DOI: 10.25338/b8ck8s
title: Data from: Systematics of the plumeweeds: The genus Carminatia (Eupatorieae, Asteraceae)

description: The genus Carminatia, which occurs from the southwestern United States to
 Central America, comprises annuals with cordate to broadly ovate leaves,
 paniculiform capitulescences that are often spike-like or narrowly
 raceme-like and with mostly fasciculate capitula, and a pappus of plumose
 bristles. We used nuclear ribosomal DNA (ITS and ETS) and the plastid
 psbA-trnH spacer DNA sequences to estimate phylogenetic relationships
 among the species of Carminatia, which have not been fully investigated
 using molecular data to date. All of our analyses supported the monophyly
 of the genus and most of them supported Brickelliastrum as sister to
 Carminatia. The analyses also supported the recognition of a new species,
 C. balsana, which is confined to the Balsas Basin in Mexico. The new
 species is more similar to C. recondita but has shorter capitula,
 corollas, and cypselae, and the capitula are oriented to more than one
 side of the capitulescence. An updated taxonomic revision of the genus,
 including morphological descriptions, a key to the species, distribution
 maps, and images are provided.
description: Field work was conducted in Mexico to study the morphology of the
 species. Pictures were taken and fresh leaves were collected and
 immediately dried in silica gel. Voucher specimens were collected and
 deposited in the National Herbarium of Mexico. Duplicates and leaf samples
 were imported to UC Davis with valid permits. Duplicates were deposited at
 the UC Davis herbarium. In the laboratory, leaf samples were ground and
 5–20 mg of dry leaf tissue was used for DNA extractions using the DNeasy
 Plant Kits (Qiagen, Valencia, California), following the manufacturer’s
 protocols with minor modifications. The concentration of DNA samples was
 measured with a spectrophotometer. Graphs of UV absorbance and the ratio
 of absorbance values (A260/A280) were also checked to determine the purity
 of the extracted DNA.  Taq PCR Core Kits (Qiagen,
 Valencia, California) in conjunction with Taq DNA polymerase from Thermo
 Fisher Scientific were used to amplify target genomic regions. The ITS
 region was amplified and sequenced using the forward and reverse primers
 from White et al. (1990) as described in Baldwin (1992):
 ITS<sub>5</sub> (GGAAGTAAAAGTCGTAACAAGG) and
 ITS<sub>4</sub> (TCCTCCGCTTATTGATATGC). To amplify and
 sequence the ETS region, we used the primers employed by Rivera et al.
 (2016), which were modified versions of those used by Baldwin and Markos
 (1998): forward primer <em>Ast–1m</em>
 (CGTAAAGGTGTGTGAGTGGTTT) and reverse primer <em>18S–Alt</em>
 (TGAGCCATTCGCAGTTTCACAGTC). The <em>psbA-trnH </em>spacer
 region was amplified and sequenced using the forward and reverse primers
 <em>psbAF</em> (GTTATGCATGAACGTAATGCTC) and trnHR
 (CGCGCATGGTGGATTCACAAATC), respectively, from Sang et al. (1997). Each
 50μl PCR reaction contained 41μl of double-distilled water, 5μl of 10×
 CoralLoad PCR Buffer, 1μl of dNTPs mixed solution (in an equimolar ratio
 of 10 mM), 0.25μl each of forward and reverse primer solution, 0.5μl of
 Taq DNA polymerase solution, and 2μl of the template DNA. In each
 amplification experiment, positive and negative (water) controls were
 included. The thermocycler was programmed mostly following Rivera et al.
 (2016), with an initial denaturation of 4 minutes at 95°C, followed by 34
 cycles, each consisting of an initial denaturation of 1 minute at 95°C, an
 annealing phase of 45 seconds at 50°C, and an extension period of 1 minute
 at 72°C, with a final extension period of 8 minutes at 72°. PCR products
 were checked and separated by agarose gel electrophoresis. The amplified
 DNA was extracted from the gel slices using the QIAquick Gel Extraction
 kit (Qiagen, Valencia, California), following the manufacturer’s protocols
 with a few minor modifications, such as using 0.25μl of dd-water to elute
 the DNA instead of 50μl of Buffer TE. The amplified DNA
 samples were submitted for sequencing to the College of Biological
 Sciences UCDNA Sequencing Facility at UC Davis where an ABI Prism 3730
 Capillary Electrophoresis Genetic Analyzer and associated software (ABI
 Prism 3730 Data Collection Software v. 3.0, ABI Prism DNA Sequencing
 Analysis Software v. 5.2) were used for sequencing and data
 analyses. Sequencher 5.4.6 (Gene Codes Corporation) was used to assemble
 contigs and edit the sequences. Taxon and marker identities for all
 sequences generated were corroborated by performing BLAST searches. The
 limits of the ITS, ETS, and <em>psbA-trnH</em> sequences were
 determined using sequences downloaded from GenBank as guidance.
 Nucleotides outside these regions were trimmed in MEGA 7.0 (Kumar et al.
 2016). In addition, about 20 of the first nucleotides of the 5’ end of the
 ETS sequences were trimmed to keep only readable sequences. The sequences
 were aligned using MUSCLE (Edgar 2004) as implemented in MEGA 7.0 using
 the default settings (-400 gap open penalty and 0 gap extension penalty).
 The alignments were checked and slightly edited manually.
 <em>Carminatia</em> and <em>Brickelliastrum</em>
 species shared a 26 bp inversion in the <em>psbA-trnH</em>
 spacer (Schilling et al. 2013). We used MEGA 7.0 to re-invert this region,
 and the inversion was then scored as a single character for phylogenetic
 analyses following Schilling et al. (2013). The alignments were exported
 as Fasta files and Mesquite 3.6 (Maddison and Maddison 2018) was used to
 export the alignments in different file formats.