DOI: 10.5061/dryad.69p8cz94x
title: A turn in species conservation for hairpin banksias: Demonstration of oversplitting leads to a better management of diversity

description: We generated SNP genotype data and chloroplast genomic data to test the
 current taxonomy and infer a population-scale evolutionary scenario for
 the Hairpin Banksias (B. collina, B. cunninghamii, B.
 neoanglica, B. spinulosa and B. vincentia) and
 outgroups using a sample-set comprehensive in its representation of
 morphological diversity and a two-and-a-half thousand kilometer
 distribution. Here, we provide an archive of these SNP genotype
 and chloroplast sequence alignment data.
description: Leaf material from silica
 dried material or herbarium specimens lodged at the National Herbarium NSW
 were sampled and are identified by the insitutional database number.
 For
 nDNA analysis, DNA was extracted from each sample using the Plant DNA
 Extraction Protocol for DArT available from the Diversity Arrays
 Technology Pty Ltd (DArT PL) website (Rossetto et al. 2019).
 Samples were sent to DArT PL (Canberra,
 Australia) for the DArT PL genotype by sequencing analysis, according to
 the documented in-house procedure. All specimens were
 co-analysed first to create a total dataset. Following this, two
 additional datasets were created through separate reanalysis of the raw
 data for two separate subclades identified from network analysis of the
 total dataset. For cpDNA analysis,  leaf material was
 sent to Deakin Genomics Research and Discovery Facility at Deakin
 University (Geelong, Australia) for DNA extraction, preparation of genomic
 libraries consisting of paired-end reads (2 x 150 bp) and paired-end
 sequencing using the Illumina NextSeq 500 platform.
 Consistent in silico assembly of chloroplast genomic DNA SNP
 detection across all Illumina Nextseq libraries were performed to generate
 a comparable dataset from paired-end libraries. Library-specific
 chloroplast genome sequences were constructed using de novo assembly of
 the NGS libraries relevant to each species using Organelle Assembler
 (http://metabarcoding.org/org.asm; Coissac et al., 2016). With the
 library-specific chloroplast genomic sequence, a consensus sequence was
 then created from each of the relevant paired-end libraries using CLC Bio
 Genomics Workbench 8.0 (CLC; http://www.clcbio.com). This first involved
 trimming the raw paired-end reads with the Quality Trimming Tool, then
 mapping the trimmed reads to the library-specific chloroplast genomic
 sequence using default settings. The resulting library-specific consensus
 sequence was remapped with the same quality trimmed reads using more
 stringent mapping parameters of 0.8 for the similarity and 0.9 for the
 length fraction to maintain a high-quality library-specific consensus
 sequence with read coverage greater than 5x. Each sequence was annotated
 using the gene prediction tool GeSeq (Tillich et al., 2017). The
 annotations were inspected manually using Geneious by making sure the
 position of the start and stop codons was correct.
   An alignment of relevant library-specific
 consensus sequences was generated using MAUVE (Darling et al., 2004) in
 Geneious Pro 9.1.8. The alignment was checked by removing areas of low
 coverage and where dubious SNPs exist (i.e., SNPs in the first or second
 codons and repetitive regions). REFERENCE:
 Coissac, E., P. M. Hollingsworth, S. Lavergne and P. Taberlet.
 2016. From barcodes to genomes: Extending the concept of DNA barcoding.
 Molecular Ecology 25: 1423–1428. https://doi.org/10.1111/mec.1354 Darling, A. C., B. Mau, F. R. Blattner and N. T. Perna. 2004. Mauve: multiple alignment of conserved genomic sequence with rearrangements. Genome research 14: 1394–1403. https://doi.org/ 10.1101/gr.2289704 Tillich, M., P. Lehwark, T. Pellizzer, E. S. Ulbricht-Jones, A. Fischer, R. Bock, and S. Greiner. 2017. GeSeq–versatile and accurate annotation of organelle genomes. Nucleic Acids Research 45: W6–W11. https://doi.org/10.1093/nar/gkx391
description: All datafiles may be opened using text
 editing software such as wordpad and microsoft word. The resulting reads
 can be read by the open access DArT proprietary analysis pipeline (KDDart)
 described at (https://www.diversityarrays.com/products-and-services/information-technology/kddart/) and available at GitHub (https://github.com/kddart). The chloroplast dataset is a nexus file format and can be opened by sequence editing programs.