant surfaces

(Juniper and Jeffree 1983).

The transverse sections were stained with Safrablue (Kraus and Arduin 1997, modified from

Bukatsh 1972), which is a double staining process using the association of Astra blue 1% and

Safranin 1%, 9:1 (v/v). It highlights the cellulose cell wall, which stains blue, and the lignified cell

7 © The Author(s) or their Institution(s) Botany Page 8 of 42

wall, which stains red. The presence of calcium oxalate crystals was confirmed by dissolution of crystals with hydrochloric acid solution 10%, according to Arduin and Kraus (1997), and modified from Chamberlain

(1932). All sections were mounted in glycerinated gelatin 50%, analyzed, and photographed.

Trichomes terminology follows Seibert (1948), Payne (1978) and Nogueira et al. (2013), while epidermal classification follows Dilcher (1974).

For Scanning Electron Microscopy (SEM) analyses, dehydrated samples (approximately 0.5 cm²) of the median portions of leaflets (both abaxial and adaxial faces) of each species were attached to aluminum stubs using double adhesive tape, coated with a gold layer of ca. 24-nm thick by a sputter coating (EMITECH, model K550X), examined and micrographed using a SEM (JEOL JSM-5600) at 15 KV. The terminology ofDraft the epicuticular waxes follows Barthlott et al. (1998).

Stomata type, density, and stomatal indices

Three leaves with two or three leaflets were analyzed for each species. The number of epidermal cells and stomata were counted in two distinct fields of each leaflet, which were observed at the same magnification, for a total of 18 fields analyzed for each species (n = 18). The classification of stomatal morphology follows Dilcher (1974).

The stomatal index and density were obtained using Anati Quanti software (Aguiar et al.

2007) using an optical microscope (Leica, model DM750), with Qwin system attached to a video camera (Leica ICC50 HD). The statistical analyses were performed using GraphPad Prism 7.00 software. Univariate ANOVA was used as a test of variance analysis, with statistical significance determined by the Tukey method (p < 0.05).

Results

Epicuticular waxes and epidermal cuticles

8 © The Author(s) or their Institution(s) Page 9 of 42 Botany

Different features of epicuticular waxes were observed on leaflet blade epidermal surfaces,

trichomes, and stomata, some of them on a single phylloplane, as a wax syntopism. Granular

epicuticular waxes were observed on at least one surface of the leaflet epidermises in all 13 species

(see Table 2). Seven species showed granules on both surfaces: A. citrinum (Figs. 1G and 1H), A.

paniculatum (Figs. 1A and 1B), B. ramentacea (Fig. 7E), D. unguis-cati (Fig. 7J), F. pubescens

(Figs. 1C and 1D), P. venusta (Figs. 1E and 1F), T. dichotomum (Figs. 2H and 7B), and X.

heterocalyx (Fig. 2A). Five species showed granules only on the adaxial surface: C. lateriflora

(Fig. 2B), T. cyrtanthum (Fig. 2C), T. parviflorum, and A. crucigerum (Fig. 2D). Epicuticular waxes

as granules were the most common type, being observed on the adaxial surfaces of approximately

85% of the species (11 spp.), but present on the abaxial surfaces of approximately 62% of the

species (8 spp.). Epicuticular waxes as crusts were on the epidermises of the leaflet blades of six species (Table 2): on both surfaces of B. ramentacea,Draft on the adaxial surface of A. citrinum (Fig. 1G), and X. heterocalyx (Fig. 2A), and on the abaxial surface of P. venusta (Figs. 1E), and A.

crucigerum (Fig. 2D). Platelets were recorded on the leaflet epidermises of five species: on both

surfaces of B. ramentacea and P. venusta, and on the adaxial surface of A. paniculatum, T.

cyrtanthum and X. heterocalyx. Fissured layers of epicuticular waxes were observed on both

surfaces of T. cyrtanthum (2C and 2E), and on the adaxial surface of P. venusta (Fig. 1E).

Epicuticular waxes as threads were present only on the adaxial surface of C. lateriflora (Fig. 2B).

The occurrence of different epicuticular waxes on the same epidermal surface or in one cell is

known as syntopism, and was observed in eight species (Table 2). Five species showed epicuticular

waxes syntopism only on the adaxial surface; two species, B. ramentacea and P. venusta (Figs. 1E

and 1F), on both surfaces; and A. crucigerum only on the abaxial surface (Fig. 2F). Epicuticular

waxes were also on the ornamented surface epidermis of simple trichomes of six species: T.

parviflorum, F. pubescens (Fig. 1C), C. lateriflora (Fig. 2B), A. crucigerum (Figs. 2D and 2F), A.

citrinum (Fig. 1G), and T. dichotomum (Fig. 2H).

9 © The Author(s) or their Institution(s) Botany Page 10 of 42

Bignonieae species showed smooth, striate, rugose and papillose cuticles on their leaflet blade epidermises, and in stomatal edges. Striate cuticles were observed on the epidermises of eight species (Table 2). Of those, five species from different genera showed striate cuticles on both surfaces: F. pubescens (Figs. 1C and 1D), P. venusta (Figs. 1E and 1F), A. citrinum (Figs. 1G and

1H), C. lateriflora (Fig. 2B), and A. crucigerum (Figs. 2D and 2F). Three species showed striate cuticles on their abaxial surfaces only: T. parviflorum, T. cyrtanthum (Fig. 2E), and D. unguis-cati

(Fig. 3A). The smooth layer type was present in four species: two were observed on both surfaces of B. ramentacea and D. unguis-cati, and on the adaxial surface of T. dichotomum and T. parviflorum. Two species, X. heterocalyx (Fig. 2A) and B. sciuripabulum (Fig. 2G), showed rugose cuticle type on both surfaces, and T. parviflorum showed only on the abaxial surface. In addition, A. paniculatum (Figs. 1A and 1B) and T. cyrtanthum (Figs. 2C and 2E) showed papillose epidermises on both surfaces. Some species such as A.Draft citrinum (Figs. 1G and 1H), showed areas of epidermis with different degrees of striate cuticle. Moreover, the cuticle on the epidermis and, especially, around the stomata (Fig. 7) showed quite distinctive morphological characters, which are taxonomically significant for the studied species.

Leaflet blade epidermis

The Bignonieae species studied here in frontal view showed three patterns of anticlinal cell walls of the epidermis of the leaflet blade: curved, straight, and sinuous. All data on the leaflet epidermises are presented in Table 3. The anticlinal cell walls were predominantly sinuous, as observed in eleven species. Five species showed sinuous walls on both surfaces: Dolichandra unguis- cati (Figs. 3A and 3B), Fridericia pubescens (Figs. 3C and 3D), Tanaecium dichotomum (Figs. 3E and 3F), Tanaecium parviflorum (Figs. 3G and 3H), and Xylophragma heterocalyx (Figs. 4A and

4B). In five species a sinuous pattern was only observed on the abaxial surface: Anemopaegma citrinum (Fig. 4D), Bignonia sciuripabulum (Fig. 4F), Cuspidaria lateriflora (Fig. 4H), Pyrostegia venusta (Fig. 5B), and Tanaecium cyrtanthum (Fig. 5D). Amphilophium crucigerum (Fig. 5E) showed

10 © The Author(s) or their Institution(s) Page 11 of 42 Botany

a sinuous pattern of anticlinal cell walls only on its adaxial surface. Anticlinal cell walls varied in

different species and were often more sinuous in abaxial than in adaxial surface of the same leaf, as

was observed in F. pubescens (Figs. 3C and 3D), A. citrinum (Figs. 4C and 4D), B. sciuripabulum

(Figs. 4E and 4F), C. lateriflora (Figs. 4G and 4H), and T. cyrtanthum (Figs. 5C and 5D.

