Introduction

The taxonomy of neotropical Salicaceae, a family that now includes the majority of the former Flacourtiaceae, has been problematic, especially because they display very diverse morphology and have several characteristics in common with many other families (Chase et al. 2002). Although there have been few recent taxonomic revisions (e.g., Sleumer 1980; Lemke 1983; Alford 2006, 2008), there are a number of works at the family level about general morphology (Hutchinson 1967; Sleumer 1954, 1980; Lemke 1983, 1987a, b, 1988), floral anatomy and development (van Heel 1977; Bernhard and Endress 1999; Endress et al. 2013), vegetative anatomy (Metcalfe and Chalk 1950; Metcalfe 1952; Golycheva 1975; Miller 1975; Baas 1984; Klucking 1992; Wilkinson 2007), seed morphoanatomy (Duke 1969), palynology (Schaeffer 1972; Keating 1973, 1975; Krishnan 1981), karyology (Mukherjee 1975; Raven 1975; Morawetz 1981; Krishnan 1986), chemistry of secondary compounds (Rehfeldt et al. 1980; Spencer and Seigler 1985) and relationships inferred from DNA data (APG 1998; Nandi et al. 1998; Chase et al. 2002; Alford 2005; Xi et al. 2012). Many of these works sought to elucidate the relationships of the former Flacourtiaceae by comprehensive sampling of particular features, but ultimately their significance remained unclear until a strong phylogenetic foundation was provided by analysis of DNA data.

Originally, Salicaceae Mirb. was composed solely of the genera Populus and Salix, which are both predominantly rich species in the northern hemisphere and amentiferous (Angus 1997; Judd 1997). Phylogenetic analyses based on morphological and molecular data have indicated relationships with members of the Malpighiales, and new circumscriptions to Salicaceae have been proposed including most or all of the non-cyanogenic Flacourtiaceae, which are not amentiferous (Chase et al. 2002; Alford 2005).

In the most comprehensive traditional revision that includes neotropical Salicaceae as outlined today (Sleumer 1980), seven tribes were recognized (Oncobeae, Abatieae, Homalieae, Prockieae, Flacourtieae, Lacistemeae, and Casearieae [=Samydeae]), including 880 species and 86 genera (most of them with low number of species). Lacistemeae has since been segregated from the family and recognized as its own family, Lacistemataceae, and much of Sleumer’s Oncobeae has been transferred to Achariaceae (Chase et al. 2002; Alford 2005). Casearia, Homalium, and Xylosma are the ones with the greatest number of species genera and account for approximately half of the total tropical species (Lemke 1988). In Brazil alone, where we collected our representative samples, there are records for 19 genera and around 90 species (Barroso et al. 2002).

Morphoanatomical studies have been helpful to aid in species identification (Solereder 1908; Metcalfe and Chalk 1950, 1979; Araújo et al. 2010; Coutinho et al. 2013). Anatomical and micromorphological data have proved to be useful as a tool to support taxonomic studies in different families (Lersten and Curtis 1994; Moraes and Paoli 1999; Scatena et al. 1999; Kong 2001; Alves et al. 2002; Sartori and Tozzi 2002; Guevara et al. 2011) and could also help to clarify the taxonomic and phylogenetic problems for Salicaceae. In addition, the anatomical characters can also assist to recognize species even without flowers and have been tools for the certification and quality control of medicinal plants (Teixeira et al. 2005).

The present study aims to describe the leaf anatomy of Abatia, Banara, Casearia, Prockia, and Xylosma and the closely related Carpotroche (Achariaceae) to determine how morphoanatomical characters of the leaves provide useful data for the taxonomy and phylogeny of Salicaceae.

Materials and methods

Branches of Banara brasiliensis (Schott) Benth., Carpotroche brasiliensis (Raddi) Endl., Casearia arborea (Rich.) Urb., C. decandra Jacq., C. gossypiosperma Briq., C. obliqua Spreng., C. sylvestris Swartz, C. ulmifolia Vahl ex Vent., Prockia crucis P. Browne ex L., and Xylosma prockia (Turcz.) Turcz. were collected in the field in three different areas of forest vegetation (20º45′52″S 42º51′43″W, 20º45′21″S 42º51′41″W, and 20º47′49″S 42º50′51″W), Abatia americana (Gardner) Eichler was collected in a rock field vegetation (20º42′55″S 42º26′51″W). These areas are in Minas Gerais state (Brazil). Voucher materials (“Appendix”) were deposited in the Herbarium VIC of the Universidade Federal de Viçosa (UFV). Samples of mature leaves (3rd to 5th node of the stem) from these specimens were fixed for 48 h in FAA (formaldehyde, acetic acid, 50 % ethanol, 1:1:18, v/v) and stored in 70 % ethanol (Johansen 1940). For this study, samples from herbarium specimens were also used with the purpose of obtaining replicates. Herborized samples were boiled in distilled water for 10 min, treated with 2 % potassium hydroxide for 2 h at room temperature (Smith and Smith 1942, modified), rinsed in tap water five times, dehydrated in an ethanol series (30–70 %) and stored in 70 % ethanol.

