SYSTEMATICS AND BIOGEOGRAPHY OF RENEALMIA L.F. (ZINGIBERACEAE) BASED ON NUCLEAR AND CHLOROPLAST DNA SEQUENCES MARION JANNES Master Sc. in Botanical Systematics and Evolution Gothenburg University / Royal Botanical Garden Edinburgh / University of Oxford Supervisors: Alexandre Antonelli, [email protected] Claes Persson, [email protected] Toby Pennington, [email protected] Tiina Sarkinen, [email protected] Front page: Picture of Renealmia pluriplicata (Zingiberaceae), Heredia: Sarapiqui La Selva Biological Station, near the town of Puerto Viejo 60 m, COSTA RICA. © 2008 by Robbin Moran. Used with permission. ABSTRACT Species of Renealmia exist in Africa as well as in the Americas. Sequence data from two chloroplast markers (psbA-trnH intergenic spacer, and trnL-F region) along with the internal transcribed spacer region (ITS) of nuclear ribosomal DNA were used to infer relationships within the geographically divided Renealmia. The psbA-trnH marker rendered low sequence divergence and adding it to previous ITS/ trnL- F data did not resolve outstanding problems. The psbA-trnH intergenic marker does not appear as a suitable region to unravel Renealmia or other recent radiated genera on the species level. The marker detected the recognised subfamilies Zingiberioideae and Alpinioideae of Zingiberaceae. Molecular divergence age estimates and the low sequence divergence confirm recent speciation proposed in Sarkinen et al. (2007) and long distance dispersal events (LDD). CONTENTS ABSTRACT INTRODUCTION RELATIONSHIPS AND CLASSIFICATIONS AIMS MATERIALS AND METHODS TAXON SAMPLING OUTGROUP SELECTION PLANT MATERIAL SELECTED GENETIC MARKER DNA EXTRACTION, GENE AMPLIFICATION, AND SEQUENCING PHYLOGENETIC ANALYSES RESULTS AMPLIFICATION OF PSBA-TRNH INTERGENIC SPACER PHYLOGENETIC ANALYSES DISCUSSION MOLECULAR ANALYSES PHYLOGENTIC ANALYSES CONCLUSIONS AKNOWLEDGMENTS REFERENCES APPENDIX I INTRODUCTION The subgenus Renealmia belongs to the Gingers (Zingiberaceae), the largest family in the monocotyledon order Zingiberales, which additionally includes the Heliconiacae (Heliconias), the Musaceae (Bananas) and the Strelitziaceae (Birds of Paradise). Gingers are mainly understorey herbs in tropical lowland forests and they reach their largest species diversity in Southeast Asia (Sarkinen, 2005). The family has long been classified primarily based on morphology (Petersen, 1889; Schumann, 1904; Burtt and Smith, 1972; Larsen et al, 1998) but during the beginning of the 2000th century a substantional reclassification using molecular data revealed four subfamilies (Kress et al., 2002); - the Siphonochiloideae W.J.Kress (Siphonochilus), - the Tamijioideae W.J. Kress (Tamijia), - the Alpinioideae , Link (former Alpinieae , - the Zingiberoideae (former Hedychiacae, Zingibereae and Globbeae). Additional molecular phylogenetic analyses of the Ginger family have been performed (Kress et al., 2005; Pedersen, 2005) as well as analyses of the shallow relationships in a number of genera (Afromomum: Harris et al., 2000, Aulotandra: Harris et al., 2006; Amomum: Xia et al., 2004; Boesenbergia: Techaprasan et al., 2006; Globba: Williams et al., 2004; Renealmia: Sarkinen et al., 2007; and Roscoea: Ngamriabsakul et al., 2000 as to name a few). While molecular techniques developed, more and more available DNA regions emerged, and a more consistent view of the family’s internal relationships were attained. Zingiberaceae is however far from being fully resolved, and still has problematic and contradicting relationships. THE SUBFAMILY ALPINIOIDEAE The Alpinioideae is the second largest subfamily after Zingiberioideae in Zingiberaceae and is distinguished morphologically from the other subfamilies by two major morphological attributes. First Alpinioideae has largely reduced or absence of the two lateral staminodes (Kress et al., 1992). These represent two modified stamens of the outer staminal whorl of the Zingiberaceae floral plan; see illustration below (fig. 1). (A) (B) Fertile stamen QuickTime och en Sepal QuickTime och en -dekomprimerare -dekomprimerare krävs för att kunna se bilden. Petal krävs för att kunna se bilden. krävs f Lateral stamens Carpels Fig. 1 Schematic illustration of floral morphology. A: Zingiberaceae, and B: solely the subfamily Alpinioideae. * indicating absent stamen. Figure modified from P,J. Rudall (2004) Used with permission. Second Alpinioideae has a transverse plane of distichy of leaves relation to rhizomes; see illustration below (fig. 2). Alpinioideae are divided in two main groups, Alpinieae A. Rich and Redelieae of which the former is the largest (fig. 3). The tribe Alpinieae is polyphyletic and largely made up of its type genus Alpinia. The tribe is problematic and contains genera with unsolved relationships both within and beside the genera included. Recently has two closely related genera, Renealmia and Afromomum of the tribe been investigated phylogenetically (Sarkinen et al., 2007, and Harris et al., 2000). These analyses have revealed very few informative characters, which is proposed to origin from recent speciation events. 