Comparison of Floral Structure and Ontogeny in Monoecious and Dioecious Species of the Palm Tribe Chamaedoreeae (Arecaceae; Arecoideae)

COMPARISON OF FLORAL STRUCTURE AND ONTOGENY IN MONOECIOUS AND DIOECIOUS SPECIES OF THE PALM TRIBE CHAMAEDOREEAE (ARECACEAE; ARECOIDEAE)

Felipe Castaño,* Xavier Marquínez,† Michèle Crèvecoeur,‡ Myriam Collin,* Fred W. Stauffer,§ and James W. Tregear1,*

*Institut de Recherche pour le Développement (IRD), Unité Mixte de Recherche–Diversité, Adaptation, et Développement des Plantes, Centre IRD Montpellier, BP 64501, 911, avenue Agropolis, cedex 5, 34394 Montpellier, France; †Departamento de Biología, Universidad Nacional de q1 Colombia, sede Bogotá, Carrera 30 45-03, Ediﬁcio 421, Bogotá, Colombia; ‡Université de Genève, Faculté des Sciences, Département de Botanique et Biologie Végétale, quai Ernest Ansermet 30, 1211 Genève 4, Switzerland and §Conservatoire et Jardin Botaniques de la Ville de Genève, Université de Genève, Laboratoire de Systématique Végétale et Biodiversité, CP 60, CH-1292 Chambésy, Switzerland

Editor: Bruce K. Kirchoff

Premise of research. The sexuality of flowers is an important reproductive character in angiosperms. An insight into the evolutionary events that led to the appearance of monoecious and dioecious species can be gained by comparing closely related groups with contrasting characters. For this study, we focused on the tribe Chamaedoreeae, within which dioecy appears to have evolved twice from a monoecious ancestor. Methodology. To improve our knowledge of flower structure and ontogeny in this group, SEM and anatomical sectioning were performed on inflorescences and flowers of the dioecious species Chamaedorea tepejilote and the monoecious species Hyophorbe lagenicaulis at different developmental stages. Pivotal results. Our data highlighted that the higher degree of spatial sexual separation seen in the dioecious C. tepejilote, compared to the monoecious H. lagenicaulis, is accompanied by a more accentuated dimorphism between male and female flowers. More specifically, in the case of C. tepejilote, the vestigial reproductive organs (staminodes of the female flower and pistillode of the male flower) are more rudimentary structures, in terms of their developmental differentiation, than their homologs in H. lagenicaulis. Conclusions. Our data suggest that the unisexual flowers already present in the monoecious ancestor of the Chamaedoreeae underwent further modifications either shortly before or since the appearance of dioecy in the genus Chamaedorea. These structural changes were presumably the result of genomic mutations causing earlier developmental arrest of the vestigial reproductive organs and are likely, in turn, to have conferred en- hanced resource-allocation efficiency.

Keywords: ﬂower development, Chamaedoreeae, Hyophorbe, Chamaedorea, monoecy, dioecy.

Introduction duction in the possible negative consequences of hermaphroditism (e.g., gamete wastage), collectively referred to as sexual Flowers are the complex reproductive structures produced interference (Barrett 2002). Second, unisexual flowers provide by angiosperms and are considered to have played a central role a means to favor outbreeding and therefore heterosis or hybrid in the evolutionary success of this group (Endress 1994). They vigor (Freeman et al. 1997). Male and female flowers may be contain the organs necessary to produce pollen and ovules for produced on either the same plant (monoecious species) or sep- seed production. Depending on the species, male and female arate plants (dioecious species). Dioecy is a mechanism that reproductive organs may be separated in different ways in time ensures total outbreeding but can also be considered in some and space. In contrast to the situation in animals, plants are respects as inefficient in that only about half of the population mostly hermaphrodites. However, some species, representing bears seeds (Richards 1997). In monoecious species, the tempo- approximately 10% of flowering plants, produce unisexual ral separation of male and female functions, a condition known male or female flowers (Ainsworth 2000). Sexual separation be- as dichogamy, has also been interpreted as a mechanism to re- tween pistillate (female) and staminate (male) flowers is consid- duce self-fertilization (Bertin and Newman 1993). ered to confer several advantages. First, the diversification of Sexual differentiation and breeding systems are important roles between male flowers devoted to pollen production and factors in the formation of fruits and seeds, which are the most female flowers producing ovules and seeds results in a more common products harvested from crop plants. Moreover, sex- efficient use of available resources and, more generally, a re- ual systems are of key importance in population genetics and evolution. In the case of dioecy, a number of observations have 1 Author for correspondence; e-mail: [email protected]. been made regarding its geographical and phylogenetic distri- Manuscript received May 2015; revised manuscript received September 2015; bution. Examples of general tendencies observed for dioecious electronically published February XX, 2016. clades include tropical distribution, inconspicuous flower or

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42822.proof.3d 1 Achorn International 01/13/16 01:35 000 INTERNATIONAL JOURNAL OF PLANT SCIENCES inflorescence production, lower species richness, and wind pol- appear to have evolved independently on numerous occasions lination (Heilbuth 2000). Dioecy has evolved numerous times in various different lineages within the family (Weiblen et al. in plants, and more than one evolutionary mechanism can ex- 2000). It is estimated that dioecy appeared at least nine times plain how it may originate from hermaphroditism (Weiblen during the radiation of the Arecaceae, from monoecious or her- et al. 2000). Once dioecy evolves from gender monomorphism, maphrodite ancestors. These multiple transitions make palms an the sexual morphs have different roles and are often observed ideal model to study the evolutionary pathways of sexual sys- to diverge in their characteristics, resulting in sexual dimor- tems at a morphological and a molecular level; however, at pres- phism (Barrett and Hough 2013). It has been suggested that ent, little is known about the molecular processes that reg- the most common evolutionary pathways involve an interme- ulate the sex of palm flowers. There is considerable interest in diate stage where plants are gynodioecious (separate female characterizing these pathways, however, since this would fa- and hermaphrodite plants) or monoecious (Barrett 2013). cilitate the genetic improvement of cultivated species and the Although plant sexuality has been studied for relatively few understanding of the population dynamics of wild ones, many species at the molecular genetic level, much progress has been of which are threatened. The best characterized species at the made in understanding the wider process of flower development molecular level are the monoecious African oil palm (Elaeis in recent years. The availability of whole-genome sequences for guineensis) and the dioecious date palm (Phoenix dactylifera), some model plants has allowed detailed studies of the molecular genome sequences having been obtained for both (Al-Dous determination of their flower structure. The ABC model, pro- et al. 2011; Al-Mssallem et al. 2013; Singh et al. 2013). Studies posed to explain the interaction of homeotic genes to control of flowerstructureandsexdeterminationhavealsobeencar- floral organ determination (Coen and Meyerowitz 1991) and ried out on both of these species (Adam et al. 2007, 2011; Daher later modified to include additional functions (Gutierrez and et al. 2010; Cherif et al. 2013). Davies 2000; Pelaz et al. 2000) has been fundamental for the This study focuses on two members of the tribe Chamae- understanding of floral development in angiosperms. The ma- doreeae, belonging to the almost entirely monoecious subfamily jority of genes implicated in the ABC model encode members Arecoideae (Asmussen et al. 2000, 2006; Asmussen and Chase of the MADS-box family of transcription factors (Becker and 2001; Lewis and Doyle 2002). The tribe Chamaedoreeae has Theissen 2003), the latter being involved in most aspects of been resolved as monophyletic in several phylogenetic analyses the plant’s life cycle (Gramzow and Theissen 2010). In spite using both morphological and molecular data (Thomas et al. of the considerable progress made in understanding the molec- 2006; Cuenca and Asmussen 2007; Cuenca et al. 2008, 2009). ular basis of flower development in monocots, attention has The key synapomorphy that defines the tribe is the arrange- tended to focus on the Poaceae (Poales), since the latter group ment of flowers in distinctive clusters called acervuli (Cuenca contains several model species such as maize and rice that are et al. 2009). These structures are considered to be a reversed easily accessible to genetic studies and also have major economic cincinnus adnate to the rachilla (Uhl and Moore 1978; Uhl importance. Among the other monocot orders, the largely trop- and Dransfield 1987; Ortega and Stauffer 2011). ical palm family (Arecaceae), which forms the order Arecales on The Chamaedoreeae are composed of five genera (Hender- its own, is of great interest as a case study to investigate the evo- son et al. 1995; Dransfield et al. 2008), two of which are dioe- lution of reproductive morphology and sexual differentiation. It cious: Chamaedorea Willd., the richest palm genus of the Neo- contains 190 currently recognized genera and around 2600 spe- tropics with a distribution ranging from southern Mexico to cies (Govaerts and Dransfield 2005; Dransfield et al. 2008), con- northwest South America, and Wendlandiella Dammer, which fined mostly to the tropics. The Arecaceae rank with grasses and is monotypic and confinedtothewesternAmazonregion.Three legumes as one of the most useful plant groups worldwide (Balick additional monoecious genera complete the tribe: Gaussia H. and Beck 1990; Johnson 2011); in the case of indigenous cul- Wendl., with five species endemic to the Caribbean region; tures in the Americas, palms are considered to be by far the Hyophorbe Gaertn., composed of five species endemic to the most important plants (Henderson et al. 1995; Johnson 1996). Mascarene Islands (Moore 1978; Dransfield et al. 2008); and The Arecaceae exhibit an enormous variability in their sexual- Synechanthus H. Wendl., which contains two species distrib- expression patterns and reproductive structures, from the sol- uted throughout the Central American region from southern itary flower to numerous different types of flower clusters. Mexico to western Ecuador (Moore 1971; Henderson et al. Aggregations of flowers on the inflorescence axes are diverse 1995). The subfamily Arecoideae has been traditionally distin- in structure and variable throughout the family but are highly guished by flowers arranged in triads (two lateral staminate diagnostic and taxonomically informative for some groups flowers and one central pistillate flower) or groups reduced (Tomlinson 1990). Studies of floral structure in the family have from triads (Moore 1973; Uhl and Dransfield 1987; Dransfield dealt with a relatively small number of species, even though those and Uhl 1998; Dransfield et al. 2008). The notable exception published so far have been valuable in determining the func- is the tribe Chamaedoreeae, in which flower clusters display tions of floral organs and the evolution of floral structure in the aforementioned acervulus structure (Uhl and Moore 1978; the context of a phylogenetic framework (Uhl and Moore 1971; Ortega and Stauffer 2011) and some dioecious species bearing sol- Uhl and Dransfield 1987; Dransfield and Uhl 1998). Flowers itary flowers occur. The monoecious genera in Chamaedoreeae of many genera have yet to be studied anatomically; such stud- (Gaussia, Hyophorbe, Synechanthus) produce unisexual flowers ies seem certain to yield data of taxonomic, biological, and evo- arranged in acervuli, whereas Chamaedorea and Wendlandiella lutionary significance (Dransfield et al. 2008). share the dioecious character and the presence of solitary flow- Sex determination is an important character in palms. About ers. These two features have been considered important when 80% of palms produce single-sex male or female flowers, on inferring the evolutionary relationships among the genera of the either dioecious or monoecious plants. Monoecy and dioecy tribe. Nevertheless, recent studies have suggested that transitions

