DOI: 10.5061/dryad.79cnp5j13
title: Resolving relationships in Mentzelia section Bartonia (Loasaceae) in the face of cytonuclear discordance

description: Phylogenetic studies based on two nuclear ribosomal markers created the
 first phylogenetic framework in which to understand evolutionary
 relationships in Mentzelia section Bartonia (Loasaceae), but low molecular
 variation resulted in several large polytomies and an incomplete
 understanding of species relationships. We applied a genome-skimming
 approach to determine whether additional genetic variation generated from
 high-throughput sequencing could resolve relationships in one of the
 largest polytomies in the section. Among 20 species sequenced, a group of
 six species that have pinnatisect leaf morphology were previously
 hypothesized to be monophyletic and we tested whether additional data
 would resolve the group as monophyletic. For the chloroplast genome, reads
 were assembled using both de novo and reference-guided approaches, whereas
 reference-guided approaches were taken for the nuclear ribosomal cistron
 region and a single anonymous nuclear locus. Significant discordance was
 identified among all three gene trees. Exhaustive measures were taken to
 ensure phylogenetic and assembly-based errors were not responsible for the
 observed discordance among gene trees. We attribute much of the
 incongruence to a low phylogenetic signal-to-noise ratio that is likely
 due to the group radiating recently and rapidly and perhaps unique
 evolutionary histories among genomes. Despite incongruence, several
 well-supported relationships emerged across data sets, and all hypothesis
 tests for a monophyletic pinnatisect group among gene trees failed to
 reject monophyly of the group, suggesting the pinnatisect leaf form is a
 synapomorphy for the clades.
description: Genomic DNA was extracted using the CTAB method (Schenk et al.,
 in press, their appendix 1). The DNA was quantified using an Agilent 2100
 Bioanalyzer (Agilent Technologies) and Qubit 4 fluorometric quantitation
 (Invitrogen; Thermo Fisher). Whole genome sequencing was carried out in
 two reactions. The first reaction constructed libraries from total
 sonicated genomic DNA for 14 species with Illumina Nextera XT (Illumina
 Inc., San Diego CA U.S.A.) and was sequenced for 150 bp pair-end reads
 with the Illumina NextSeq platform (see Appendix 1) at the Georgia
 Genomics and Bioinformatics Core. The second reaction sequenced libraries
 for six additional species using NEB NEBNext® Ultra™ II DNA Library Prep
 Kit (New England Biolabs, Ipswich MA, U.S.A.) with enzymatic fragmentation
 and 150 bp pair-end reads were sequenced with the Illumina MiSeq platform
 at the Ohio University Genomics Facility.
 <strong>Assembly and Alignment</strong>—Raw reads
 were filtered using Geneious v.10.2.2 (Biomatters, Ltd., Auckland, New
 Zealand). Reads were removed if they were duplicates, contained greater
 than four ambiguities, or were shorter than 75 bp. <em>Mentzelia
 conspicua</em> was first assembled using the Geneious de novo
 feature because it generated the greats number of reads, and resulting
 contigs that corresponded to the chloroplast and five anonymous loci were
 used in the reference guided assemblies. Regions that had less than 5x
 coverage were removed. Both de novo and
 reference-guided methods were used to assemble the chloroplast genomes
 (see Table 1). The 14 NextSeq <em>Mentzelia</em> chloroplast
 genomes were assembled using GetOrganelle (Jin et al. 2020) with default
 settings and the <em>M. conspicua</em> de novo assembly as a
 seed. Reference-guided assemblies were performed in Geneious using
 <em>M. conspicua</em> as a reference for the six species
 sequenced with MiSeq because read depth was not great enough for de novo
 assembly. All reference-guided assemblies were conducted under medium/fast
 sensitivity for 25 iterations. A consensus sequence was assembled for each
 gene region or genome separately, filtering out sequences that had more
 than four ambiguities. A coverage filter of 65% was applied when
 assembling the consensus species.
description: Geneious and GetOrganelle.