1 2 4 5 3 A B C D Fig. 2 Illustrations of A: transverse plane of distichy of leaves to rhizome (Alpiniodeae/Tamijiodeae), B: parallel plane of distichy of leaves to rhizome (Zingiberoideae/Siphonochiloideae), and C: Renealmia flower composition. 1: tubular calyx, 2: anther and style, 3: erect, petaloid and 3-lobed labellum, 4: tubular bracteole, 5: corolla. D: stellate hair on leaves (A) (B) Fig. 3 A: Phylogenetic tree representing the four subfamilies of Zingibereceae, and B: showing subfamily Alpinioideae. Modified from Krees et al, 2002. THE GENUS RENEALMIA The genus Renealmia is distinguished from the other members of Alpinoideae in its Neotropical and African distribution and morphologically from its closely related genera Afromomum by having a corolla longer than the calyx and stellate hairs on leaf lamina (Hutchinson and Dalziel, 1968; Maas, 1977; Larsen et al., 1998), see fig 1 and table 1. Renealmia comprises 70-83 species of wet tropical forest understorey herbs and is postulated to be monophyletic (Harris et al., 2000; Kress et al., 2002, 2005; Pedersen, 2004; and Sarkinen et al., 2007) with three potential inclusive monophyletic groups (Sarkinen et al., 2007; Kress et al., 2005): I. The Alpinia fax / A. abundiflora clade (A. fax clade), II. the Afromomum clade, and III. the Elattariopsis / Amomum clade. In addition to its distinctly low molecular informative characters, the genus Renealmia is proposed to have accomplished one or possibly two long distance dispersal (LDD) events between its geographically widespread locations on both sides of the Atlantic sea (Sarkinen et al., 2007). CLASSIFICATION The current classification of the genus is based on morphology and divided by its Neotropical and African distribution. The Neotropical species are divided into four major groups: . RENEALMIA CERNUA GROUP . RENEALMIA ALPINIA GROUP . RENEALMIA ACREANA GROUP . RENEALMIA AROMATICA GROUP Three unplaced taxa (Renealmia jamaicensis (Gaertn) Horan, R. pyramidalis, and R. variegata Maas & Maas is also contained in this Neotropical group (Maas et al., 1977, 1979, 1987, 1990). In Africa, the genus is divided into two main species complexes (Thiselton,- Dyer, 1898; Kochlin, 1964, 1965; Hepper 1968; Lock, 1985; Poulsen and Lock, 1997; and Turner, 1999): . RENEALMIA AFRICANA SPECIES COMPLEX, . RENEALMIA CONGOENSIS SPECIES COMPLEX. Aside of these complexes there are eleven unplaced species; Renealmia battenbergiana Cummins ex Baker, R. bracteata de Wild. & T.Durand, R. cincinnata (K. Schum.) Baker, R. densispica J.Koechlin, R. engleri K.Schum., R. longifolia K.Schum., R. maculata Stapf., R. mannii Hook.f., R. polyantha K.Schum., R. polyantha K.Schum., R. polypus Gagnep., and R. sancti-thomae I.M.Turner. Table 1 Morphological characteristics of Renealmia and closely related genera in Alpineae (Larsen et al., 1998). Inflorescence Flowers Bracteole Labellum Calyx Corolla Colour Region Renealmia Lax, Single Tubular 3-lobed, Tubular Longer White to Neotropics sometimes, or erect or to than yellow and Africa branched, cincinni spreading, turbinate calyx terminal, longer terminal, on a than separate petals, leafless shoot petaloid or prostrate, rarely terminal in leafy shoot Amomum Congested, Single Open to Spathulate, Tubular, ± calyx White, Australasia terminal on a or base or longer 3- lenght yellow, separate cincinni tubular than dentate orange or, leafless shoot petals, red variously lobed Afromomum Congested, Single Open to Broad, Split Narrow, Violet, Africa terminal on a or base suberect or down 1 almost red, separate cincinni narrower side as long yellow, leafless shoot thicker and as calyx rarely decurved, white petaloid Alpinia Terminal, lax In Open to Erect or Tubular, = calyx Red, Asia or congested, cincinni base or concave, 3- lenght yellowish, sometimes tubular, sometimes dentate, white to surrounded sometimes spreading, often greenish by sterile clompletely variously split bracts, enclosing lobed or down 1 sometimes cincinnus entire side branched Elattariopsis Terminal on a Single Open to Broad, Tubular, Slender, White Indomalesia separate or base erect, 3- longer with red leafless rarely variously dentate than and shoot, two lobed or calyx yellow congested entire, and erect, or longer lax branched than petals and prostrate AIMS The purpose of this study is to: 1. Further investigate phylogenetic relationships within Renealmia by combining the data sets of Sarkinen et al. (2007)