42822.proof.3d 2 Achorn International 01/13/16 01:35 CASTAÑO ET AL.—FLOWER DEVELOPMENT IN CHAMAEDOREEAE 000 to solitary flowers and dioecy have each occurred more than mens were chosen for SEM. They were dehydrated, critical- once within the tribe (Thomas et al. 2006; Cuenca and Asmussen point dried, and sputter-coated with gold at the Muséum 2007; Cuenca et al. 2009). d’Histoire Naturelle de la Ville de Genève (Geneva, Switzerland) In this study, we wished to investigate sexual differentiation and the Centre de Ressources en Imagerie Cellulaire (CRIC; in palms by comparing two contrasting species of the Chamae- Montpellier, France). Micrographs were obtained using a JEOL doreeae. First, we chose the dioecious palm Chamaedorea te- (JSM-6510LV) SEM at the Bioimaging Center at the Faculty of pejilote Liebm., an understory palm distributed from southern Sciences (University of Geneva) and a Hitachi S4000 SEM at Mexico through Central America to northern South Amer- the CRIC laboratory in Montpellier. ica (fig. 1A). The male inflorescences of this species are edible when immature and are used as a traditional food, called pa- Anatomical Analysis caya, in Central America, especially Guatemala, El Salvador, and southern Mexico (Castillo Mont et al. 1994). The genus Anatomical studies of inflorescence and flowers were car- Chamaedorea contains approximately 110 species, all of which ried out using paraffin embedding, staining with Astra blue are dioecious (Dransfield et al. 2008). It is therefore likely that and fuchsin, and mounting with Eukitt medium in the Labo- the genomic rearrangement(s) that conferred dioecy occurred ratory of Histotechnology, Department of Biology, at the Uni- in the common ancestor of all Chamaedorea species. For our versidad Nacional de Colombia. Cross and longitudinal sec- comparative study, we also chose the monoecious species Hyo- tions were obtained using a rotary microtome (Leitz 1512) at phorbe lagenicaulis (L.H. Bailey) H.E. Moore, commonly known 5–10-mm thickness. Further details on this technique are ex- as the bottle palm. This is a solitary, moderate-size palm (fig. 3A) plained by Igersheim and Cichocki (1996) and Stauffer et al. native to the Round Island of the Mascarene Archipelago and (2002). Photos of the anatomical sections were obtained using probably also Mauritius (Moore 1978). Together, these two a Leica DMIRE2 microscope and a Leica DC 300F camera. Per- species provide an interesting case study to investigate sexual di- manent slides of the microtome sections are stored at the De- morphism in palms. For practical reasons, only one species was partment of Biology at the Universidad Nacional de Colombia. studied from each of the two genera. The chosen species can be considered to be representatives of their genera for the key Results characters described here, namely, those associated with the fertile and sterile androecium and gynoecium (Cuenca et al. Flowering in Chamaedorea tepejilote 2009). Two main approaches were used, namely, morphological observations of dissected samples using SEM and anatom- Chamaedorea tepejilote is a dioecious species, with sepa- ical studies of stained paraffin–embedded sections using LM. rate individuals producing male and female inflorescences. Both kinds of inflorescence are infrafoliar, appearing below the crown of leaves at the anthetic stage; they are usually branched Material and Methods to one order and protected by four or five tubular, green, cori- aceous peduncular bracts (fig. 1B). The rachis and rachillae Plants are whitish before the opening of the bracts. The male rachillae become yellow-cream at anthesis (fig. 1B), whereas the fe- Flowers of Chamaedorea tepejilote and Hyophorbe lageni- male rachillae are green at this stage (fig. 2A). Male and female caulis were collected from the living collections of the Mont- flowers are generally referred to as being solitary, in that they gomery Botanical Center and the Fairchild Tropical Botanic are not grouped together in acervuli as in other species of the Garden, both located in Miami, Florida. Additional develop- tribe Chamaedoreeae, nor do they form triads as is commonplace mental stages for C. tepejilote were observed using flowers orig- within the subfamily Arecoideae as a whole. Male and female inally collected in Panama, on loan from the Laboratory of Mi- flowers are evenly distributed on the rachilla, sessile and sunken cromorphology at the Conservatoire et Jardin Botaniques of in shallow pits. In the case of the male inflorescence, flowers Geneva, Switzerland. occur in large numbers and show a dense distribution on the rachilla at maturity. No bracts were observed to subtend the Harvesting, Fixation, and Dissection flowers. Flowers are trimerous, appearing more or less triangular when viewed from above. Inflorescence samples, in the form of rachillae or sections thereof, were harvested from leaf axils at a range of different developmental stages for each species. Fixation was carried out The Male (Staminate) Flower of C. tepejilote as described by Adam et al. (2005), and observations were also Morphology. Early after their initiation, male flowers were fi made on fresh material. Dissection was performed on the xed seen to be arranged in loose spirals on the rachillae; however, fl owers as required for later microscopic studies. Sepals and they become tightly disposed as development proceeds. At early fl petals were removed from some owers to facilitate observation stages of development, the flowers are clearly sunken in pits fi and in ltration of the inner organs. (fig. 1C). At intermediate stages, the flowers appear to still be partially sunken, compounded by the fact that they are sessile (fig. 1F,1G). Nevertheless, the pits become inconspicuous Morphological Analysis in mature rachillae. Each flower is initiated as a small mound of Flowers were dissected for morphological study under a ste- about 0.1 mm in diameter in the bottom of the shallow pit reomicroscope (Wild M3B). Some parts of the dissected speci- (fig. 1C). This floral primordium elongates and emerges from

