Floral vasculature and its variation for carpellary supply in Anthurium (Araceae, Alismatales)

Introduction and Aims Anthurium is the largest genus of Araceae, with 950 species distributed in the neotropics. Despite the great diversity of the genus, the knowledge of its floral vasculature is based on observations in only two species, viz. A. denudatum and A. lhotzkyanum, with remarkable variation in vascular carpellary supply: carpels are either vascularized by ventral bundles alone or by reduced dorsal bundles in addition to the ventral ones. Our main objective is to test this peculiar variation through a detailed anatomical study of the floral vasculature in taxa belonging to some sections of Anthurium designated as monophyletic groups in recent phylogenies. Methods We compare the floral vasculature of 20 neotropical species belonging to distinct sections of Anthurium, using both light and confocal laser scanning microscopies. Results The number and position of vascular bundles are constant within the tepals and stamens, regardless of the species and sections studied. However, the gynoecium vasculature exhibits variation between species belonging to the same or different sections. Our results reveal two patterns of vasculature: carpels vascularized by synlateral bundles alone (Pattern A) and carpels vascularized by both dorsal and synlateral bundles (Pattern B). Pattern A is shared by the majority of species studied here and corroborates the previous data in the literature. Pattern B occurs in three species: A. affine (Anthurium sect. Pachyneurium series Pachyneurium), A. obtusum and A. scandens (Anthurium sect. Tetraspermium), described here for the first time for the genus. Conclusions The variation in the supply to the carpels in Anthurium is corroborated here. However, our results in addition to those from the available literature suggest the existence of three patterns (A, B and C) of carpellary vasculature. Based on the recent phylogeny of Anthurium it is possible to notice that the three patterns of carpellary vasculature occur in representatives of Clade B and deserve to be investigated in a larger number of species. Pattern A could be a plesiomorphy for the genus and the occurrence of dorsal bundles could be a derived character. Our data contributes to the taxonomy and to the understanding of the floral evolution of the largest neotropical genus of Araceae.

Historically, representatives of Anthurium were circumscribed in 18-19 sections (Engler, 1905;Croat & Sheffer, 1983) (Table 1), based on vegetative characteristics. The recent phylogenetic study by Carlsen & Croat (2013), based on molecular data, revealed 18 clades, many of which lack a corresponding classification in the sections suggested by Engler (1905) and Croat & Sheffer (1983) (Table 1). However, four of these sections are well represented in Brazil (Boyce & Croat, 2016) and present definite correspondence with clades of the phylogeny.
Floral anatomical studies in Araceae indicate variation in the gynoecium vasculature and point out its applicability to the understanding of certain evolutionary aspects (e.g., Eyde, Nicolson & Sherwin, 1967;Buzgo, 2001). An example is the reduction of dorsal vascular bundles in the gynoecium of Schismatoglottis Zoll. & Moritzi, designated by Hotta (1971) as a characteristic associated with variation in the number of carpels and ovules and with sub-basal placentation. Also emphasized is the identification of additional vascular bundles supplying the carpels, leading to the confirmation of pseudomonomery in Calla L. (Barabé & Labrecque, 1983), Lysichiton Schott (Barabé & Labrecque, 1984), Orontium L. (Barabé & Labrecque, 1985) and Symplocarpus Salisb. (Barabé, Forget & Chrétien, 1986). In studies of Monstera Adans (Barabé & Chrétien, 1985) and Spathiphyllum Schott , the variation in the pattern of gynoecium vasculature was related to the size and volume of the carpels.
Despite these studies on floral anatomy and vasculature in Araceae, there is still a lack of information with regard to the genus Anthurium, in which all knowledge is limited to only two species: Anthurium denudatum Engler (A. sect. Belolonchium Schott) (Carvajal, 1977) and A. lhotzkyanum Schott (A. sect. Urospadix) (Barabé, Forget & Chrétien, 1984). In A. denudatum, Carvajal (1977) verified the occurrence of underdeveloped dorsal bundles restricted to the gynoecium base, with each carpel vascularized solely by ventral bundles. In A. lhotzkyanum, synonymous of A. augustinum K. Koch & Lauche (Cardozo et al., 2014), Barabé, Forget & Chrétien (1984) reported only ventral complexes and placental traces, which diverge from four complexes at the floral base. Thus, an infrageneric variation has been observed in relation to the carpellary vasculature as well as previously indicated for other floral characteristics (Carvajal, 1977;Higaki, Rasmussen & Carpenter, 1984;Carvell, 1989;Poli, Temponi & Coan, 2012;Poli, Temponi & Coan, 2015). These characteristics are contributing to new data toward the understanding of the floral morphology of the genus and may help in the still widely debated infrageneric classification.
The study of floral vasculature in more representatives of Anthurium, specifically in traditionally recognized groups such as the four previously described, is expected to point out new anatomical data that may be used in their delimitation. Thus, the present study proposes a comparative analysis of floral vasculature of Anthurium species, with emphasis on gynoecial aspects, seeking to answer the following questions: (1) Are the patterns of carpellary vasculature previously reported for Anthurium found in the taxa analyzed here; (2) Do the floral vascular characteristics aid in the delimitation of Anthurium sect. Dactylophyllium, A. sect. Pachyneurium series Pachyneurium, A. sect. Tetraspermium and A. sect. Urospadix?