The straight pattern of anticlinal cell walls in leaflet epidermises was observed in eight

species. Amphilophium paniculatum (Figs 5G and 5H) and B. ramentacea (Figs. 5I and 5J)

demonstrated straight patterns on both leaflet surfaces. Anemopaegma citrinum (Fig. 4C), B.

sciuripabulum (Fig. 4E), C. lateriflora (Fig. 4G), P. venusta (Fig. 5A) and T. cyrtanthum (Fig. 5C)

showed a straight pattern only on their adaxial surface, and A. crucigerum only on the abaxial

surface (Fig. 5F). A curved pattern of anticlinal cell walls in leaflet epidermises was observed only

on the adaxial surface of A. citrinum (Fig. 4C) and T. cyrtanthum (Fig. 5C), and on the abaxial surface of B. sciuripabulum (Fig. 4F), C. Draftlateriflora (Fig. 4H), and A. crucigerum (Fig. 5F). With respect to thickenings of the cell walls, eight species showed thicker anticlinal cell walls

on the adaxial surfaces: D. unguis-cati (Fig. 3A), T. parviflorum (Fig. 3G), X heterocalyx (Fig. 4A),

B. sciuripabulum (Fig. 4E), C lateriflora (Fig. 4G), P. venusta (Fig. 5A), A. paniculatum (Fig. 5G),

and B. ramentacea (Fig. 5I). Pits were observed at the angles of the anticlinal cell walls on the

adaxial surface of A. citrinum (Fig. 4C). In transverse section, the Bignonieae species showed

epidermal cells of four different types. The tabular type on both surfaces of B. sciuripabulum (Fig.

6A); the flat type was observed on the abaxial surface of F. pubescens (Fig. 6C); the voluminous

type was present on the adaxial surface of A. paniculatum (Fig. 6H); and the papillose type was

observed in three species: on both surfaces of A. paniculatum (Figs. 1A, 1B and 6B) and T.

cyrtanthum (Figs. 2C and 2E), and on the abaxial surface of T. dichotomum (Fig. 6I).

Types of trichomes

All species of Bignonieae (see Table 3) showed non-glandular and glandular trichomes on

their epidermises. Two distinct types of non-glandular trichomes were observed: branched (Fig. 6C)

11 © The Author(s) or their Institution(s) Botany Page 12 of 42

type, and simple, uniseriate type (Figs. 6D and 6E). The simple trichomes varied in terms of their numbers of cells (from two to six) and also by the type of cuticle (either thick, smooth, striate, warty, or rugose). Five types of glandular trichomes were observed in the Bignonieae species: capitate (Fig. 6F), stipitate (Fig. 6G), peltate (Figs. 6H), patelliform (Fig. 6I) and patelliform/cupular (Figs. 1E).

Simple trichomes were observed on the leaflet blades of nine species belonging to eight genera (Table 3). They were observed on both leaflet blade surfaces of eight species: F. pubescens

(Figs. 1C and 1D), A. citrinum (Fig. 1G), B. ramentacea, C. lateriflora (Fig. 2B), A. crucigerum

(Fig. 2D and 2F), T. dichotomum (Fig. 2H), T. parviflorum, and X. heterocalyx. They also were observed on the abaxial leaflet surface of P. venusta. Simple trichomes with cuticular warts were present on both surfaces of A. crucigerum (Figs. 2D, 2F and 5F), C. lateriflora (Fig. 2B), and on the abaxial surface of T. parviflorum. BranchedDraft trichomes were observed on both surfaces of the leaflet blade of F. pubescens (Fig. 6C).

The glandular-stipitate trichomes were observed only on the adaxial epidermis surface of T. dichotomum (Fig. 2H) and C. lateriflora (Fig. 6G), and the capitate glandular trichomes were present on both epidermal surfaces of F. pubescens (Fig. 6F). All species showed glandular peltate trichomes on both leaflet blade epidermis [ e.g. P. venusta (Fig. 1F), X. heterocalyx (Fig. 2A), C. lateriflora (Fig. 2B), F. pubescens (Fig. 1D), A. crucigerum (Figs. 2D, 2F, 5F and 6E), T. cyrtanthum (2E), B. sciuripabulum (Fig. 2G)] (Table 3). Glandular patelliform trichomes were observed in all species, mainly on their abaxial epidermal surface [e.g., A. paniculatum (Figs. 1A and 1B), T. cyrtanthum (Fig. 2C), and T. dichotomum (Fig. 6I)]. The glandular patelliform/cupular type was observed on the abaxial surface of P. venusta (Fig. 1F), A. citrinum (Fig. 1H), and on the adaxial surface of A. crucigerum (Fig. 6E).

Stomatal types, densities, and stomatal indices

12 © The Author(s) or their Institution(s) Page 13 of 42 Botany

All species showed hypostomatic leaflet epidermises. Four to nine types of stomata were

observed occurring on the same leaflet. Table 4 lists all of the different stomatal types observed on

the leaflet blade epidermises. The largest number of stomatal types was observed in the genus

Bignonia, with nine types in B. ramentacea (Fig. 5J showing five types), and seven in B.

sciuripabulum (Fig. 4F showing two types). Two species had six types of stomata each:

Anemopaegma citrinum (Fig. 4D, with two types), and T. dichotomum. Five species showed the

lowest numbers (four) of different stomatal types: D. unguis-cati (Fig. 1B), C. lateriflora (Fig. 4H,

showing two types), P. venusta (Fig. 5B, with two types), A. crucigerum (Fig. 5F, showing three

types), and A. paniculatum (Fig. 5H., with three types).

The anomotetracytic stomata type was predominant, and was observed in all 13 species

(Table 4, Figs. 3B, 4B, 5B, 5D, 5F, 5H and 5J). Anomocytic stomata were observed in twelve species (Figs. 3B, 3F, 3H, 4B, 4D, 4F, 4H,Draft 5B, 5D, 5F and 5H), except in F. pubescens. The anisocytic type was observed in ten species (Table 4). The brachyparacytic stomatal type was

observed in ten species (Table 4). The staurocytic pattern (Fig. 4D) was recorded in nine species,

and the cyclocytic type (Figs. 5F and 5H) was observed in five (Table 4). Hemiparacytic stomata

(Figs. 3H, 4H and 5D) were present in five species, and paracytic stomata were present in F.

pubescens (Fig. 3D) and three species of Tanaecium (Fig. 3H), while brachyparahexacytic stomata

(Fig. 4F) were observed only in species of Bignonia, thus representing a character for the genus and

their species. Amphicyclocytic stomata were only observed in B. ramentacea (Fig. 5J), thus

constituting a distinctive character for that species. Stomatal complexes were observed in all

species, throughout the epidermis or in part, with the stomata sharing two or more subsidiary cells,

as was observed for example in T. dichotomum (Fig. 3F), T. parviflorum (Fig. 3H), T. cyrthanthum

(Fig. 5D) and A. crucigerum (Fig. 5F).

Some species exhibited stomata between prominent cells, with evident striations such as T.

cyrtanthum (Fig. 7A). In others, they are surrounded by prominent and smooth cells as T.

dichotomum (Fig. 7B) and T. parviflorum (Fig. 7C); or they are between flat cells with rugose

13 © The Author(s) or their Institution(s) Botany Page 14 of 42

cuticle as in X. heterocalyx (Fig. 7D) and B. ramentacea (Fig. 7E). In addition, some stomata are encrusted in the rugose epidermis as was observed in B. sciuripabulum (Fig. 7F), or striate in C. lateriflora (Fig. 7G). Striate cuticle surrounding the stomata was observed in P. venusta (Fig. 7H), and randomic striae on the stomatal apparatus were observed in A. citrinum (Fig. 7I), D. unguis-cati

(Fig. 7J), F. pubescens (Fig. 7K), and A. crucigerum (Fig. 7L), sometimes reaching the guard cells.

Four species have stomata with differentiated parietal thickening of the guard cells: F. pubescens

(Fig. 3D), T. dichotomum (Fig. 3F), T. parviflorum (Fig. 3H) and T. cyrtanthum (Fig. 5D), and these types of thickenings such as grass-type and butterfly-type are differential characters for these four species of Fredericia and Tanaecium. All 13 species of Bignonieae showed stomata at the level of the epidermal cells, in transverse section, as was observed in B. sciuripabulum (Fig. 6A), for example. Five species, however, also showed stomata elevated above the epidermal cells when the stomatal pore is opened: T. cyrtanthum (Fig.Draft 7A), A. paniculatum (Fig. 6B and 7M), A. citrinum, C. lateriflora, and P. venusta. The alternation of the state of this character depends on the opening and closing movement of the stomatal pore.

Data concerning stomatal indices (SI), as observed under light microscopy, are presented in

Table 5 and Fig. 8A. The lowest SI index (6.21%) was observed in B. ramentacea, and the highest

(23.52%) in T. parviflorum. Species with the most similar values of their stomatal indices were members of the genus Tanaecium (T. dichotomum and T. cyrtanthum, with indices of 20.28% and

20.30%, respectively). Stomatal densities showed wide variations among the studied species (Table

5 and Fig. 8B). The highest density was observed in T. parvilorum (752.9 stomata/mm²), while A. citrinum and P. venusta had the lowest densities (averaging 81 and 76 stomata/mm² respectively).