Wherever possible, at least three specimens for each species were analyzed. To ensure standardization of the effect of light condition on field specimens, only leaves from lower branches were evaluated. The specimen identifications were verified by a taxonomist (Roseli B. Torres) and are listed in the "Appendix".

Light microscopy

Several pieces from the middle area of the leaf (midvein, margin with or without teeth, and the area between the margin and midvein) and petiole (apical, middle and basal regions) were dissected and used for structural characterization. These samples were dehydrated in an ethanol series and embedded in methacrylate (Historesin, Leica Instruments, Heidelberg, Germany). Cross and longitudinal sections (6 μm thick) were made using an automatic microtome (Leica—RM 2155) and glass knives. The slides were stained in Toluidine blue pH 4.0 (O’Brien and McCully 1981) and then mounted in synthetic resin (Permount®, Fisher Scientific, USA).

Whole mature leaves of field collected and herbarium materials were cleared or submitted to epidermis dissociation, and both were used to describe the surface characters, such as the venation pattern, stomata, and trichome types. From those samples stored in 70 % ethanol, several were rehydrated, cleared using 10 % sodium hydroxide and 20 % hypochlorite solutions, and stained with diluted safranin (Johansen 1940). Others were submitted to dissociation by nitric and chromic acid (Jensen 1962), and, after washing with water, these pieces were stained with Astra blue and basic fuchsin (Kraus et al. 1998). The slides were mounted in glycerinated gelatin.

All the observations, imaging, and paper photographic documentation were performed using a light microscope (model AX70TRF, Olympus Optical, Tokyo, Japan) equipped with a U-Photo system and digital camera (model Spot Insight color 3.2.0, Diagnostic Instruments Inc., New York, USA). The observations were also recorded by means of hand drawings made with the aid of a camera lucida attached to a light microscope (Olympus CBA, Olympus Optical, Tokyo, Japan).

Leaf architecture was described using the terminology of Hickey (1973), Hickey and Wolfe (1975) and Ellis et al. (2009). Stoma types and the cuticular patterns were described using the terminology of Dilcher (1974). The vascular bundle arrangement, both in the petiole and in the blade, was classified according to Dickison (2000).

Scanning electron microscopy

Samples of marginal teeth of the leaf were fixed in a solution of 2.5 % glutaraldehyde and 4 % paraformaldehyde in 0.1 M cacodylate buffer, pH 7.2 (Karnovsky 1965), and postfixed with osmium tetroxide 1 % made in the same buffer (Bozzola and Russel 1992). Subsequently, these materials were dehydrated in an ethanol series and critical point-dried with CO2 in a Bal-Tec 020 CPD dryer (Bal-Tec, Balzers, Liechtenstein). Samples were mounted on stubs and coated with gold using an FDU 010 sputter coater (Bal-Tec, Balzers, Liechtenstein). Examinations and photography were carried out using a Leo 1430VP scanning electron microscope (Zeiss, Cambridge, England) at the Microscopy and Microanalysis Center, UFV.

Phenetic analysis

To demonstrate graphically the patterns of similarity and dissimilarity between species and/or genera, we performed a multivariate analysis. All species were compared according to their analyzed structural characteristics using a presence/absence matrix (Tables 1, 2, 3, 4) or Euclidian distance (Sneath and Sokal 1973). This matrix used Sorensen’s index and an average group linkage technique (also known as unweighted pair-group method using arithmetic averages, UPGMA) with the MVSP (Multi-Variate Statistical Package) 3.13 m program (Mueller-Dombois and Ellenberg 1974).

Table 1 Venation characters of the leaves of neotropical Salicaceae
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Table 2 Epidermal characters of the leaf of neotropical Salicaceae
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Table 3 Leaf characters of neotropical Salicaceae
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Table 4 Petiole characters of neotropical Salicaceae
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Results

In the sampled species (ten species of neotropical Salicaceae and Carpotroche brasiliensis), the leaves are simple with marginal teeth (Fig. 1). Theoid teeth were observed in Carpotroche brasiliensis (Achariaceae, outgroup) and Casearia species (Fig. 1a–d). Salicoid teeth were found in Abatia americana, Banara brasiliensis, Prockia crucis (Fig. 1e), and Xylosma prockia (Fig. 1f). Theoid teeth were vascularized by just one central vascular bundle (Fig. 1b), whereas in salicoid teeth several vascular bundles merge and branch toward the tooth, forming abundant vascularization (Fig. 1e).

