DOI: 10.6078/d1h13d
title: Phylogeny of species, infraspecific taxa, and forms in Iris subgenus Xiphium (Iridaceae) that have centers of diversity in the Mediterranean Basin biodiversity hotspot

description: Iris subgenus Xiphium is a small group of taxa that mostly occur in the
 Mediterranean Basin, a long-recognized biodiversity hotspot. Phylogenetic
 relationships among these Iris were reconstructed based on sequence data
 from 110 nuclear markers and whole plastomes using Bayesian inference and
 maximum likelihood methods. Best trees based on plastome and combined
 datasets resolved subgenus Xiphium and I. xiphium as not monophyletic
 while nuclear data resolved the subgenus as monophyletic but I. xiphium as
 not monophyletic. Topology tests indicated that the alternative hypothesis
 of a monophyletic subgenus cannot be rejected while a monophyletic I.
 xiphium can be rejected. We hypothesize that the subgenus is monophyletic
 based on these analyses, morphology, and biogeography and that uneven
 patterns of missing data is a likely reason for topological incongruence
 among datasets. A previously suggested informal group within the subgenus
 was supported. Patterns of relationships among species suggest multiple
 exchanges between the African and European continents but also the
 importance of the Strait of Gibraltar as a barrier to genetic exchange.
 Bayesian analyses, biodiversity hotspots, constraint trees, Iberian
 Peninsula, Iridaceae, maximum likelihood analyses, Mediterranean Basin,
 missing data, North Africa, nuclear markers, Strait of Gibraltar, targeted
 enrichment, whole plastomes
description: Genomic DNA was isolated from silica-dried leaf materials using
 protocols modified from the CTAB method of Doyle and Doyle (1987).
 Modifications from this procedure included RNase treatment and an ethanol
 precipitation with ammonium acetate following the initial isopropanol
 precipitation. Prior to library preparation, extracted DNA (0.4–1.2 µg)
 was fragmented to an average length of 500 bp with sonication (Bioruptor
 UDC-200, Diagenode, Denville, NJ, USA). Single index library construction
 and DNA enrichment followed Meyer and Kircher (2010).
 Targeted markers were captured and enriched with a custom
 myBaits-hyb capture kit designed for <em>Iris </em>(Daicel
 Arbor Biosciences, Ann Arbor, MI, USA) following the manufacturers
 recommendations except that kit blockers were replaced with blocking
 oligos from Xgen (Integrated DNA Technologies Inc., Coralville, IA, USA)
 and SeqCap plant capture enhancer (Roche, Burgess Hill, UK). Data for
 targeted markers and un-enriched plastomes was obtained using NGS 100 bp
 paired-end read sequencing run on an illumina 4000 (Illumina Inc., San
 Diego, CA, USA). DNA extraction, library preparation, and sequencing were
 performed at the University of California, Berkeley, California,
 USA. A pipeline was developed and executed on the Savio
 high-performance computing cluster at the University of California,
 Berkeley (available from communicating author). The pipeline used
 Trimomatic (Bolger et al. 2014) to filter and remove index sequences with
 parameters settings of 40 bp minimum length and a 10:20 sliding window.
 Using a minimum read depth of four and quality of 20%, plastomes were
 assembled against the <em>I. gatesi</em> R.C. Foster plastome
 (Wilson 2014) and nuclear markers were assembled against the 635 markers
 developed from exome data that are described above. Data for each nuclear
 marker was examined in Geneious 9.14 (Biomatters, Ltd., New Zealand) to
 select markers &gt; 900 bp in length with &lt; 25% missing data
 for each sample. Final datasets were assembled in in Geneious 9.14
 (Biomatters, Ltd., New Zealand) and included plastomes, 110 selected
 nuclear exome markers, combined nuclear markers (nuclear), combined
 plastome and nuclear markers (combined), and combined nuclear and plastid
 coding regions (all-genes). Sites with &gt; 50% of n’s were excluded
 from datasets with reading frames preserved by excluding bp in multiples
 of three within coding sequences. Plastomes were
 partitioned by coding, intron, and intergenic spacer regions and each
 coding region of plastome and nuclear datasets was partitioned by codon
 position resulting in 379 and 330 partitions, respectively. Partitions were merged and modeled for nuclear, plastome,
 all-gene, and combined datasets using PartitionFinder
 (Lanfear et al. 2012) executed in IQ-TREE v2.1.3 (Nguyen et al. 2015)
 resulting in 12, 12, 18, and
 25 partitions, respectively. RAxML v. 8 (Stamatakis 2014) and
 IQ-TREE v2.1.3 (Nguyen et al. 2015) maximum likelihood (ML; Felsenstein
 1981) and ML bootstrap (Felsenstein 1985) analyses on nuclear, plastome, all-gene, and
 combined datasets were each performed with one thousand
 replicates for each bootstrap. MrBayes Version 3.1.2 (Huelsenbeck and
 Ronquist 2001) was used to perform Bayesian Inference (BI) which was run
 for four million generations, with two runs and six chains that were
 sampled every 1,000 generations with a burn-in rate of 0.25.
description: The dataset uploaded includes the plastome, nuclear, combined,
 and all-gene datasets.  Also included are partition files for each
 dataset, constraint trees that test monophyly of<em> Iris
 xiphium</em>, a monophyletic subgenus with a nested monophyletic
 <em>I. xiphium</em>, and a monophyletic Xiphium group +
 <em>I. juncea</em>. Final trees include those
 based on the combined, all-gene, plastome, and nuclear datasets. Tables
 1-3 provide information on datasets and trees.