MATERIALS AND METHODS
Neotropical representatives of four traditional sections of Anthurium, mainly distributed in Brazil, were selected for the present study (Table 2; Fig. 1). Vouchers were deposited at the herbaria HRCB, RB, SPF, UB, UFP, VIC (acronyms according to Index Herbariorum, Thiers, 2016) and at the herbarium of the Universidade Estadual do Oeste do Paraná (Cascavel, Paraná, Brazil). The material examined is listed in Table 2, based on the following field permits: SisBio permanent permit no. 40816-2 (April 2011 to present) to LP Poli; SisBio collecting permits no. 28776 (June 2011-May 2014) and 28686 (June 2011-May 2014) to LG Temponi.
At least two samples of inflorescences from different specimens were analyzed, whenever possible from different localities ( Table 2). The inflorescences were collected at different stages of development and fixed in Transeau solution (Bicudo & Menezes, 2006) or FAA 50 (Johansen, 1940. For the study using light microscopy (LM), samples were dehydrated through a n-butyl alcohol series (Feder & O'Brien, 1968), embedded in 2-hydroxyethyl methacrylate (Leica Historesin Embedding Kit), and sectioned at 7-10 µm on a rotatory microtome (Leica). The anatomical sections were stained with periodic acid Schiff (PAS reaction) and 0.05% Toluidine blue O in 0.1 M sodium phosphate buffer (pH 6.8) (Feder & O'Brien, 1968), or only with 0.05% Toluidine blue O in 0.1 M sodium phosphate buffer (pH 6.8) (O'Brien, Feder & McCully, 1965), and mounted in Entellan (Merck). Samples of gynoecia were also individualized and cleared using the technique of Shobe & Lersten (1967). The results were documented in photomicrographs obtained with the image digitization program LAS (Leica Application Suite v. 4.0; Leica), using an image capture apparatus Leica) attached to the microscope (DM 4000B, Leica).
Diagrams were produced using CorelDRAW X7 (Corel Corporation) software.

RESULTS
The results are presented in two main topics: ''Floral organography'' and ''Floral vasculature.'' In the first topic a single floral description is presented for all species herein studied because they generally share many anatomical aspects; differences, when present, are emphasized throughout the text. The second topic includes the description of the floral vascular supply in Anthurium and is divided into two subtopics: ''Pattern A: Carpels vascularized by only synlateral bundles,'' and ''Pattern B: Carpels vascularized by synlateral and dorsal bundles.''

Floral organography
The flowers of Anthurium are sessile ( Fig. 2A) and are arranged spirally along the spadix (Figs. 1B, 1D, 1F and 1H). The flowers are bisexual and dimerous, possessing two external tepals, two internal tepals, two external stamens, two internal stamens and a bicarpellary gynoecium (Figs. 2B and 2C). All species studied are protogynous; thus, the majority of the anatomical description of the sterile floral parts corresponds to the pistillate stage (Figs. 1D and 1F).
The tepals are free, with a cucullate shape (Figs. 2B and 2C) due to the congested disposition of the flowers on the spadix. Of the external tepals, around two thirds of their adaxial surface come into contact with the external stamens, and the upper third is in contact with the apical portion of the internal tepals (Figs. 2B and 2C); about two thirds of the abaxial surface come into contact with the same region of the adjacent flower, and the upper third is oriented toward the environment (Fig. 2C).
The internal tepals have about two thirds of their adaxial surface in contact with the internal stamens, and the upper third is in contact with the style and stigma (Figs. 2B and 2C); about two thirds of their abaxial surface are found in contact with the same region of the adjacent flower, and the upper third is partly covered by the external tepals and partly oriented toward the environment (Figs. 2B and 2C).
The gynoecium is differentiated into stigma, style and ovary (Fig. 2C). The stigma is composed of secretory trichomes (Fig. 2C). The style is formed by external epidermis composed of isodiametric cells (

