The Evolution of Violaceae from an Anatomical and Morphological Perspective Author(s): Saúl E. Hoyos-Gómez Source: Annals of the Missouri Botanical Garden, 100(4):393-406. Published By: Missouri Botanical Garden DOI: http://dx.doi.org/10.3417/2012027 URL: http://www.bioone.org/doi/full/10.3417/2012027 BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/ page/terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and noncommercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder. BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. THE EVOLUTION OF VIOLACEAE FROM AN ANATOMICAL AND MORPHOLOGICAL PERSPECTIVE1 Saúl E. Hoyos-Gómez2 ABSTRACT Recent work suggests that Fusispermum Cuatrec. and Rinorea Aubl. form small clades that are sister to the rest of the Violaceae and that the Goupiaceae is sister to the Violaceae. However, little is known about the morphology and anatomy of these phylogenetically critical groups. In this paper I present aspects of the morphology and anatomy of stem, node, leaf, flower, and seed of three species of Fusispermum and seven species of Rinorea, as well as Goupia glabra Aubl. (Goupiaceae), which is the outgroup. Placing this variation in the context of hypothesized phylogenetic relationships, I found Fusispermum to have unique pentalacunar nodes, heterogeneous pith, and elongated seeds, while Goupia Aubl. has a unique 5-carpellate gynoecium with marginal styles and a tegmen with U-shaped thickenings. Furthermore, variation in androecium and nectary links the distinctive androecium so common in the Violaceae with more conventional structures found in other taxa of the parietal placentation group of Malpighiales. Strengthening our basic knowledge of anatomy and morphology in these groups is an essential prerequisite for understanding the evolution and diversification not only of Violaceae but of Malpighiales as a whole. Key words: Anatomy, flower, Fusispermum, Goupia, Goupiaceae, leaf and seed morphology, Malpighiales, Rinorea, stem, Violaceae. The violet family (Violaceae) is predominantly tropical in distribution and contains 23 genera and more than 900 species (Feng, 2005; Tokuoka, 2008). The family is supported as monophyletic (Feng, 2005; Tokuoka, 2008), although phylogenetic relationships within it are still unclear (Feng, 2005; Tokuoka, 2008). The Violaceae encompasses a great diversity of vegetative and floral variation. Habits may vary from herbs to lianas to trees, with flowers ranging from zygomorphic (monosymmetric) and strongly spurred to actinomorphic (polysymmetric) and unspurred. Anther variation is considerable, and there is variation in shape and structure of the androecium in the Neotropical Rinorea Aubl., especially in the connective scales. The fruits range from an indehiscent nut or fleshy berry to loculicidal capsules, sometimes explosively dehiscent; the seeds are small to noticeably large and may be winged or carunculate (Hekking, 1988). Recent studies (Wahlert et al., 2014) have changed our understanding of the phylogeny of Violaceae by elucidating unsuspected or lesserknown groups that are prime candidates for the study of morphology and anatomy. Within the Violaceae, clades for Fusispermum Cuatrec. and Rinorea apiculata Hekking (Wahlert & Ballard, 2012) are strongly supported as being successive sisters to the rest of the family (Fig. 1). The uncommon Fusispermum is a Neotropical genus with three species, ranging from Costa Rica to Peru. In contrast, Rinorea is a common pantropical genus with 49 species in the Neotropics (Wahlert & Ballard, 2012). Another development that affects our understanding of the Violaceae is their phylogenetic association with the Goupiaceae, which contains Goupia Aubl. with two species, one from Central America (G. guatemalensis Lundell), and the other (G. glabra Aubl.) from northern South America to the north of the Amazon basin. The phylogenetic position of the Goupiaceae has long been poorly understood. Goupia was included in the Celastraceae by Bentham and Hooker (1862) as the subfamily Goupioideae, a view later accepted by Cronquist (1981). The genus has been recognized as its own family (e.g., Simmons et al., 2001). However, Goupia has not previously been associated with the Violaceae or with any other family 1 I thank the grants and institutions that made this research possible: Fulbright Colombia, Corporación Fragmento, University of Missouri–Saint Louis, Whitney R. Harris World Ecology Center, The Madidi Project–Bolivia (Missouri Botanical Garden), and Idea Wild. I also want to thank Peter F. Stevens, Richard C. Keating, Peter Møller Jørgensen, David Bogler, Sara Fuentes, and Juan Carlos Penagos (Missouri Botanical Garden). Also thanks to Reinaldo Aguilar (Costa Rica); Marcos Rios, Elvis Valderrama, and Asunción Alipo (Peru); Marta Villalobos and Alfredo Fuentes (Bolivia); and Alvaro Cogollo (Colombia). Their helpful assistance in the field was inestimable. The scanning electron microscope was provided by National Science Foundation (NSF) grant DBI-MRI 0958873 to David Bogler. Finally, I’d like to thank the Missouri Botanical Garden and the New York Botanical Garden herbaria for facilitating access to material for my research. 2 Corporacion Fragmento, Carrera 77 # 33 A 61, Medellin, Colombia. saulhoyos@fragmento.org, saulhoyos@gmail.com. doi: 10.3417/2012027 ANN. MISSOURI BOT. GARD. 100: 393–406. PUBLISHED ON 10 AUGUST 2015. 