Dolichandra unguis-cati and X. heterocalyx had very similar stomatal density values (92.6 stomata/mm² and 93.2 respectively).

Inorganic idioblasts

14 © The Author(s) or their Institution(s) Page 15 of 42 Botany

Another important character observed on the leaf epidermises of the Bignonieae species

studied was the presence of inorganic idioblasts of different types, which were observed in nine

species (Table 4). Prismatic crystals were observed in the epidermal cells on both surfaces of seven

species: A. crucigerum, C. lateriflora, D. unguis-cati, F. pubescens, T. cyrtanthum, T. dichothomum

and T. parviflorum. The prismatic crystals were present in the adaxial leaflet blade surface of

Bignonia ramentacea (Fig. 5I) and in T. dichotomum.

Crystals sands were observed in six genera and eight species. Three species (A. crucigerum,

D. unguis-cati, and T. cyrtanthum) had crystal sands in the cells of the epidermis, which were

visible on both sides of the leaflets. In Cuspidaria lateriflora, T. parviflorum and P. venusta the

crystal sands were only visible abaxially, while in Bignonia ramentacea and T. dichotomum the

crystals sands were visible adaxially. Druses crystals are present in the epidermalDraft cells of P. venusta (Fig. 5A), and idioblasts as styloids crystals in the epidermal cells of F. pubescens (Fig. 3C). In addition, raphides were only

observed in the intracellular spaces of A. crucigerum (Fig. 5E). The presence of raphides in A.

crucigerum and styloids in F. pubescens constitute the first record of these types of idioblasts for

Amphylophium and Fridericia.

Discussion

All studied individuals from Pico do Jabre are lianas except for T. parviflorum. The habit of

X. heterocalyx in our study site is a liana, which differs from the shrub habit of this species as

documented in the Flora do Brasil (2020, under construction).

According to Barthlott (1981), epicuticular waxes often show high variability between

morphologically related species or even between infra-specific categories. However, their high

micromorphological diversity, make them a valuable diagnostic tool for the taxonomy of various

groups, which serve to characterize genera, families or even higher categories. Additionally, they

play important roles in structuring the epidermal surface on a sub-cellular scale (Barthlott et al.

15 © The Author(s) or their Institution(s) Botany Page 16 of 42

2017). The epicuticular waxes observed here in all 13 species of Bignonieae showed different patterns and syntopism which have taxonomic significance and can provide a diagnostic value to the foliolar epidermis of the Bignonieae studied, using Barthlott’s (1998) classification of epicuticular waxes. Epicuticular waxes on the leaf and stem epidermises are thought to protect them from biotic and abiotic factors (Ahmad et al. 2015).

Morphologic and taxonomic considerations of epicuticular waxes in Bignoniaceae, such as those reported by Ogundipe and Wujek (2004) for the African species, have been less frequent.

Epicuticular waxes as granules were the most common type, observed on either one or both blade surfaces. Epicuticular waxes as granules are not exclusive of tribe Bignonieae, they are also found in some African Bignoniaceae genera belonging to other tribes: Crescentia L., Kigelia DC.,

Markhamia Seem. ex Baill., Newbouldia Seem. ex Bureau, Parmentiera DC., Rhodocolea Baill., Spathodea P.Beauv., Stereospermum Cham.,Draft and Tabebuia (Ogundipe and Wujek 2004). Epicuticular waxes of thread type, were exclusive to the epidermis of C. lateriflora

The presence of sinuous anticlinal cell walls on the abaxial surface of the leaflet epidermis in eleven species were also observed in other species of Dolichandra and Fridericia by González

(2013), in Pyrostegia venusta (Duarte and Jurgensen 2007; González 2013), and in Tanaecium

(Souza et al. 2007). African species of Bignoniaceae, on the contrary, have straight and curved anticlinal cell walls (Ogundipe and Wujek 2004; Ugbabe and Ayodele 2008). Epidermal cells with strongly sinuous anticlinal walls on both leaflet surfaces were observed in species of the genera

Fridericia, Tanaecium, and Xylophragma which also have taxonomic affinities (Lohmann and

Taylor 2014). The straight anticlinal cell wall pattern on the adaxial surface seen in Anemopaegma citrinum was also reported in Mauro et al. (2007) and Firetti-Ligieri et al. (2014) for other Brazilian species of Anemopaegma. The curved anticlinal cell walls in the adaxial surface, and the sinuous cell walls in the abaxial surface of Tanaecium cyrtanthum is a pattern similar to that recorded in

Souza et al. (2007) for Arrabidaea mutabilis Bureau & K.Schum. [now Tanaecium mutabile

(Bureau & K.Schum.) L.G.Lohmann in Lohmann and Taylor (2014)].

16 © The Author(s) or their Institution(s) Page 17 of 42 Botany

The smooth and striate cuticle patterns observed in Bignonieae in the present study were in

agreement with the Bignoniaceae pattern already recorded in Metcalfe and Chalk (1950) and in

Paliwal (1970). The thick cuticle observed on the leaf epidermises of most of the Bignonieae

species studied here, occurred mainly on cells with straight anticlinal cell walls. The thickening of

cellular walls in species that grow in very dry habitats is a character that helps to protect the internal

structures of the leaves from unfavorable environmental conditions (Krauss 1949).

Epidermal attachments, such as trichomes, exhibit wide variations within the Bignoniaceae,

especially in terms of their positions and structures (Seibert 1948). The epidermises of the 13

species analyzed here showed five different types of glandular and non-glandular trichomes,

corroborating the observations of Metcalfe and Chalk (1950) and Nogueira et al. (2013) for

Bignonieae species. Glandular and non-glandular trichomes are extremely common in species of Bignonieae (Nogueira et al. 2013). However,Draft according to Seibert (1948), the presence of “glands as represented in the family Bignoniaceae, may be utilized to advantage in a taxonomic treatment”.

In our work, the taxonomic contribution of trichome morphology was relevant at the specific level,

distinguishing F. pubescens with an exclusive type of branched trichomes, C. lateriflora with

glandular stipitate trichomes, and F. pubescens with capitate glandular trichomes, for example.

All Bignonieae species are hypostomatic and, according to Metcalfe and Chalk (1950), that is

a consistent pattern for Bignoniaceae. Santos et al. (2009) and Gonzalez (2013), however, recorded

amphistomatic leaves in species of Bignoniaceae belonging to the tribe Tecomeae (Tecoma) and the

Tabebuia alliance (Tabebuia and Handroanthus) and Firetti-Ligieri et al. (2014) observed

amphistomatic leaves in Anemopaegma (Bignonieae) – differing from the patterns observed in our

work (including Anemopaegma citrinum, which we classified as having hypostomatic leaves).

According to Metcalfe and Chalk (1950), stomata can have varying numbers of subsidiary

cells in Bignoniaceae, including Bignonieae. We observed ten different types of stomata in the 13

species of Bignonieae studied here, as based on the classification of Dilcher (1974) – which allowed

a more refined analysis of stomatal complexes. Five new types of stomata are recorded here for the

17 © The Author(s) or their Institution(s) Botany Page 18 of 42

first time in Bignoniaceae: amphicyclocytic, anomotetracytic, brachyparacytic, brachyparahexacytic, and hemiparacytic.

Brachyparahexacytic stomata were observed only on the leaflet epidermises of Bignonia species (B. ramentacea and B. sciuripabulum) and constitute a distinctive character for that genus and its species. Bignonia ramentacea also showed amphicyclocytic stomata, a differential character for that species. The presence of paracytic stomata only in species of Fridericia and Tanaecium (T. dichotomum and T. parviflorum) was previously reported by Metcalfe and Chalk (1950) who recognize them as one of the few groups of Bignoniaceae having stomata that type. In other species, stomata types did not have taxonomic relevance due to their overlapping types.

Diacytic stomata are common in the leaflet epidermises of African Bignoniaceae (Ogundipe and Wujek 2004; Ugbabe and Ayodele 2008), but there are no records for Bignonieae. Anomocytic type stomata have been the most cited forDraft different Bignoniaceae clades, as observed in the works of Mauro et al. (2007), Duarte and Jurgensen (2007), Ortolane et al. (2008), and Gonzalez (2013), corroborating the present report, as the anomocytic type is only slightly less common than the anomotetracytic type.

