Computational protocol: Integrating environmental, molecular, and morphological data to unravel an ice-age radiation of arctic-alpine Campanula in western North America

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Protocol publication

[…] We generated a multi-locus nuclear DNA (nDNA) dataset of anonymous noncoding markers for estimating the evolutionary history of Campanula of interest. Our approach to designing the anonymous locus primer sets (ALPS) for plants followed DeChaine et al. (), using Invitrogen's (Carlsbad, CA) TOPO® Shotgun Subcloning Kit on genomic DNA extracted from leaf tissue of C. lasiocarpa (accession WWB 22731) using Qiagen's (Valencia, CA) DNeasy® Plant Extraction kit. To select the appropriate loci for use as ALPS, we ran blastn and blastx searches against the nucleotide databases of the National Center for Biotechnology Information. We discarded DNA fragments matching organelle or protein coding sequences and removed sequences that had long open reading frames. Fifteen sequences were selected for designing ALPS, and primer pairs were optimized in Primer3-web 0.4.0 (Rozen and Skaletsky ). Ultimately, 10 ALPS reliably produced clean PCR products and were chosen for sequencing (Table; see for taxon/locus matrix). We included the internal transcribed spacer (ITS) region as an eleventh locus following rigorous quality control (Wendling et al. ).Anonymous locus primer sets (ALPS) were PCR amplified (following the protocol of DeChaine et al. ), cloned using TOPO® TA cloning kits, and three clones per sample (when possible) were sequenced in both directions by the University of Washington's High-Throughput Genomics Service Unit. In preparation for the analyses, all genetic datasets were edited on SEQUENCHER v4.8 (GeneCodes Corp., Ann Arbor, MI), aligned using CLUSTALX 2.0 (Larkin et al. ), and alignments were manually edited in MACCLADE 4.08a (Maddison and Maddison ). We coded indels (most of which separated the Cordilleran Campanula from C. rotundifolia) as simple binary characters (Simmons and Ochoterena ) and excluded all sites of ambiguous alignment from further analyses. We tested each locus for recombination with the RDP (Martin and Rybick ), GENECONV (Padidam et al. ), MAXCHI, and CHIMAERA (Posada and Crandall ) algorithms implemented in the software package RDP3 (Martin and Rybick ). These approaches were chosen because the first two detection methods identify recombinants as regions where the percent similarity is higher than others, while the latter two use the proportion of variable and nonvariable sites to identify breakpoints. Recombinant individuals and/or segments that were detected by >1 detection method were excluded from the study: Approximately 5% of all sequences were discarded, and ALPS 14, 19, and 24 were trimmed by 212, 367, and 38 bp, respectively.Using DNASP v. 5 (Librado and Rozas ), we calculated haplotype diversity (h), the number of segregating sites (θS), and nucleotide diversity (π), and performed neutrality tests for each ALP by comparing Tajima's D (Tajima ) and Fu and Li's D* and F* (Fu and Li ) statistics to 1000 coalescent simulations of a large, neutrally evolving population. […]

Pipeline specifications

Software tools BLASTN, BLASTX, Primer3, Sequencher, Clustal W, MacClade, DnaSP
Databases AS-ALPS
Application qPCR