Fig. 1
figure 1

Description of Salicaceae teeth. Casearia arborea. a Stereoscope view (bar 50 mm). b Theoid tooth cleared, highlighting water pore and vascularization with prevalence of xylem (bar 60 μm). c SEM view from a tooth where the setae is still present (bar 60 μm). d Cross section of theoid tooth (bar 50 μm). e Salicoid tooth of Prockia crucis, highlighting the abundant vascularization (bar 350 μm). f Cross section of salicoid tooth of Xylosma prockia (bar 50 μm). wp water pore, pe palisade-like epidermis, pc prismatic crystals, st seta, vc vascularization, xy xylem

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The anatomical structure of theoid teeth follows the pattern described for hydathodes. However, we were neither able to observe an epithem nor a parenchymatic sheath in the hydathode of the theoid teeth of the studied species (Fig. 1d). The salicoid teeth show an anatomic structure that resembles extrafloral nectaries, as they display a secretory epidermis consisting of a single-layered palisade that covers a concavity, a secretory parenchyma (Fig. 1f) and a profuse vascularization, predominantly formed by phloem (Fig. 1e).

Camptodromous–brochidodromous is the most common type of venation, observed in nine species (Fig. 2a, b). Only Banara brasiliensis and Prockia crucis (tribe Prockieae) display the acrodromous–brochidodromous type. The arrangement of the marginal ultimate venation followed two patterns. The incomplete type was observed in Casearia (Fig. 2a), with freely ending veinlets directly adjacent to the margin. The looped type was present in the remaining genera (Fig. 2b), with the major portion of the marginal ultimate venation recurved to form loops. The tertiary veins have a ramified transverse pattern, branching into higher orders without rejoining the secondary veins and oriented across intercostal area (Fig. 2c). The secondary and tertiary veins have determined imperfect areole development, with irregular shape and random distribution (Fig. 2d). Although this arrangement was uniform in the studied species, differences in the veinlet branching pattern were observed: some are branched (Fig. 2e) and others are unbranched—simple (Fig. 2f).

Fig. 2
figure 2

Salicaceae foliar venation. a Incomplete marginal ultimate venation of Casearia ulmifolia (bar 200 μm). b Looped marginal ultimate venation of Carpotroche brasiliensis (bar 300 μm). c Transverse ramified third order veins of Casearia arborea (bar 600 μm). d Imperfect areole development, with irregular shape and randomly distributed of Casearia arborea (bar 300 μm). e Casearia gossypiosperma areole, photo under polarized light highlighting numerous druses all over the mesophyll and branched veinlets (bar 400 μm). f Simple veinlets of Xylosma prockia, prismatic crystals in association with vascular bundles (bar 100 μm). mv midvein, sv secondary vein, tv tertiary vein, pc prismatic crystals

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Brachyparacytic stomata (Fig. 3f, j) occur only on the abaxial surface (Fig. 3a, d, g, k), and are level with the leaf surface (Fig. 4h, k). Carpotroche brasiliensis and Casearia ulmifolia do not follow the pattern. Carpotroche brasiliensis has anomocytic stomata (Fig. 3c), and Casearia ulmifolia is amphistomatic, with stomata on both surfaces (Figs. 3k, l, 4j, k).

Fig. 3
figure 3

Surface epidermis view of Salicaceae leaves. a–c Carpotroche brasiliensis, undulate anticlinal walls. d–f Prockia crucis, abaxial surface with undulate anticlinal walls and adaxial surface with straight to slightly undulate walls. g–j Xylosma prockia, undulate anticlinal walls. k, l Casearia ulmifolia, adaxial surface with straight anticlinal walls and abaxial surface with straight to slightly undulate walls. a, d, g, k Abaxial surface (bars 140 μm). b, e, h, l Adaxial surface (bars 140 μm). f, j Stomata brachyparacytic in the abaxial surface (bars 30 μm). c Stomata anomocytic in the abaxial surface (bar 30 μm). i adaxial surface highlighting striate periclinal walls (bar 30 μm). Arrows indicate epidermis crystals at figure j and stomata at figure l. tt tector trichome