We report the first record of stomata with “grass-type” and “butterfly” thickenings of the guard-cells in species of the genera Fridericia and Tanaecium, which was also had not previously reported in Bignonieae. Due to the specificity of this feature, it constitutes a significant taxonomic criterion for those genera and, consequently for tribe Bignonieae. Similar results pointing to the taxonomic importance of these types of thickening in Angiosperms were reported for the Lauraceae

(Christophel 1996).

Regarding stomatal indices and stomatal densities, it was possible to verify that species within the same genus share closer values (Table 5), than species of different genera that are taxonomically closely related, as seen in the treatments of Fridericia, Tanaecium, and Xylophragma in Lohmann

(2006) and Lohmann and Taylor (2014). Fridericia pubescens, T. cyrtanthum, T. dichotomum, and

T. parviflorum showed the highest stomatal indices (exceeding 20%) and high stomatal densities

18 © The Author(s) or their Institution(s) Page 19 of 42 Botany

exceeding 381 stomata / mm²) and can be easily separated from the others species, which show a

maximum stomata index of 12% and a density of 212 stomata/mm² (Table 3).

Salisbury (1927) considered whether stomatal frequency was influenced by internal or

external factors (such as humidity), and suggested that plants with higher stomatal frequencies are

more common in xeric environments. Xu and Zhou (2008) demonstrated the influence of different

environmental parameters on stomatal density, and suggested that water deficits lead to density.

That observation could explain the presence of the high densities and stomatal indices observed in

our work. It does not, however, explain the lower indices and stomatal densities in species such as

X. heterocalyx, collected in the same area and at the same altitude as the species with the highest

indices.

Inorganic idioblasts were reported for Bignoniaceae in previous works (Metcalfe and Chalk 1950; Gardner 1977), although the presenceDraft of inorganic idioblasts as raphides in A. crucigerum and styloids in F. pubescens constituted the first records for those genera, and also in Bignonieae.

The occurrence of idioblasts as druse type in P. venusta is an exclusive character for the species,

which was already registered in material from the south of Brazil (Duarte and Jurgensen (2007).

Thus, in the Bignonieae species analyzed here, the presence of druses, raphides, and styloids

showed taxonomic relevance due to their specificity of occurrence and their different characteristics

in each species. According to Gardner (1977) and Prychid and Rudall (1999), inorganic idioblasts

(such as calcium oxalate crystals) are the products of secondary metabolic responses to pathogens

and herbivores, while Cutler et al. (2008) viewed them as being part of a species' physiology.

Metcalfe and Chalk (1950) recorded the presence of nuclear crystalloids in the leaflet

epidermis of Arrabidea species. Thus, this work expanded records for different types of crystals for

the epidermal characterization of different species of Bignonieae, as well as of Bignoniaceae. The

presence of the cellular inclusions can often provide valuable taxonomic characters, according to

Franceschi and Horner (1980), and also reported by Prychid and Rudall (1999) and Prychid et al.

(2003) in some groups of monocotyledons. In the Bignonieae species analyzed here, the presence of

19 © The Author(s) or their Institution(s) Botany Page 20 of 42

druses, raphides, and styloids showed taxonomic relevance due to their specificity of occurrence and their different characteristics in each species.

Conclusions

The leaflet blade of 13 Bignonieae species from Pico do Jabre showed a set of distinctive characters that could be used to separate them taxonomically. Some characters were relevant to characterize genera, while other were relevant to characterize species. A wide variety of epicuticular waxes was observed on the leaf epidermis of the species studied here, and even within species (showing syntopism). These epicuticular waxes showed relative taxonomic importance for the species studied here with some exceptions. The cuticle morphology on the epidermis and, especially, around the stomata is very distinctive, and taxonomically significant at the specific level. The thickening of the cuticle of the guard-cells,Draft as grass-type, was relevant at the generic level to characterize species of Fridericia and Tanaecium. The different types of stomata were also taxonomically significant, such as the brachyparahexacytic type in Bignonia species, and the amphyciclocytic type only present in Bignonia ramentacea. The stomatal indices and stomatal densities were also relevant for distinction between Fridericia and Tanaecium, which showed the highest indices. Other characters were relevant at the specific level, such as the presence of idioblasts as druses in P. venusta, raphides in A. crucigerum, and styloids in F. pubescens. Some types of trichomes were useful to separate species, such as the branched and the capitate-glandular types only observed in Fridericia pubescens, the glandular-stipitate type present in Cuspidaria lateriflora. The set of different characters of cuticles, epicuticular waxes, idioblasts, stomata and trichomes provided an additional tool to support the taxonomy of Bignonieae species from Pico do

Jabre.

Acknowledgements

20 © The Author(s) or their Institution(s) Page 21 of 42 Botany

The authors thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

(CAPES) for the scholarships awarded to RFLS, ALS and EAVS. The Conselho Nacional de

Desenvolvimento Científico e Tecnológico (CNPq) awarded a grant to MFA. We are grateful to

Maria de Fátima de Araújo, for her support during the fieldwork, Annelise Frazão for collaborating

with the identification of T. dichotomum, Leonardo Félix, Julissa Roncal, Christian R. Lacroix, and

the anonymous reviewers for their helpful comments and suggestions that greatly improved our

manuscript, Meyson Cassio do Nascimento for technical support with SEM, Roy Funch and Alain

Chautems for their valuable revisions of the English and French languages, respectively.

References

Agra, M.F., Barbosa, M.R.V., and Stevens, W.D. 2004. Levantamento florístico preliminar do Pico do Jabre, Paraíba, Brasil. In BrejosDraft de altitude em Pernambuco e Paraíba: História natural, Ecologia e Conservação. Edited by K.C. Porto., J.J.P. Cabral and M. Tabarelli, Ministério do

Meio Ambiente, Brasília. pp. 123—138.

Aguiar, T.V., Sant’anna-Santos, B.F., Azevedo, A.A., and Ferreira, R.S. 2007. ANATI QUANTI:

software de análises quantitativas para estudos em anatomia vegetal. Planta daninha, 25(4):

649—659.

Ahmad, H.M., Rahman, M.U., Ali, Q., and Awan, S.I. 2015. Plant cuticular waxes: a review on

functions, composition, biosyntheses mechanism and transportation. Life Sci. J. 12(4): 60—67.

Alcantara, S., and Lohmann, L.G. 2010. Evolution of floral morphology and pollination system in

Bignonieae (Bignoniaceae). Am. J. Bot. 97(5): 782—796. doi:10.3732/ajb.0900182

Barthlott, W., Neinhuis, C., Cutler, D., Ditsch, F., Meusel, I., Theisen, I., and Wilhelmi, H. 1998.

Classification and terminology of plant epicuticular waxes. Bot. J. Linn. Soc. 126(3): 227—236.

doi: 10.1111/j.1095-8339.1998.tb02529.x

Barthlott, W., Mail, M., Bhushan, B., and Koch, K. 2017. Plant Surfaces: Structures and Functions

for Biomimetic Innovations. Nano-micro Lett. 9(23):1—40. doi: 10.1007/s40820-016-0125-1

21 © The Author(s) or their Institution(s) Botany Page 22 of 42

Barthlott, W. 1981. Epidermal and seed surface characters of plants: systematic applicability and

some evolutionary aspects. Nord. J. Bot. 1: 345—355. doi: 10.1111/j.1756-

1051.1981.tb00704.x.

BFG - The Brazil Flora Group 2015. Growing knowledge: an overview of Seed Plant diversity in

Brazil. Rodriguésia, 66(4): 1085—1113. doi: 10.1590/2175-7860201566411.

Bridson, D., and Forman, L. 1992. The herbarium handbook. Kew: Royal Botanic Gardens.

Bukatsh, F. 1972. Benerkungen zur doppelfarbung astrablau-safranin. Microkosmos, 61: 1—255.

Carlquist, S. 1961. Comparative Plant Anatomy. Holt, Rinehart and Winston.

Chamberlain, C.l. 1932. Methods in plant histology. 5th edition. The University of Chicago Press,

Chicago.

Christophel, D.C., Kerrigan, R., and Rowett, A.I. 1996. The use of cuticular features in the taxonomy of the Lauraceae. Ann. Mo. Bot. Gard.Draft 419—432. doi: 10.2307 / 2399871. Cunha, M.C., and Silva-Júnior, M.C. 2014. Flora e estrutura de floresta estacional semidecidual

montana nos estados da Paraíba e Pernambuco. Nativa, 2(2): 95—102. doi: 10.14583/2318-

7670.v02n02a06

Cutler, D.F., Botha, C.E.J., and Stevenson, D.W. 2008. Plant anatomy: an applied approach.