394 Annals of the Missouri Botanical Garden Figure 1. Summary tree diagram for relationships between Fusispermum Cuatrec. and Rinorea Aubl. (Violaceae) and the outgroup exemplar Goupia glabra Aubl. (Goupiaceae). Morphological and anatomical character states useful for understanding relationships among these taxa are mapped on the tree. Those character states found at the stem of this tree (1, 3, 4, 5, 7, 9, 11, 13, 15; see Table 2) may be found in other members of the parietal placenta clade. Mapped characters include (1) cylindrical stem, (2) fluted stem, (3) homogeneous pith, (4) heterogeneous pith, (5) trilacunar nodal anatomy, (6) pentalacunar nodal anatomy, (7) nectary disc, (8) gland adnate to filament, (9) connective scale apical, (10) connective scale basal, (11) style straight, (12) style curved, (13) seed with exotegmen with sclereids (one layer), (14) seed with exotegmen with fibers (four to six layers), (15) stigma area not expanded, and (16) stigma area flared outward. in the parietal placentation group within the Malpighiales. Placentation in the Goupiaceae has been distinguished as basal-axile (Stevens, 2012). Simmons et al. (2001), in their study of the relationships of the Celastraceae, first suggested that its subfamily Goupioideae belonged to the Malpighiales. Feng (2005) and Wurdack and Davis (2009) placed it, as the Goupiaceae, as sister to the Violaceae, and both analyses find strong support for this position. The Goupiaceae are part of a clade that includes the Achariaceae, Lacistemataceae, Passifloraceae, and Salicaceae. Goupia remains the only taxon in this group that has axile placentation, with all the others presenting parietal placentation (Wurdack & Davis, 2009). Few comprehensive studies have been published on the Violaceae, and the morphology of the family, particularly Fusispermum species and the Rinorea apiculata clade, is largely unknown (Wahlert & Ballard, 2012). Even less is known about floral evolution and development (but cf. Arnal, 1945; Feng, 2005). Most studies have focused on Viola L., a genus widely distributed across both hemispheres. But Viola is atypical in the family in being largely herbaceous, with derived character states, and of largely temperate distribution (Metcalfe & Chalk, 1972; Corner, 1976; Feng, 2005). In contrast, the majority of the family are woody and tropical taxa, so extrapolation from the derived states in Viola to the other genera in the family may be inappropriate or misleading. Metcalfe and Chalk (1972) summarized what little is known of the vegetative anatomy of Goupia, Fusispermum, and Rinorea. For seeds, Hekking (1984) briefly described the external morphology of the seeds of Fusispermum. Corner (1976) investigated the seed anatomy of Viola, Hybanthus Jacq., and two species of Malesian Rinorea. The broadest survey of seed anatomy of Violaceae is by Plisko (1992), but only 14 species were studied there. The seed anatomy of G. glabra was described by Melikian and Sarinov (2000; but see below). The seed anatomy of Fusispermum and the R. apiculata group has not been previously described. Volume 100, Number 4 2015 Hoyos-Gómez The Evolution of Violaceae 395 In an attempt to develop a better understanding of the evolutionary history of Violaceae, I examine here the stem, leaf, flower, and seed of three species of Fusispermum, of the Rinorea apiculata clade (R. apiculata and R. crenata S. F. Blake), and Rinorea s. str. (five species considered), as well the outgroup exemplar Goupia glabra (Goupiaceae). For these taxa, I describe anatomical and morphological characters of the stem, node, leaf, androecium, gynoecium, and seed anatomy and morphology using scanning electron microscopy (SEM) and light microscopy (LM). I then place this information in the context of proposed phylogenetic relationships among malpighiacous taxa in the Violaceae and Goupiaceae. Olympus BX40 microscope (Olympus, Tokyo, Japan) was used to record all light microscopic images. For SEM imagery, plant material was dehydrated in an increasing ethanol series and then critical-point dried (using an EMITech-K 850 critical-point dryer [EMITech Inc., Fall River, Massachusetts, U.S.A.]). The samples were affixed to supports using carbon adhesive tape and were gold-coated (using a Denton vacuum sputter coater [Denton, Moorestown, New Jersey, U.S.A.]) and were examined using a JEOL Neoscope JCM 5000 SEM (JEOL, Tokyo, Japan). The SEM was used to study cuticle and stomatal microdetail, as well as pollen, fruit, and flower ultrastructure. RESULTS MATERIALS AND METHODS During the summer of 2010 several field trips were made to Central and South America to obtain plant material. In Bolivia, collection and observations were made in Departamento de la Paz, Provincia Franz Tamayo, Parque Nacional Madidi, Laguna Chalalan, 14825 0 39.49 00 S, 67854 0 57.68 00 W, at an altitude of 350 m. In Colombia, our collection site was ´ ˜ Antioquia, Municipio de San Luis, Canon de Rıo Claro, Reserva Natural, 05853 0 N 074839 0 W, 350 m. In Costa Rica, exemplars were taken from Puntarenas, Osa, Sierpe, Reserva Forestal Golfo Dulce, Estación Biológica Los Charcos de Osa, 08840 0 18 00 N 83830 0 17 00 W, 70 m. In Peru, field work occurred in Distrito de Loreto, Provincia de Maynas, carretera Bella Vista–Mazán, 3831 0 41.9S, 73805 0 45.8W, 86 m. Voucher specimens (cf. Table 1) of the newly collected material were deposited in Costa Rica (INB), Bolivia (LPB), Peru (USM), Colombia (COL, HUA, JAUM), and in the United States (MO). When additional material was needed, I used collections from the Missouri Botanical Garden (MO). No material of Rinorea oraria Steyerm. & A. Fernández, which is the third known member of the R. apiculata group, was available. In the field, stem sections, leaves, flowers, and fruits were preserved in 70% ethanol. Leaves from herbarium specimens were used to complete the taxonomic sampling (Table 1). Dried leaves were rehydrated by boiling in water with a drop of detergent before examination. Cross sections of leaves and petioles were handmade, using a razor blade. Microtome sections of seeds were also made. Cresyl violet acetate (CVA) was used for tissue staining, and a 20% calcium chloride solution (CaCl2; Ogburn & Edwards, 2009) was used as a mounting