Floral vasculature
Based on our sample of 20 species of Anthurium from four distinct sections (Table 2), two patterns of floral vasculature were observed, related primarily to the carpellary supply.
These patterns are illustrated through diagrams, based on median longitudinal sections (Figs. 3A and 3B) and on transverse sections at different heights of the flower (Figs. 4 and 5), and photomicrographs (Figs. 6-11).

Pattern A: carpels vascularized by only synlateral bundles (Figs. 3A and 4)
Pattern A was observed in the majority of the studied species, which correspond to the representatives of A. sect. Dactylophyllium, A. sect. Urospadix, and three out of four studied species of A. sect. Pachyneurium series Pachyneurium.   The vasculature of the axis of the spadix diverges to the flowers ( Fig. 2A-white arrows), branching into two (Fig. 6A) and, posteriorly, four (Figs. 4A and 6B) vascular complexes that are visible at the base of the flower. Two complexes vascularize the external tepal and stamen whorls (Figs. 4A and 6B), referred to here as external complexes (c1); the other two vascularize the internal tepal and stamen whorls and the carpels (Figs. 4A and 6B), referred to here as the internal complexes (c1').
Initially, each of the two external complexes (c1) (Fig. 4A) diverges into two traces that vascularize the external tepals and stamens (Figs. 4B and 6C). The trace of the external tepal Dorsal bundles were not observed in the carpels.

Pattern B: carpels vascularized by synlateral and dorsal bundles (Figs. 3B and 5)
Pattern B was observed in three of the studied species: A. affine (A. sect. Pachyneurium series Pachyneurium), A. obtusum and A. scandens (A. sect. Tetraspermium). As in pattern A, the vasculature of the axis of the spadix diverges to the flower, branching into two (Fig. 9A); at the base of the flower, four vascular complexes are visible, referred to here as external (c1) and internal complexes (c1') (Figs. 5A and 9B). However, the destination differs for these two complexes, as described below. Initially, each of the two external complexes (c1) (Figs. 5A and 9B) develops two others (c2 and c2') (Fig. 5B). Each complex (c2) diverges into two traces: one to the external tepal and the other to the external stamen (Figs. 5B, 5C, 9C and 9D). The trace of the external    ( Fig. 5L). In the transverse section of the carpel, it is possible to observe the variation in the location of the dorsal bundle in relation to the epidermis and the mesophyll: in A. obtusum and A. scandens (Fig. 11C), the dorsal bundle is located next to the internal epidermis; in A. affine (Fig. 11D), it is located in the median plane of the mesophyll, equidistant from the internal and external epidermis. The dorsal bundle of the carpel is no longer visible at the height of the stigma (Fig. 5M).
At the base of the flower, each of the other two vascular complexes (c1') (Figs. 5A and 5B) originates two smaller complexes: one peripheral (pc) and one carpellary (cc) (Figs. 5C and 9C). Each peripheral complex (pc) (Fig. 5C) diverges into two traces: one to the internal tepal and the other to the internal stamen (Figs. 5D-5F and 9C). The trace of the internal tepal (itt) remains singular for almost its entire length (Figs. 5E-5K), except in the upper third, where it branches into two (itt') (Figs. 5L, 5M and 10B). The trace of the internal stamen (ist) extends to the connective (Figs. 5D-5J and 10B).
The placental complex (pl) extends through the ovarian septum (Figs. 5H, 5I, 10D and 10E) and diverges at the height of the placenta into four placental bundles in A. obtusum and A. scandens (Figs. 2F and 5J), and into two placental bundles in A. affine (Figs. 2C and 11A).