Blackwell Publishing, Malden, Massachusetts, USA.

Dickison, W.C. 1975. The bases of angiosperm phylogeny: vegetative anatomy. Ann. Mo. Bot.

Gard. 62(3): 590—620.

Dilcher, D.L. 1974. Approaches to the identification of angiosperm leaf remains. Bot. Rev. 40(1):

1—157.

Duarte, M.R., and Jurgensen, I. 2007. Diagnose Morfoanatômica de Folha e Caule de Pyrostegia

venusta (Ker Gawl.) Miers, Bignoniaceae. Lat. Am. J. Pharm. 26(1): 70—75.

Firetti-Leggieri, F., Lohmann, L.G., Semir, J., Damarco, D., and Castro, M.M. 2014. Using leaf

anatomy to solve taxonomic problems within the Anemopaegma arvense species complex

22 © The Author(s) or their Institution(s) Page 23 of 42 Botany

(Bignonieae, Bignoniaceae). Nord. J. Bot. 32(1): 620—631. doi: 10.1111/j.1756-1051.

2013.00275.x

Flora of Brazil 2020 under construction. Jardim Botânico do Rio de Janeiro. Available at:

. Accessed on: May. 2020.

Fróes, F.F.P.C., Gama, T.S.S., Feio, A.C., Demarco, D., and Aguiar-Dias, A.C.A. 1015. Structure

and distribution of glandular trichomes in three species of Bignoniaceae. Acta Amaz. 45(4):

347—354. doi: 10.1590/1809-4392201404393.

Franceschi, V.R., and Horner, H.T. 1980. Calcium oxalate crystals in plants. Bot. Rev. 46(1): 361—

427. doi: 10.1007/BF02860532

Gardner, R.O. 1977. Systematic distribution and ecological function of the secondary metabolites of

the Rosidae-Asteridae. Biochem. Syst. Ecol. 5(1): 29—35. doi: 10.1016/0305-1978(77)90015- 1 Draft Gentry, A.H. 1980. Bignoniaceae, Part I, Tribes Crescentieae and Tourretieae. Flora Neotrop.

Monogr. 25(1):1—131.

Gentry, A.H. 1992. A synopsis of Bignoniaceae ethnobotany and economic botany. Ann. Mo. Bot.

Gard. 79(1): 53—64. doi: 10.2307/2399809

Gerolamo, C.S., and Angyalossy, V. 2017. Wood anatomy and conductivity in lianas, shrubs and

trees of Bignoniaceae. IAWA J. 38(3): 412—432. doi: 10.1163/22941932-20170177

Gonzalez, A.M. 2013. Indumento, nectarios extraflorales y anatomía foliar em Bignoniáceas de la

Argentina. Bol. Soc. Argent. Bot. 48(2): 221—245. doi: 10.31055/1851.2372.v48.n2.6207

Johansen, D.A. 1940. Plant Microtechnique. McGraw-Hill Book. New York.

Juniper, B.E., and Jeffree, C.E. 1983. Plant surfaces. London: Edward Arnold. London, UK. 93pp

Kraus, J.E., and Arduin, M. 1997. Manual básico de métodos em morfologia vegetal. Serope´dica:

Editora da Universidade Rural do Rio de Janeiro, Brasil. 198 pp.

Krauss, B.H. 1949. Anatomy of the vegetative organs of the pineapple, Ananas comosus (L.) Merr.

(Continued) II. The leaf. Int. J. Plant Sci. 110(3): 333—404.

23 © The Author(s) or their Institution(s) Botany Page 24 of 42

Lohmann, L.G. 2006. Untangling the phylogeny of Neotropical lianas (Bignonieae, Bignoniaceae).

Am. J. Bot. 93(2): 304—315. doi: 10.3732/ajb.93.2.304

Lohmann, L.G., and Taylor, C.M. 2014. A new generic classification of Bignonieae (Bignoniaceae)

based on molecular phylogenetic data and morphological synapomorphies. Ann. Mo. Bot.

Gard. 99(3): 348—489. doi: 10.3417/2003187

Mauro, C., Pereira, A.M.S., Silva, C.P., Missima, J., Ohnuki., and Rinaldi, R.B. 2007. Estudo

anatômico das espécies de cerrado Anemopaegma arvense (Vell.) Stellf. ex de Souza (catuaba),

Zeyheria montana Mart. (bolsa-de-pastor) e Jacaranda decurrens Chamisso (caroba) –

Bignoniaceae. Rev. Bras. Farmacogn. 17(2): 262—265. doi: 10.1590/S0102-

695X2007000200021

Metcalfe, C.R. 1946. The systematic anatomy of the vegetative organs of the Angiosperms. Biol. Rev. Camb. Philos. Soc. 21(4): 159—172.Draft doi: 10.1111/j.1469-185X.1946.tb00322.x Metcalfe, C.R., and Chalk, L. 1950. Anatomy of the dicotyledons: leaves, stem and wood in relation

to taxonomy, with notes on economic uses. Clarendon Press, Oxford.

Metcalfe, C.R., and Chalk, L. 1979. Anatomy of dicotyledons. London: Oxford University Press.

Nogueira, A., El Otrra, J.H.L., Guimarães, E., Machado, S.R., and Lohmann, L.G. 2013. Trichome

structure and evolution in Neotropical lianas. Ann. Bot. 112(7): 1331—1350.

doi:10.1093/aob/mct201

Ogundipe, O.T., and Wujek, D.E. 2004. Foliar anatomy on twelve genera of Bignoniaceae

(Lamiales). Acta Bot. Hung. 46(3): 337—361. doi: 10.1556/ABot.46.2004.3-4.7

Olmstead, R.G., Zjhra, M.L., Lohmann, L.G., Grose, S.O., and Eckert, A.J. 2009. A molecular

phylogeny of Bignoniaceae. Am. J. Bot. 96(9): 1731—1743. doi:10.3732/ajb.0900004

Ortolani, F.A., Mataqueiro, M.F., Moro, J.R., Moro, F.V., and Damião-Filho, C.F. 2008. Morfo-

anatomia de plântulas e número cromossômico de Cybistax antisyphilitica (Mart.) Mart.

(Bignoniaceae). Acta Bot. Bras. 22(2): 345—353. doi: 10.1590/S0102-33062008000200005

Pace, M.R., Lohmann, L.G., and Angyalossy, V. 2009. The rise and evolution of the cambial

variant in Bignonieae (Bignoniaceae). Evol. Dev. 11(5): 465—479. doi: 10.1111/j.1525-

142X.2009.00355.x

Pace, M.R., Lohmann, L.G., Olmstead, R.G., and Angyalossy, V. 2015. Wood anatomy of major

Bignoniaceae clades. Plant Syst. Evol. 301(3): 967—995. doi: 10.1007/s00606-014-1129-2

Paliwal, G.S. 1970. Epidermal structure and ontogeny of stomata in some Bignoniaceae. Flora,

159(1/2): 124—32. doi: 10.1016/S0367-2530(17)31021-6

Payne, W.W. 1978. A glossary of plant hair terminology. Brittonia, 30(2): 239—255. Doi:

10.2307/2806659

Prychid, C.J., and Rudall, P.J. 1999. Calcium oxalate crystals in monocotyledons: a review of their

structure and systematics. Ann. Bot. 84(1): 725—739. doi.org/10.1006/anbo.1999.0975 Prychid, C.J., Rudall, P.J., and Gregory, M.Draft 2003. Systematics and biology of silica bodies in monocotyledons. Bot. Rev. 69(1): 377—440. doi: 10.1016/j.sajb.2014.06.004

Rocha, E.A., and Agra, M.F. 2002. Flora of the Pico do Jabre, Paraíba, Brazil: Cactaceae Juss. Acta

Bot. Bras. 16(1): 15—21. doi:10.1590/S0102-33062002000100004

Salisbury, E.J. 1927. On the cause and ecological significance of stomatal Frequency with special

reference to the woodland Flora. Philos. Trans. R. Soc. Biol. Sci. 216(1): 1—65.

Santos, S.R. 2017. A atual classificação do antigo gênero Tabebuia (Bignoniaceae), sob o ponto de

vista da anatomia da madeira. Balduinia, 58: 10—24. doi: 10.5902/2358198028146

Santos, G.M.A. 1995. Wood Anatomy, Chloroplast DNA, and Flavonoids of the Tribe Bignonieae

(Bignoniaceae). Ph.D. Dissertation, University of Reading, Reading, U.K.