42822.proof.3d 3 Achorn International 01/13/16 01:35 Fig. 1 Adult form, macroscopic view of male inflorescence and developmental stages of the male flower in Chamaedorea tepejilote. A, Adult specimen. B, Male inflorescence at anthesis. C, Male floral primordium at an early stage (SEM upper view). D, Portion of rachilla showing male flowers in early bud starting their elongation (SEM upper view). E, Male flower in early bud showing differentiation of the calyx (SEM lateral view). F, Male flower bud at an intermediate developmental stage showing early-differentiating perianth whorls (SEM lateral view). G, Longi- tudinal section of male flower bud. H, Male flower in late bud (SEM upper view, perianth removed). I, Transverse section of the basal region of the male flower in late bud. J, Male flower close to anthesis, revealing anther dehiscence and young pistillode (SEM lateral view, perianth removed). K, Male flower close to anthesis (SEM upper view, perianth removed). L, Male flower at anthesis (SEM lateral view, one petal removed). M, Transverse section of male flower at a late bud stage corresponding to the basal region of the pistillode. N, Transverse section of the male flower at a late bud stage around pistillode midheight. O, Transverse section of the male flower a late bud stage near the apex of the pistillode, revealing detail of the androecium and the pistillode. Pe p petal; Pi p pistillode; R p raphide idioblast; S p stamen; Se p sepal; V p vascular strand. Scale bars: C p 50 mm; D p 250 mm; E p 75 mm; F p 200 mm; G–I p 100 mm; J, K, M–O p 500 mm; L p 1 mm.

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Fig. 2 Macroscopic view of the female inflorescence and developmental stages of the female flower in Chamaedorea tepejilote. A, Rachillae displaying some flowers at anthesis. B, Flower at an early bud stage (SEM lateral view). C, Flower in early bud at onset of calyx differentiation (SEM lateral view). D, Flower in early bud showing early differentiation of the corolla (SEM upper view). E, Flower at an intermediate developmental stage showing the imbricate sepals (SEM upper view). F, Flower at a late bud stage (SEM upper view). G, Detail of the gynoecium shortly after its initiation (SEM upper view, perianth removed). H, Detail of the gynoecium shortly before anthesis (SEM upper view, perianth removed). I, Flower at anthesis (SEM lateral view). J, Gynoecium at anthesis, revealing the papillate stigma (SEM lateral view, perianth removed). K, Transverse section of the flower at an intermediate bud stage toward the transition region between the stigma and style. L, Transverse section of the flower at an intermediate bud stage around midheight of the style. M, Transverse section of the flower at a late bud stage in the upper region of the ovary. N, Transverse section of the flower at a late bud stage in the middle region of the locules. O, Longitudinal section of the flower at anthesis (arrowhead p pollen tube transmission tract). P, Detail of the ovule (lateral view). C p carpel; Et p epithelial tissue; Fb p floral bud; Pe p petal; R p raphide idioblast; Se p sepal; Sg p stigma; St p staminode; Ti p tannin idioblast; V p vascular strand. Scale bars: B, C p 50 mm; D p 100 mm; E p 250 mm; F, I, J, M–O p 500 mm; G, L, P p 100 mm; H, K p 200 mm. the pit (fig. 1D), and then the differentiation of the perianth though they remain free toward the acuminate apex (fig. 1F, and inner whorls starts centripetally in an alternating se- 1G,1I). They reach 0.5 mm at anthesis (fig. 1J,1L). quence (fig. 1E). At anthesis, the pits are no longer evident, The initiation of the corolla is similar to that of the calyx, and the flowers reach 2–3 mm in length and 3–4 mm in diam- with petals displaying more or less the same size as the sepals eter (fig. 1K,1L). in the early floral bud (fig. 1F). At this developmental stage, The calyx is the first whorl to be initiated as three very small the petals tightly enclose the inner sexual organs; this is facil- knob-shaped sepals (fig. 1E). The sepals are fused toward the itated by their fleshy constitution, valvate aestivation, and the base from an early developmental stage. Shortly after, when layer of epidermal papillae on their margins (fig. 1F–1I). The the inner whorls have started their differentiation, they become three free petals continue their elongation and become larger concave, reaching about 0.2 mm in length (fig. 1F,1G). As the than the sepals. At anthesis, they are concave, orbicular, and sepals elongate, their fused bases form a thick ring of tissue, about 2–2.5 mm wide and 2–2.5 mm long (fig. 1L–1O).

