DOI: 10.5061/dryad.1rn8pk0sc
title: DNA matrix combined (nuclear and indels coded) datasets for Hyptidinae (Lamiaceae)

description: Hyptidinae, ca. 400 species, is an important component of Neotropical
 vegetation formations. Members of the subtribe possess flowers arranged in
 variously modified bracteolate cymes and nutlets with an expanded areole
 and all share a unique explosive mechanism of pollen release, except for
 Asterohyptis. In a recent phylogenetic study, the group had its generic
 delimitations rearranged with the recognition of 19 genera in the
 subtribe. Although the previous phylogenetic analysis covered almost all
 the higher taxa in the subtribe, it lacked a broader sampling at the
 species level. Here we present a new expanded phylogenetic analysis for
 the subtribe comprising 153 accessions of Hyptidinae sequenced for the
 nuclear nrITS, nrETS, and waxy regions and the plastid markers trnL-F,
 trnS-G, trnD-T, and matK. Our results widely support the previous
 phylogenetic results with some changes in the support and relationship
 between genera. It also uncovers the need for a new combination of Eriope
 machrisae in Hypenia and the phylogenetic position of Hyptis sect.
 Rhytidea, which was demonstrated to be part of Mesosphaerum. The generic
 delimitation in Hyptidinae is discussed, and we recommend that further
 studies with more markers are needed to confirm the monophyly of
 Hyptidendron and Mesosphaerum, as well as to support taxonomic changes on
 the infrageneric delimitation within Hyptis s. s.
description: <b><i>DNA Amplification and
 Sequencing</i></b><b>—</b>Total genomic DNA was
 extracted from fresh or silica-gel dried leaf material and sodium
 chloride/CTAB preserved material (Chase and Hills 1991) or from herbarium
 specimens. The fragments, when extracted from herbarium, were removed from
 HUEFS and K collections. A rescaled version of the Doyle and Doyle (1987)
 protocol was used for genomic DNA extractions. We chose the nuclear
 ribosomal internal transcribed spacer region (nrITS) including both ITS1
 and ITS2 and intervening 5.8S and nuclear ribosomal external transcribed
 spacer region nrETS, and the nuclear low copy waxy granule-bound starch
 synthase I (GBSS). The plastid regions used were
 <i>3’trnK</i>-<i>matK</i> (including partial
 <i>trnK</i> intron and <i>matK</i> coding region),
 <i>trnL-F</i> region<i> </i>(including the
 <i>trnL</i> intron and the
 <i>trnL</i>-<i>trnF</i> intergenic spacer),
 <i>trnD-T</i> and <i>trnS-G</i> region (including
 <i>trnS-psbZ</i> intergenic spacer, partial sequence;
 <i>psbZ</i> gene, complete cds; <i>psbZ-trnG</i>
 intergenic spacer, complete sequence; and tRNA-Gly
 (<i>trnG</i>) gene). The nrITS region was amplified using the
 primers 17SE and 26SE of Sun et al. (1994). The 3′ 18S-IGS primer of
 Baldwin and Markos (1998) and the 5′ primer ETS-B (Beardsley and Olmstead,
 2002) were used to amplify a portion of the 3′ end of the nrETS. The
 nuclear region waxy were sequenced between GBSSI bd9f-bd11r, using the
 same methodology designed by Drew and Sytsma (2013). Therefore, for the
 GBSSI gene, we used a nested PCR approach to amplify the region between
 (and including parts of) exons 7–11. The initial PCR reaction was used the
 primers bd7f and bd12r Drew and Sytsma (2013). The PCR product from the
 above amplification was then used (after 1: 20 dilution) as a template for
 the additional PCR reaction, using the primers bd9f and bd11r. The product
 of this amplification was then sequenced with the same primers used in the
 nested PCR. The partial <i>matK/trnK</i> locus was amplified
 using 390F and 1326R (Cuénoud et al. 2002). The whole
 <i>trnL-trnF</i> region was amplified using primers “c” and
 “f”, with the use of internal primers “d” and “e” for some problematic
 samples, of Taberlet et al. (1991). For amplifying the
 <i>trnS-G</i> spacer we used the set of primers described in
 Shaw et al. (2007). The spacer <i>trnD-T</i> was amplified for
 most taxa using the primers of Demesure et al. (1995),
 <i>trnD</i> GUC and <i>trnT</i> GGU. For some
 samples which could not be amplified using these primers, we used the
 internal primer <i>trnY</i> GUA (Shaw et al. 2005). The
 plastid loci were sequenced using the same set of primers used for the
 amplification, whereas the nuclear nrITS was sequenced using internal
 primers ITS92 (Desfeux and Lejeune 1996) and ITS4 (White et al. 1990) with
 the same PCR program. All PCR amplifications were
 performed in a final volume of 10 µL containing: 5 µL of TopTap master mix
 kit (Qiagen, Valencia, California), 2.25 pMol primers each, 5–10 ηg of
 genomic DNA, and ultrapure H<sub>2</sub>O (enough to complete
 the volume to 10 µL). For the ITS amplification, we added 2% DMSO
 (dimethyl sulfoxide) and 1M of betaine. All regions were amplified using
 initial denaturation at 94°C (5 min), 28 (ITS) or 32 (plastid loci) cycles
 of denaturation at 94°C (1 min), annealing 52°C (ITS) or 54°C (plastid
 loci) (1 min), elongation at 72°C (2 min), and a final elongation of 4
 min. Amplified products were purified using precipitation with 11%
 solution of polyethylene glycol (PEG) 8000 and ethanol cleaning.
 Sequencing reactions in both directions were performed using BigDye
 Terminator 3.1 (Applied Biosystems, Carlsbad, California) chemistry and
 analyzed on an ABI3130XL sequencer (Applied Biosystems/Life Technologies
 Corporation, Carlsbad, California) following the manufacturer’s protocol
 at Universidade Estadual de Feira de Santana, Bahia, Brazil. Some PCR
 products were sequenced at the Interdisciplinary Center for Biotechnology
 Research at the University of Florida, Gainesville.
 <b><i>Sequence Assembly and
 Alignment—</i></b>The sequences were edited using Geneious
 6.1.8 (https://www.geneious.com) and aligned using the program Clustal2X
 (Larkin et al. 2007); alignments were checked by eye. Gaps were coded
 according to the "simple coding" criterion of Simmons and
 Ochoterena (2000) using the software Seqstate v.1.4.1 (Müller
 2005).
description: The matrix includes 230 taxa and 2557 characters. DNA:1-2191
 (nrETS = 1-635, nrITS = 636-2191); and standard (indels coded)
 2192-2557.