DOI: 10.5061/dryad.w0vt4b8pc
title: Molecular systematics of the tribe Physarieae (Brassicaceae) based on the nuclear ITS, Luminidependens, and chloroplast ndhF: Sequence alignments, trees, and supplemental figures

description: Physarieae is a small tribe of herbaceous annual and woody perennial
 mustards that are mostly endemic to North America, with its members
 including a large amount of variation in floral, fruit and chromosomal
 variation. Building on a previous study of Physarieae based on morphology
 and ndhF plastid DNA, we reconstructed the evolutionary history of the
 tribe using new sequence data from two nuclear markers, and compared the
 new topologies against previously published cpDNA-based phylogenetic
 hypotheses. The novel analyses included ca. 420 new sequences of ITS and
 LUMINIDEPENDENS (LD) markers for 39 and 47 species, respectively, with
 sampling accounting for all seven genera of Physarieae, including
 nomenclatural type species, and 11 outgroup taxa. Maximum parsimony,
 maximum likelihood, and Bayesian analyses showed that these additional
 markers were largely consistent with the previous ndhF data that supported
 the monophyly of Physarieae and resolved two major clades within the
 tribe, i.e. DDNLS (Dithyrea, Dimorphocarpa, Nerisyrenia, Lyrocarpa, and
 Synthlipsis) and PP (Paysonia and Physaria). New analyses also increased
 internal resolution for some closely related species and lineages within
 both clades. The monophyly of Dithyrea and the sister relationship of
 Paysonia to Physaria was consistent in all trees, with the sister
 relationship of Nerisyrenia to Lyrocarpa supported by ndhF and ITS, and
 the positions of Dimorphocarpa and Synthlipsis shifted within the DDNLS
 Clade depending on the employed data set. Finally, using the strong, new
 phylogenetic framework of combined cpDNA + nDNA data, we discussed
 standing hypotheses of trichome evolution in the tribe suggested by ndhF.
description: <b><i>Taxon
 Sampling</i></b>—The study
 included a total of 47 putatively diploid species of Physarieae, each
 represented by two different accessions, selected to capture a broad range
 of morphological, geographic and cytogenetic variation. Total sampling
 consisted of 110 accessions for the tribe and 11 outgroup species chosen
 based on family-level analyses and belonging to tribes Alysseae,
 Boechereae, Camelineae, Crucihimalayeae, Descurainieae, Halimolobeae, and
 Lepidieae (see Appendix 1 for details; Bailey et al. 2006; Beilstein et
 al. 2006, 2008; Warwick et al. 2008; Couvreur et al. 2010; Warwick et al.
 2010; Huang et al. 2015; Nicolov et al. 2019). Sequences of seven outgroup
 taxa were downloaded from GenBank including <i>Arabidopsis thaliana
 </i>(L.) Heynh.<i>, Capsella bursa-pastoris </i>(L.)
 Medik.<i>, Descurainia sophia </i>(L.) Webb ex
 Prantl<i>, Exhalimolobos berlandieri </i>(E.Fourn.) Al-Shehbaz
 & C.D.Bailey, <i>Neslia paniculata </i>(L.) Desv.,
 <i>Pennellia longifolia</i> (Benth.) Rollins and
 <i>Transberingia bursifolia </i>(DC.) Al-Shehbaz &
 O'Kane. <b><i>DNA
 Isolation</i></b>—DNA was
 extracted from silica-gel dried leaves following the standard CTAB
 protocol (Doyle and Doyle 1987) and purified in cesium-chloride –
 ethidium-bromide gradients by ultracentrifugation or from herbarium
 material following Stefanović et al. (2002). <b><i>DNA
 Amplification</i></b>—The ITS
 nuclear data set comprised the 5.8S gene flanked by the internal
 transcribed spacers ITS1 and ITS2, and was amplified with the primers ITS4
 (White et al. 1990) and ITS18S (Howarth et al. 2003). The
 <i>LD</i> sequences extend from intron 4 to exon 7 and were
 amplified with LD-D1F and LD-XC4R primers (Slotte et al. 2006).
 <i>ITS</i> and
 <i>LD</i> PCR reactions were performed in 25 µL total volume
 including 5 µL of 5× reaction buffer, 2 µL of 2.5 mM
 MgCl<sub>2</sub>, 2 µL (ITS) or 3 µL (LD) of 2.5 mM dNTPs, 3
 µL of each primer 10 µM, 0.5 µL of Taq polymerase (5 units/µL) (Promega,
 Madison, Wisconsin), and 0.5 µL of DMSO. Cycling reactions for ITS and
 <i>LD</i> were 4 minutes at 95°C (<i>ITS</i>) or 2
 minutes at 94°C (<i>LD</i>), 34 (ITS) or 35
 (<i>LD</i>) cycles of 30 seconds at 94°C, 1 minute at 55°C
 (ITS) or 1.5 minutes at 57°C (<i>LD</i>), 1.5 (ITS) or 2
 (<i>LD</i>) minutes at 72°C, and finally 7 (ITS) or 9
 (<i>LD</i>) minutes at 72°C. The PCR products of both nDNA
 regions were purified with a QIAquick gel extraction kit (Qiagen Inc.,
 Redwood City, California). <b><i>Cloning and
 Sequencing</i></b>—In a pilot
 study, 20 accessions representing ten species of Physarieae and exhibiting
 a wide range of morphological variation were investigated to identify
 locus copy number, orthologous regions and variation in nuclear loci.