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Fig. 4
figure 4

Salicaceae leaf blade highlighting dorsiventral mesophyll and hypostomatic leaves (except figure j) in cross section. Abatia americana. a Presence of hypodermis, the arrows indicate smaller vascular bundles (bar 50 μm). b Uniseriate unbranched trichome (bar 100 μm). c Smaller vascular bundle and thick cuticle in Banara brasiliensis, asterisk indicates sclerenchyma (bar 50 μm). d Carpotroche brasiliensis, arrows indicate druses on epidermis and asterisk indicates sclerenchyma (bar 50 μm). Xylosma prockia. e Smaller vascular bundle and striated cuticle, asterisk indicates sclerenchyma (bar 25 μm). f Arrows indicate prismatic crystals in epidermis (bar 50 μm). g Branched trichome of Casearia arborea (bar 25 μm). h Stomata of Casearia decandra (bar 25 μm). i Casearia arborea, mesophyll with cavities (filled diamond) all over the blade (bar 50 μm). j, k Casearia ulmifolia, mesophyll with two layers of palisade parenchyma, amphistomatic leaves and cavities (filled diamond) only in the interface of palisade and spongy parenchyma (bars 50 μm). hy hypodermis

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The epidermal cells show straight to slightly undulate anticlinal walls in the adaxial side for most species (Fig. 3e, l). Carpotroche brasiliensis (Fig. 3b) and Xylosma prockia (Fig. 3h) exhibited undulate anticlinal walls. For the abaxial surface, straight to slightly undulate anticlinal walls were observed in Abatia americana, Banara brasiliensis, Casearia decandra, C. gossypiosperma, C. sylvestris, C. obliqua, and C. ulmifolia (Fig. 3k), while undulate walls were observed in Carpotroche brasiliensis (Fig. 3a), Casearia arborea, Prockia crucis (Fig. 3d), and Xylosma prockia (Fig. 3g).

The outer periclinal walls are thick, as observed in most studied species, being thickest in Banara brasiliensis (Fig. 4c) and Casearia decandra. Striate cuticular ornamentation occurs only in Xylosma prockia (Figs. 3i, 4e).

Unbranched, non-secretory trichomes (Figs. 3d, e, 4b) were found in all studied species (Table 2). These trichomes are formed by only one cell, and sometimes the apex is curved, like a hook. Only Casearia arborea also displays branched-multicellular trichomes (Fig. 4g). The largest number of trichomes was observed in leaves of Abatia americana and Casearia arborea.

Single round cells or groups of 2–4 small, round cells occur among the ordinary epidermal cells of the leaves of Carpotroche brasiliensis and Xylosma prockia. Such cells contain calcium oxalate crystals (Fig. 3j) and are most easily observed on the veins (Figs. 2f, 3g). The crystals are druses in C. brasiliensis (Fig. 4d) and are prismatic in X. prockia (Fig. 4f). A hypodermis with 1–4 layers was observed only in the adaxial surface of Abatia americana (Fig. 4a).

Dorsiventral mesophyll was observed as a common feature (Fig. 4a, d, f, i–k). Variation in number of cell layers for palisade parenchyma was observed, varying between one and two (Fig. 4a, c, d, f, i–k).

Crystalliferous idioblasts with druses and prismatic monocrystals are frequently dispersed over the blade (Fig. 2e), sometimes close to phloem. Xylosma prockia has only prismatic crystals (Fig. 2f), and Abatia americana does not bear such idioblasts. These crystals can be inside sclerenchyma cells and/or form a crystalliferous sheath surrounding the bundles (Fig. 4c).

Secretory ducts and cavities are a common feature for Casearia. Ducts occur in the midvein (Fig. 5a, b) and scattered among the ground parenchyma cells on the adaxial and abaxial side of the blade (Fig. 6c, d, g, h, j, k). C. arborea and C. sylvestris display secretory ducts related to phloem tissue (Figs. 5a, 6d, j). C. ulmifolia bears secretory cavities in the mesophyll and leaf edge, placed between the palisade and spongy parenchyma (Fig. 4k). In all other species, the secretory cavities were observed randomly distributed in the mesophyll (Fig. 4i).

Fig. 5
figure 5

Midvein of Salicaceae leaves, cross (a, c) and longitudinal (b) sections; and cross sections of petiole distal portion (d, e). Casearia sylvestris. a Vascular bundle in flat arc, arrows indicate phloem ducts (bar 70 μm). b Duct at ground parenchyma in longitudinal section (bar 40 μm). c Duct at ground parenchyma in transverse section, asterisk lumen of the duct (bar 40 μm). d Petiole of Casearia decandra. The arrows indicate phenolic idioblasts in the ground parenchyma and (filled diamond) ducts (bar 150 μm). e Vascular bundle of Casearia obliqua. The arrows indicate phenolic idioblasts in the phloem and (filled diamond) ducts (bar 40 μm). cb cambium, co collenchyma, sc sclerenchyma, ph phloem, pd periderm, xy xylem

