1 Verhuellia is a segregate lineage in Piperaceae: more evidence from flower, fruit and 2 pollen morphology, anatomy and development 3 4 Marie-Stéphanie Samain1*, Alexander Vrijdaghs2, Michael Hesse3, Paul Goetghebeur1, 5 Francisco Jiménez Rodriguez4, Alexandra Stoll5, Christoph Neinhuis6, Stefan Wanke6 6 7 1 Ghent University, Department of Biology, Research Group Spermatophytes, B-9000 Gent, 8 Belgium 9 2 Katholieke Universiteit Leuven, Institute of Botany and Microbiology, Laboratory of Plant 10 Systematics, Kasteelpark Arenberg 31, B-3001 Leuven, Belgium 11 3 University of Vienna, Institute of Botany, Department of Palynology and Structural Botany, 12 A-1030 Vienna, Austria 13 4 Jardín Botánico Nacional Dr. Rafael M. Moscoso, Herbarium, Santo Domingo, Dominican 14 Republic 15 5 Centro de Estudios Avanzados en Zonas Aridas – CEAZA, Casilla 599, Campus Andres 16 Bello, Colina El Pino s/n., La Serena, Chile 17 6 Technische Universität Dresden, Institut für Botanik, D-01062 Dresden, Germany 18 19 short title: Morphology, anatomy and development in Verhuellia 20 *Corresponding author: 21 Marie-Stéphanie Samain 22 Ghent University – Department of Biology 23 Research Group Spermatophytes 24 K.L. Ledeganckstraat 35 25 B-9000 Gent 26 Phone: +32 (0) 9 264 50 58 27 Fax: +32 (0) 9 264 53 34 28 [email protected] 29 30 Morphology, anatomy and development in Verhuellia: 1 (07/01/2010) 1 Abstract 2 Background and Aims The perianthless Piperales, i.e. Saururaceae and Piperaceae, have 3 simple reduced flowers strikingly different from the other families of the order (e.g. 4 Aristolochiaceae). Recent molecular phylogenies proved Verhuellia to be the first branch in 5 Piperaceae, making it a promising object to study the detailed structure and development of 6 the flowers. Based on recently collected material, the first detailed study since 1872 was 7 conducted with respect to morphology, anatomy and development of the inflorescence, pollen 8 ultrastructure and fruit anatomy. 9 Methods Original Scanning Electron Microscopy (SEM), Transmission Electron Microscopy 10 (TEM) and Light Microscopy (LM) observations on Verhuellia lunaria were compared with 11 Piperaceae, Saururaceae and fossils. 12 Key results The inflorescence is an indeterminate spike with sessile flowers, each in the axil 13 of a bract, developing in acropetal, helical succession. Flowers consist of two (occasionally 14 three) stamens with basifixed tetrasporangiate anthers and latrorse dehiscence by a 15 longitudinal slit. The gynoecium lacks a style but has three to four stigma branches and a 16 single, basal orthotropous, and unitegmic ovule. The fruit is a drupe with large multicellular 17 epidermal protuberances. The pollen is very small, inaperturate, and areolate with 18 hemispherical microechinate exine elements. 19 Conclusions Despite the superficial similarities with different genera of Piperaceae and 20 Saururaceae, the segregate position of Verhuellia revealed by molecular phylogenetics is 21 supported by morphological, developmental and anatomical data presented here. Unitegmic 22 ovules and inaperturate pollen, which are synapomorphies for the genus Peperomia, are also 23 present in Verhuellia. 24 Morphology, anatomy and development in Verhuellia: 2 (07/01/2010) 1 Key words: Verhuellia lunaria, Piperales, Peperomia, Appomattoxia ancistrophora, floral 2 development, floral anatomy, fruit morphology, pollen morphology, unitegmic ovule, 3 inaperturate pollen. 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Morphology, anatomy and development in Verhuellia: 3 (07/01/2010) 1 INTRODUCTION 2 Verhuellia is a Piperaceae genus of three species, known from very few collections and 3 localities on Cuba and Hispaniola (Haiti and Dominican Republic). It is a small, herbaceous, 4 saxicolous perennial with monopodial architecture, whorled leaves and perianthless flowers 5 organised in solitary lateral spikes (Fig. 1). These flowers, each subtended by a bract, consist 6 of a gynoecium with three to four stigmas and two tetrasporangiate stamens. With the 7 exception of Schmitz (1872a, b), who only described some morphological characters based on 8 herbarium specimens, detailed morphological, anatomical or developmental studies have 9 never been conducted. This is probably due to the very limited number of collections and its 10 absence from living collections and also to the presumed close relationship with the genus 11 Peperomia. 12 The absence of some floral organs in the flowers of perianthless Piperales has recently re- 13 enhanced the focus on the representatives of the sister families Saururaceae and Piperaceae, 14 presenting them as an interesting model for studying floral development and evolution 15 (Jaramillo and Kramer, 2007; Arias and Williams, 2008; Madrid and Friedman, 2009). 