The nuclear SSU and ITS regions learn more were amplified using the primers EAF3 and ITS055R, and sequenced using additional internal primers, such as 528F, 920F, EBR, 920R, 536R (Marin et al. 2003), a and b from Coleman et al.

(1994). Plastidal psaA was amplified and sequenced using the primers psaA130F and psaA970R (Yoon et al. 2002). The mitochondrial cox1 was amplified using the primer pair GazF2 and GazR2 (Saunders 2005). To amplify and sequence desmarestialean rbcL, we designed the specific primers rbcL77F (5′-TGG GNT AYT GGG ATG CTG A-3′) and rbcL1471R (5′-ATS AGG TGT ATC TGT TGA TGT-3′). PCR amplification was performed in a total volume of 50 μL, containing 0.5 units · μL−1of Taq DNA Polymerase, 1 ×  Qiagen PCR Buffer, 1.5 mM MgCl2, and 200 μM of each dNTP, 1 μM of each primer (except for cox1, for which 300 nM of each primer were used), and 1–10 ng of template DNA. PCR of the SSU-ITS region was carried out with an initial denaturation at 95°C for 3 min, followed by 30 cycles of amplification (denaturation at 95°C for 1 min, annealing at 50°C for 2 min and extension at 68°C for 3 min) with a final extension step at 72°C

for 5 min. PCR of cox1 was performed as follows: initial denaturation at 94°C for 5 min followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 1 min with one final extension at 72°C for 5 min. Amplified DNA was purified with the QIAquick™ RG7422 ic50 PCR Purification Kit (Qiagen) and sent to commercial sequencing at the NERC Biomolecular Analytics Facility in Edinburgh. Electropherogram

outputs for each were edited using the Temsirolimus Chromas v.1.45 (http://www.technelysium.com.au/chromas.html). Assembled sequences of nuclear SSU and ITS were aligned using ClustalW implemented in SeaView v.4.3.3 (Gouy et al. 2012; http://pbil.univ-lyon1.fr/software/seaview.html) then refined by eye with Se-Al™ v2.0a11 (Sequencing Alignment Editor Version 2.0 alpha 11; http://tree.bio.ed.ac.uk/software/seal/). The plastid and mitochondrial protein coding genes were aligned manually with Se-Al™ based on inferred amino acid sequences. Two data sets were used for phylogenetic analyses. First, in the DNA data set (a total of 5,138 bp; c5dna data), we combined all DNA alignments of psaA (675 bp), rbcL (1,257 bp), cox1 (655 bp), SSU (1,720 bp), and ITS (831 bp). Second, in the protein + DNA mixed data set (3,413 characters; c5mix data), translated psaA (225 aa), rbcL (389 aa), and cox1 (218 aa) were combined with SSU and ITS DNA sequences to avoid possible artifacts of phylogenetic calculations such as homoplasy at the third codon position. We used an independent evolution model for each partition (five individual genes) to minimize the effect on phylogeny of heterogeneity among genes.