DOI: 10.5061/dryad.nvx0k6dtg
title: Sequence alignments for ITS, rpl16, and trnL-trnF

description: The two data files consist of sequence alignments in FASTA format. The
 file 'Distichium_alignment_ITS.txt' is the alignment used for
 the analysis resulting in the network in Fig. 1A in the paper and the file
 'Distichium_alignment_3 markers.txt' is the alignment used for
 the analysis resulting in the network in Fig. 1B. The GenBank numbers
 corresponding with the sequence numbers can be found in Appendix 1 in the
 paper.
description: The core portion of this study includes 86 samples of <i>D.
 capillaceum</i> (ITS, rpl16, trnL-trnF). Sixty-nine come from
 Sweden, 16 from mainland Norway, and one from Svalbard. The samples cover
 its phenotypic variation in Scandinavia. To explore
 <i>Distichium</i> relationships in a wider context I
 downloaded Internal Transcribed Spacers 1 and 2 (ITS) sequences from
 GenBank for eight additional <i>D. capillaceum</i> specimens,
 from mainland Norway (3 samples), Jan Mayen (1), Svalbard (1), Greenland
 (2), and Antarctica (1), and two sequences of <i>D.
 hagenii</i> Ryan ex H. Philib. The beginnings of the downloaded ITS
 sequences were less complete than the newly generated ones. Two specimens
 of <i>D. inclinatum</i> were used as outgroup based on its
 position as sister to the other two species of the genus.
 <b>Molecular methods</b> Total DNA
 was extracted using the Mag-Bind® Plant DNA Plus 96 Kit (Omega Bio-tek)
 with the KingFisher Flex and Duo magnetic particle processors. Double
 stranded DNA templates were prepared by polymerase chain reaction (PCR).
 PCR was performed using IllustraTM Hot Start Mix RTG (GE Healthcare) in a
 25 µl reaction volume according to the manufacturer’s
 instructions. In all cases, the specified PCR programs
 were initiated by a denaturation step of 5 min at 95º C and followed by a
 final extension period of 8 min at 72º C. The PCR programs were, for ITS
 and for the plastid <i>trn</i>LUAA intron plus
 <i>trn</i>LUAA-<i>trn</i>FGAA spacer
 (<i>trn</i>L-<i>trn</i>F), 4 cycles of 30 sec at
 95º C, 40 sec at 57º C, and 1 min at 72º C, 4 cycles of 30 sec at 95º C,
 30 sec at 55º C, and 1 min at 72º C, 35 cycles of 30 sec at 95º C, 30 sec
 at 52º C, and 1 min at 72º C. The primers ‘ITSbryoR’ and ‘ITS4bryo’ were
 used to amplify ITS and the primers ‘trnC’ and ‘trnF’ to amplify
 <i>trn</i>L-<i>trn</i>F. For the plastid
 <i>rpl</i>16 G2 intron (<i>rpl</i>16) the PCR
 program was 43 cycles of 30 sec at 95º C, 40 sec at 58º C, and 1 min 15
 sec at 72º C, with the primers ‘F71’ and ‘rpl16-antR2’.
 The amplified PCR products were purified from excess primers and
 nucleotides by adding 1µl of Exonuclease I (20U/µl) and 4µl of FastAP
 Thermosensitive Alkaline Phosphatase (1U/µl) (Thermo Scientific™) and
 incubating at 37° C for 30 min followed by an enzyme inactivation step at
 80° C for 15 min. The purified PCR products, together with the same
 primers used for PCR amplification, were subsequently sent to Macrogen
 Europe B.V (Amsterdam, The Netherlands) for single-stranded sequencing on
 an Applied Biosystems 3730XL sequencer.
 <b>Sequence editing and analysis</b>
 Nucleotide sequence fragments were edited and assembled for each
 DNA region using PhyDE® 0.9971 (http://www.phyde.de/index.html). The assembled sequences were aligned manually in PhyDE®. Regions of partially incomplete data in the beginning and end of the sequences were identified and were excluded from subsequent analyses. Gaps were coded using the simple indel coding of Simmons and Ochoterena (2000) in SeqState (Müller 2005). Gaps provided additional information, and this was included in the analyses. The sequence alignments used in the analyses are uploaded in Dryad.
description: The two data files consist of sequence alignments in FASTA
 format. In the file
 'Distichium_alignment_ITS.txt': positions 1-762 correspond with
 ITS and positions 764-810 with indel codings for ITS.
 In the file 'Distichium_alignment_3 markers.txt':
 positions 1-759 correspond with ITS, positions 761-800 with indel codings
 for ITS; positions 802-1448 with rpl16, positions 1450-1457 with indel
 codings for rpl16; positions 1459-2007 with trnL-trnF, positions 2009-2015
 with indel codings for trnL-trnF.