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Fig. 6
figure 6

Midvein of Salicaceae leaves, drawings of cross sections depicting the vascular system, the distribution of sclerenchyma and phenolic compounds. a Abatia americana. b Banara brasiliensis. c Casearia decandra. d Casearia arborea. e Carpotroche brasiliensis. f Prockia crucis. g Casearia gossypiosperma. h Casearia obliqua. i Xylosma prockia. j Casearia sylvestris. k Casearia ulmifolia. Circles represent ducts. d, j Ducts in the phloem. Bars 200 μm

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The midvein vascular pattern forms a flat arc (Figs. 5a, 6b–d, g–k), with sclerenchyma both on the adaxial and abaxial sides (Fig. 6b–d, g, i–k). Casearia obliqua (Fig. 6h) is the only species where sclerenchyma is not present. When the leaves are bigger, as in Abatia americana (Fig. 6a) and Carpotroche brasiliensis (Fig. 6e), the vascular system of the midvein is the siphonostele type. In Prockia crucis, an arc with invaginated ends is formed (Fig. 6f). Fibers are the main type of sclerenchyma cells (Fig. 5a), but sometimes sclereids also occur. In the smaller veins, the vascular bundle follows the same pattern described for the midvein and display fibers on both adaxial and abaxial sides (Fig. 4c–e).

Idioblasts with green-colored compounds were observed in the midvein phloem (Figs. 5a, 6d, g, h, k) and petiole phloem (Figs. 5d, e, 7a, c, d, f, h, i, k) of some of the species. Such idioblasts are also dispersed in the ground parenchyma of the petiole (Figs. 5d, 7c, h, i, l) and sometimes in the xylem (Figs. 6c, d, g, 7c, d, f, g, h, k).

Fig. 7
figure 7

Distal region of the petiole of Salicaceae, drawings of cross sections depicting the vascular system, the distribution of sclerenchyma and phenolic compounds. a Abatia americana. b Banara brasiliensis. c, g Casearia decandra. d Casearia arborea. e Carpotroche brasiliensis. f Prockia crucis. h Casearia gossypiosperma. i Casearia obliqua. j Xylosma prockia. k Casearia sylvestris. l Casearia ulmifolia. g Petiole with secondary vasculature. Circles represent ducts. d, k Ducts in the phloem. Bars 200 μm

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The anatomical characteristics of the petiole did not change according to the region of the petiole (base, middle, or apex) in any of the analyzed species. The epidermis is uniseriate with thin outer periclinal walls and trichomes, the collenchyma takes a subepidermic position, and the ground parenchyma displays few crystal cells (Fig. 5d).

Secretory ducts were observed in the petiole of all Casearia species studied. They occur among the ground parenchyma cells (Figs. 5d, 7c, d, h, i, k, l) and also in the phloem (Figs. 5e, 7d, i, k), in the same way as related to the leaf blade. It is interesting to emphasize that although ducts may be present in the phloem of the petiole of Casearia obliqua (Fig. 5e), they are not present in the leaf blade phloem. The petiolar vascular patterns are similar to those described for the midvein.

In this study, we described anatomically ten species of Salicaceae, highlighting shared and limited characters which could be important at both generic and specific levels. Based on these characters and our sampling, an identification key to the neotropical species based on anatomy is provided. This represents an important additional tool for species identification even without flowers.

Identification key for neotropical Salicaceae based on morphoanatomical characters of leaves

  1. 1.

    Leaf margin with theoid teeth ··· 2

  2. 1.

    Leaf margin with salicoid teeth ··· 8

  3. 2.

    Internal secretory structures (ducts and cavities) absent; druses in the epidermis present; stomata anomocytic; marginal ultimate venation looped; midvein vascular bundle has a radial structure, resembling a siphonostele ··· Carpotroche brasiliensis

  4. 2.

    Internal secretory structures (ducts and cavities) present; crystals in the epidermis absent; stomata brachyparacytic; marginal ultimate venation incomplete; midvein vascular bundle as a flat arc ··· 3

  5. 3.

    Leaves amphistomatic; mesophyll cavities localized between palisade and spongy parenchyma ··· Casearia ulmifolia

  6. 3.

    Leaves hypostomatic; cavities dispersed all over the mesophyll ··· 4

  7. 4.

    Ducts in the phloem present ··· 5

  8. 4.

    Ducts in the phloem absent ··· 7

  9. 5.

    Phenolic idioblasts all over the petiole ground parenchyma ··· Casearia obliqua

  10. 5.

    Phenolic idioblasts in the petiole ground parenchyma absent ··· 6

  11. 6.

    Phenolic idioblasts all over the parenchyma attached to the petiole vascular bundle ··· Casearia sylvestris

  12. 6.