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The androecium comprises six stamens. Three are initiated Raphide idioblasts were observed in all floral organs but not opposite the sepals and three opposite the petals (fig. 1H,1I). at the base. They were present from early developmental stages The stamens are seen to be smaller in size than the pistillode in the perianth mesophyll (fig. 1G,1I ), becoming larger and in late floral buds (fig. 1G,1H). The differentiation of the an- more numerous as development progressed (fig. 1M–1O). In ther tissues precedes that of the filaments. Each anther starts the androecium, raphide idioblasts were restricted to the an- as a very small rounded mound (fig. 1G), which soon becomes ther and intermixed with pollen grains shortly before anthesis. bilobed, reaching 0.1 mm in width and about the same in height The pistillode was also characterized by the presence of raph- (fig. 1H). Shortly thereafter, each lobe cleaves to produce the ide idioblasts, which were formed during later developmental pollen sacs, concomitant with the differentiation of the con- stages, mostly in the upper half of this organ and around the nective region (fig. 1I). The anthers reach maturity before com- central apical duct (fig. 1N). The apical lobes of the pistillode plete elongation of the filaments (fig. 1J,1K). At this stage, the were devoid of raphides. Tannin idioblasts were not observed microsporangia are still located at the level of the pistillode in floral organs at any developmental stage. apex; however, due to filament elongation shortly thereafter, the mature anthers are then raised further so as to be slightly The Female (Pistillate) Flower higher than the pistillode (fig. 1L). In mature flowers, the filaments are fleshy, cylindrical, and completely free (fig. 1L, Morphology. From an early developmental stage, female 1M). The connective region is thick and prominently developed flowers are borne in close spirals that will become looser as (fig. 1K,1N). The anthers are about 1 mm long, all fertile, and the rachillae elongate (fig. 2A). Female flower initiation starts dorsifixed, displaying latrorse dehiscence through a longitudi- with a small protuberance of about 0.1 mm in diameter in the nal stomium (fig. 1L,1N,1O). center of a shallow pit (fig. 2B) that becomes deeper as devel- The differentiation process culminates in the development opment progresses. Pistillate flowers reach 2–3 mm in length of a sterile gynoecium or pistillode. The latter is composed of and 2–3 mm in diameter when mature (fig. 2I,2O). three carpellodes located in the center of the flower, each one Development of the floral whorls starts with the calyx, which, opposite the sepals (fig. 1H). Early after initiation, the three shortly after initiation, is composed of three very small bulges wedge-shaped carpellode primordia are free (fig. 1H). From a concentrically disposed around the floral apex (fig. 2C). Al- very early developmental stage, these organs undergo a post- though they start their differentiation as three free append- genital fusion on their lateral flanks (fig. 1I). This results in ages, shortly afterward their bases undergo postgenital fusion the gradual consolidation of an erect cylindrical pistillode of when they are about 0.2 mm wide, concomitant with the ini- about 0.3–0.4 mm in diameter and 1–1.5 mm in length shortly tiation of the petals (fig. 2D) and an increase in depth of the before anthesis (fig. 1J,1K). Nevertheless, the carpellodes re- pit. The sepals continue their elongation and become imbri- main free toward the apical portion, displaying a trilobed stig- cate, enveloping the inner organs (fig. 2E). The consecutive ex- matic zone distinguishable throughout development (fig. 1J– pansion of the petals and development of the sexual organs 1L,1O). This zone is also characterized by the presence of a takes place while they are still covered by the sepals. However, short conducting tract in the center (fig. 1I–1N) composed of the petals subsequently expand to exceed the size of the sepals three confluent ventral slits (fig. 1O). This structure, known (fig. 2F), which at this point are about 1 mm wide (fig. 2M). At as a compitum, provides a connection that allows pollen land- anthesis, the sepals are ca. 1 mm long and 2 mm wide, concave, ing on any one stigma to fertilize ovules in any carpel. The and fleshy, forming a thick ring with their fused bases (fig. 2I, pistillode reaches 1.5 mm in length and 0.5 mm in diameter at 2N,2O). After elongation, the three free, imbricate, fleshy pet- anthesis (fig. 1L–1O). als reach about 2–2.5 mm in length and 2.5–3 mm in width; Anatomy. Although the vasculature was seen to be con- they are concave and acuminate with reflexed apices (fig. 2I, solidated during development, the vascular tissue of the male 2K,2M–2O). Six staminodes, seen as very small bulges, are ini- flower was notably inconspicuous, and the procambial strands tiated between the corolla and the gynoecium whorls (fig. 2G). were composed of only a few cells at all stages. The vascular Three are seen opposite the sepals and three opposite the pet- bundles destined to serve all the floral whorls could be ob- als. In this case, the process of organogenesis is arrested very served passing through the floral base, arranged in three con- early, and only one staminode was observed in each flower centrically disposed rings (not shown) once the primordial flo- shortly before anthesis. It was very small, ca. 0.1–0.15 mm ral organs were in place. First, the outer ring was composed long, flattened, and capitate, showing no sign of anther differ- of nine bundles corresponding to the vascularization of the co- entiation. The staminode was observed to be opposite one of rolla. Second, two internal rings composed of three bundles the petals (fig. 2N). each that irrigate the two whorls of the androecium could be Three distinct carpels form the gynoecium from its early ini- distinguished. No vascularization was observed at any stage tiation, each carpel being opposite a sepal (fig. 2G,2N). When for either the calyx or the pistillode. Three (or sometimes four) still primordial, the carpels display a laminar and slightly con- vascular strands were seen to irrigate the petals from the base cave shape, with a width of about 0.1 mm (fig. 2G). As their to the apex (fig. 1M–1O). One central vascular bundle irri- elongation progresses, the carpels become basally ascidiate, and gates each filament throughout its length, reaching the con- the flanks of contiguous carpels fuse postgenitally (fig. 2N), giv- nective region (fig. 1M–1O), which is approximately four cell ing the gynoecium a characteristic pyriform shape. Only the layers thick at anthesis (fig. 1N). Apart from the epidermal apical region of each carpel remains free throughout the differ- cells and raphide idioblasts, the pistillode was observed to be entiation process. In the late floral bud, the gynoecium is more composed entirely of ground parenchyma. or less globose and 1 mm in diameter, and the three free api-

42822.proof.3d 6 Achorn International 01/13/16 01:35 CASTAÑO ET AL.—FLOWER DEVELOPMENT IN CHAMAEDOREEAE 000 cal lobes are inflexed (fig. 2H). When mature, the gynoecium epidermis (fig. 2O,2P). The ovule is anatropous. It is attached reaches approximately 2 mm in height and 2 mm in diameter. to the upper half of the locular cavity, which is almost entirely At this stage, it is almost entirely syncarpic, albeit apocarpic filled by it (fig. 2O,2P). Toward the micropyle, the outer and in the upper part of the style and the stigmatic region (fig. 2J, the inner integuments are eight and four cell layers thick, re- 2O). The stigmatic branches are reflexed, equally developed, spectively. At ovule midlength, the two integuments display the and densely covered with unicellular papillae (fig. 2J,2O). same thickness. The nucellus is one or two cell layers thick. The Anatomy. As in the male flower, the vascular bundles that micropyle is straight and narrow (fig. 2P). will serve all the primordial floral organs are disposed through Raphide idioblasts were rarely observed within the floral the floral base, where three concentric rings of vascular bundles base and in the basal region of perianth organs during floral could be observed from early developmental stages. The outer development. Raphides became frequent, however, as develop- ring contains the bundles that will vascularize the perianth or- ment progressed in the mesophyll at the midheight of sepals gans; it branches into two separate rings that extend upward and petals, mostly associated with the abaxial side, distributed to irrigate the calyx and the corolla. The next inner ring is as far as the apical region (fig. 2K,2M–2O). In spite of the fact formed by the vascular strands that will supply the staminodes, that raphides were virtually absent from the basal half of the and the innermost ring contains the vascularization for the locular region, their frequency was seen to be greater in the carpels. The latter ring later divides into three different com- upper regions, forming a dense layer throughout the style, while plexes of strands that vascularize the three carpels. As develop- becoming rare in the stigmatic region (fig. 2O). ment progresses, the vascularization of the calyx increases in its Some scattered tannin idioblasts were seen to be present complexity, and shortly before anthesis it is composed of ap- toward the center of the floral base. In the perianth, tannini- proximately 20 vascular strands in the basal region where ferous idioblasts were observed throughout the mesophyll in the sepals are congenitally united (fig. 2N). Nevertheless, only the sepals, but they were scarcely present in the petals (fig. 2M– two or three strands were seen to be present in the free upper 2O). Tannins were also present in the gynoecium, mostly in region of the sepals through to the apex (fig. 2M). Similar de- the style, the locular walls, and the funiculus, but they also velopment was observed in the corolla, where eight or nine formed a continuous layer in the inner integument surround- vascular bundles irrigate the basal part of the petals in the late ing the nucellus (fig. 2O,2P). Oil bodies were observed mostly bud (fig. 2N), whereas in the apical region only three or four in the stigmatic branches and farther downward around the were observed (fig. 2M). The vascular bundles in the perianth ventral slits, as far as the upper level of the style (fig. 2K,2O). organs were seen to be exclusively associated with their ad- They were also distributed close to the inner epidermis of the axial faces. No vascular bundles were observed to supply the carpels in the locular region (fig. 2P) and petal margins. The staminodes. The three vascular bundles that will vascularize presence of vascular bundle–associated stegmata with hat- the gynoecium, observed in the floral base at early stages, sub- shaped silica bodies was noted in both sepals and petals (fig. 2M, sequently differentiate into three branches for each carpel. Each 2N). The presence of tannins, oil bodies, and stegmata was ob- branch divides as development progresses to constitute, just served at intermediate developmental stages, progressively in- before anthesis, a complex of one dorsal and four lateral vascu- creasing toward anthesis. The abaxial epidermis of the peri- lar bundles around the middle region of each ovary (fig. 2N). anth organs was observed to be characterized by the presence However, the vascular supply of each carpel was seen to be re- of densely grouped unicellular papillae (fig. 2M). duced to only one dorsal and two lateral bundles in the region between the base and the upper level of the style (fig. 2M). The Flowering in Hyophorbe lagenicaulis stigmatic region was devoid of vasculature throughout its development (fig. 2K). Two vascular strands were observed to Hyophorbe lagenicaulis is a monoecious palm species pro- penetrate into the ovule through the funiculus shortly before ducing unisexual male and female flowers in infrafoliar inflo- anthesis. Shortly before anthesis, the carpels are uniovulate, rescences that are branched to three orders, at least in basal ascidiate toward the ovary (fig. 2N–2P), and plicate from the and midheight rachillae, and are protected by five or six woody basal region of the style to the stigma (fig. 2K–2M,2O). The peduncular bracts (fig. 3A). Flowers are disposed on the ra- gynoecium is characterized by the presence of a basal synasci- chillae in spirally arranged acervuli (fig. 3B). Each acervulus diate portion (fig. 2N) and a synplicate distal region (fig. 2L, is composed of generally six or seven distal male flowers and 2M) at the final stages of its development. one basal female flower. Development of the acervulus is prot- As the differentiation of the gynoecium tissues progresses, a androus (fig. 3B,3C). In the acervulus, the flowers are borne compitum is formed in the style among the confluent ventral in a zigzag pattern, giving the appearance of two lines (fig. 3B, slits of the three carpels. It differentiates from the base of the 3C). The male flowers are initiated first, from the axil of a style and extends as far as the stigmatic region (fig. 2K,2L). proximal, very small subtending bract (fig. 3D), and are shed The pollen tube transmitting tract (PTTT) divides downward once they reach anthesis. This process is simultaneous with a dis- in three separate branches (one for each locule) in the zone of placement of the floral bud toward the distal end of the acer- confluence between the base of the style and the upper part of vulus. The female flower is the last to be formed, reaching anthe- the ovary (fig. 2M). From the internal walls of the locule, in par- sis after all the male flowers have been shed. Floral subtending ticular, at the base of the placenta and throughout the length of bracts were not observed. Flowers are borne on a short pedicel, the ventral slits (compitum), the PTTT is covered by a contin- which is wider in the female flower (fig. 4G). The rachis and uous layer of epithelial tissue. The PTTT reaches the ovule by rachillae are creamy white before the opening of the bracts, surrounding the short funiculus with a continuous papillate turning green shortly before anthesis (fig. 3B). The initiation