solution (Keating, 1996, 2000). A Canon Power Shot A640 camera (Canon, Tokyo, Japan) coupled to an YOUNG STEMS All species studied had lenticels on the stem and a cork cambium that developed early and was superficial in position. A band of collenchyma tissue ca. five to 10 cells broad was observed in the outer cortex. Goupia glabra (Goupiaceae) lacked crystals and druses in the collenchyma; however, all Violaceae studied had rhombic crystals and druses of calcium oxalate there. No sclereids or other idioblastic cells were observed in the stem cortex. The stem vasculature of all species studied was eustelic. The central vascular cylinder was noted as more or less circular in transverse section, except in G. glabra, where the vascular cylinder was fluted (Fig. 2A). The outermost phloem was completely surrounded by a band of pericyclic fibers. Stem pith was well developed in all species and was usually made up of small, unlignified cells that were more or less similar in size. In G. glabra, pith cells were ca. 20–50 lm in diameter (Fig. 2A); in all Rinorea species studied, pith cells were 15–40 lm across; in all species the walls of the pith cells were fairly thin, ca. 4–5 lm across (Fig. 2B, C, R. crenata, R. paniculata (Mart.) Kuntze, respectively). In contrast, in Fusispermum laxiflorum Hekking (Fig. 2D), F. minutiflorum Cuatrec., and F. rubrolignosum Cuatrec., the pith cells were larger and variable in size, 50–200 lm in diameter; the cell walls were thinner, ca. 2–3 lm across. Pith in Fusispermum can be considered heterogeneous in contrast to the more homogeneous condition confirmed in both Goupia and Rinorea exemplars. STEM NODES Goupia glabra (Fig. 2E) and Rinorea crenata (Fig. 2F), as did all Rinorea exemplars, had three-trace, trilacunar nodes. However, Fusispermum laxiflorum 396 Annals of the Missouri Botanical Garden Figure 2. Light microscopic images of stem and leaf blade sections among representative Rinorea Aubl. and Fusispermum Cuatrec. (Violaceae) and Goupia Aubl. (Goupiaceae, outgroup). —A. Transverse stem section, G. glabra Aubl. (Thomas 3992). —B. Transverse stem section, R. crenata S. F. Blake (S. E. Hoyos-Gómez 1115). —C. Transverse stem section, R. paniculata (Mart.) Kuntze (S. E. Hoyos-Gómez 1110). —D. Transverse stem section, F. laxiflorum Hekking (S. E. Hoyos-Gómez 1119). —E. Internodal stem anatomy, G. glabra (Thomas 3992). —F. Internodal stem anatomy, R. crenata (S. E. Hoyos-Gómez 1115). —G. Internodal stem anatomy, F. laxiflorum (S. E. Hoyos-Gómez 1119). —H. Leaf blade midrib, F. laxiflorum (S. E. Hoyos-Gómez Volume 100, Number 4 2015 Hoyos-Gómez The Evolution of Violaceae (Fig. 2G), F. minutiflorum, and F. rubrolignosum had pentalacunar nodes, with the vascular traces widely spaced from the central cylinder. In all the species the stipules were vascularized from the lateral traces. vascular strands. Finally, toward the apex of the petiole leading into the blade, the vasculature became a more or less complete cylinder surrounding a smaller elliptical cylinder of xylem with phloem on the inside; in addition, there are two small lateral wing bundles on either side of the central cylinder. In all the Fusispermum and Rinorea species studied, the leaves were dorsiventrally oriented, with scattered uniseriate hairs on both sides but principally on the abaxial side; Goupia glabra had scarce uniseriate hairs on the abaxial side only. Leaf blades varied in thickness from the thickest, ca. 270 lm, in F. laxiflorum (Fig. 3F), to ca. 190 lm in G. glabra and ca. 150–180 lm among the seven sampled species of Rinorea (Fig. 3G, H, R. apiculata and R. squamata, respectively). In both G. glabra and F. laxiflorum (Fig. 3F) there was well-defined palisade tissue: with two palisade layers seen in the former and two to three layers in the latter. However, in the seven Rinorea species sampled, the palisade tissue was irregularly organized as two to four layers and was not strongly differentiated from the rest of the mesophyll (Fig. 3G, H, R. apiculata and R. squamata, respectively). No sclereids or other idioblastic cells were observed in either Fusispermum or Rinorea exemplars. There was some chlorophyllous tissue on the adaxial side of the midrib among the seven species of Rinorea but not in F. laxiflorum or G. glabra. Otherwise the midrib was transcurrent, and smaller veins were embedded. The vascular tissue in the midrib of the 11 species studied consisted of two arcuate bands of fibers surrounding the vascular tissue. In Goupia glabra and Fusispermum laxiflorum alone there were also fibers in the medullary tissue of the bundle, and in F. laxiflorum they were made up of thin-walled cells like those of the stem pith. In F. laxiflorum there were two additional bands of vascular tissue in the midrib that were continuous with the elliptical central cylinder in the petiole. All 11 species studied had hypostomatic leaves and anomocytic stomata, the stomata being ca. 16–20 lm long. In the seven species of Rinorea there were usually three epidermal cells that were directly adjacent to the guard cells, although these adjacent cells were not otherwise distinguishable from the surrounding epidermis. This differed in other taxa in that Goupia glabra had four to five epidermal cells adjacent to the stomata, and in Fusispermum laxiflorum there were three to five cells directly abutting the stomata. LEAVES In transverse sections taken at the midpoint of the petiole, peripheral collenchyma tissue was found in all species exemplars. The cells were tanniniferous and contained rhombic crystals and druses, except in Goupia glabra (Fig. 3A), which lacked rhombic crystals but did have druses. In all sampled species except Rinorea apiculata (Fig. 3B), the vascular tissue formed a more or less closed and somewhat dorsiventrally flattened cylinder with phloem