A molecular phylogenetic study of Graptopetalum (Crassulaceae) based on ETS, ITS, RPL16, and TRNL‐F nucleotide sequences

Abstract

Nuclear ETS and ITS, as well as plastid rpl16 and trnL-F DNA sequences were used to determine relationships among species of Graptopetalum (Crassulaceae) and closely related genera. Graptopetalum is member of a group of taxa restricted to North America, one of the centers of diversity of Crassulaceae; however, their phylogenetic relationships are not yet understood. Nineteen species of Graptopetalum and 24 species from nine other genera of Crassulaceae were sampled for use in three separate parsimony analyses: ITS alone, ETS alone, and a combined nuclear + plastid DNA analysis using all four gene regions. The ETS data set had the highest number of parsimony-informative sites, about 30% more than in ITS, but the most fully resolved tree resulted when the four DNA regions were combined. Only four subclades of the tree received moderate to strong bootstrap support, one of which includes all species of Graptopetalum having a single whorl of stamens. However, Graptopetalum is not monophyletic. Instead, Tacitus bellus and select species of Cremnophila, Sedum, and Echeveria are interspersed among species of Graptopetalum and show evidence of grouping according to geographical range of distribution more so than habit or floral morphology.

Phylogeny, Biogeography, and Evolution of Edaphic Association in the European OreophytesSempervivumandJovibarba(Crassulaceae)

“…For cycle sequencing, primers ITS A and ITS-4 (White et al 1990;Hungerer and Kadereit 1998) were used. Since published primers for external transcribed spacer (ETS) of Crassulaceae did not yield PCR products in our study group (Acevedo-Rosas et al 2004), we first amplified the entire IGS region using primers 26S-II and 18S-II (Ochsmann 2000) in a 50 mL PCR reaction with 200 mM dNTPs, 0.5 mM of each primer, 0.5 U Phusion DNA Polymerase (Finnzymes, Espoo, Finland), and 1# HF buffer, with 45 s at 987C, 30 cycles with 10 s at 987C, and 3 min at 727C, followed by a final elongation step at 727C for 10 min. PCR products were gel purified, excised, and cleaned with the NucleoSpin Gel and PCR Clean-Up kit (Macherey-Nagel, Düren, Germany), following the manufacturer's protocol.…”

“…First, we obtained selected matK sequences of Crassulaceae ( Johnson and Soltis 1995;Fishbein et al 2001;Mort et al 2001Mort et al , 2002Schaefer et al 2011; various barcode projects and unpublished data) as well as sequences of selected outgroup families (Fishbein et al 2001;Moody and Les 2007) from GenBank. ITS sequences were collected in the same way (Gehrig et al 2001;Mort et al 2002;Acevedo-Rosas et al 2004;Mayuzumi and Ohba 2004;Schultheis and Donoghue 2004;Moody and Les 2007;Carrillo-Reyes et al 2008, 2009de Lange et al 2008;Vargas and García 2008;Gao et al 2012;Xiang et al 2012; various barcode projects and unpublished data). As alignment across this large sample was problematic in the ITS1 and ITS2 spacer regions, we pruned the alignment and used only the 5.8S ribosomal gene and those parts of the spacer regions that contained no indels 13 bp.…”

Molecular phylogeny, divergence time estimates, and historical biogeography of Circaea (Onagraceae) in the Northern Hemisphere

“…Therefore we combined the two data sets to explore whether resolution and support would be improved by increasing the amount of sequencing data. This approach was performed by direct inspection of nodes with bootstrap scores above 70% in the separate analyses (Kluge, 1989;Nixon and Carpenter, 1996;Muellner et al, 2003;Acevedo-Rosas et al, 2004;Berry et al, 2004;Inda et al, 2008). Phylogenetic analyses were undertaken for the nrITS, for the concatenated plastid data set, and for all molecular markers combined using maximum parsimony (MP) and Bayesian inference (BI) methods (Rannala and Yang, 1996).…”

Phylogenetic Relationships and Evolution in <I>Dudleya</I> (Crassulaceae)

“…Extractions were made using the DNeasy plant mini kit (QIAGEN , Valencia, California) according to the manufacturer's instructions. Target DNA regions were amplified via PCR using the primer combinations ETS-IGSf/18S-ETS (Baldwin and Markos 1998;Acevedo-Rosas et al 2004), ITS1/ITS4 (White et al 1990), and the universal trn primers C and F (Taberlet et al 1991). The PCR was performed using GoTaq green master mix (Promega, Fitchburg, Wisconsin).…”