42822.proof.3d 7 Achorn International 01/13/16 01:35 Fig. 3 Adult form, macroscopic view of a rachilla and development of the male flower in Hyophorbe lagenicaulis. A, Adult specimen. B, Portion of a rachilla displaying acervuli. C, Young acervulus with six flower buds (SEM upper view, perianth manually unfolded). D, Longitu- dinal section of the floral primordium still subtended by the bract. E, Flower in bud showing early differentiation of the calyx (SEM upper view). F, Flower in bud showing the calyx enveloping the floral apex (SEM upper view). G, Flower in bud showing early differentiation of the corolla and floral apex (SEM upper view, calyx manually unfolded). H, Longitudinal section of the flower at an early bud stage. I, Flower in bud showing early differentiation of the androecium and pistillode (SEM upper view, corolla manually unfolded). J, Longitudinal section of a floral bud at an intermediate developmental stage. K, Detail of the androecium and pistillode at an intermediate developmental stage (SEM upper view, perianth removed). L, Detail of the androecium and pistillode at a late bud stage (SEM upper view, perianth and one stamen removed). M, Detail of the androecium and pistillode shortly before anthesis (SEM upper view, perianth removed). N, Flower at anthesis (SEM upper view). O, Flower at anthesis (SEM lateral view, one petal removed). P, Transverse section of the ovary at the level of the septal nectary. Q, Transverse section of a late floral bud in the basal region of the pistillode. R, Transverse section of a late floral bud in the apical region of the pistillode showing the free lobes. Br p bract; F p female bud; M p male bud or flower; N p nectary; Pe p petal; Pi p pistillode; R p raphide idioblast; S p stamen; Se p sepal; V p vascular strand. Scale bars: C, D, G, H, J, L, P p 100 mm; E, F, I, K p 50 mm; M p 200 mm; N p 1 mm; O, Q, R p 500 mm.

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42822.proof.3d 8 Achorn International 01/13/16 01:35 Fig. 4 Development of the female flower of Hyophorbe lagenicaulis. A, General appearance of a rachilla with a pistillate flower in bud (SEM lateral view). B, Detail of the sterile androecium and gynoecium at an early bud stage (SEM upper view, perianth removed). C, Detail of the sterile androecium and gynoecium at an intermediate bud stage (SEM upper view, perianth removed). D, Detail of the sterile androecium and gynoecium at a late bud stage (SEM lateral view, perianth removed). E, Detail of the sterile androecium and gynoecium shortly before anthesis (SEM upper view, perianth removed). F, Detail of the gynoecium at anthesis (SEM upper view, perianth removed). G, Longitudinal section of the flower at a late bud stage (arrowhead p staminode). H, Detail of the stigmatic region in longitudinal section. I, Detail of the three apical lobes of the stigma (transverse section). J, Transverse section of the style showing ventral slits. K, Transverse section in the upper region of the ovary showing the openings of the septal nectary. L, Detail of the ovule (longitudinal section of the locule). C p carpel; Od p opening duct; Pe p petal; R p raphide idioblast; Se p sepal; St p staminode; Ti p tannin idioblast; V p vascular strand. Scale bars: A p 1 mm; B–D, L p 100 mm; E, G p 500 mm; F, I–K p 200 mm; H p 400 mm.

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42822.proof.3d 9 Achorn International 01/13/16 01:35 000 INTERNATIONAL JOURNAL OF PLANT SCIENCES and early development of the perianth has been observed to nate only at the basalmost level, forming a fleshy ring together be similar in both male and female flowers in terms of mor- with the corolla, which envelops the base of the pistillode (not phological and anatomical characters. Therefore, we provide shown). the description of this process here only for male flowers. The pistillode (sterile gynoecium) is composed of three carpellodes, which are initially differentiated as three small lobes from the triangular floral apex (fig. 3J,3K) after the initiation of the stamens. From its early initiation, the apical region of The Staminate Flower the pistillode displays three equally developed free lobes of Morphology.Thefirst floral primordium was observed to about 0.1–0.2 mm in height and 0.1 mm in width (fig. 3L). Nev- be formed underneath the bract that subtends the acervulus. ertheless, this pattern of development does not remain con- It is a very small bulge of about 0.1–0.2mmindiameter(fig. 3D). stant, and the carpellodes undergo a postgenital fusion that Shortly afterward, the differentiation of the floral whorls takes starts a few microns down from the apical lobes (fig. 3M,3O). place in a centripetal sequence. At anthesis, the flowers are In mature flowers, the pistillode is very conspicuous and about 3–4 mm long and 2–3mmindiameter(fig. 3N,3O). The floral 1–1.5 mm long (fig. 3N,3O). Apically, it displays three papil- base has the form of an inverted shallow cup and is about 0.5 mm late lobes, which are notably angular and free (fig. 3O,3R). long (fig. 3O). Anatomy. At least one vascular-bundle complex serves all The three sepals that comprise the calyx are initiated asyn- the perianth organs as well as the floral apex from the very early chronously as very small excrescences (fig. 3E). As they con- developmental stages. The vascularization of these organs was tinue their differentiation, apical imbrication occurs; as a result, seen to increase in complexity during development. The ba- they envelop the floral apex, aided by their strongly concave sally connate sepals constitute a ring of tissue vascularized by shape (fig. 3F,3G). The remaining floral organs initiate while 17 or 18 bundles grouped near the abaxial epidermis in anthetic still covered by the sepals (fig. 3H,3J). Nevertheless, as the in- flowers; however, five or six bundles were observed at mid- ner organs start their elongation, the calyx opens and is sub- level and only one or two toward the apex (fig. 3Q). The same sequently surpassed in size by them. At anthesis, sepals are pattern of vascular tissue development was observed for the about 1–1.5 mm long, 1.5–2 mm wide at midlength, basally petals, at least one vascular strand being present in early buds, fleshy, and connate for about 1 mm, with three free, imbricate whereas seven to nine vascular bundles were observed at mid- lobes and an abaxial epidermis that is conspicuously papillate length and four to six toward the apex at anthesis (fig. 3R). (fig. 3O,3Q). A single vascular bundle was seen to irrigate each filament Once the floral apex is covered by the calyx, the three petals from its early initiation, reaching as far as the connective re- are initiated asynchronously in an alternate position (fig. 3G, gion at mature stages (fig. 3Q). The connective region is very 3H). At this stage, the petals have reached about 0.1 mm in short but includes a layer of sclerenchymatous cells just be- diameter, and the floral apex is clearly triangular. Shortly af- neath the abaxial epidermis. The tapetum was entirely de- terward, the petals become cordate and somewhat acuminate graded just before anthesis in the late floral bud (fig. 3Q,3R), at their apex (fig. 3I), displaying a valvate aestivation that, in whereas apart from the endothecium and the epidermis, no conjunction with the epidermal papillae, results in the inner de- middle layers were observed at this stage. veloping organs being hermetically protected (fig. 3J). Shortly No vascular tissue was observed during the early develop- before anthesis, the free petals are strongly concave, fleshy, 3– mental stages of the pistillode. In the late floral buds, vascu- 4 mm long, and 1–2 mm wide (fig. 3N,3O,3Q,3R). The dor- larization of the pistillode was restricted to the floral base and sal face is densely covered with unicellular papillae, which are the flanks of the nectary, where three vascular strands were also present throughout the margins and act as a zipper, tightly observed (fig. 3P). At midlength, the lobes were seen to begin closing the corolla when the flower is still in bud (fig. 3N,3O, their postgenital fusion through their ventral faces (which at 3Q,3R). this level appear notoriously papillate), forming a narrow con- The androecium is initiated in two whorls, each consisting duct in their confluent zone. Below this level, the carpellodes of three stamens, which early in development appear as very are completely fused, showing a triangular cross section, com- small mounds (fig. 3I,3J). The first three stamen primordia are posed of compact parenchyma, with no tract in the middle located in an antesepalous position, the second stamen whorl (fig. 3Q). At the basal level, a nectary-like tissue was seen to being antepetalous (fig. 3I). Later in development, the stamens form at the center of the pistillode (fig. 3P). The nectary is com- have the appearance of being attached at the same level (fig. 3K), posed of secretory epithelial cells forming three small conducts and the floral apex becomes notably three lobed. The anthers with openings to the surface of the carpels (fig. 3P,3Q). start to differentiate before the filaments, splitting into two lobes Within the floral base, idioblasts were infrequently observed that are afterward divided to form the four microsporangia throughout floral development. However, small raphide idio- (fig. 3L), reaching about 0.2 mm in width. In late bud, shortly blasts scattered within the bases of the petals, filaments, and before anthesis, the anthers have doubled their size, and the con- pistillode were observed to become larger and more abundant nective structures have consolidated, although the filaments from intermediate stages onward. Raphides remained frequent have not yet elongated (fig. 3M). Anthers are notoriously an- in these organs throughout their length, except in the apex of gular and versatile at anthesis, when they reach about 0.6– the carpellodes (fig. 3P–3R). They were, however, completely 0.7 mm in width and 1 mm in height (fig. 3N,3O). They absent in the calyx. In the androecium, some scattered raph- are dorsifixed with extrorse dehiscence through a longitudinal ides were present in the microsporangia at late stages, derived stomium (fig. 3N,3O,3Q,3R). The stamens are adnate to from tapetal cells (fig. 3Q,3R). Tannin idioblasts were not ob- the corolla at their base (fig. 3N,3O ). The filaments are con- served in any organs of the flower. Some scattered oil bodies