on the outside, xylem on the inside, and the vascular cylinder surrounded by fibers. In R. apiculata the main petiole bundle was arcuate, and even there the edges of the arc were strongly incurved. The petiole pith was usually unlignified, but there were dispersed fibers in the pith of the petioles in G. glabra (Fig. 3A). All the examined species in Fusispermum and Rinorea had druses in the pith cells, but G. glabra did not. As was seen in the stem cross section, F. laxiflorum had much larger and thinner-walled pith cells (Fig. 3C, D) than those of the other examined species. There were rib traces on either side of the main petiole bundle, usually in adaxial to lateral positions; these traces consisted of arcuate bands of vascular tissue accompanied by a few fibers, especially adjacent to the phloem. There were four rib traces, two on each side of the main bundle, typically seen in Rinorea apiculata (Fig. 3B), R. crenata, and R. paniculata. However, in R. viridifolia Rusby, R. dasyadena A. Robyns, and R. squamata S. F. Blake, only two rib traces were present, one on each side, while in all four specimens of R. lindeniana (Tul.) Kuntze (Fig. 3E) examined there were three adaxial rib traces, two on one side and one on the other side above the vascular cylinder. Fusispermum laxiflorum (Fig. 3C, D), F. minutiflorum, and F. rubrolignosum have a rather complex petiole vasculature. At the very base of the petiole, immediately after traces depart to the stipules, the vasculature consists of a larger central vascular cylinder and two lateral cylinders on either side, the latter representing the lateral traces. The lateral and central bundles merge and reorganize, becoming broadly incurved and arcuate with a pair of adaxial 397 1119). Scale bars in A, D, E, G ¼ 500 lm; scale bars in B, C, F ¼ 350 lm; scale bar in H ¼ 700 lm. Asterisks indicate nodes in E–G. All collections are at MO. 398 Annals of the Missouri Botanical Garden Figure 3. Light microscopic images of leaf petiole and blade sections among representative Rinorea Aubl. and Fusispermum Cuatrec. (Violaceae) and Goupia Aubl. (Goupiaceae, outgroup). —A. Transverse petiole section, G. glabra Aubl. (Thomas 3992). —B. Transverse petiole section, R. apiculata Hekking (F. Hurtado 2964). —C, D. Transverse petiole section, F. laxiflorum Hekking (S. E. Hoyos-Gómez 1119). —E. Hand section of leaf blade, R. lindeniana (Tul.) Kuntze (S. E. Hoyos-Gómez 1135). —F. Hand section of leaf blade, F. laxiflorum (S. E. Hoyos-Gómez 1119). —G. Hand section of leaf blade, R. apiculata ˜ 6026). —H. Hand section of leaf blade, R. squamata S. F. Blake (S. E. Hoyos-Gómez 1114). Scale bars in A ¼ 150 lm; (P. Nunez B, E ¼ 200 lm; C, D ¼ 300 lm; F–H ¼ 50 lm. All collections are at MO. Volume 100, Number 4 2015 Hoyos-Gómez The Evolution of Violaceae 399 Figure 4. SEMs of androecia and gynoecia among representative Rinorea Aubl. and Fusispermum Cuatrec. (Violaceae) and Goupia Aubl. (Goupiaceae, outgroup). —A. Androecium, G. glabra Aubl. (B. E. Hammel 21278). —B. Androecium, F. laxiflorum Hekking (G. S. Hartshorn 2929). —C. Androecium, R. crenata S. F. Blake (M. H. Grayum 10069). —D. Androecium, R. lindeniana (Tul.) Kuntze (S. E. Hoyos-Gómez 999). —E. Gynoecium, G. glabra (B. E. Hammel 21278). —F. Gynoecium, F. laxiflorum (Hartshorn 2929). —G. Gynoecium, R. crenata (M. H. Grayum 10069). —H. Gynoecium, R. viridifolia Rusby (S. E. Hoyos-Gómez 1003). —I. Gynoecium, R. lindeniana (S. E. Hoyos-Gómez 1127). Scale bars in A, B ¼ 100 lm; C, F, I ¼ 500 lm; D, E, G ¼ 200 lm; H ¼ 1000 lm. All collections are at MO. ANDROECIA In all 11 species studied, stamen filaments were glabrous. In Goupia glabra these filaments were ca. 200 lm long, and the anther thecae were ca. 260 lm long; in Fusispermum laxiflorum they were ca. 500 lm and 240 lm long, respectively. In the seven Rinorea species examined the filaments ranged from ca. 300 lm to 950 lm long, and the anther thecae were comparatively longer than in G. glabra and F. laxiflorum, ranging from ca. 530 lm to 1100 lm long. Goupia glabra was the only species to have trapezoid anther shapes (Fig. 4A); the remaining 10 species exemplars had ellipsoid to ovoid anthers. In all species dehiscence was introrse via longitudinal slits in each theca. In Goupia glabra, the androecial connective formed a thick, blunt prolongation beyond the thecae ca. 90 lm long, with cylindrical hairs ca. 230 lm long that were scattered on both the connective and thecae (Fig. 4A). In Fusispermum laxiflorum (Fig. 4B), F. minutiflorum, and F. rubrolignosum, the prolongation of the connective was less massive, ca. 80 lm long, with an irregular fringe apically, while in Rinorea apiculata and R. crenata (Fig. 4C) the connective formed a broad apical prolongation that exceeded the thecae in length, ca. 930–1100 lm long, with a fimbriate margin. In the remaining five Rinorea species sampled, the thecae were not evident from the dorsal side; the connective formed a slightly fringed acute to triangular projection ca. 1300–2000 lm long. Only in R. apiculata and R. crenata (Fig. 4C) were there flattened hairs on both the adaxial and abaxial surfaces of the anther thecae; in R. squamata 400 Annals of the Missouri Botanical Garden there were scattered cylindrical hairs adaxially on the thecae. The remaining Violaceae exemplars had glabrous anthers. In Goupia glabra and Fusispermum laxiflorum, F. minutiflorum, and F. rubrolignosum, an apparently warty disc-like structure with epidermal stomata surrounded the stamens but was quite separate from the anthers. In G. glabra this lobed disc completely surrounded the flower (Fig. 4E), with the lobes alternating with the staminal radii. In F. laxiflorum (Fig. 4F), F. minutiflorum, and F. rubrolignosum, the massive disc consists of five lobes alternating with the stamens. By contrast, the seven