 After this initial assessment a minimum of two clones was screened for all
 103 accessions representing the remaining species. PCR fragments from at
 least two separate PCR reactions were cloned to eliminate labeling and
 pipetting errors and sequenced following Sambrook et al. (1989) and
 Mathews et al. (2000). Sequence reactions used the fluorescent ABI Prism
 Big Dye 3.1 (Applied Biosystems, Foster City, California) to label the DNA
 for analysis in an ABI 3100 (Applied Biosystems) sequencer at the
 University of Missouri-St. Louis or at the PennState University Nucleic
 Acid Facility (State College, Pennsylvania). Universal primers T7 and SP6
 were used for sequencing both ITS and
 <i>LD</i>.<b><i>Sequence Editing and
 Alignment</i></b>—SeqMan version
 4 (DNASTAR, Madison, Wisconsin) and GENEIOUS version 4.0.2 (Drummond et
 al. 2009) were used for editing and contig assembly. Only double-stranded
 sequences with at least 85% overlap and Phred scores above 20 as estimated
 by PhredPhrap (Ewing and Green 1998; Ewing et al. 1998) and 4peaks version
 1.7 (Griekspoor and Groothuis 2005) were considered good quality sequences
 and accepted for analysis. Base pairs with scores below 20 were eliminated
 from the analysis except when they matched the complementary strand with
 Phred scores above 20. Sequence identities were confirmed
 by comparing sequences of available Brassicaceae accessions deposited in
 GenBank. Nucleotide sequences of both ITS and <i>LD</i>
 sequences were initially aligned in MUSCLE (Robert 2004), followed by
 manual alignment using MacClade version 4 (Maddison and Maddison 2005).
 Alignment of <i>LD</i> exons was guided by identification of
 open reading frames, exon positions and stop codons in MacClade and
 protein alignment using <i>Arabidopsis</i>
 <i>thaliana</i> as a reference species in MUSCLE and
 GENEIOUS. <b><i>Phylogenetic
 Analyses</i></b>—The
 g<sub>1</sub> statistics were obtained from each data set to
 distinguish phylogenetic signal from random noise (Hillis and Huelsenbeck
 1992), and the test was performed with 10,000 replicates as implemented in
 PAUP version 4.04b. Data sets were examined individually and combined.
 Pairwise comparisons of data sets included only those taxa in common for
 the combined partitions. Conflict among data sets was evaluated before
 merging them using a partition homogeneity test or incongruence length
 difference (ILD) test (Farris et al. 1994). The ILD tests were conducted
 in PAUP version 4.04b with all invariant characters removed (Cunningham
 1997), simple addition sequence, TBR branch swapping, and MAXTREES set to
 500 random partitions. For each of the pairwise data partitions, 500
 random partitions were analyzed as recommended by Johnson and Soltis
 (1998).             Phylogenies were constructed using
 maximum parsimony (MP) with all characters equally weighted, maximum
 likelihood (ML), and Bayesian analysis (BI), and indels coded as missing
 data. Analyses were run on the Cyberinfrastructure for Phylogenetic
 Research (CIPRES) cluster computer housed at the San Diego Supercomputer
 Center, University of California (http://www.phylo.org)
 and on the Beowulf computer cluster at the University of Missouri-St.
 Louis. Parsimony ratchet searches (Nixon 1999) were conducted using
 PAUPMacRat (Sikes and Lewis 2001) implemented in PAUP version 4.04b 10 for
 UNIX (Swofford 2002). Searches consisted of 20 independent replicates of
 200 iterations, each with 15% of the characters re-weighted per iteration,
 and the strict consensus of the resulting trees was generated in PAUP.
