Diversification History of Adesmia Ser. Psoraleoides
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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector South African Journal of Botany 89 (2013) 257–264 Contents lists available at ScienceDirect South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb Diversification history of Adesmia ser. psoraleoides (Leguminosae): Evolutionary processes and the colonization of the southern Brazilian highland grasslands J.R.V. Iganci a,⁎, S.T.S. Miotto b, T.T. Souza-Chies b, T.E. Särkinen c, B.B. Simpson d, M.F. Simon e, R.T. Pennington f a Universidade Federal de Santa Catarina, Campus Universitário de Curitibanos, Caixa Postal 101, Rod. Ulysses Gaboardi, Km 3, 89520-000 Curitibanos, SC, Brazil b Universidade Federal do Rio Grande do Sul, Programa de Pós-Graduação em Botânica, Av. Bento Gonçalves, 9500, Prédio 43433, Bloco 4, Sala 214, Campus do Vale, Porto Alegre, RS 91501-970, Brazil c Department of Botany, Natural History Museum, Cromwell Road, London SW7 5BD, UK d Department of Botany, University of Texas, Austin, TX 78713, United States e Embrapa Recursos Genéticos e Biotecnologia, PqEB, Caixa Postal 02372, 70770-917 Brasília, DF, Brazil f Royal Botanic Garden Edinburgh, Tropical Diversity Section, 20a Inverleith Row, Edinburgh EH3 5LR, UK article info abstract Available online 22 July 2013 A molecular phylogeny is used to analyze the diversification history of Adesmia ser. psoraleoides, and its implica- tions for understanding the historical assembly of the grasslands in the highlands of southern Brazil. All species of Edited by B-E Van Wyk A. ser. psoraleoides were sampled, including multiple accessions for each species, plus representative species of the rest of Adesmia covering its geographic distribution. Phylogenetic analyses were based on nuclear and plastid DNA Keywords: sequences, and a plastid matK phylogeny was dated. A. ser. psoraleoides is a well-supported monophyletic group, Biogeography nested within the series bicolores, muricatae, subnudae, longisetae and candidae.ThestemofA. ser. psoraleoides is Campos de Cima da Serra c. 11 Mya, but most extant species diversified c. 3–5 Mya, suggesting that the flora of the subtropical grasslands of Dated phylogeny Endemism southern Brazil was assembled recently, co-incident with the expansion of other tropical grassland systems Adesmia globally. Fabaceae © 2013 SAAB. Published by Elsevier B.V. All rights reserved. 1. Introduction with an unappreciated level of plant species endemism as high as 25%. A principal goal of this study is to make the first investigation of Analyses of phylogenetic trees integrated with developing knowl- the age of origin of the CCS grasslands by evaluating the age of origin edge of the timing and nature of climatic and geological changes have of species of A. ser. psoraleoides, which are confined to this area. We been providing new perspectives on the evolutionary history of biomes test the hypothesis that the CCS are ecologically young, as is the (Byrne et al., 2008). In the Neotropics, such studies have provided case for other C4 grasslands globally (Cerling et al., 1997; Beerling new insights into the historical assembly of the floras of Amazonia and Osborne, 2006; Stromberg, 2011) and the neotropical savannas (e.g., Pennington and Dick, 2004), the savannas of the Brazilian cerrados (“cerrados”) that are situated further north in Brazil (Pennington et (Simon et al., 2009) and seasonally dry tropical forests (e.g., Särkinen et al., 2006; Simon et al., 2009; Simon and Pennington, 2012). al., 2011). This paper presents the first phylogenetic biogeographic The geographical affinities of the CCS flora are still poorly known. study of the “Campos de Cima da Serra” (CCS grasslands), focusing Relationships of the CCS flora with the Andes have been suggested by on a clade of Leguminosae, Adesmia ser. psoraleoides,withthegoalof several workers and explored floristically (Rambo, 1951, 1953a,b, illuminating the history of this poorly known biome. 1956; Smith, 1962; Safford, 1999, 2007; Iganci et al., 2011), but no The CCS is a subtropical grassland located in the highlands of phylogenetic studies have been done thus far. Around one-third of the southern Brazil. The CCS was often considered as a northern extension genera found in the southern Brazilian flora as a whole are related of the more southerly and low altitude temperate pampas grasslands to lineages assumed to be temperate Austral–Antarctic and Andean (“Campos Sulinos”—southern grasslands), but Iganci et al. (2011) in origin (Rambo, 1951, 1953a,b, 1956; Smith, 1962; Safford, 1999, made a strong case that the CCS is a distinct subtropical formation 2007; Waechter, 2002; Iganci et al., 2011). Many authors have assumed that eastern South American species in these groups are secondary radi- ations of Andean and Austral–Antarctic lineages (Rambo, 1953a,b; ⁎ Corresponding author. Tel.: +55 49 99643996. Safford, 1999, 2007; Waechter, 2002). One aim of this paper is to test E-mail address: [email protected] (J.R.V. Iganci). this scenario in a phylogenetic context for Adesmia. 