examined species of Rinorea lacked a disc but did have an abaxial gland-like structure at the base of the filaments; this has hairs in R. lindeniana (Fig. 4D), R. viridifolia, and R. paniculata. In both R. apiculata and R. crenata (Fig. 4C), this gland was massive, ca. 190 lm long, hemispherical, and joined at the very base with adjacent glands. In the other five species of Rinorea, glands were distinct from one another and borne on the filament. In the 11 species studied, there were stomata on the disc and glands consistent with their being nectariferous. stigmatic apex resembled that of the other species of Rinorea examined. GYNOECIA Goupia glabra has a glabrous gynoecium of five carpels with five styles borne toward the margin of the carpels (Fig. 4E). Each style, only ca. 200 lm long, has an adaxial furrow and tapers somewhat toward a perhaps hollow but otherwise undistinguished apex. Placentation is axile with ca. five to six ovules per carpel. All 10 species exemplars of the Violaceae had three fused carpels with parietal placentation. There were many ovules per carpel in Fusispermum laxiflorum, F. minutiflorum, and F. rubrolignosum, but only one ovule per carpel in the seven species of Rinorea examined. In F. laxiflorum (Fig. 4F), F. minutiflorum, F. rubrolignosum, R. apiculata, and R. crenata, the ovary was glabrous, but in other species such as R. viridifolia (Fig. 4H), the ovary was pubescent. Styles of all exemplars were continuous with the ovary. In F. laxiflorum (Fig. 4F), F. minutiflorum, and F. rubrolignosum the straight style was narrowed distally to the apex, which had an inconspicuous depression, while in R. apiculata and R. crenata (Fig. 4G) the style was conspicuously flared at the apex. Rinorea viridifolia and R. squamata had straight styles and stigmas rather like those of F. laxiflorum (Fig. 4F). Rinorea lindeniana and R. paniculata had sigmoid styles, subapically swollen in R. lindeniana, but in both species the SEEDS The outgroup representative Goupia glabra had dark brown ovate seeds ca. 2–3 mm long (Fig. 5A), with the anticlinal walls of the exotesta forming a reticulum (Fig. 5B, C). In transverse section the exotesta was two, perhaps three, cell layers thick and ca. 60–80 lm across; the outer periclinal walls of the exotesta were slightly thickened. There was also a single layer of exotegmic sclereids, these cells being ca. 60 3 100 lm and massively thickened on the anticlinal and inner periclinal walls and also pitted (Fig. 5B, C). Goupia glabra lacked crystals in the seed coat. Its straight embryo occupied half the length of the seed and was surrounded by copious oily endosperm. In contrast, Fusispermum minutiflorum (Fig. 5D) and F. rubrolignosum had pale brown elongated seeds, ca. 2–3 mm long. The seed had what appears to be a wing down one side where the seed coat was much thicker and was ca. 11 to 16 cells across. There was also a single layer of exotegmic sclereids, the cells being ca. 100 3 10 3 15 lm, and the cell walls being massively lignified all around and pitted (Fig. 5E). There were druses in the testa, as were seen in all seven Rinorea species studied. The straight embryo occupied 3/5 the length of the seed, and an oily endosperm surrounded the embryo (Fig. 5D). Rinorea apiculata and R. crenata (Fig. 5G) have light brown ovoid seeds, ca. 4–5 mm long. In transverse section, the testa consisted of unlignified, more or less isodiametric cells three to four layers thick. There was a single layer of exotegmic cells ca. 150 3 10 3 70 lm (Fig. 5F, R. apiculata). These cells had more or less pitted walls that were all equally thickened. Only in R. apiculata and R. crenata among the seven species studied here were there rhombic crystals in the testa. The straight embryo occupied four fifths of the length of the seed and was surrounded by copious oily endosperm surrounding the embryo. Rinorea squamata, R. viridifolia (Fig. 5H), and R. dasyadena had brown globose seeds ca. 6–9 mm across. The testa consisted of eight to 10 layers of unlignified flattened cells, with individual cells isodiametric and ca. 20 lm across (Fig. 5H, R. viridifolia). There is a middle layer of lignified cells about four to six cells across, the cells being ca. 190 3 15 3 30 lm (Fig. 5H). The cell walls were equally thickened and more or less consistently pitted. The embryo occupied four fifths of the length of the seed, Volume 100, Number 4 2015 Hoyos-Gómez The Evolution of Violaceae 401 Figure 5. Light microscopic images of seeds and transverse sections among Fusispermum Cuatrec. and Rinorea Aubl. (Violaceae) and Goupia glabra Aubl. (Goupiaceae). —A. Two intact seeds, G. glabra. —B. Transverse seed section, G. glabra. —C. Transverse seed section, G. glabra. (A–C taken from T. W. Henkel 3245.) —D, E. Transverse seed sections, F. minutiflorum Cuatrec. (J. Espina 1330). —F. Transverse seed section, R. apiculata Hekking (C. E. Cerón 5478). —G. Three intact seeds, R. crenata S. F. Blake (B. E. Hammel 18113). —H. Transverse seed section, R. viridifolia Rusby (S. E. HoyosGómez 1004). Scale bars in A ¼ 3000 lm; B–E ¼ 250 lm; F, H ¼ 50 lm; G ¼ 2000 lm. All collections are at MO. 402 Annals of the Missouri Botanical Garden and the cotyledons had several sinuous foldings; copious oily endosperm surrounded the embryo. As Howard (1974, 1979a, 1979b) emphasizes, the vascular anatomies of stem, petiole, and blade midrib cannot be considered in isolation. Bearing this in mind, I nevertheless particularize some observed variations to facilitate the discussion. Sinnott (1914) previously reported three-trace, three-gap nodal anatomy for the Violaceae, Passifloraceae, and Flacourtiaceae s.l., in general for plants with stipules. Such trilacunar stem nodes are found throughout the Malpighiales, with the exception of the Bonnetiaceae–Calophyllaceae clade (cf. Stevens, 