10.5061/DRYAD.NVX0K6DTG
Hedenäs, Lars
0000-0003-1763-1696
Swedish Museum of Natural History
Sequence alignments for ITS, rpl16, and trnL-trnF
Dryad
dataset
2021
FOS: Biological sciences
Internal institutional funds*
2021-11-06T00:00:00Z
2021-11-06T00:00:00Z
en
262068 bytes
2
CC0 1.0 Universal (CC0 1.0) Public Domain Dedication
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.
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.
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.