Seibert, R.J. 1948. The use of glands in a taxonomic consideration of the family Bignoniaceae. Ann.

Mo. Bot. Gard. 35(2): 123—137. doi: 10.2307/2394389

Silva, A.M.L., Costa, M.F.B., Leite, V.G., Rezende, A.A., and Teixeira, F.P. 2009. Anatomia foliar

com implicações taxonômicas em espécies de ipês. Hoehnea, 36(2): 329—338. doi:

10.1590/S2236-89062009000200010

Smith, F.H., and Smith, E.C. 1942. Anatomy of the inferior ovary of Darbya. Am. J. Bot. 29: 464—

471. doi: 10.1002/j.1537-2197.1942.tb10236.x

Souza, L.A., Lopes, W.A.L., and Almeida, O.J.G. 2007. Morfoanatomia da plântula e do tirodendro

de Arrabidaea mutabilis Bureau & K. Schum. (Bignoniaceae). Acta Sci. Biol. Sci. 29(2):

131—136. doi: 10.4025/actascibiolsci.v29i2.599

Sousa-Baena, M.S., Sinha, N.R., and Lohmann, L.G. 2014. Evolution and development of tendrils

in Bignonieae (Lamiales, Bignoniaceae). Ann. Mo. Bot. Gard. 99(3): 323—347. doi:

10.3417/2011018

Thiers, B. (2019) [continuously updated]. Index Herbariorum: A global directory of public herbaria

and associated staff. New York Botanical Garden's Virtual Herbarium.

http://sweetgum.nybg.org/science/ih/. Ugbade, G.E., and Ayodele, A.E. 2008. FoliarDraft epidermal studies in the Family Bignoniaceae Juss. in Nigeria. Afr. J. Agric. Res. 3(2): 154—166. Available from

http://www.academicjournals.org/AJAR

Wilkinson, H.P. 1979. Plant surface. In Anatomy of the Dicotyledon. Edited by C.R. Metcalfe and L.

Chalk. Clarendon Press, Oxford, pp. 97—162.

Xu, Z., and Zhou, G. 2008. Responses of leaf stomatal density to water status and its relationship

with photosynthesis in a grass. J. Exp. Bot. 59(12): 3317—3325. doi:10.1093/jxb/ern185

Figure Captions

Fig. 1. Epicuticular waxes, cuticles and trichomes on leaflet blade epidermises of Bignonieae

observed with scanning electron microscopy: (A and B) Amphilophium paniculatum (Agra et al.

7112): patelliform trichomes on adaxial and abaxial surfaces. (C and D) Fridericia pubescens (Agra

et al. 7115): (C) simple trichomes on adaxial surface, (D) peltate and simple trichomes on abaxial

surface. (E and F) Pyrostegia venusta (Lopes 257): patelliform trichome on adaxial surface, and

patelliform/cupular and peltate trichomes on abaxial surface. (G and H) Anemopaegma citrinum

(Lopes 239): simple trichomes on adaxial surface and patelliform/cupular trichomes on abaxial

surface. Legends: pa= patelliform trichome, pc = patelliform/cupular trichome, pt = peltate trichome,

tr = simple trichome. Arrowheads (epicuticular waxes): cr = crusts, fl = fissured layer, gr = granules, pl = platelets. Scale bars: (A, C, E, G) = 20μm,Draft (B, D, F, H) = 10μm.

Fig. 2. Epicuticular waxes and trichomes on leaflet blade epidermises of Bignonieae observed with

scanning electron microscopy: (A—D) Adaxial surfaces (A) Xylophragma heterocalyx (Lopes 261):

peltate trichome. (B) Cuspidaria lateriflora (Agra et al. 3932): peltate and simple trichomes. (C)

Tanaecium cyrtanthum (Lopes 263): patelliform trichome. (D) Amphilophium crucigerum (Lopes

135): peltate and simple trichomes. (E—H) Abaxial surfaces. (E) Tanaecium cyrtanthum (Lopes 263):

peltate trichome. (F) Amphilophium crucigerum (Lopes 135): peltate and simples trichomes. (G)

Bignonia sciuripabulum (Lopes 238): peltate trichome. (H) Tanaecium dichotomum (Lopes 259):

simple and glandular-stipitate trichomes. Legends: gs = glandular-stipitate, pa = patelliform trichome,

pt = peltate trichome, tr = simple trichome. Arrowheads (epicuticular waxes): cr = crusts, fl = fissured

layer, gr = granules, pl = platelets. Scale bars: (A, B, C, E, F, G) = 10μm, (D and H) = 20μm.

Fig. 3. Anticlinal cell walls, idioblasts and stomata on leaflet blade of Bignonieae observed with light

microscopy: (A and B) Dolichandra unguis-cati (Lopes 246): sinuous anticlinal cell walls on adaxial

and abaxial surfaces. (C and D) Fridericia pubescens (Agra et al. 7115): sinuous anticlinal cell walls on adaxial and abaxial surfaces, styloids (arrowheads) in adaxial surface. (E and F) Tanaecium dichotomum (Lopes 259): sinuous anticlinal cell walls on adaxial and abaxial surfaces, (G and H)

Tanaecium parviflorum (Lopes 240): sinuous anticlinal cell walls on adaxial and abaxial surfaces.

Stomata: As = anisocytic, An = anomocytic, At = anomotetracytic, Bp = brachyparacytic, Hp = hemiparacytic, Pr = paracytic. Scale bars = 50μm.

Fig. 4. Anticlinal cell walls and stomata on leaflet blade of Bignonieae observed with light microscopy, in front view: (A and B) Xylophragma heterocalyx (Lopes 261): sinuous anticlinal cell walls on adaxial and abaxial surfaces. (C and D) Anemopaegma citrinum (Lopes 239): curved and straight anticlinal walls on adaxial surface, and sinuous type on abaxial surface. (E and F) Bignonia sciuripabulum (Lopes 238): straight and sinuousDraft anticlinal cell walls on adaxial and abaxial surfaces. (G and H) Cuspidaria lateriflora (Agra et al. 3932): straight, curved and sinuous anticlinal cell walls on adaxial and abaxial surfaces. Stomata: An= anomocytic, At= anomotetracytic, Bh= brachyparahexacytic, Hp= hemiparacytic, St= staurocytic. pt= peltate trichome, ts= trichome scar.

Scale bars = 50μm.

(arrowhead). (G and H) Amphilophium paniculatum (Agra et al. 7112): straight anticlinal cell walls on adaxial and abaxial surfaces, detail of an anomotetracytic stoma on the abaxial surface. (I and J)

Bignonia ramentacea (Agra et al. 4022): straight anticlinal cell walls on adaxial and abaxial surfaces,

prismatic crystals (arrowheads) on adaxial surface. Stomata: Ap = amphicyclocytic, As = anisocytic,

An = anomocytic, At = anomotetracytic, Bp = brachyparacytic, Cc = ciclocytic, Hp = hemiparacytic,

pa = patelliform trichome, pt = peltate trichome, tr = simple trichome. Scale bars = 50μm.

Fig. 6. Leaflet epidermis observed with light microscopy, in transverse section: (A) Tabular

epidermal cells of Bignonia sciuripabulum (Lopes 238). (B) Stomata above the level of papillose

epidermis of Amphilophium paniculatum (Agra et al. 7112). (C) Branched and simple trichomes on

abaxial surface of Fridericia pubescens (Lopes 256). (D) Simple trichome of Fridericia pubescens.

(E) Simple and patelliform/cupular trichomes on adaxial surface of Amphilophium crucigerum (Lopes

243). (F) Capitate glandular trichome of Fridericia pubescens (Agra et al. 7115). (G) Glandular

stipitate trichome of Cuspidaria lateriflora (Agra et al. 3932). (H) Peltate trichome in the voluminous epidermal cells on adaxial surface of A. paniculatumDraft. (I) Patelliform trichome on abaxial surface of Tanaecium dichotomum (Lopes 259). Legends: br = branched trichome, cg = Capitate glandular

trichome, ct = cuticle, ep = epidermis, pa = patelliform trichome, pc = patelliform/cupular trichome,

pp = palisade parenchyma, gs = glandular-stipitate trichome, sp = spongy parenchyma, st = stomata,

tr = simple trichome. Scale bars: (A) = 100μm and (B, C, D, E, F, G, H) = 50 μm.