42822.proof.3d 10 Achorn International 01/13/16 01:35 CASTAÑO ET AL.—FLOWER DEVELOPMENT IN CHAMAEDOREEAE 000 were seen to be present, mostly associated with the nectary and number is reduced to five to seven vascular bundles toward the abaxial epidermis toward the margins of the petals. Vascu- the apical region of the sepals. In the petals, six or seven vascular bundles were characterized by the presence of stegmata with lar bundles were observed between the base and midheight, hat-shaped silica bodies in all the organs of the perianth toward whereas in the apical region there were only three or four (not the onset of (but not before) anthesis. shown). Each staminode was vascularized by only one central basal vascular bundle, observed only toward the time of anthesis. In the gynoecium, at least one dorsal and two lateral vascu- The Pistillate Flower lar bundles were observed in each carpel (fig. 4J), of which only Morphology. The female floral primordium was also ob- the dorsal one remained constant in the style and the stigmatic served to be formed underneath the acervulus-subtending bract. zone. The vascularization of the floral organs was established The female flower is initiated in an identical fashion to that during the middle period of development, though it became of the male flower, with the development of the perianth or- clearly defined shortly before anthesis. gans also being essentially the same. However, as described The stigmatic branches were observed to have ventral faces below, the development of the sexual organs and the resulting covered with unicellular papillae (fig. 4H,4I). In the style, a floral structure are notably different. Near anthesis, the pistil- compitum is formed by the confluent ventral slits of the three late flowers are slightly triangular in cross section and about carpels. Therefore, the ventral slits are confluent in a common 1.5–2 mm long and 2–2.5 mm in diameter (fig. 4A), being wider stylar canal as far as the upper region of the ovary (fig. 4J), and shorter than the staminate flowers. where the PTTT divides shortly before reaching the three locu- Although sepals are initiated in the same pattern as in the lar cavities (fig. 4K). In the synascidiate zone of the gynoecium, male flower, these organs become conspicuously fleshy at their the three postgenitally fused carpels form three locules, each base as development progresses. At anthesis, the three concave with a fertile ovule. A septal nectary is formed in the middle re- sepals reach about 1.5 mm in length and 2 mm in width, region of the ovary, specifically, between the locules. The nec- maining congenitally united toward the base (fig. 4A). Apically, tary is a branched and secretory cavity with opening ducts be- the free lobes of the sepals are imbricate. Initiation of the pet- tween the lateral surfaces of the fused carpels toward the upper als is similar to that of the male flower. In the late floral bud, level of the ovary (fig. 4K). A continuous layer of epitheliar tis- the three free, valvate petals are approximately 2–3 mm long sue forms the inner surface of the nectary (not shown). The and 1–2 mm wide, markedly concave, and ovate (not shown). ovules are hemianatropous, bitegmic, and crassinucellar, but The sterile androecium is initiated in two whorls. The first they do not completely fill the locular cavity (fig. 4L). The prox- whorl to be formed is composed of three very small staminodes imal end of the funiculus, which may correspond to the pla- opposite the sepals. The second whorl of three staminodes is centa region, has a secretory surface formed by epithelial cells. initiated soon afterward opposite the petals; however, once Toward the micropyle region, the outer and inner integuments both whorls are formed, they have the appearance of being in- are five and three cell layers thick, respectively. At ovule mid- serted at the same level (fig. 4B–4E). From the early develop- length, the thickness of the two integuments is the same. The nu- mental stages, the staminodes are shortly connate toward their cellus is two or three cell layers thick. The micropyle is straight bases and slightly adnate to the corolla, especially in the case and narrow (fig. 4L). of the antepetalous staminodes (fig. 4D,4E). In late floral buds, Tanniniferous cells were observed only in the ovule, more shortly before anthesis, the staminodes are ca. 0.5–1 mm long precisely forming a continuous one-cell-thick layer in the inner and 0.5 mm wide at the base. They are subulate, with flat- integument surrounding the nucellus (fig. 4L). Although raph- tened, fleshy filaments (fig. 4E,4G). In spite of the fact that the ides were scarce in the floral base and the sepals (fig. 4G), they staminodes form a small apical lobe similar to an anther, they were seen to be more abundant in other regions of the flower. are completely sterile. The basal region of the petals was filled with abundant and The gynoecium is composed of three fertile carpels, which at large raphides, which were also frequent in the stylar region the time of initiation are opposite the sepals. At this moment, and the basal region of the staminodes (fig. 4G,4J). In the up- carpel primordia start their development as three free, very per regions of the gynoecium, raphide idioblasts were occa- small cuneiform mounds of about 0.1–0.2 mm in diameter sionally observed. Oil bodies were seen to be abundant mostly (fig. 4B). The carpels become slightly globose shortly after- in the stigma, the style, the locules, and the ovule, mostly asso- ward; however, the apical lobes remain free (fig. 4C ). The ven- ciated with the funiculus and both integuments (fig. 4H–4L). tral slits appear very soon afterward in the apex of each lobe Within the petals, oil bodies were seen to be scattered in the (fig. 4D). Nevertheless, the carpels later undergo a postgenital marginal regions (fig. 4H). fusion in their basal regions, producing a gynoecium that is basally synascidiate but free and plicate in the upper part of the fi style and the stigmatic region ( g. 4F). Shortly before anthe- Discussion sis, the gynoecium is 1.5–2 mm long, 1 mm in diameter, and mostly globose, with a very short style. The carpels are ascid- Comparative Development of Male and Female Flowers in iate and connate in their basal region (fig. 4F,4J,4K). Three Chamaedorea tepejilote and Hyophorbe lagenicaulis free, slightly reflexed branches could be distinguished at the apex of the stigma (fig. 4F–4I). On account of their respectively monoecious and dioecious Anatomy. The fleshy ring formed after the fusion of the characters, both H. lagenicaulis and C. tepejilote display aborted basal portion of the sepals is vascularized by 26–28 bundles, development in the residual reproductive organs of their uni- which are mainly associated with the adaxial surface. This high sexual flowers. The developmental basis of unisexual flower dif-