2012). The eight species studied here for Rinorea and Goupia shared these trilacunar nodes, with the exception of Fusispermum, which had pentalacunar stem nodes. In the context of Malpighiales, these pentalacunar nodes are likely to be an apomorphy and are mapped as such (Fig. 1). The most conspicuous gross variation for leaf midrib anatomy in the species studied here was observed for the number of adaxial to lateral bundles and the tissue configuration in the central vascular bundle of the midrib. Although Goupia glabra and Fusispermum laxiflorum had medullary plates of vascular tissue associated with the central bundle, the midrib vasculature was very different in morphology. Metcalfe and Chalk (1972) mention that in G. glabra, xylem was adaxially presented and phloem abaxial (1972). However, in F. laxiflorum, xylem entirely surrounded phloem in the latter, which is rather unusual and probably an apomorphy (Fig. 2H). Rinorea apiculata has an open central bundle, while the midrib vasculature in the other Rinorea species examined is annular. Although this anatomical variation was not optimized on the summary tree, the structure of the petiole bundle (and associated nodal anatomy) throughout the family will require further examination. For the herbaceous Viola, Metcalfe and Chalk (1972) report an arcuate central vascular strand in the petiole accompanied by one or two rib strands on each side. There are possible correlations with leaf insertion. Thus, Fusispermum laxiflorum, F. minutiflorum, F. rubrolignosum, Rinorea apiculata, R. crenata, and R. paniculata, all with distichous phyllotaxy, have two pairs of rib bundles. It will be interesting to see if other species in Rinorea s. str. that have been reported to have such ranked or alternate leaves have similar vasculature. It is confirmed here that R. viridifolia, R. dasyadena, and R. squamata, which have opposite leaves and symmetric lamina bases, have only a single rib bundle on either side of the petiole. Rinorea lindeniana, with two rib bundles on one side and one on the other (Fig. 3E), presents an interesting DISCUSSION The results of this study suggest a number of morphological and anatomical characters that are useful for understanding the Neotropical relationships among the Goupiaceae (i.e., Goupia glabra) and the two basalmost genera among the Violaceae (i.e., Fusispermum laxiflorum, F. minutiflorum, and F. rubrolignosum; Rinorea apiculata, R. crenata, R. lindeniana, R. squamata, R. viridifolia, R. dasyadena, and R. paniculata). Several significant character states (16) are mapped on a summary tree diagram for phylogenetic relationships among these Violaceae and Goupiaceae (Fig. 1). Observations are confirmed here, often for the first time, and are integrated with the sparse literature available (cf. Metcalfe & Chalk, 1972, anatomy; Corner, 1976, seeds). Overall, these results show the value of anatomical studies when related to a phylogeny. Nodal anatomy is a good example. Goupia glabra was considered to belong to Celastraceae by some authors based on gross morphology (Cronquist, 1981). However, the nodes of Celastraceae are overwhelmingly unilacunar (Sinnott, 1914; Cronquist, 1981). The three-trace, three-gap nodes of G. glabra are more consistent with its new position, more so since other Celastraceae with other than unilacunar nodes (e.g., Perrottetia Kunth, Bhesa Buch.-Ham. ex Arn.) have also been removed from Celastraceae to clades where these nodes are common (Stevens, 2012). Furthermore, the pentalacunar nodes of Fusispermum laxiflorum are uncommon in Malpighiales and are a potential apomorphy for the genus, and it can be noted here that nodal anatomy is associated with a distinctive petiole anatomy. The fluted stem pith of Goupia glabra is an apparently trivial difference, which is nevertheless consistent within the Goupiaceae and contrasts with the cylindrical pith seen within those Violaceae analyzed (see also Metcalfe & Chalk, 1972). Although little more is known about pith shape in the Achariaceae, it has been characterized as circular (Metcalfe & Chalk, 1972), so that the fluted stem pith of Goupia may be regarded as an apomorphy, and stem pith shape is mapped on Figure 1 (character states 1, 2). Metcalfe and Chalk (1972: 103) say of Violaceae that ‘‘the pith is solid in woody species, but frequently becomes hollow in herbs’’; however, there is no mention of heterogeneous pith. The heterogeneous pith of Fusispermum is potentially an apomorphy for the genus. Volume 100, Number 4 2015 Hoyos-Gómez The Evolution of Violaceae 403 anomaly. It has distichous leaves with very strongly asymmetric lamina bases, which may account for the odd number of vascular bundles. All 10 species of the Violaceae (Rinorea, Fusispermum) studied here had calcium oxalate crystals (as rhombic crystals and druses) in the collenchyma. Only Goupia glabra lacked such rhombic crystals of calcium oxalate in the collenchyma tissue. The thickness of the leaf lamina found here for the 10 Violaceae sampled may correlate with environmental conditions (Dickison & Weitzman, 1996; Alvarenga & Lombardi, 2010). Fusispermum laxiflorum, with the thickest blades at 270 lm, can reach the forest canopy, being a tree up to 20 m or more, while those Rinorea species studied are understory species from 2 to 10 m tall. In the sampled species, the leaves of Rinorea ranged from 150 to 180 lm, and these trees never reached the canopy. Both Goupia glabra and F. laxiflorum had similar leaf blades with well-developed palisade tissue as sampled, and both were trees that reach the canopy. The Rinorea species studied have poorly organized palisade tissue, and all were collected from smaller plants. It should be noted that R. apiculata can grow to heights of 30 m (Hekking, 1988). Palisade tissue tends to be better developed under