Fig. 7. Stomata on leaflet blade epidermises of Bignonieae observed with scanning electron

microscopy: (A) Tanaecium cyrtanthum (Lopes 243). (B) Tanaecium dichotomum (Lopes 259). (C)

Tanaecium parviflorum (Lopes 240). (D) Xylophragma heterocalyx (Lopes 261). (E) Bignonia

ramentacea (Agra et al. 4022). (F) Bignonia sciuripabulum (Lopes 238). (G) Cuspidaria lateriflora

(Agra et al. 3932). (H) Pyrostegia venusta (Lopes 257). (I) Anemopaegma citrinum (Lopes 239). (J)

Dolichandra unguis-cati (Lopes 246). (K) Fridericia pubescens (Agra et al. 7115). (L) Amphilophium

crucigerum (Lopes 263). (M) Amphilophium paniculatum (Agra et al. 7112). Scale bars = 2μm.

Fig. 8. (A) Stomatal indices and (B) stomatal densities in Bignonieae species.

Draft

Fig. 1. Epicuticular waxes, cuticles and trichomes on leaflet blade epidermises of Bignonieae observed with scanning electron microscopy: (A and B) Amphilophium paniculatum (Agra et al. 7112): patelliform trichomes on adaxial and abaxial surfaces. (C and D) Fridericia pubescens (Agra et al. 7115): (C) simple trichomes on adaxial surface, (D) peltate and simple trichomes on abaxial surface. (E and F) Pyrostegia venusta (Lopes 257): patelliform trichome on adaxial surface, and patelliform/cupular and peltate trichomes on abaxial surface. (G and H) Anemopaegma citrinum (Lopes 239): simple trichomes on adaxial surface and patelliform/cupular trichomes on abaxial surface. Legends: pa= patelliform trichome, pc = patelliform/cupular trichome, pt = peltate trichome, tr = simple trichome. Arrowheads (epicuticular waxes): cr = crusts, fl = fissured layer, gr = granules, pl = platelets. Scale bars: (A, C, E, G) = 20μm, (B, D, F, H) = 10μm.

61x82mm (300 x 300 DPI)

Draft

Fig. 2. Epicuticular waxes and trichomes on leaflet blade epidermises of Bignonieae observed with scanning electron microscopy: (A—D) Adaxial surfaces (A) Xylophragma heterocalyx (Lopes 261): peltate trichome. (B) Cuspidaria lateriflora (Agra et al. 3932): peltate and simple trichomes. (C) Tanaecium cyrtanthum (Lopes 263): patelliform trichome. (D) Amphilophium crucigerum (Lopes 135): peltate and simple trichomes. (E—H) Abaxial surfaces. (E) Tanaecium cyrtanthum (Lopes 263): peltate trichome. (F) Amphilophium crucigerum (Lopes 135): peltate and simples trichomes. (G) Bignonia sciuripabulum (Lopes 238): peltate trichome. (H) Tanaecium dichotomum (Lopes 259): simple and glandular-stipitate trichomes. Legends: gs = glandular-stipitate, pa = patelliform trichome, pt = peltate trichome, tr = simple trichome. Arrowheads (epicuticular waxes): cr = crusts, fl = fissured layer, gr = granules, pl = platelets. Scale bars: (A, B, C, E, F, G) = 10μm, (D and H) = 20μm.

61x81mm (300 x 300 DPI)

Draft

Fig. 3. Anticlinall cell walls, idioblasts and stomata on leaflet blade of Bignonieae observed with light microscopy in front view:(A and B) Dolichandra unguis-cati (Lopes 246): sinuous anticlinal cell walls on adaxial and abaxial surfaces. (C and D) Fridericia pubescens (Agra et al. 7115): sinuous anticlinal cell walls on adaxial and abaxial surfaces, styloids (arrowheads) on adaxial surface. (E and F) Tanaecium dichotomum (Lopes 259): sinuous anticlinal cell walls on adaxial and abaxial surfaces, (G and H) Tanaecium parviflorum (Lopes 240): sinuous anticlinal cell walls on adaxial and abaxial surfaces. Stomata: As = anisocytic, An = anomocytic, At = anomotetracytic, Bp = brachyparacytic, Hp = hemiparacytic, Pr = paracytic. Scale bars = 50μm.

39x63mm (300 x 300 DPI)

Draft

Fig. 4. Anticlinal cell walls and stomata on leaflet blade of Bignonieae observed with light microscopy, in front view: (A and B) Xylophragma heterocalyx (Lopes 261): sinuous anticlinal cell walls on adaxial and abaxial surfaces. (C and D) Anemopaegma citrinum (Lopes 239): curved and straight anticlinal walls on adaxial surface, and sinuous type on abaxial surface. (E and F) Bignonia sciuripabulum (Lopes 238): straight and sinuous anticlinal cell walls on adaxial and abaxial surfaces. (G and H) Cuspidaria lateriflora (Agra et al. 3932): straight, curved and sinuous anticlinal cell walls on adaxial and abaxial surfaces. Stomata: An= anomocytic, At= anomotetracytic, Bh= brachyparahexacytic, Hp= hemiparacytic, St= staurocytic. pt= peltate trichome, ts= trichome scar. Scale bars = 50μm.

39x62mm (300 x 300 DPI)

Draft

Fig. 5. Anticlinal cell walls and stomata on leaflet blade epidermises of Bignonieae observed with light microscopy, in front view: (A and B) Pyrostegia venusta (Lopes 257): straight and sinuous anticlinal cell walls on adaxial and abaxial surfaces, with druses (arrowheads) in the epidermis of adaxial surface. (C and D) Tanaecium cyrtanthum (Lopes 263): curved and sinuous anticlinal cell walls on adaxial and abaxial surfaces. (E and F) Amphilophium crucigerum (Lopes 243): sinuous and straight anticlinal cell walls on adaxial and abaxial surfaces, and adaxial surface with raphides (arrowhead). (G and H) Amphilophium paniculatum (Agra et al. 7112): straight anticlinal cell walls on adaxial and abaxial surfaces, detail of an anomotetracytic stoma on the abaxial surface. (I and J) Bignonia ramentacea (Agra et al. 4022): straight anticlinal cell walls on adaxial and abaxial surfaces, prismatic crystals (arrowheads) on adaxial surface. Stomata: Ap = amphicyclocytic, As = anisocytic, An = anomocytic, At = anomotetracytic, Bp = brachyparacytic, Cc = ciclocytic, Hp = hemiparacytic, pa = patelliform trichome, pt = peltate trichome, tr = simple trichome. Scale bars = 50μm.

39x78mm (300 x 300 DPI)

Draft

Fig. 6. Leaflet epidermis observed with light microscopy, in transverse section: (A) Tabular epidermal cells of Bignonia sciuripabulum (Lopes 238). (B) Stomata above the level of papillose epidermis of Amphilophium paniculatum (Agra et al. 7112). (C) Branched and simple trichomes on abaxial surface of Fridericia pubescens (Lopes 256). (D) Simple trichome of Fridericia pubescens . (E) Simple and patelliform/cupular trichomes on adaxial surface of Amphilophium crucigerum (Lopes 243). (F) Capitate glandular trichome of Fridericia pubescens (Agra 7115). (G) Glandular stipitate trichome of Cuspidaria lateriflora (Agra et al. 3932). (H) Peltate trichome in the voluminous epidermal cells on adaxial surface of A. paniculatum. (I) Patelliform trichome on abaxial surface of Tanaecium dichotomum (Lopes 259). Legends: br = branched trichome, cg. Capitate glandular trichome, ct = cuticle, ep = epidermis, pa = patelliform trichome, pc = patelliform/cupular trichome, pp = palisade parenchyma, gs = glandular-stipitate trichome, sp = spongy parenchyma, st = stomata, tr = simple trichome. Scale bars: (A) = 100μm and (B, C, D, E, F, G, H) = 50 μm.

107x102mm (300 x 300 DPI)

Draft

Fig. 7. Stomata on leaflet blade epidermises of Bignonieae observed with scanning electron microscopy: (A) Tanaecium cyrtanthum (Lopes 243). (B) Tanaecium dichotomum (Lopes 259). (C) Tanaecium parviflorum (Lopes 240). (D) Xylophragma heterocalyx (Lopes 261). (E) Bignonia ramentacea (Agra et al. 4022). (F) Bignonia sciuripabulum (Lopes 238). (G) Cuspidaria lateriflora (Agra et al. 3932). (H) Pyrostegia venusta (Lopes 257). (I) Anemopaegma citrinum (Lopes 239). (J) Dolichandra unguis-cati (Lopes 246). (K) Fridericia pubescens (Agra et al. 7115). (L) Amphilophium crucigerum (Lopes 263). (M) Amphilophium paniculatum (Agra et al. 7112). Scale bars = 2μm.

