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Data for: Alignments of chloroplast DNA and ITS of tribe Adonideae


Tremetsberger, Karin (2022), Data for: Alignments of chloroplast DNA and ITS of tribe Adonideae, Dryad, Dataset,


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.


Three chloroplast markers (atpI-atpH intergenic spacer, matK, rpl16 intron) and the nuclear rDNA internal transcribed spacer (ITS) region were sequenced in all available Adonis and Trollius 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 atpI-atpH with primers atpI and atpH and for rpl16 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 matK 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).

Usage Notes

BioEdit Sequence Alignment Editor.


Austrian Science Fund, Award: I 3002-B25