DISCUSSION
Our results, based on the analysis of 20 species belonging to Anthurium sect. Dactylophyllium, A. sect. Pachyneurium series Pachyneurium, A. sect. Tetraspermium and A. sect. Urospadix, broaden the knowledge on floral vasculature of this neotropical genus of Araceae and reveal the homogeneity of this characteristic in the stamens and tepals, irrespective of the species and sections studied.
The greatest variations observed here are in relation to the carpels, corroborating the heterogeneity of the gynoecium vasculature of Anthurium, as reported by previous studies (Carvajal, 1977;Barabé, Forget & Chrétien, 1984). Although the vasculature of only two species of the genus has been studied previously, such results demonstrate the existence of two distinct vascular patterns. Our data allow the addition of a third vascular pattern and contribute to the characterization of the carpel of the genus in Araceae.
The first description of floral vasculature in Anthurium was presented by Carvajal (1977) in their study with A. denudatum (A. sect. Belolonchium). In this species, two ventral bundles and two dorsal bundles were observed at the base of the ovary. In the transverse section of the median plane of the ovary, only placental bundles were observed, though without knowledge of their origin, whether they were formed from ventral bundles or from dorsal bundles of the carpels. This pattern was not observed in any of the species studied in the present work.
The second description of the floral vasculature of the genus was presented by Barabé, Forget & Chrétien (1984) in their study with Anthurium lhotzkyanum (=A. augustinum) (A. sect. Urospadix). In the gynoecium of this species, two ventral complexes and two placental bundles were observed both at the base and in the median plane of the ovary. In tranverse sections at the height of the style, the ventral bundles were observed surrounding the stylar canal. There is no report of dorsal bundles of the carpels. This pattern of gynoecium vasculature was corroborated by the majority of the species we analyzed that belong to A. sect. Dactylophyllium, and in some species of A. sect. Pachyneurium series Pachyneurium and A. sect. Urospadix, here called as Pattern A. However, in the present study another terminology is proposed to refer to this ventral vascular supply of the carpels which will be further discussed.
For the first time for this genus, our data show the existence of a third pattern of carpellary vasculature, called as Pattern B. In our sample, the occurrence of this pattern is restricted to only one of the studied species of A. sect. Pachyneurium series Pachyneurium (A. affine) and to all of the studied species of A. sect. Tetraspermium (A. obtusum and A. scandens). These three species all have carpels vascularized by both synlateral and dorsal bundles.
Within Alismatales, Araceae present some representatives, as Pothos (Eyde, Nicolson & Sherwin, 1967;Buzgo, 2001), Anthurium denudatum (Carvajal, 1977), Lysichiton camtschatcensis (L.) Schott (Barabé & Labrecque, 1984), Spathiphyllum wallisii Regel , and Zamioculcas zamiifolia (Lodd.) Engl. (Barabé & Forget, 1988), in which the carpels are vascularized by dorsal bundles and by other bundles that run along their margins. In the ovary these latter bundles are located on the opposite side of the ovarian septum and have been interpreted as pertaining to adjacent carpels (Eyde, Nicolson & Sherwin, 1967;Barabé & Labrecque, 1984;Barabé, Forget & Chrétien, 1984;Igersheim, Buzgo & Endress, 2000;. Although there is a consensus on the nature of the carpellar bundles, distinct terminologies have been used to refer to them, for exafmple: ventral bundle complex (Barabé & Labrecque, 1984;Barabé, Forget & Chrétien, 1984;), ventral bundle (Carvajal, 1977Barabé & Forget, 1988), synlateral bundle (Igersheim, Buzgo & Endress, 2000), and septal vascular bundle  (Buzgo, 2001). This vascular supply may branch out at the height of the style, forming two other ventral bundles (Barabé & Labrecque, 1984;Barabé, Forget & Chrétien, 1986;. In the species studied of Anthurium, the characteristics of the bundles to vascularize the margins of the carpels, either branching (Pattern A) or not (Pattern B) in the style, suggest that they resemble (in position and function) those bundles located opposite the ovarian septum described in previous studies. Anthurium also presents a set of vascular bundles located in the ovarian septum that derive the traces that supply the ovules. In previous studies, this vascular supply is referred to as ventral bundle (Carvajal, 1977) or placental column (Barabé, Forget & Chrétien, 1984) and in the present study, as a placental complex. Our data reveal that this placental complex has the same origin as the bundles located on the margins of the carpels, deriving differently from distinct complexes in Patterns A and B.
