Sequence alignments for ITS, rpl16, and trnL-trnF

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.

Methods
The core portion of this study includes 86 samples of D. capillaceum (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 Distichium relationships in a wider context I downloaded Internal Transcribed Spacers 1 and 2 (ITS) sequences from GenBank for eight additional D. capillaceum specimens, from mainland Norway (3 samples), Jan Mayen (1), Svalbard (1), Greenland (2), and Antarctica (1), and two sequences of D. hagenii Ryan ex H. Philib. The beginnings of the downloaded ITS sequences were less complete than the newly generated ones. Two specimens of D. inclinatum were used as outgroup based on its position as sister to the other two species of the genus.

Molecular methods

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 trnLUAA intron plus trnLUAA-trnFGAA spacer (trnL-trnF), 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 trnL-trnF. For the plastid rpl16 G2 intron (rpl16) 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.

Sequence editing and analysis

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.