Sep 13, 2012 ... Platyhelminthes: Proseriata. Pseudomonocelis Monocelis lineate. Pse. 18S. 31. 9
. 2. 4. 3. 5. N/A. 1. 0.010. 0.06. G. Rotifera: Bdelloidea. Rotaria.
Supporting Information Tang et al. 10.1073/pnas.1209160109 SI Materials and Methods DNA Extraction. DNA was extracted from individual animals; these were isolated into sterile tubes, and 40 μl of Chelex (InstaGene Matrix; Bio-Rad, http://www.bio-rad.com) was added to each sample. Sample mixtures were vortexed for a minimum of 5 s each and heated as per the manufacturer’s protocol (56 °C for 20 minutes followed by 96 °C for 20 minutes) but then stored at −20 °C overnight and heated at 56 °C for 20 minutes followed by 96 °C for 20 minutes. The suspended DNA was directly used in subsequent PCR amplification. DNA Amplification. PCRs were performed in a total volume of 25 μl using the illustra Ready-To-Go RT-PCR Beads (GE Healthcare); 2 μl of each primer (10 mM), 2 μl of DNA (10-50 ng) and ddH20 up to 25 μl. COI was amplified using optimized Folmer primers Hcox1 (5’GGT CAA CAA ATC ATA AAG ATA TTG G-3’) and Lcox1 (5’-TAA ACT TCA GGG TGA CCA AAA AAT CA-3’). Cycle conditions comprised initial denaturing at 95 °C for 5 min, followed by 35 cycles of 95 °C for 1 min, 46.4 °C for 1 min and 72 °C for 90 seconds, and a final extension step of 72 °C for 5 min. Nested PCRs were performed where necessary using the primers COIrot (5’-ATT ATT CGT ACT GAR TTA GG-3’) and COICT1R (5’-TAM ACT TCY GGA TGM CCR AAR AAT CA-3’) and the same cycle conditions but for only 25 cycles. 18S rRNA was amplified using a nested approach; the total fragment was amplified using 18SFnew (5’-AGA TTA AGC CAT GCA TGT CT-3’) and 9R (5’-GAT CCT TCC GCA GGT TCA CCT AC-3’). Cycle conditions comprised initial denaturing at 95 °C for 5 min, followed by 38 cycles of 95 °C for 1 min, 55 °C for 1 min and 72 °C for 2 min and a final extension step of 72 °C for 10 min. Three nested PCRs were performed using these primer pairs: 18SFnew (5’-AGA TTA AGC CAT GCA TGT CT-3’) – 4R (5’-GAA TTA CCG CGG CTG CTG G-3’), 18SCT1 (5’TGG AGG GCA AGT CTG GTG CCA GC-3’) – 18Sbi (5’GAG TCT CGT TCG TTA TCG GA-3’), and 18Sa2.0 (5’-ATG GTT GCA AAG CTG AAA C-3’) – 9R (5’-GAT CCT TCC GCA GGT TCA CCT AC-3’). The nested PCR cycle conditions comprised an initial denaturation step of 95 °C for 5 min, followed by 25 cycles of 95 °C for 1 min, 55 °C for 1 min and 72 °C for 1 min, and a final extension step of 72 °C for 5 min. All PCR amplicons were purified and concentrated using Illustra GFX PCR DNA and Gel band purification kit as per manufacturer’s protocol with a final volume ranging from 10 μl to 20 μl depending on the concentration of the amplicon. Cycle 1. Drummond AJ, et al. (2006) Geneious v2.4.2. Available from http://www.geneious.com. 2. Librado P, Rozas J (2009) DnaSP v5: A software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25:1451–1452. 3. Katoh K, Asimenos G, Toh H (2009) Multiple alignment of DNA sequences with MAFFT. Methods in Molecular Biology 537:39–64. 4. Paradis E, Claude J, Strimmer K (2004) APE: Analyses of Phylogenetics and Evolution in R language. Bioinformatics 20:289–290. 5. Chambers J, et al. (2011) R: A language and environment for statistical computing. Available from: http://www.R-project.org.
Tang et al. www.pnas.org/cgi/content/short/1209160109
sequencing reactions were set up using purified amplicons, 1:10 of the PCR primers and the ABI Big Dye Terminator v1.1 kit and run on an ABI 3770 automated sequencer. The sequences were checked and assembled by eye using Geneious Pro 5.4.2 (1). Phylogenetic Analysis. Sequences were collapsed into unique types using DnaSP (2) and aligned using MAFFT (3) with the default settings, followed by checking and manual editing in Geneious Pro 5.4.2 (1). A matrix of pairwise uncorrected distances was generated using the package ape 2.8 (4) in R 2.14.0 (5). Maximum likelihood trees were constructed using PhyML 3.0 (6) using the default settings with the best evolutionary model as determined using jModeltest (7) and a suitable outgroup (Table S1). Gene trees were made ultrametric using penalized likelihood in r8s 1.71 (8), where cross-validation analysis was performed to identify the best level of smoothing.
Nucleotide Divergence Threshold Script for Delimiting Species align