0254-6299/$ – see front matter © 2013 SAAB. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.sajb.2013.06.016 258 J.R.V. Iganci et al. / South African Journal of Botany 89 (2013) 257–264 Adesmia comprises around 240 species, and is formally subdivided Dalbergioid legumes were used as outgroups. Plant material was mostly into two subgenera (Adesmia subgen. Adesmia and Adesmia subgen. sampled from the wild, and from cultivated specimens obtained from Acanthadesmia Burkart) and 43 series based on morphology (Burkart, seeds collected in the wild or held in national collections. All collections 1967; Lewis et al., 2005). Most species are concentrated along the plus georeferenced herbarium records for A. ser. psoraleoides were Andes spreading in temperate areas from the north of Peru to “Tierra mapped using DIVA-GIS (Hijmans et al., 2004) to indicate the most spe- del Fuego”, the southern tip of South America, and it is the most cies rich areas. species-rich genus of Leguminosae in temperate South America (Burkart, 1967). The northeast limit of Adesmia is in the CCS. Almost 2.2. DNA extraction, amplifications and sequencing all 11 species of A. ser. psoraleoides Burkart are endemic to the CCS, growing mainly in patches of grassland and rock outcrops amongst for- Total DNA was isolated from 0.3 g of silica gel-dried leaf tissue ests dominated by Araucaria angustifolia (Bertol.) Kuntze (Miotto and using a modified CTAB extraction method (Doyle and Doyle, 1987). Leitão Filho, 1993; Miotto and Waechter, 1996; Iganci and Miotto, The molecular markers analyzed were the nuclear ribosomal 5.8S 2011). Given the high level of endemism in the CCS and the occurrence and flanking internal transcribed spacers — ITS1 and ITS2 (Delgado- of diversity in other biomes, including the Andes and more southern Salinas et al., 1999), and three chloroplast (cpDNA) regions: the trnK grasslands, A. ser. psoraleoides makes an excellent study group to inves- intron including matK (Lavin et al., 2000, 2001; Wojciechowski et al., tigate the biogeographic affinities of the CCS flora and its diversification. 2004), rbcL (Olmstead et al., 1992; Kajita et al., 2001) and rps16 Species of A. ser. psoraleoides are unarmed, perennial herbs or (Oxelman et al., 1997). Polymerase chain reaction (PCR) was con- shrubs that in common with other Adesmia species have decorative ducted with a reaction volume of 25 μL containing ~20 ng of DNA yellow flowers with brownish nectar guides and fruits that are a template, 1× buffer, 2 μM MgCl2,1μM of each dNTP, 10 pmol of plane hemicraspedium with arillate seeds (Burkart, 1967; Miotto each primer and 1 U of Taq DNA Polymerase (CenBiot, Universidade and Leitão Filho, 1993)(Fig. 1). Such morphological characters and Federal do Rio Grande do Sul, Porto Alegre, Brazil). For ITS amplifica- others have collectively been used to circumscribe the series, but tions, 1 μL of DMSO, and for matK amplifications, 1 μL of BSA were they are not synapomorphies because all are shared with other also added. PCR conditions were as follows: ITS: 95 °C for 4 min, Adesmia species (Burkart, 1967). One aim of this study – a key prereq- 35 cycles of 30 s at 94 °C, 30 s at 58 °C and 2 min at 72 °C, followed uisite of the subsequent biogeographic analysis – is to evaluate the by a final extension of 7 min at 72 °C; matK: 95 °C for 4 min, 35 cycles monophyly of A. ser. psoraleoides and how it relates to other species of 30 s at 94 °C, 30 s at 50 °C and 2 min at 72 °C, followed by a final of Adesmia from other geographic areas. extension of 7 min at 72 °C; rbcL: 95 °C for 2 min, 30 cycles of 30 s Here we present a phylogenetic analysis of the diversification histo- at 95 °C, 30 s at 53 °C and 1:30 min at 72 °C, followed by a final exten- ry of A. ser. psoraleoides, providing a time frame for the diversification sion of 7 min at 72 °C; rps16: 95 °C for 4 min, 35 cycles of 30 s at of major clades. We address the following questions: (1) Is A. ser. 94 °C, 30 s at 50 °C and 2 min at 72 °C, followed by a final extension psoraleoides a monophyletic group and when did this group originate?; of 7 min at 72 °C. PCR products were cleaned using exonuclease I (2) What is the sister group of A. ser. psoraleoides and where is it from?; and shrimp alkaline phosphatase (Exo/SAP) and sequenced using and finally (3) What can be inferred from the species diversification BigDye v.3.1 chemistry (Applied Biosystems, Warrington, UK). For pattern of A. ser. psoraleoides in terms of the evolutionary history of the matK region, the samples were amplified in two parts and se- the CCS? Species diversification pattern is analyzed based on a time- quenced in eight reactions, using both the two outer and the two in- calibrated, densely sampled plastid and nuclear ribosomal internal nermost primers for PCR amplification, and four additional internal transcribed spacer (ITS) phylogeny of A.