well-insulated conditions (e.g., Dickison & Weitzman, 1996; Alvarenga & Lombardi, 2010). Some species of Viola have been noted to have no clearly differentiated palisade tissue (Metcalfe & Chalk, 1972), but the genus is herbaceous and may occur in somewhat shaded conditions. The structure of the midrib largely mirrored that of the petiole for the 11 species sampled. In Rinorea apiculata, with its incurved-arcuate primary petiole bundle, the midrib was fully bilayered, as in the other species of Rinorea s. str. examined. In Goupia glabra the midrib was also bilayered, evidence of the petiolar medullary plate being lost except for the presence of a few medullary fibers. In Fusispermum laxiflorum, F. minutiflorum, and F. rubrolignosum, the vascular tissue in the midrib consisted of two arcuate bands of fibers surrounding the vascular tissue, with two additional bands of vascular tissue in the midrib that were continuous with the elliptical central cylinder in the petiole. The stamens of many Violaceae are notable for the abaxial nectar glands on some or all filaments and the complex, flattened connective (Arnal, 1945). Species of Rinorea s. str. have glands adnate to the androecial filament that can be either fused or free, and the variation is taxonomically significant. Assuming that the Goupiaceae are sister to Violaceae, there appears to be an increasing elaboration of the androecial connective and a shift in the nectary from a structure more or less separate from other parts of the flower to its adnation to the filament in basal Violaceae, as is seen in the Rinorea exemplars, even though polarization of the anatomical variation was difficult to distinguish. The rather massive disc surrounding the staminal whorl in Goupia glabra and Fusispermum laxiflorum is similar to the nectaries of other members of the parietal placentation group in the Passifloraceae, Violaceae, and Salicaceae (Stevens, 2012), with the nectary lobes alternating with the filaments. For the seven species of Rinorea examined, the nectary was more specifically associated with the abaxial part of the filament. The enlarged, hemispherical disc of the R. apiculata group (R. apiculata, R. crenata, and R. oraria) is morphologically similar to the thickened disc of Fusispermum and Goupia, while in the other species of Rinorea s. str. the nectariferous tissue is more closely associated with the filament, which is the more common condition seen elsewhere in the family. These characters—the nectary lobes alternating with or opposite to filaments, the nectary disclike, and the gland adnate to the filament—are included on the tree (see Fig. 1). However, more detailed comparative studies on the stamens/nectaries in Violaceae are likely to disclose more phylogenetically interesting information. The anther connective is stout and rounded in Goupia glabra but flattened and relatively thin in all other species sampled. Fusispermum laxiflorum, F. minutiflorum, and F. rubrolignosum have a very short, erose connective ca. 80 lm long, while the longest connective occurs in Rinorea viridifolia (ca. 2 mm long). In the R. apiculata group (R. apiculata, R. crenata, and R. oraria), the anther thecae are visible in adaxial view, while in the other species they are concealed. The connective scales are known to vary in size and shape among other Violaceae (Engler, 1925; Arnal, 1945). Variation in the gynoecia of the 11 species sampled is of considerable interest. The axile placentation and basal ovules of the outgroup Goupia glabra (see, e.g., Miers, 1862) are unique among the parietal placentation group in the Malpighiales and may be hypothesized as apomorphies, as are the 5carpellate gynoecium and marginal styles (uncommon in Malpighiales). However, details of the stigmatic area of both Goupia and those Violaceae sampled may well be similar: the stigmatic area is not expanded, and there is a central depression or pore noted in all (Fig. 4E, F, H). More noteworthy in the Rinorea apiculata and R. crenata (Fig. 4G), the apex of the style flares outward a likely apomorphy. 404 Annals of the Missouri Botanical Garden Most Violaceae studied had straight styles (Fig. 4H), but in Rinorea lindeniana the style is curved at the base (Fig. 4I), and in R. paniculata the style is sigmoid at the base. In genera such as Hybanthus, Amphirrhox Spreng., Paypayrola Aubl., and Gloeospermum Triana & Planch., the styles are straight, consistent with the family (Steyermark et al., 2005). In Rinoreocarpus ulei (Melch.) Ducke and other species of Rinorea and Viola, the style has been noted as slightly curved (Engler, 1925; Arnal, 1945; Hekking, 1988). The taxonomic significance of the style curvature needs further investigation within the family. Of the species studied, only Goupia and Fusispermum have more than a single ovule per carpel (Hekking, 1984), which is probably the plesiomorphic condition for Violaceae as a whole. Although the seven species of Rinorea examined consistently had only one ovule per carpel, there is considerable variation in ovule number in other Violaceae, and the phylogenetic utility of the character is not clear. It has been suggested that the flowers of Rinorea (see Arnal, 1945) and Fusispermum (Hekking, 1988) species are actinomorphic. Recent studies of floral development in Rinorea demonstrate that the polysymmetry of this actinomorphy is achieved late in the ontogeny. Almost actinomorphic to very weakly zygomorphic flowers are noted in Gloeospermum (Steyermark et al., 2005), and flowers that are actinomorphic in early stages of development in Leonia Ruiz. & Pav. become zygomorphic later (Feng, 2005). Feng and Ballard (2005) suggest that the family had a most recent common ancestor with zygomorphic monosymmetric, rather than actinomorphic, flowers. However, early developing flower buds of F. laxiflorum