42822.proof.3d 11 Achorn International 01/13/16 01:35 000 INTERNATIONAL JOURNAL OF PLANT SCIENCES ferentiation has been reviewed by numerous authors, with the protection of the floral organs against herbivory nor the of- literature extending as far back in time as the observations of fer of a reward for potential pollen transporters is primordial. Charles Darwin (1877). The simplest way to classify develop- Thus, our observations are compatible with a putative ane- mental arrest (Mitchell and Diggle 2005) is in terms of whether mophilous mode of pollination. Interestingly, Ríos et al. (2014) the nonfunctional organ is initiated (type I development) or not found in a multispecies study of the genus that the consump- (type II development). In this scheme, all the unisexual flowers tion of tissues by insects was more evident on staminate inflo- described in this study can be considered to show type I devel- rescences, particularly in C. tepejilote, suggesting that this con- opment. A more detailed survey of developmental diversity in sumption might be related to low levels of anatomical defenses. residual reproductive organ arrest was carried out by Diggle As described here, this is not the case in the female flower, where et al. (2011), who classified androecium and gynoecium devel- raphides, tannins, stegmata, and oil bodies are found to accu- opment into eight stages while distinguishing six different pro- mulate in profusion in the perianth organs and gynoecium, even cesses that can lead to the loss of reproductive organ function. surrounding the nucellus. Our observations fit with the gen- Using this scheme, we can see a distinction in female flower dif- eral pattern of the family, whereby pistillate flowers are usu- ferentiation between H. lagenicaulis, in which staminode de- ally more heavily protected than staminate ones (Uhl and velopment proceeds as far as the stage of anther lobe and fila- Moore 1973). The Chamaedoreeae appear to be one of the ment distinction (albeit weakly), and C. tepejilote, in which few major groups of palms where wind might play an impor- staminode development ceases earlier, at the stage of the anther tant role in pollination, according to Henderson (2002). Indeed, primordium. In the case of the male flower, there is less con- C. tepejilote has been found to be a wind-pollinated species in trast between the two species. The gynoecium is initiated, and several studies (Bawa et al. 1985; Oyama 1990; Otero-Arnaiz carpel formation is seen to occur in male flowers of both C. and Oyama 2001). Interestingly, it is not uncommon for ap- tepejilote and H. lagenicaulis; however, there is no sign of ovule parently wind-pollinated species to receive appreciable quanti- initiation in either. Although the pistillode of C. tepejilote is ties of insect-dispersed pollen, as observed in several species of somewhat larger than that of H. lagenicaulis, the degree of dif- Chamaedorea (Listabarth 1993; Berry and Gorchov 2004). ferentiation can be considered as slightly greater in the monoe- Ríos et al. (2014) suggested that there is not a dichotomy of pol- cious species for two reasons. First, whereas no vascularization lination mechanisms between entomophilous and anemophi- is seen in the pistillode of the male flower in C. tepejilote, its lous species in the genus Chamaedorea, since most species (in- homolog in H. lagenicaulis displays vascular tissues in its basal cluding C. tepejilote) exhibit a combination of floral traits for region. Second, nectary differentiation is seen in the latter, both insect and wind pollination. again with some accompanying vascular elements. This con- Although raphide crystals have been interpreted as a pos- trasts with the situation in the C. tepejilote pistillode, which sible herbivore deterrent (Uhl and Moore 1973, 1977; Prychid develops as a structure consisting of only parenchymatous tis- and Rudall 1999; Tomlinson et al. 2011), their potential bio- sue and scattered raphide idioblasts, enclosed by an epidermis. logical significance, particularly in pollination and dispersal, is To summarize, the floral developmental features observed in still unknown (Henderson and Rodriguez 1999). Tannins ap- this study collectively reveal a stronger degree of differentia- pear to act as repellents, growth inhibitors, and direct toxic tion in the staminodes and pistillode of the monoecious species agents (Uhl and Moore 1973), although they have rarely been H. lagenicaulis compared to the dioecious species C. tepejilote. reported in Chamaedorea flowers (Uhl and Moore 1971; Ask- Conversely, developmental arrest is observed to be earlier in gaard et al. 2008). Stegmata or silica cells are widespread in the case of the residual reproductive organs of C. tepejilote,es- monocots, where they are thought to have different functions pecially the staminode. (Prychid et al. 2003). Such cell types have previously been described in the vegetative tissues of species of the Chamaedo- reeae (Tomlinson 1961; Tomlinson et al. 2011), as well as in the flowers of Chamaedorea (Askgaard et al. 2008) and Gaus- Anatomical Divergence between C. tepejilote and sia (Castaño et al. 2014). Oil bodies are rarely present in mono- H. lagenicaulis and Its Possible Significance cots (Lersten et al. 2006) and to date have been reported for Species of the tribe Chamaedoreeae are often characterized the Arecaceae only in Gaussia attenuata (Castaño et al. 2014) by the occurrence of raphide-containing idioblasts in their flow- and H. lagenicaulis (this study). ers (Uhl and Moore 1973; Dransfield and Uhl 1998; Hender- In the case of H. lagenicaulis, the histological features de- son and Rodriguez 1999; Askgaard et al. 2008). Idioblasts of scribed in this study showed certain distinctions from those this kind, as well as tanniniferous cells and stegmata, have been of C. tepejilote. All floral organs were seen to possess special- considered to be involved in defense mechanisms within the ized defense-related cells in their tissues, including the sterile family (Uhl and Moore 1973; Tomlinson 1990; Tomlinson et al. and fertile gynoecia of male and female flowers, respectively. 2011). In this study, we found contrasting patterns in the dis- These characters, combined with basipetal floral development, tribution of these cell types in the flowers, which could be re- protandrous acervulus development, and nectar secretion by lated to some extent to the hypothetical pollination syndromes male and female flowers, could be an indication of insect pol- of the species studied (i.e., anemophily in C. tepejilote and en- lination as previously hypothesized for Gaussia (Castaño et al. tomophily in H. lagenicaulis). Tanniniferous cells, stegmata, 2014) and Synechanthus (Siefke and Bernal 2004). The nectar- and oil bodies were seen to be absent in the male flowers of ies observed in this study in H. lagenicaulis correspond to the C. tepejilote. These features, added to the absence of a septal gynopleural- or septal-type nectary form, which is the most nectary in the pistillode, suggest that in this species neither the common type seen in the palms (Schmid 1983) and more gener-

