DOI: 10.5061/dryad.crjdfn37h
title: Data for: Alignments of chloroplast DNA and ITS of tribe Adonideae

description: The Euro-Siberian steppe flora consists of warm- and cold-adapted species,
 which may have responded differently to Pleistocene glacials and
 interglacials. Genotyping-by-sequencing individuals from across the
 distribution range of the pheasant’s eye (Adonis vernalis), we aimed to
 gain insight into steppe florogenesis based on the species’ evolutionary
 history. Although the primary area of origin of the species group
 comprising A. vernalis, A. villosa and A. volgensis is in Asia, our
 results indicate that A. vernalis itself is not of Asian origin but
 evolved in southern (incl. Southeastern) Europe during the Pleistocene,
 with Spanish populations being clearly genetically distinct from the
 Southeastern European populations. We inferred that A. vernalis migrated
 eastwards from the sub-Mediterranean forest-steppes of Southeastern Europe
 into the continental forest-steppe zone. Eastern European
 populations had the highest private allelic richness, indicating
 long-term large population sizes in this region. As a thermophilic
 species, A. vernalis seems unlikely to have survived in the cold deserts
 of the Last Glacial Maximum in Western Siberia, so this region was
 likely (re)colonized postglacially. Overall, our results
 reinforce the importance of identifying the area of origin and the
 corresponding ecological requirements of steppe plants in order to
 understand the composition of today’s steppe flora.
description: Three chloroplast markers
 (<em>atp</em>I-<em>atp</em>H intergenic spacer,
 <em>mat</em>K, <em>rpl</em>16 intron) and the
 nuclear rDNA internal transcribed spacer (ITS) region were sequenced in
 all available <em>Adonis</em> and
 <em>Trollius</em> species. For each PCR reaction, Red HS Taq
 Master Mix (10 μl; Biozym Scientific, Hessisch Oldendorf, Germany),
 forward and reverse primer (0.8 μl, 10 μM each), DNA extract (1 μl), and
 water (7.4 μl) was used. The PCR program for
 <em>atp</em>I-<em>atp</em>H with primers atpI and
 atpH and for <em>rpl</em>16 with primers rpL16F71 and
 rpL16R1516 started at 95°C for 1.5 minutes, followed by 35 cycles of 95°C
 for 15 seconds, 53°C for one minute, and 72°C
 for one minute, followed by 72°C for seven minutes and hold at 10°C.
 Amplification conditions for <em>mat</em>K with primers
 matK-413f-4 and matK-1227r-4 were 95°C for 1.5 minutes, 35 cycles of 95°C
 for 30 seconds, 44°C for one minute and 72° for one minute, followed by
 72°C for ten minutes and hold at 10°C. The program for ITS with primers
 ITS5 and ITS4 was 94°C for two minutes, 35 cycles of 94°C for 30 seconds,
 50°C for 30 seconds, and 72°C for two minutes, followed by 72°C for five
 minutes and 10°C hold. Amplification products were purified by enzymatic
 treatment with Exonuclease I (1 μl) and FastAP Thermosensitive Alkaline
 Phosphatase (2 μl; Thermo Fisher Scientific, Waltham, Massachusetts, USA)
 at 37°C for 15 minutes and at 85°C for 15 minutes thereafter. Both DNA
 strands were sequenced. Cycle sequencing reactions consisted of BigDye
 Terminator v3.1 Ready Reaction Mix (1 μl; Thermo Fisher Scientific), 5×
 sequencing buffer (1.5 μl), primer (1 μl, 3.5 μM; forward or reverse) and
 PCR product (6.5 μl). Cycling conditions after an initial denaturation at
 96°C for 1 minute were 35 cycles of 96°C for 10 seconds, 50°C for
 5 seconds and 60°C for 4 minutes. Excess dye-labelled nucleotides from the
 sequence reaction were removed using Sephadex G-50 Fine (GE Healthcare,
 General Electric, Boston, Massachusetts, USA) columns prepared in
 MultiScreen-HV (Merck Millipore, Merck, Darmstadt, Germany) filter plates
 followed by a run on a 3500 Genetic Analyzer (Applied Biosystems, Thermo
 Fisher Scientific). The forward and reverse sequences were assembled,
 edited, and aligned in Geneious 6.1.8 (Biomatters, Auckland, New
 Zealand).
description: BioEdit Sequence Alignment Editor.