16 Furthermore, several phylogenetic studies in the order Piperales are now available as a robust 17 framework to interpret character evolution (Wanke et al. 2007a, b). With the exception of the 18 two Piperaceae genera Verhuellia and Manekia, floral characters of nearly all genera have 19 been extensively studied in earlier years (Tucker, 1975, 1976, 1979, 1980, 1981, 1982a, b, 20 1985; Liang and Tucker, 1989, 1990, 1995; Igersheim and Endress, 1998; Lei and Liang, 21 1998, 1999; Tucker et al., 1993). In addition, Smith and Stockey (2007) studied the pollen of 22 the four Saururaceae genera. Detailed observations on the fruit morphology in this family are 23 lacking. In Piperaceae, pollen and fruit development have not been studied extensively despite 24 the large number of species. Morphology, anatomy and development in Verhuellia: 4 (07/01/2010) 1 Verhuellia was generally considered to be part of or at least closely related to Peperomia (e.g. 2 Tebbs, 1993), due to its superficial similarities with this genus. However, Wanke et al. 3 (2007b) recently showed that Verhuellia is sister to all other Piperaceae, a position formerly 4 thought to be occupied by Zippelia and Manekia (Jaramillo et al., 2004; Wanke et al., 2007a). 5 As a consequence, the traditional subdivision of the family Piperaceae into Piperoideae and 6 Peperomioideae has been revised, resulting in three subfamilies congruent with the clades 7 recovered by Wanke et al. (2007 b): Verhuellioideae (with Verhuellia), Zippelioideae (with 8 Zippelia and Manekia) and Piperoideae (with Piper and Peperomia) (Samain et al., 2008). 9 Figure 2 shows a summary cladogram of the perianthless Piperales. 10 Given the hypothesis that Verhuellia is sister to the four other Piperaceae genera (Wanke et 11 al., 2007b), intermediate characters between Piperaceae and Saururaceae could be expected. 12 This is supported by the fact that Zippelia begoniifolia, placed in the next branching clade in 13 Piperaceae (Jaramillo et al., 2004; Wanke et al., 2007a, b), also shows some similarities to 14 Saururaceae (Igersheim & Endress 1998). Tucker et al. (1993) and Liang and Tucker (1995) 15 consider these characters as plesiomorphic. Zippelia has sometimes been considered a 16 member of Saururaceae (e.g. Blume, 1830; Wu and Wang, 1957; Heywood, 1993) whereas it 17 has never been questioned that Verhuellia belongs to Piperaceae. 18 The aims of this paper are: 1) to present a detailed study on morphology, anatomy and 19 development of the Verhuellia inflorescence, flower, pollen and fruit based on fresh material 20 to overcome the lack of knowledge on this genus and 2) to discuss the floral ontogenetic 21 findings in the light of its new position in the family Piperaceae and in relation to available 22 data of other perianthless Piperales, including the presumed fossil relative Appomattoxia 23 ancistrophora. 24 25 Morphology, anatomy and development in Verhuellia: 5 (07/01/2010) 1 MATERIAL AND METHODS 2 Plants of Verhuellia lunaria (Ham.) C.DC., the type species of the genus, originating from the 3 Dominican Republic were cultivated in the Botanical Gardens of Ghent University and 4 Dresden Technical University. Inflorescences, flowers and fruits of all stages were collected 5 and preserved. Voucher specimens are deposited in the herbaria of Ghent University (GENT) 6 and Dresden Technical University (DR). 7 Material for Scanning Electron Microscopy (SEM) was prepared by fixation in Kew mix 8 (53% industrial methylated spirit, 37% water, 5% formaldehyde solution and 5% glycerol), 9 followed by dehydration and critical point drying with carbon dioxide in a BAL-TEC CPD 10 030 critical-point-dryer. Subsequently, samples were fixed to aluminium stubs (Plano GmbH) 11 using a carbon adhesive tape (LEIT-TABS, Plano GmbH), sputter-coated with gold (20 nm) 12 under argon atmosphere using an EMITECH K550 sputter-coater. Images were obtained with 13 a LEO 420 scanning electron microscope at acceleration voltage of 15 kV. 14 Light microscopy (LM) samples were prepared in 70% ethanol and subsequently gradually 15 transferred to 100% ethanol. The samples were then transferred to LR White Resin, hard 16 grade (London Resin Company Ltd, Reading, England) in a graded LR White Resin/ethanol 17 series using solutions of 25/75, 50/50, 75/25, 100/0 resin/100% ethanol for at least 5 h each. 18 Subsequently, the samples were placed in a closed capsule filled with fresh resin and 19 hardened at 60°C for 48h. Sections of 2 µm were made with a rotation microtome (Microm 20 HM360 Waldorf, Germany) and subsequently stained with 0,1% toluidine blue. The stained 21 sections were fixed on microscopy slides using Eukitt© quick hardening mounting medium 22 (Fluka Chemie GmbH, Switzerland). Observations were done with a light microscope (Leitz 23 Dialux 20, Van Hopplynus, Brussels, Belgium) equipped with a camera (PixeLINK PL- 24 B622CF, Ottawa, Canada) with specially developed software (Microscopica v1.3, Orbicule, 25 Leuven, Belgium). Morphology, anatomy and development in Verhuellia: 6 (07/01/2010) 1 For TEM pictures pollen of Verhuellia lunaria was fixed in 50% alcohol, embedded in Agar 2 Low Viscosity Resin (Agar Scientific Ltd., Stansted, Essex, England); ultrathin sections were 3 poststained using uranyl acetate – lead citrate, KMnO4, and the Thièry Reaction for 4 polysaccharaides. Images were obtained with a Zeiss EM-900 transmission electron 5 microscope.