    Phenolic idioblasts loosely dispersed in the parenchyma attached to the petiole vascular bundle ··· Casearia arborea

  13. 7.

    Sclerenchyma on the petiole vascular bundle present ··· Casearia decandra

  14. 7.

    Sclerenchyma on the petiole vascular bundle absent ··· Casearia gossypiosperma

  15. 8.

    Prismatic crystals in the epidermis present; cuticular ornamentations striate ··· Xylosma prockia

  16. 8.

    Crystals in the epidermis absent; striate cuticular ornamentations absent ··· 9

  17. 9.

    Hypodermis present; crystals in the mesophyll and phloem absent ··· Abatia americana

  18. 9.

    Hypodermis absent; crystals in the mesophyll and phloem present ··· 10

  19. 10.

    Midvein vascular bundle as a flat arc; petiole vascular bundle as an arc with invaginated ends ··· Banara brasiliensis

  20. 10.

    Midvein vascular bundle as an arc with invaginated ends and accessory traces; petiole vascular bundle has a radial structure, resembling a siphonostele ··· Prockia crucis

Tables 1, 2, 3 and 4 summarize the results of this paper for venation patterns, epidermis, leaf blade, and petiole of Salicaceae leaves. In the phenetic multivariate analysis (Fig. 8), two well-defined groups were formed: one for Casearia and another clustering Xylosma prockia, Banara brasiliensis, Prockia crucis, and Carpotroche brasiliensis. Banara brasiliensis and Prockia crucis formed a small group. Abatia americana was the only species that did not cluster with any other one (Fig. 8).

Fig. 8
figure 8

Dendrogram generated by UPGMA cluster analysis of foliar anatomical characters of species of Salicaceae using Euclidean distance

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Discussion

According to Hickey and Wolfe (1975), the type of marginal tooth of the ‘dicotyledon’ leaves is a stable character in larger clades, being an important tool for taxonomic approaches. Some authors have recorded the presence of theoid teeth and its salicoid derivate type as a common character to neotropical Salicaceae (Hickey and Wolfe 1975; Charlton 1994; Judd 1997; Nandi et al. 1998; Bernhard and Endress 1999; Chase et al. 2002; Alford 2003, 2005, 2006; Wilkinson 2007). The main difference between these two secretory structures is the presence of an opaque and deciduous seta in the theoid tooth and a non-deciduous and persistent bright dark seta in the salicoid tooth, which forms a spherical callosity fused to the tooth apex (Hickey and Wolfe 1975; Hickey 1973).

The anatomical structure of theoid teeth follows the pattern described for hydathodes. Hydathodes are structures that discharge liquid water with dissolved substances from the interior of the leaf to its surface, a process called guttation (Fahn 1979). The usual structure of an hydathode consists of terminal tracheids of one to three vein endings, the epithem (composed of thin-walled, chloroplast deficient parenchyma cells located above or distal to the vein endings), a sheath continuation of the bundle sheath that extends to the epidermis, and openings, called water pores, in the epidermis. Presence of theoid teeth is one reason why some authors (Wurdack and Davis 2009; Alford 2005) have proposed the segregation of Casearia and relatives into Samydaceae, a family separated from Salicaceae sensu lato (Chase et al. 2002; APG III 2009).

The salicoid teeth of Prockia crucis have been anatomical and chemically described as extrafloral nectaries (Thadeo et al. 2008). Although the similarity between the salicoid teeth and extrafloral nectaries of Abatia americana, Banara brasiliensis and Xylosma prockia is evident, the secretion produced by this structure needs to be analyzed to confirm this description. Extrafloral nectaries with an anatomical structure very similar from these were reported in species of Salicaceae from the temperate region, in Populus (Curtis and Lersten 1974, 1978; Pemberton 1992; Wilkinson 2007) and Salix (Curtis and Lersten 1980; Wilkinson 2007), and also in tropical species of the tribe Flacourtieae (Charlton 1994; Wilkinson 2007). These data suggest that these glands could be homologous and reinforce the statement of Wilkinson (2007), who considers that simple glands occur in neotropical species while complex glands are found in the genera Populus and Salix. The anatomical data should be analyzed in combination with molecular data to make an up-to-date assessment of the ancestral conditions for this character in this group and to identify homologies or homoplasies. The anatomical similarity between salicoid teeth observed in the neotropical Salicaceae studied here is additional evidence that shows how close Flacourtiaceae (sensu Lemke) are to the temperate genera of Salicaceae.

