Genome-wide RAD sequencing resolves the evolutionary history of serrate leaf Juniperus and reveals discordance with chloroplast phylogeny

We sampled leaf material from 68 Juniperus individuals representing 22/22 serrate juniper species, 2/5 serrate juniper varieties, and 14 additional outgroup taxa. Leaf samples were dried and stored in silica gel, and DNA was extracted with Qiagen DNeasy Plant Mini Kits (Qiagen Inc., Valencia, CA, USA). Reduced representation libraries for Illumina sequencing were constructed using a ddRADseq method. Libraries were size-selected for fragments between 350 and 450 bp in length and two lanes of single-end 100-base sequencing were executed using an Illumina HiSeq 2500 platform. We used the tapioca pipeline (https://github.com/ncgr/tapioca) to identify and discard Illumina primer/adaptor sequences and potential biological sequence contaminants (e.g., PhiX, E. coli) and discarded all reads which aligned to the Juniperus squamata chloroplast genome (GenBank Accession Number MK085509). We demultiplexed reads to individual using a custom Perl script which trims restriction site-associated bases and parses the reads according to unique barcode sequences assigned to each individual. We used ipyRAD (Eaton 2014) to de novo assemble our reads according to the parameter settings provided in the ipyrad param files. Two alignments were generated, one including all sampled taxa, and the other including a subset of only serrate juniper taxa: 1) All 68 individual samples were included in an assembly in which much more missing data was permitted and 2) 22 individual samples representing the 22 serrate juniper species were included in an assembly in which loci with missing data were more rigorously filtered.