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.

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): ITS₅ (GGAAGTAAAAGTCGTAACAAGG) and ITS₄ (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 Ast–1m (CGTAAAGGTGTGTGAGTGGTTT) and reverse primer 18S–Alt (TGAGCCATTCGCAGTTTCACAGTC). The psbA-trnH spacer region was amplified and sequenced using the forward and reverse primers psbAF (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 psbA-trnH 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. Carminatia and Brickelliastrum species shared a 26 bp inversion in the psbA-trnH 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.