In previous work, pinnate venation with secondary veins semicraspedodromous, brochidodromous or eucamptodromous were described for Salicaceae (Klucking 1992; Wilkinson 2007; Ellis et al. 2009). The palmate type was described as characteristic for Prockieae (Judd 1997). However, further sampling is necessary to determine how important this character is for characterizing taxa or for phylogenetic inference. The arrangement of the marginal ultimate venation can be useful to distinguish Casearia from the other neotropical Salicaceae and represent anatomical evidence that corroborates the proposal to recognize a separate family for Casearia (and close relatives) (Alford 2003, 2005; Wurdack and Davis 2009; Xi et al. 2012). The pattern of foliar venation has been used in several studies as a good anatomical character for taxonomy (Moraes and Paoli 1999; Reis et al. 2004; Cardoso and Sajo 2006). However, the relevance of this character depends on the taxon that is being considered, once variation can be observed both in fossil and recent plants (Dilcher 1974). As this character was not evaluated as a whole in Salicaceae, it is important to determine how variable the character is.

The stomata type is in accord with Wilkinson (2007), who described brachyparacytic stomata for the tribe Flacourtieae. Salicaceae also have paracytic and anomocytic stoma types reported in the literature (Solereder 1908; Metcalfe and Chalk 1950, Merida and Medina 1967; Judd 1997). However, the recognition of paracytic stoma type may not always be obvious, since sometimes the subsidiary cells do not reach the end of the guard cell of stoma. For this case, Dilcher (1974) proposed the term brachyparacytic, which we consider appropriate for these species. Epidermal cells with undulate anticlinal walls have been correlated with more strength, increasing mechanical protection against cell collapse during drought conditions (Haberlandt 1928). Therefore, this character has limited use for taxonomic approaches.

Occurrence of striate cuticular ornamentation as for Xylosma prockia is also recorded for Oncoba spinosa (Wilkinson 2007). Before analyses of DNA data, Oncoba was always allied with the cyanogenic Flacourtiaceae (Lemke 1988), the ones that are now placed in the family Achariaceae. In the most recent molecular analysis (Chase et al. 2002; Alford personal communication), Oncoba forms a clade with some representatives of tribe Flacourtieae (where Xylosma is placed). Therefore, the presence of striate cuticular ornamentation may indicate a relationship with Xylosma.

Trichomes have been considered as an important character in systematic studies of angiosperms. However, the shape, the size, and the amount of these structures can vary according to environmental conditions (Metcalfe and Chalk 1950); thus, it is necessary to be careful about the utility of trichomes in taxonomic studies. It is clear that trichomes play a role in plant defense, especially with regard to phytophagous insects (Levin 1973), and in prevention of water loss (Fahn 1986). In numerous species, there is a negative correlation between trichome density and insect feeding and oviposition responses. Specialized hooked trichomes may impale adults or larvae as well (Levin 1973).

The presence of epidermal crystals in part agrees with Solereder (1908) and Metcalfe and Chalk (1950), who related them to Xylosma and Casearia species. However, these structures have not been observed in any of the six species of Casearia studied here. The presence of crystals in epidermal cells can represent an adaptive advantage for bright light conditions, as demonstrated for some Peperomia species (Franceschi 2001).

Although hypodermis was not observed in other studied species except in Abatia americana, this character has been reported for Banara, Casearia, and Xylosma (Solereder 1908; Metcalfe and Chalk 1950). Such genera occur in different types of vegetation and have been considered as having high adaptive capacity (Klein and Sleumer 1984). The hypodermis may play a role in water retention due to its considerable ability to absorb water and to protect the photosynthetic apparatus against the risk of photo oxidative destruction (Fahn 1979; Fahn and Cutler 1992; Feller 1996; Al-Tardeh et al. 2008). The presence of hypodermis only in Abatia americana may have been induced by the extreme conditions present at “campos de altitude”, where this species occurs. A similar result was suggested for Chamaecrista species (Coutinho et al. 2013). However, it is important to verify if this character is present in other species of Banara (which bears only 35 species) and in Casearia and Xylosma (which have greater numbers of species), and to evaluate the value of this character for the taxonomy of Salicaceae.

In general, epidermal characters have proven to be of systematic value in different plant lineages (Dickison 2000). Although the cell size and shape, stomata, and trichome frequency can vary according to the environment, stoma and trichome type, cellular contents, and pattern of epicuticular waxes represent valuable sources of systematic data (Dilcher 1974; Dickison 2000). For Malpighiales, epidermal characters have been used successfully in Euphorbiaceae (Raju and Rao 1977). The presence of crystals in the epidermal cells, anomocytic stomata, and striae on the cuticle have been important characters to distinguish species (see identification key).