The common origin of the bundles of the margins of the carpels and of the ovarian septum suggests the use of a single term to denominate this carpellary supply. Given the different terminologies found in the literature, in the present study we chose to denominate the bundles and placental complex as synlateral bundles, since the traces from which they derive come from different carpels. This term is in accordance with the previous study by Igersheim, Buzgo & Endress (2000) in representatives of basal monocotyledons, including Araceae.
In Acorus (Acoraceae, Alismatales), a genus previously included as early-divergent in Araceae, carpels are supplied by vascular bundles of a central column . Thus, the absence of the dorsal bundles is compensated by the transference of its function to the vascular bundles of central column, which vascularizes the ovules and branch supplying the style ; in other words, the loss of the vascular tissues during floral development leads to a reorganization of the existing bundles for the supply to the floral organs.
On the other hand, in species of Anthurium which gynoecia lack dorsal bundles (Barabé, Forget & Chrétien, 1984; described in the present study as Pattern A), their absence is compensated by splitting the synlateral bundles. These synlateral bundles branch into four bundles (sy') adjacent to the stylar canal, suggesting their equivalence (in position and function) to the dorsal bundles observed in A. affine (A. sect. Pachyneurium series Pachyneurium), A. obtusum and A. scandens (A. sect. Tetraspermium), also studied in the present work.
Also in relation to the floral structure, another highlight regarding the heterogeneity of the carpellary vasculature is the variation in the location of the synlateral bundles. In the majority of the species studied here, the synlateral bundles are located between the margin of the carpel and the ovarian wall. However, only in A. obtusum and A. scandens (A. sect. Tetraspermium) are the synlateral bundles located in the ovarian wall. This variation in the location of the synlateral bundles in A. sect. Tetraspermium may be related to the supply to the apical and ovarian septa found in this group, in addition to the existence of two ovules per locule. The apical septum was reported for A. scandens by Poli, Temponi & Coan (2015) as being related to the ovary's reduced size to accommodate the ovules.
More important than highlighting the existence of these three patterns of carpellary vasculature in Anthurium is to emphasize the variation in terms of the origin of the dorsal and synlateral bundles. Our results provide the first evidence that the carpellary bundles in Anthurium possess a mixed nature; they originate from two distinct vascular complexes and present a relationship intrinsic to the branching, or not, of the external complex (referred to here as c1) of the floral base into an additional complex, referred to here as c2.
The results presented here, together with those already reported, show that the vasculature of the carpels in Anthurium follow three main patterns: Pattern A, in which the gynoecium is vascularized only by synlateral bundles, as verified in the majority of the species of A. sect. Dactylophyllium, A. sect. Pachyneurium series Pachyneurium and A.sect. Urospadix (Barabé, Forget & Chrétien, 1984;Fig. 12); Pattern B, in which the gynoecium is vascularized by synlateral and dorsal bundles, as observed in A. affine, a species belonging to A. sect. Pachyneurium series Pachyneurium, and in A. scandens-A. sect. Tetraspermium (Fig. 12); and Pattern C, in which the gynoecium is vascularized only by ventral bundles (here interpreted as synlateral bundles), while the dorsal bundles are vestigial (Carvajal, 1977), as reported for a single species of A. sect. Belolonchium (Fig. 12).
While analyzing the patterns of vasculature described here, and the possible use of the vascular characteristics for better delimitation of the sections of Anthurium, we noted that the presence of an apical septum and carpels vascularized by dorsal and synlateral bundles (Pattern B)-observed in all of the studied species of A. sect. Tetraspermium (50% of all the species of this group)-might represent synapomorphies of this section. In the evolutionary hypothesis presented in the Bayesian analysis by Carlsen & Croat (2013) (see adapted Fig. 12 in the present study), Anthurium is divided into Clades A and B that contain the large majority of the species. The three patterns of carpel vasculature occur in Clade B and deserve to be investigated in a larger number of species, particularly from A. sect. Pachyneurium, since this section presents this carpel variation and was already designated as being more than one group of species, both by works of classical taxonomy (Croat, 1991) as well as by the phylogenic study of the genus, conducted by Carlsen & Croat (2013). A possibility is that Pattern A could be a plesiomorphy for all Anthurium species, given that it is also found in other species of A. series Pachyneurium, and that the appearance of dorsal bundles is derived within the genus. However, the lack of samples from other sections precludes a clear distinction between these two patterns and reinforces the importance of vasculature data in the genus.