DOI: 10.5061/dryad.zkh18937v
title: A revised classification of Glossopetalon (Crossosomataceae) based on restriction site-associated DNA sequencing

description: Glossopetalon inhabits arid regions in the American west and northern
 Mexico on limestone substrates. The genus comprises four species: G.
 clokeyi ; G. pungens ; G. texense ; and G. spinescens . Three of the
 species are narrow endemics. The fourth, G. spinescens , is a widespread
 species with six recognized varieties. All six varieties are intricately
 branched shrubs that have been difficult to identify due to a lack of
 clearly delineating morphological characters. Characters typically used to
 differentiate the varieties of G. spinescens, such as stem coloration,
 leaf blade size, and presence of stipules, are highly variable within and
 among populations. A custom protocol of double digest restriction-site
 associated DNA sequencing (ddRAD) was used to resolve the phylogeny of
 Glossopetalon and address if population genetic data analyses (such as
 STRUCTURE, SVDquartets, and phylogenetic networks) support the recognition
 of six varieties of G. spinescens . Glossopetalon was fully supported as
 monophyletic and G. pungens was resolved sister to the remaining taxa in
 the genus. The varieties of G. spinescens were resolved as two distinct
 lineages corresponding to their biogeography – one to the northwest
 (lineage 1) and one to southeast (lineage 2). Glossopetalon clokeyi was
 resolved at the base of lineage 1 and G. texense was embedded within
 lineage 2 sister to var. spinescens . Taxonomic changes include the
 recognition of G. texense and G. clokeyi as varieties of G. spinescens and
 description of a unique population from northern Arizona as a new variety
 – G. spinescens var. goodwinii .
description: Genomic DNA was
 isolated from silica dried leaf material and herbarium vouchers using an
 amended Sorbitol protocol (Štorchová et al. 2000) with the exception of
 <i>G. texense</i>, which was extracted using a CTAB protocol
 with the addition of pvp-40 (Doyle and Doyle 1987). Preliminary DNA
 quality was assessed with 1% agarose gel electrophoresis. DNA
 quantifications and purity determinations were conducted via a Nanodrop
 1000 Spectrophotometer (Thermo Scientific, Carlsbad, California) and
 PicoGreen quantification was conducted with a Synergy HTX Multi-Mode
 Microplate Reader (BioTek Instruments, Winooski, Virginia). All samples
 were normalized to 10ng/uL using 10mM Tris-Cl pH 8.0 before library
 preparation. The libraries were
 prepared using an amended protocol of Peterson et al. (2012). Template DNA
 was digested with restriction enzymes <i>Msp</i>I and
 <i>Eco</i>RI (NEB, Inc.). Adapter ligation was simultaneously
 conducted during the same reaction. Preparation of the adapters (Eurofins
 Genomics, USA) were as follows: the P1.1 <i>Eco</i>RI Adapter
 5’-CCTATGTGGAGAGCCAGTAAGCGATGCTATGGT-3’ was annealed to P1.2
 <i>Eco</i>RI Adapter
 5’-[PHO]AATTACCATAGCATCGCTTACTGGCTCTCCACATAGG-3’ using a PTC-100
 Programmable Thermal Cycler heated to 95°C for 5 minutes followed by a
 cool down to 25°C. Afterwards the <i>Eco</i>RI adapter was
 diluted to a concentration of 0.05μM with sterile water. The
 P2.1-<i>Msp</i>I Adapter
 5’-GTCAACGCTCACTACTGCGATTACCCAAGTCAG-3’ was likewise annealed to P2.2
 Adapter 5’-[PHO]GCCCTGACTTGGGTAAGATAGCAC-3,’ but subsequently diluted to a
 concentration of 0.5μM using sterile water. The differences in
 concentration of the adapters were to account for the higher frequency of
 <i>Eco</i>R1 restriction enzyme sites. T4 DNA Ligase (NEB,
 Inc) was employed to ligate the adapters to digested DNA fragments.
 Furthermore, the reagents utilized for this reaction were: BSA (100x),
 <i>Eco</i>R1 10x Buffer, T4 DNA Ligase 10x Buffer with 10μM
 ATP, and sterile water. The reaction underwent 6 cycles of 37°C for 20
 mins followed by 25°C for 20 mins and remained in the thermal cycler
 overnight at 10°C. A 1:1 bead cleanup was
 performed with 25% PEG before the PCR indexing reaction. This
 amplification reaction consisted of Phusion HS II (Thermal Scientific),
 MgCl<sub>2</sub>, custom primers, template DNA, and sterile
 water. Each sample was double indexed using distinctive forward and
 reverse indices. Indexing was performed over 25 cycles of 95°C for 1 min,
 35°C for 15 secs, 55°C for 15 secs, 72°C for 30 secs, and 72°C for 7 mins.
 Now that samples were indexed, all samples were pooled and underwent a 1:1
 bead clean up with 18% PEG. Samples were subsequently quantified using a
 Nanodrop 1000 Spectrophotometer and analyzed on an Advanced Analytical
 Fragment Analyzer (Advanced Analytical Technologies GmbH, Heidelberg,
 Germany). Based on the high presence of the fragments from 200-550 base
 pairs, a size selection at that range was conducted using the Pippen Prep
 (Sage Science, Beverly, Massachusetts) for the ddRAD library. These
 libraries were sequenced on a single lane on a HiSeq 4000 instrument
 (Illumina, San Diego, California) at the University of Oregon’s Genomics
 and Cell Characterization Core Facility using custom primers to produce
 single-end 150 base pair reads. <b><i>Sequence Data Preparation</i></b>— Demultiplexing of raw data was conducted in accordance with akutils RADseq utility protocol using the module fastq-multx from EA-UTILS (Aronesty 2011; Andrews 2018). The demultiplexed data has been uploaded to Dryad. 
description: The demultiplexed data files are named by sample number. For example,
 r1.C1.fq, are the reads for sample tube C1. I uploaded a separate file
 indicating the taxon identified, collection locations, and collection
 date, for each sample tube ID. Each sample tube corresponds to an
 individual plant.