The morphology and distribution of crystals have found use as taxonomic characters (Prychid and Rudall 1999; Lersten and Horner 2000) and indicate tight genetic regulation of crystal deposition (Franceschi and Nakata 2005). The internal secretory types observed in the present study and their position in the leaves are important diagnostic features in Casearia, both at species and generic level (Tables 3, 4). The variations in the morphology, anatomy, function, and position of the secretory structures have shown to be very good tools for taxonomic studies in several families (Solereder 1908; Metcalfe and Chalk 1950; 1983; Fahn 1979), as well as for Casearia decandra (Thadeo et al. 2009).

The green color present in the idioblasts is a result of the Toluidine Blue stain and indicates the presence of phenolic compounds, as shown in Souto and Oliveira (2008). In the present study, the vascular system of both the midvein and the petiole was valuable to identify species, which is in accordance with Howard (1979) and Metcalfe and Chalk (1979). In another study, which evaluated species of Malpighiaceae, the importance of petiole vascular patterns tothe taxonomy was highlighted (Araújo et al. 2010), reinforcing the potential for this character in Malpighiales.

In the phenetic multivariate analysis Banara brasiliensis and Prockia crucis grouped, corroborating the taxonomy, which places them together in the tribe Prockieae (Chase et al. 2002). Abatia americana has been included in Passifloraceae because of the resemblance in the floral morphology, interpreting the extra-staminal disk of their flowers as a corona (Lemke 1988; Takhtajan 1997) and as a tribe of Flacourtiaceae/Salicaceae on the basis of phytochemistry, other floral characteristics (presence of staminodes and valvate sepal estivation) and molecular data (Bernhard and Endress 1999; Sleumer 1980; Chase et al. 2002; Alford 2008). The anatomical data show that this species is different from all others in the study. Abatia Ruiz & Pav. (including Aphaerema Miers) is a genus that occurs in high-elevation areas from Mexico and Central and South America (Alford 2006). Such special feature is uncommon for the studied species, as Salicaceae members are usually found in forests. The anatomical differences observed in Abatia could be interpreted as an adaptive response to high-elevation conditions. However, further studies in Abatia are necessary.

In this study, Carpotroche brasiliensis formed a group with Xylosma, Prockia, and Banara. Carpotroche was transferred to Achariaceae based on molecular and morphological data. The genus has cyanogenic glycosides, lack of salicoid teeth, wood with mostly solitary pores and no tracheids, many more petals than sepals without any specific orientation of parts and sometimes whorled and indistinct, lack of a disk, and typically linear anthers (Chase et al. 2002). Given the close relationships of these families and their similarity, additional observations, with more samples of Achariaceae, are necessary.

Casearia comprises about 200 species which display much morphological variation, although many are vegetatively very similar (Alford 2003). The present anatomical data show clearly how such characters can assist the taxonomical approaches. Therefore, further studies are planned to evaluate a larger number of species and include other genera in the new circumscription of Samydaceae.

The genus Casearia is characterized by the common occurrence of pellucid dots or striations in the leaves and the absence of salicoid teeth and cyanogenic glycosides (Alford 2003). According to molecular data, the genus was transferred from Flacourtiaceae (sensu Lemke) to Salicaceae (sensu APG III 2009). Recently, as mentioned previously, there are additional studies that evaluate a larger number of species (since Chase et al. 2002 only included one species of Casearia in their molecular data) and combine them with morphological data (Alford, unpublished data; Xi et al. 2012). They consider to resurrect the family Samydaceae for Casearia and its relatives, solving some taxonomic issues for the group.

The taxonomic relationships in Salicaceae are still very controversial. According to Chase et al. (2002), there is a lot of heterogeneity in the group and it is not surprising that tribal limits need to be revised. The molecular data have been very helpful but, there is no consensus on the family-level classification, and the tribal-level classification needs modification (Alford 2003). In the past, many authors highlighted Flacourtiaceae and Salicaceae as close relatives (Metcalfe and Chalk 1950; Hickey and Wolfe 1975; Meeuse 1975; Miller 1975; Cronquist 1988; Leskinen and Alström-Rapaport 1999; Azuma et al. 2000; Chase et al. 2002; Alford 2003, 2005). In this work, it was possible to recognize similar characters for the neotropical Salicaceae (former Flacourtiaceae) and Salicaceae sensu stricto such as the presence of salicoid teeth, brachyparacytic stomata, secondary growth in petiole, abundance of crystals, collateral and arc-shaped vascular system at the midrib and sclerenchyma accompanying the bundles.

These anatomical studies in the neotropical Salicaceae allow us to conclude that the foliar anatomy is able to provide data to assist with the taxonomy of Salicaceae, at both generic and specific levels. The data obtained are promising and should be emphasized in future studies involving combined analysis with phylogenetic trees to assess the evolution of characters, especially secretory structures.