 Bootstrap analysis was used to evaluate the support of specific branches
 and clades (Felsenstein 1985). Bootstrap values were calculated with 1000
 full heuristic bootstrap replicates, one random sequence addition,
 tree-bisection-reconnection (TBR) branch swapping, and MULTREES = yes
 options.             For each individual gene data set
 ML analyses used the best-fitting evolutionary model selected by Modeltest
 version 3.6 (Posada and Crandall 1998) according to the Akaike Information
 Criteria (AIC), TrN + G for ITS and HKY85 + I + G for
 <i>LD</i>. Likelihood replicates (1000) were run on CIPRES and
 using RAxML bootstrapping (Stamatakis et al. 2008). The ML analyses of
 combined data sets were estimated as a single partition under the GTR + G
 model of evolution in RAxML, and as partitioned data sets following the
 method proposed by Meerow et al. (2009) using models of evolution and
 Treefinder scripts generated in KAKUSAN4 version 2 (Tanabe 2007). Scripts
 were implemented in Treefinder to run ML analyses (Jobb 2008). The latter
 strategy allowed parameters to be optimized independently among different
 genes included in a combined data set.             In
 the BI analyses, MrModeltest 2.2 (Nylander 2004) selected models GTR + G
 for ITS and HKY + I + G for <i>LD</i>, and those algorithms
 were implemented for single gene and combined data sets in MrBayes version
 3.1 (Huelsenbeck and Ronquist 2001). The BI analyses were conducted with
 two independent runs of four chains for 5,000,000 generations per run
 (sampling every 1000 generations). Convergence across runs was evaluated
 by plotting-log likelihood against the number of generations. The data
 reached convergence within the first 100,000 generations, but the first
 200,000 generations of each run were conservatively discarded as the burn
 in. Bayesian posterior probabilities were obtained from the majority-rule
 consensus trees generated in PAUP.             Initial
 phylogenetic analyses included sequences of all clones (ITS: 224 clones
 and <i>LD</i>: 219 clones). To minimize computational effort
 and reduce redundancy, clones were pruned from the original data sets
 according to the following four rules: (1) a single clone was chosen at
 random from a well resolved and monophyletic species clade to respresent a
 species if sequences differ only by no more than three base pairs (bp);
 (2) the clone resolved in the shortest branch length of a well-supported
 and resolved species clade was chosen to represent the species; (3) if
 accessions representing a species were found paraphyletic one clone for
 each accession was chosen to represent the species; (4) if accession
 representing a species were found unresolved in a polytomy with at least
 one other species, one clone per species was chosen at random to represent
 each species within that clade. When clones from the same accession
 failed to form a monophyletic group, a careful check was made to identify
 potential errors due to contamination or labeling mistakes, and for the
 presence of conflicting phylogenetic signal using Splits graphs in the
 software Splits Tree version 4.3 (Huson and Bryant 2006). Sequences with
 conflicting phylogenetic signals were included in the initial phylogenetic
 analyses to investigate their effects on the tree topologies.
 <b><i>Likelihood
 Topology Tests</i></b>—Topological
 congruence and evolutionary hypotheses were evaluated using the
 Shimodaira-Hasegawa test (S-H test, Shimodaira and Hasegawa 1999) in PAUP
 version 4.04b. To obtain full-taxon compatibility among the tree
 topologies, we reduced the nuclear and chloroplast data sets to 48 taxa
 and re-ran the phylogenetic analyses. The S-H test included comparison
 between the optimal trees (unconstrained) from the maximum likelihood
 analyses of ITS, <i>LD</i>, combined ITS +
 <i>LD</i>, and chloroplast <i>ndhF</i>. We also
 created less-resolved constraint trees that included only well-supported
 nodes either with &gt; 50% or &gt; 70% bootstrap support. Poorly
 supported nodes were defined as having less than 50% bootstrap support and
 were considered ambiguous polytomies.            
 Additional S-H tests were run using the combined data sets to test support
 for particular relationships and estimates of character evolution
 suggested by previous studies. Putative sister relationships were tested
 for <i>Dithyrea</i> + <i>Dimorphocarpa</i> as
 suggested by Rollins (1979) and supported by earlier
 <i>ndhF</i> data (Fuentes-Soriano and Al-Shehbaz 2013),
 <i>Synthlipsis</i> with all other members of the DDNLS Clade
 suggested by the <i>ndhF</i> data (Fuentes-Soriano and
 Al-Shehbaz 2013), and the alliance of <i>Synthlipsis,</i>
 <i>Nerisyrenia</i>, and <i>Lyrocarpa</i> as
 suggested by Bacon (1978). Hypotheses of trichome evolution in the tribe
 suggested by <i>ndhF</i> data (Fuentes-Soriano 2010) were also
 tested. Constraint trees were created to force the tested group to be
 monophyletic, while the rest of the taxa were placed at the base of a
 completely unresolved tree using MacClade 4.08 (Maddison and Maddison,
 2005). Constraint trees were compared to the ML unconstrained trees. If
 likelihood values for the topologies being compared were not significantly
 different, each topology was considered an equally likely phylogenetic
 hypothesis. The S-H test was run with full optimization and 1000 bootstrap
 replicates.