DOI: 10.5061/dryad.08kprr558
title: Tetramolopium stemmermanniae sp. nov. (Asteraceae), a new species found at Pōhakuloa Training Area, Hawaiʻi Island

description: A new species endemic to Hawaiʻi Island, Tetramolopium
 stemmermanniae, is described and illustrated. Molecular and morphological
 evidence support Tetramolopium stemmermanniae as being
 distinct from Tetramolopium
 arenarium var. arenarium, Tetramolopium consanguineum ssp. leptophyllum, and Tetramolopium humile ssp. humile, which occur at Pōhakuloa Training Area, Hawaiʻi Island. Tetramolopium stemmermanniae shares an upright and multibranched habit as that of Tetramolopium arenarium var. arenarium and Tetramolopium consanguineum ssp. leptophyllum. It differs in ray and disc flower color and number, and in having an open, paniculate inflorescence. We provide a description of the new taxon, include a key to the Tetramolopium species of Hawaiʻi, and a brief description of the habitat where it occurs.
description: Plant specimens were collected from 10
 individuals of the new form of <em>Tetramolopium</em> at
 Pōhakuloa Training Area, (Fig.
 1)  from 2009–2020.
 Five collections for type specimens were made from one locality in 2020
 because of the small number of individuals at the other locations.
 Collected specimens were compared with voucher specimens of
 other <em>Tetramolopium </em>taxa held at the Bishop Museum
 Herbarium (BISH). Voucher specimens of field-collected plants are
 deposited at
 Bishop
 Museum
 Herbarium, United States National Herbarium at the
 Smithsonian Institution (US), Colorado State University Center for
 Environmental Management of Military Lands Herbarium (CMML) at Colorado
 State University, and Pōhakuloa Training Area Herbarium (PTA). All
 material was legally collected with appropriate permits. Plant leaf tissue of 68 individuals from three
 <em>Tetramolopium</em> species found at PTA was collected into
 silica gel in August and September 2014 for a molecular population study.
 The collections include 32 individuals of the new form representing six
 sites, 9 individuals of <em>T. arenarium </em>representing
 three sites, and 27 individuals of <em>T. consanguineum</em>
 representing eight sites. Seven <em>Tetramolopium</em>
 species (total of 14 samples from the Hawaiian Islands including four
 samples used in population study) were obtained for the phylogenetic
 analysis; included two herbarium samples contributed by Dr. Timothy
 Lowrey; <em>Keysseria erici </em>(Forbes) Cabrera for use as
 an outgroup taxon in the phylogenetic work (Appendix 1). DNA was extracted
 from all samples and purified following previously established laboratory
 protocols described in Morden et al. (1996). Each sample was assigned an
 accession number in the Hawaiian Plant DNA Library (HPDL; Morden et al.
 1996; Randell and Morden 1999;
 http://www.botany.hawaii.edu/hawaiian-plant-dna-library; Appendix 1). The
 concentration and quality of the extracted DNA were assessed using a Nano
 Drop Spectrophotometer (ND-1000, ver. 3.6.0, Thermo Fisher Scientific,
 Waltham, Massachusetts). All DNA samples were diluted to 10–15 ng/μl and
 stored at -20°C until used. Sequence-related amplified polymorphism (SRAP)
 markers were utilized to investigate genetic variation within and among
 populations of <em>T. arenarium</em>, <em>T.
 consanguineum</em> and the new form of
 <em>Tetramolopium</em> (Li and Quiros 2001; Robarts and Wolfe
 2014; Zagorcheva et al. 2020). DNA from one to a few individuals of each
 species were surveyed with 100 combinations of 10 forward and 10 reverse
 SRAP primers (Budak et al. 2004). SRAP analyses were conducted using a 15
 μl PCR reaction mixture consisting of: 1xPCR buffer [10 mM Tris-HCl (pH
 9.0 at 25°C), 50 mM KCl and 0.1% Triton X-100, Promega, Madison,
 Wisconsin], 1.5 mM MgCl<sub>2</sub>, 0.25 mg BSA, 0.2 mM
 dNTPs, 0.5 mM of each forward and reverse primers (IDT, Coralville, Iowa),
 1 unit of <em>Taq</em> DNA polymerase (Promega), and
 approximately 10–15 ng of total DNA. All reactions were carried out in an
 MJ Research (GMI, Inc. Ramsey, Minnesota) or Eppendorf thermal cycler
 (Eppendorf AG, Hamburg, Germany) with cycling conditions following that
 established by Li and Quiros (2001). PCR amplified products were mixed
 with loading dye and separated on a 2% agarose gel, stained with ethidium
 bromide (EtBr), and visualized with a UV light source. Negative control
 reactions were run without DNA for all PCR amplifications to ensure
 reaction components were uncontaminated. Each primer combination was
 repeated at least once with selected samples to confirm the
 reproducibility of the genetic markers. Size of amplification products was
 estimated using either a 100 bp ladder (Promega) or a pBS plasmid
 (Stratgene, La Jolla, California) digested with restriction enzymes to
 produce fragments in a size range from 0.448–2.96 Kb. Final gel products
 were viewed using a Gel Doc XR (BIO-RAD, Hercules, California) and
 digitally recorded using Quantity One software (BIO-RAD, ver. 4.5.1). SRAP
 markers were scored either present (1) or absent (0). The scored data were
 entered into a binary matrix and assessed for polymorphic, unique loci.