64x64mm (300 x 300 DPI)

Draft

Fig. 8. (A) Stomatal indices and (B) stomatal densities in Bignonieae species.

214x223mm (300 x 300 DPI)

Table 1. Species and specimens of the Bignonieae used in the blade leaflets epidermis analyses.

Species Habit Collector and number (Herbarium acronym)

Amphilophium crucigerum (L.) L.G.Lohmann Liana M.F. Agra et al. 5013 (JPB)* R. Lopes 135 (EAN) R. Lopes 243 (EAN) Amphilophium paniculatum (L.) Kunth Liana M.F. Agra & P.C.Silva 4873 (JPB)* M.F. Agra et al. 2688 (JPB)*

Anemopaegma citrinum Mart. ex DC. Liana M.F. Agra et al. 2629 (JPB)* R. Lopes 239 (EAN) R. Lopes 255 (EAN) Bignonia ramentacea (Mart. Ex DC.) L.G.Lohmann Liana M.F. Agra et al. 4022 (JPB)* R. Lopes 253 (EAN) Lopes 254 (EAN) Bignonia sciuripabulum (K. Schum.) L.G.Lohmann Liana M.F. Agra et al. 3935 (JPB)* M.F. Agra et al. 4654 (JPB)* R. Lopes 238 (EAN) Cuspidaria lateriflora (Mart.) DC. Draft Liana M.F. Agra et al. 3932 (JPB)* Dolichandra unguis-cati (L.) L.G.Lohmann Liana M.F. Agra et al. 4113 (JPB)* M.F. Agra et al. 4792 (JPB)* R. Lopes 246 (EAN) Fridericia pubescens (L.) L.G.Lohmann Liana M.F. Agra et al. 1984 (JPB)* M.F. Agra et al. 7115 (JPB)* Lopes 256 (EAN) Pyrostegia venusta (Ker: Gawl.) Miers Liana M.F. Agra et al. 4371 (JPB)* M.F. Agra et al. 4398 (JPB)* R. Lopes 257 (EAN) Tanaecium cyrtanthum (Mart. ex DC.) Bureau & K.Schum. Liana R. Lopes 263 (EAN)

Tanaecium dichotomum (Jacq.) Kaehler & LG.Lohmann Liana R. Lopes 259 (EAN)

Tanaecium parviflorum (Mart. ex DC.) Kaehler & L.G.Lohmann Shrub M.F. Agra et al. 4791 (JPB)* R. Lopes 240 (EAN)

Xylophragma heterocalyx (Bureau & K.Schum.) A.H.Gentry Liana R. Lopes 260 (EAN) R. Lopes 261 (EAN)

* = Analyzed samples from herbaria

Table 2. Micromorphology of epicuticular waxes and cuticle on the leaflet epidermis of Bignonieae species. Legends: + = presence, - = absence, Cru = Crusts, Fla = Fissured layer, Gra = Granules, Pap= Papillose, Pla = Platelets, Rug = Rugose, Smt = Smooth, Str = Striate, Thr = Threads.

Epicuticular waxes Cuticle Species Adaxial surface Abaxial surface Adaxial surface Abaxial surface

Cru Fla Gra Pla Thr Cru Fla Gra Pla Thr Pap Rug Smt Str Pap Rug Smt Str

Amphilophium crucigerum - - + - - + ------+ - - - + Amphilophium paniculatum - - + + - - - + - - + - - - + - - - Anemopaegma citrinum + - + - - - - + - - - - - + - - - +

Bignonia ramentacea + - + + - + - + + - - - + - - - + - Bignonia sciuripabulum - - + - - Draft------+ - - - + - - Cuspidaria lateriflora - - + - + ------+ - - - + Dolichandra unguis-cati - - + - - - - + - - - - + - - - + + Fridericia pubescens - - + - - - - + - - - - - + - - - + Pyrostegia venusta - + + + - + - + + - - - - + - - - + Tanaecium cyrtanthum - + + + - - + - - - - + - - + - - + Tanaecium dichotomum - - + - - - - + - - - - + - + - - - Tanaecium parviflorum - - + ------+ - - + - + Xylophragma heterocalyx + - + + - - - + - - - + - - - + - -

Table 3. Anticlinal cell walls and trichomes on the leaflet epidermises of Bignonieae species. Legends: - = Absence, + = Presence, Cv = Curved, St = Straight, Sn = Sinuous, Br = Branched, Cg =Capitate glandular, Gs = Glandular stipitate, Pc = Patelliform/cupular, Pa = Patelliform, Pt = Peltate, Tr = Simple.

Contour of anticlinal walls Type of trichomes Species Adaxial Abaxial Adaxial surface Abaxial surface surface surface Cv Sn St Cv Sn St Br Cg Gs Pc Pt Pa Tr Br Cg Gs Pc Pt Pa Tr Amphilophium crucigerum - + - + Draft- + - - - + + - + - - - - + + + Amphilophium paniculatum - - + - - + - - - - + + - - - - - + + - Anemopaegma citrinum + - + - + - - - - - + - + - - - + + + + Bignonia ramentacea - - + - - + - - - - + - + - - - - + + + Bignonia sciuripabulum - - + + + - - - - - + ------+ + - Cuspidaria lateriflora - - + + + - - - + - + - + - - - - + + + Dolichandra unguis-cati - + - - + - - - - - + ------+ + - Fridericia pubescens - + - - + - + + - - + - + + + - - + + + Pyrostegia venusta - - + - + - - - - - + + - - - - + + + + Tanaecium cyrtanthum + - + - + - - - - - + + - - - - - + + - Tanaecium dichotomum - + - - + - - - + - + - + - - - - + + + Tanaecium parviflorum - + - - + - - + - - + - + - - - - + + + Xylophragma heterocalyx - + - - + - - - - - + - + - - - - + + +

Table 4. Inorganic idioblasts and stomata types in Bignonieae species. Legends: + = Presence, - = Absence, Ap = Amphyciclocytic, As = Anisocytic, An = Anomocytic, At = Anomotetracytic, Bp = Brachyparacytic, Bx = Brachyparahexacytic, Cc = Ciclocytic, Cs = Crystals sand, Dr = Druse, Hp = Hemiparacytic, Pc= Prismatic crystal, Pr = Paracytic, Rp = Raphides = St = Staurocytic, St = Styloid.

Crystal morphology Stomata type Species Adaxial surface Abaxial surface Cs AbaxialDr Pc surfaceRp St Cs Dr Pc Rp St Ap As An At Bp Bx Cc Hp Pr Sc

Amphilophium. crucigerum + - + +- - + - + - - - - + + - - + - - + - - Amphilophium paniculatum ------+ + - - + - - + - - Anemopaegma citrinum ------+ + + + - + - - + Bignonia ramentacea + - + - - Draft- - - - - + + + + + + + + - + Bignonia sciuripabulum ------+ + + + + + - - +

Cuspidaria lateriflora - - + - - + - + - - - - + + + - - + - -

Dolichandra unguis-cati + - + - - + - + - - - + + + - - - + - +

Fridericia pubescens - - + - + - - + - - - + - + + - - - + +

Pyrostegia venusta - + - - - + - + - - - + + + + - - - - -

Tanaecium cyrtanthum + - + - - + - + - - - + + + + - - + - -

Tanaecium dichotomum + - + - - - - + - - - + + + + - - - + +

Tanaecium parviflorum - - + - - + - + - - - + + + + - - + + -

Xylophragma heterocalyx ------+ + + + - - - - +

Table 5. Stomatal densities and stomatal indices in Bignonieae species from Pico do Jabre.

Species Stomatal density (mm²) Stomatal index (%)

Amphilophium crucigerum 111.5 8.12%

Amphilophium paniculatum 198 9.63%

Anemopaegma citrinum 81.4 6.63%

Bignonia ramentacea 126.2 6.21%

Bignonia sciuripabulum 104.8 6.33%

Cuspidaria lateriflora Draft212.3 11.75%

Dolichandra unguis-cati 92.6 8.44%

Fridericia pubescens 381.3 21.36%

Pyrostegia venusta 76.4 9.90%

Tanaecium cyrtanthum 473.9 20.30%

Tanaecium dichotomum 503.5 20.28%

Tanaecium parviflorum 752.9 23.52%

Xylophragma heterocalyx 93.2 6.85%