42822.proof.3d 12 Achorn International 01/13/16 01:35 CASTAÑO ET AL.—FLOWER DEVELOPMENT IN CHAMAEDOREEAE 000 ally in monocotyledons (van Heel 1988; Nepi 2007). Initially between sexual systems can provide an insight into the ecologi- reported in this species by Uhl (1978), the secretion of nec- cal drivers of floral and individual sexual specialization (Torices tar in male and female palm flowers has been linked to ento- et al. 2011). Multiple transitions can also provide useful oppor- mophily in only a few studies (Narayana 1938; Uhl and Moore tunities to investigate whether similar molecular and develop- 1977; Küchmeister et al. 1997; Rudall et al. 2003). In spite of mental mechanisms are responsible (Barrett 2008). Although the fact that the nectaries described here for the two types of the resolution of these questions is beyond the scope of this flower were only slightly different, nectar composition and se- study, some interesting earlier observations could help shed creted volume might differ between male and female flowers. light on this matter. Renner and Ricklefs (1995) found that It has been suggested that variations in these factors might in- monoecy is strongly associated with dioecy, from a phyloge- fluence the volume and diversity of insect visitors in certain netic point of view, and that the single most important predic- monoecious and dioecious species (Pacini et al. 2003). Never- tor of a group’s tendency to acquire dioecy is the presence of theless, although septal nectaries are widespread and have monoecy in the group. It has been suggested that those spe- often been referred to in the literature associated with mono- cies that evolved dioecy from monoecy may have previously cotyledon flowers, little is still known about their morphol- possessed an outbreeding device such as dichogamy (Freeman ogy and anatomy (Stauffer et al. 2009). Indeed, to our knowl- et al. 1997), which is actually the case for the acervulus of edge, no published research has as yet addressed the ecological H. lagenicaulis described here, displaying protandrous develop- and evolutionary significance of differences in nectary morphol- ment. A similar situation occurs in the other monoecious genera ogy and nectar composition between male and female flowers of the Chamaedoreeae (Moore 1971; Uhl 1978; Ortega and in the palm family. Stauffer 2011; Castaño et al. 2014). Another developmental feature that suggests a possible disposition toward the evolution of dioecy in H. lagenicaulis is the presence of exclusively Sexual Expression male acervuli, mostly toward the distal region of the inflores- In contrast to flowering plants as a whole, the production of cence (this study; Uhl 1978; Ortega and Stauffer 2011). Simi- unisexual flowers is the rule rather than the exception within lar observations have also been made for Gaussia (Quero and the palm family. Approximately 60% of palms are monoecious, Read 1986; Castaño et al. 2014) and Synechanthus (Moore 22% are dioecious, and 17% are hermaphroditic (Henderson 1971; Siefke and Bernal 2004). As suggested by Ortega and 2002). Thus, even dioecy is more prevalent than hermaphrodit- Stauffer (2011), the apparent transition from bisexual to uni- ism in palms, with about 37 genera being completely dioecious, sexual acervuli may underlie the origin of the dioecious condi- compared with 30 genera producing hermaphrodite flowers tion observed in Chamaedorea and Wendlandiella or at least (Dransfield et al. 2008). Within the subfamily Arecoideae, the the origin of unisexual male plants. Furthermore, it could rep- largest and most diverse subfamily constituted by 13 tribes resent the existence of intermediary androdioecious stages in (Dransfield et al. 2008), most species are monoecious and bear the pathway to dioecy within the tribe, although androdioecy unisexual flowers grouped in triads or derived forms including is known to be rare in nature. Nevertheless, according to pairs or solitary flowers. The only exceptions are found in the Charlesworth and Charlesworth (1978b), dioecy can evolve tribe Chamaedoreeae, with two of its five genera being dioecious from monoecy by a gradual process of alteration of the rela- (Chamaedorea and Wendlandiella). Taken overall, this phylo- tive proportions of male and female flowers rather than via genetic distribution strongly suggests that the common ancestor gynodioecy with essentially complete male sterility to give fe- of Arecoideae was also monoecious. Moreover, given that the males. The evolution of dioecy from monoecy has been en- two aforementioned genera probably represent independent visioned as the spread of alleles affecting individuals’ floral evolutionary transitions from monoecy to dioecy within the sex ratio because male and female sterility was already estab- same tribe, it seems likely that Chamaedoreeae are more pre- lished (Renner and Won 2001). disposed than other arecoid palms toward the evolution of uni- Although the production of unisexual flowers was probably sexual plants (Cuenca et al. 2009). Weiblen et al. (2000), in an not an innovation in Chamaedorea or Wendlandiella since the optimization of breeding-system characters in the phylogeny Chamaedoreeae most likely evolved from a monoecious an- of monocotyledons, concluded that dioecy probably evolved cestor, it is interesting to note that a higher degree of sexual more often from hermaphroditism than from monoecy. How- specialization is seen in C. tepejilote than in H. lagenicaulis. ever, in the same study, these authors found that the tran- This is witnessed by the extent of the reduction of sterile or- sitions of Chamaedorea and Wendlandiella were the only two gans. On one hand, in H. lagenicaulis, the female flower dis- unambiguous changes from monoecy to dioecy observed in the plays conspicuous staminodes with vestigial anthers lacking palm family. Several different pathways are thought to be in- pollen sacs, and the male flower bears a pistillode with elab- volved in the evolution of dioecy, with the most common sce- orated septal nectaries; both organs are moderately vascular- narios probably involving a gynodioecious or monoecious an- ized and cease development at a relatively late stage. In con- cestor (Charlesworth and Charlesworth 1978a; Bawa 1980; trast, the female flower of C. tepejilote bears staminodes that Freeman et al. 1997; Weiblen et al. 2000; Torices et al. 2011; are hardly detectable at anthesis, the pistillode of the male Barrett 2013). The phylogenetic structure of the Chamaedoreeae flower being uniformly composed only of ground parenchyma, strongly suggests that dioecy evolved directly from monoecy in with both organs lacking any trace of vascular tissue. The ster- the tribe; however, an important question remains: did the evo- ile organs of the dioecious C. tepejilote are thus considerably lution of dioecy follow the same pathway in Chamaedorea and more reduced than those of the monoecious H. lagenicaulis. Wendlandiella?Theclarification of the evolutionary transitions Although the exact significance of these observations remains

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to be determined, it is interesting to note that an initial loss of Acknowledgments sexual function during the evolution of dioecy may be followed by additional mutations that result in earlier termination of This work was made possible by a PhD grant from the IRD development of sterile organs (Diggle et al. 2011). This might to Felipe Castaño, by core funding from our institutions, and have the effect of enhancing the efﬁciency of resource alloca- through additional support from the Agropolis Fondation in q2 tion for the plant. the framework of the Open Science 2012 call for proposals To summarize, even though genomic rearrangements will (Male Domestics project 1202-069). We are very grateful to have occurred in the dioecious lineages of the Chamaedoreeae to the Montgomery Botanical Center and the Fairchild Tropical produce a genetic system with separate male and female individ- Botanic Garden (Miami) for allowing access to their living palm uals, it is likely that the same sex-determination genes will oper- collections for this study. Particular thanks go to Dr. Patrick ate in these clades as in the monoecious ones, given that they Grifﬁth, Dr. Carl Lewis, and Dr. Larry Noblick. We are also probably share a common monoecious ancestor. Nevertheless, indebted to Dr. André Piuz (Museum d’Histoire Naturelle, in order to assess the validity of this hypothesis and examine the Geneva, Switzerland), Chantal Cazevieille, and Cécile Sanchez likely independent origins of the two dioecious genera, compar- (CRIC, Montpellier, France) for their technical assistance with ative genomic studies will be necessary to reveal the extent of SEM. Dr. Gloria Galeano (Instituto de Ciencias Naturales, Uni- molecular divergence between the different clades. In parallel, versidad Nacional de Colombia) is thanked for providing per- the increasingly broad picture of sexual differentiation being as- manent support to the project. The team of the library of the sembled for the Chamaedoreeae will help to provide clues as to Botanical Conservatory in Geneva is thanked for providing per- the developmental basis of the sex-determination process. manent access to their collections.

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Q1. AU: For the author afﬁliation in Bogotá, Colombia, is there a postal code to include with this address? Q2. AU: Note that I have added “for” to “resource allocation the plant,” as a word seemed to be missing. Let us know if a different wording should be used. Q3. AU: Henderson 1986 is not cited in the main text. Should this be removed from Literature Cited? If not, please provide a place in the text to add a citation. Q4. AU: Núñez‐Avellaneda and Rojas‐Robles 2008 is not cited in the main text. Should this be removed from Literature Cited? If not, please provide a place in the text to add a citation. (Also note that the ﬁrst half of this reference was deleted and had to be reinserted before typesetting, so please make sure this info is as intended.)

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