 From this observed and expected heterozygosity were calculated using
 GenAlEx6.502 (Peakall and Smouse 2006, 2012). Principal coordinates
 analysis (PCO) using Gower general similarity coefficients (Gower 1971)
 were calculated using MVSP 3.1 (Kovach 2007). Pairwise similarities were
 averaged for individuals within and among species. The SRAP binary matrix
 was also used to resolve an unrooted neighbor joining (NJ) tree from a
 mean Euclidean distance matrix with 1,000 bootstrap replicates to assess
 clade support using DARwin v6.0.021 (Perrier et al. 2003). 
 DNA Sequence variation was analyzed for
 four nuclear rDNA regions and five chloroplast DNA regions. The non-coding
 nuclear regions included rDNA ITS (Baldwin 1992), ETS (Baldwin and Markos
 1998), <em>5S-NTS</em> (Cox et al. 1992; Sastri et al. 1992),
 and nuclear NIA (Levin et al. 2009). Chloroplast loci included the
 <em>rpl16</em> intron (Shaw et al. 2005), the
 <em>psbA–trnH</em> spacer (Shaw et al. 2005),
 <em>trnT-L</em> spacer, <em>trnL</em> intron, and
 the <em>trnL-F</em> spacer (Taberlet et al. 1991). Samples
 were PCR amplified in 25 µl volumes with the following components: 25 ng
 of DNA, ca. 0.2 mM each of dATP, dCTP, dGTP, dTTP, 1X <em>Taq
 </em>Polymerase buffer (10 mM Tris-HCl [pH 9.0 at 25°C], 50 mM KCl,
 and 0.1% Triton X-100 [Promega]), 1.5 mM MgCl<sub>2</sub>,
 0.50 mg BSA, 0.2 mM forward and reverse primers, and ca. 1 unit of
 <em>Taq</em> DNA Polymerase (Promega). PCR reactions were
 performed using thermocycling conditions outlined in the original primer
 references. Positive PCR amplification was confirmed and digitally
 recorded as described above. The PCR products were purified using an
 Exo-Sap-It kit (Affymetrix, Santa Clara, California) according to the
 manufacturer’s instructions. PCR products were bidirectionally Sanger
 sequenced using amplification primers on an <strong>Applied
 Biosystems 3730XL DNA Analyzer </strong>(Applied Biosystems,
 Waltham, Massachusetts, USA) at the University of Hawaii’s facility for
 Advanced Studies in Genomics, Proteomics, and Bioinformatics.
 Sanger sequence results were edited and
 contiged using Sequencher® ver. 5.0 sequence analysis software (Gene Codes
 Corporation, Ann Arbor, Michigan) and aligned using MEGA6.06-mac (Tamura
 et al. 2013) using ClustalW with default parameters. Aligned sequences
 were converted by Mesquite ver. 3.03 (Maddison and Maddison 2015) to
 Nexus format for use in phylogenetic analyses. Phylogenetic Bayesian
 analysis was conducted using MrBayes on XSEDE (3.2.6) (Ronquist et al.
 2012) from the CIPRES Portal (Miller et al. 2010). The GRT+I+G model was
 chosen for partitioned and combined data sets as proposed by Abadi et al.
 (2019) using four heated four chains run for 10 million generations with
 sampling every 1000 generations and the first 25% of trees discarded as
 burn-in. Branch support was evaluated by posterior probability. PAUP* v.
 4.0b 10 (Altivec; Swofford 2002) was used for parsimony analysis using a
 branch-and-bound search option and bootstrap resampling (1000
 pseudoreplicates) to calculate branch support (Felsenstein 1985). An
 additional set of phylogenetic analyses were conducted using a
 concatenated alignment containing only perfectly homologous and parsimony
 informative loci to ensure lineage-specific loci were not biasing the
 results. The trimmed matrix was analyzed using the BI procedures described
 above as well as an NJ method using the Tamura-Nei genetic distance model
 (Tamura and Nei 1993) and 1,000 jack knife replicates to assess clade
 support. Model Finder
 (Kalyaanamoorthy et al. 2017) and IQ-Tree (Version 2.2.0) (Nguyen et al.
 2015) (www.igtree.ort);(http://iqtree.cibiv.univie.ac.at) were used to determine the best-fit model and to construct a maximum-likelihood tree for each locus respectively. Ultrafast bootstrap approximation (UFBoot) (Hoang et al. 2018) was used to obtain branch support values. Trees were visualized using FigTree (v. 1.4.4) (http://Tree.bio.ed.ac.ul/software/figtree/).
description: Programs that can access .xls and .tif files.