and two species of Rinorea (i.e., R. lindeniana and R. dasyadena) have been seen to be zygomorphic with a more or less oblique orientation (Hoyos-Gómez, pers. obs.), which might suggest that zygomorphic flowers may indeed be a synapomorphy for the Violaceae. There is interesting variation in the seed coat among the Violaceae, and studies by Corner (1976), Plisko (1992), and Melikian and Sarinov (2000) have placed this variation in phylogenetic context. When studying seed coat anatomy, ideally one should look at developmental stages so that one can follow the development of particular portions of the integuments as incorporated into the seed coat (e.g., Corner, 1976). Such developmental series could not be observed here, so the attribution of cells to the tegmen is inferred on the number of layers to the testa, the presence of a cuticle, and so forth. The filiform seeds of Fusispermum minutiflorum have longitudinal ridges or wings 11 to 16 cells across, with the embryo occupying about half the length of the seed. This differed from the ovoid to ellipsoid seeds of the other taxa examined here and also differed from seeds of the Achariaceae, Passifloraceae, and the like (Malpighiales). The winged, filiform seed shape noted for species of Fusispermum can be considered an apomorphy and is linked to differences in seed coat anatomy. The exotegmen of the seed wall consisted of a more or less well-developed layer of lignified cells, all cell walls of which were more or less evenly thickened in the Fusispermum and Rinorea species sampled (but not in Goupia glabra), and there were fine plasmodesmata evident across the cell walls (Fig. 5E, F). In G. glabra the testa was at least two to three cell layers across, and there was a single layer of massively cubical cells, presumably exotegmic (Fig. 5C). In the R. apiculata group (with R. crenata) the testa was three to four cell layers across, and the sclerified layer consisted of a single layer of obliquely overlapping cells (Fig. 5F). In the other five species of Rinorea s. str. examined, the seed coat was eight to 10 cell layers across, of which four to six layers were lignified, the cells being elongated and flat lying (Fig. 5H). A single layer of sclerified cells in the exotegmen was found in all Violaceae studied by Corner (1976), Plisko (1992), and Melikian and Sarinov (2000), including the berry-fruited Leonia. But for species like Rinorea anguifera Kuntze (Plisko, 1992), four to five layers of sclerified cells were observed. In this study, R. dasyadena, R. lindeniana, and R. squamata have similar, multilayered sclerified tegmic tissue. Note that Melikian and Sarinov (2000) show the seed coat of Goupia glabra as having mesotestal sclereids. However, although the material examined here was limited, there was clearly more than one layer of cells above the sclerified layer, suggesting that the sclerified layer is exotegmic. This layer was also definitely longitudinally ridged, another feature not noted by Melikian and Sarinov (2000), who placed the Goupiaceae near Celastraceae, which would commonly have a lignified mesotesta. Some kind of exotegmen made up of more or less fibrous cells is common in the Malpighiales (e.g., Corner, 1976; Stevens, 2012). Details of cell elongation and the degree, and plane, of the cellular flattening have varied and need to be integrated with phylogeny across the clade. However, despite our still limited knowledge of seed coats in the Violaceae, the four or five layers of fibrous cells found in the species Volume 100, Number 4 2015 Hoyos-Gómez The Evolution of Violaceae of Rinorea s. str. studied, as a provisional finding, can be considered unique. The embryo in Goupia glabra, Fusispermum minutiflorum (Fig. 5D), and the Rinorea apiculata clade was straight, with flat cotyledons, a condition considered to be normal for the family (e.g., Plisko, 1992). However, in the other examined species of Rinorea s. str., the cotyledons exhibited complex folds. Vogel (1980) noted that Rinorea species had seedling type I (Macaranga Thouars–type), where cotyledons in most cases were more or less folded within the seed. Further investigation of embryo morphology would repay the effort with characters of phylogenetic significance. Examination of the androecium and nectary in those Violaceae and Goupiaceae examined relates the distinctive androecium so common in Violaceae with more conventional structures found in other members of the parietal placentation group within the Malpighiales. Although many features of the gynoecium of Goupia are quite different from those of the parietal classification group, the two families had not been previously associated, not until support for the sister relationship of the Goupiaceae to the Violaceae was corroborated by molecular evidence. Otherwise, in the morphology of the stigma and androecial features, as well as in basic anatomy, the Violaceae and Goupiaceae agree quite well. Without such detailed anatomical and morphological knowledge we cannot hope to understand evolution and diversification, not only of Violaceae but of Malpighiales as a whole, such an important component of the small trees that are so abundant in tropical rainforests (Davis et al., 2005). including observations on a foliar endodermis. Amer. J. Bot. 83: 405–418. Engler, A. 1925. Die natürlichen Pflanzenfamilien. Verlag von Wilhelm Engelmann, Liepzig. Feng, M. 2005. Floral Morphogenesis and Molecular Systematics of the Family Violaceae. Ph.D. Thesis, Ohio University, Athens. Feng, M. & H. E. Ballard. 2005. Molecular systematic, floral developmental and anatomical revelations on generic relationships and evolutionary patterns in the Violaceae. P. 169 in XVII International Botanical Congress Abstracts, Vienna. Hekking, W. H. A. 1984. 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