DOI: 10.5061/dryad.3bk3j9ktf
title: Data from: Rubus wahlbergii and related species in Sweden

description: We studied Rubus wahlbergii and related species in R. sect. Corylifolii,
 primarily in Sweden, using DNA microsatellites. The results show that the
 material can be divided into three species: R. wahlbergii s.s. which
 occurs along the Swedish coast from Bohuslän to Uppland, R. nordicus which
 occurs on the western coast all the way south to north-western Skåne, and
 a previously unrecognized taxon which we here describe as R.
 wahlbergioides. The latter has scattered localities in Halland, Skåne,
 Blekinge, Öland and Småland, but is common around Kivik in Skåne and
 Kalmar in Småland. It is characterised by leaves that are without felt
 underneath, terminal leaflets with a somewhat more triangular shape and
 coarser serration than those of R. wahlbergii and also has more glands on
 the stem and the inflorescence. In the northern part of the distribution,
 R. nordicus is easily distinguished morphologically from R. wahlbergii,
 e.g. by a strong tendency to form 7-foliolate leaves, but in Skåne and
 Halland, the two species are harder to distinguish. Still, genetically R.
 nordicus is more similar to R. sordirosanthus than to R. wahlbergii.
description: Samples (young leaves from the tip of the annual shoot) of
 <em>R. wahlbergii </em>and several related
 <em>Rubus</em>species were collected, mainly from Sweden, but
 also from Denmark, Norway and Germany. The localities<em>
 </em>are specified in Table S1 in the Supplementary Material.
 Vouchers are deposited in LD. The samples were stored in a freezer at
 –80˚C until DNA was extracted. DNA was extracted with
 the 2× CTAB method (Doyle and Doyle, 1987). The concentration of the DNA
 was determined by a fluorometer and the samples were diluted with
 ddH<sub>2</sub>O to ~14 ng/µL. Four pairs of microsatellite
 primers, described in Table S2, were used to analyse variable
 microsatellite loci. The three loci Ru105b, Ru117b and Ru275a were
 described in <em>R. idaeus</em> (Graham et al., 2004), whereas
 the locus mRaCIRRIV2A8 was described in <em>R.
 alceifolius</em> Poir. (Ansellem et al., 2001). They will here be
 referred to as 105b, 117b, 275a and 2A8, respectively. They have been
 successfully used also for other <em>Rubus</em> species
 (Stafne et al., 2005; Zhou, 2014; Hedrén et al., 2020; Ryde et al., 2021;
 Lewin et al., 2022). One primer in each pair was tagged with a fluorescing
 molecule (Cy5-) to enable the identification of the polymerase chain
 reaction (PCR) products in an automated sequencer (ALF Express
 II). The PCR amplification consisted of 35 cycles of
 denaturation at 94 ℃ for 30 s, annealing at varying temperatures (see
 Table S2) for 30 s and polymerisation at 72 ℃ for 1 min (10 min in the
 last cycle). Each PCR was performed in a solution consisting of (in a
 total volume of ca. 6.4 µL): 4.4 µL ddH<sub>2</sub>O, 0.66 µL
 10´ reaction buffer (100 mM Tris-HCl, pH 8.3, 500 mM KCl and 15 mM
 MgCl<sub>2</sub>), 0.56 µL dNTPs (2.5 mM for each nucleotide),
 0.25 µL Cy5-tagged primer (10 µg/mL), 0.1 µL untagged complementary primer
 (25 µmol/mL), 0.03 µL Taq polymerase (5 u/µL; Applied Biosystems) and 0.6
 µL template DNA (14 ng/µL). The PCR products were mixed
 with formamide and appropriate size markers and were then heated to 94 ℃
 for 25 min before they were separated by electrophoresis in a
 polyacrylamide gel. The DNA fragments were detected and analysed with the
 help of an ALF Express II analysis system. Samples that resulted in poor
 PCR reactions were purified with a Qiagen purification kit, after which
 the PCR and electrophoresis were redone. Samples that still resulted in
 poor amplification (unspecific amplification, dominance of short fragments
 etc.) were excluded. The relative height of the
 amplified fragments on the resulting electropherogram was recorded on a
 five-graded scale for each sample to obtain an estimate of band intensity.
 Differences in relative band intensity between plants with overlapping
 banding patterns were matched with information on ploidy levels and
 interpreted as possibly due to variation in allele copy numbers.
 Similarly, differences in relative band intensity between plants of the
 same ploidy expressing different numbers of bands were also interpreted as
 due to variation in allele copy numbers. Several samples were re-amplified
 by PCR and rerun to verify the patterns of relative band intensity. Such
 analyses gave rise to closely similar differences in band intensity as the
 original runs, with only minor variation in peak height (typically, at
 most by a single unit along the five-graded scale used by us).
 DNA-ploidy levels were estimated by flow cytometry for 22
 samples. This analysis was performed by Plant Cytometry Services
 (http://www.plantcytometry.nl). Fresh leaf material was homogenised and
 mixed with an ice-cold buffer with DAPI (Arumuganathan and Earle, 1991).
 The amount of  DNA per cell nucleus was determined in a CyFlow ML with
 iceberg lettuce (<em>Lactuca sativa</em>) as an internal
 standard. The estimates were calibrated by using
 <em>Rubus</em>samples with known chromosome numbers. The
 DNA-ploidy levels are presented as fractional chromosome numbers from this
 calibration line between the ratio in fluorescence between the samples of
 known chromosome numbers and the internal standard. The
 search for putative genetic relationships based on the microsatellite
 results was facilitated by the use of computer programs taking into
 consideration both the length of the various microsatellite alleles and
 the relative intensity of the PCR products (Lewin et al., 2022).
 Principal-coordinate (PCO) analyses were performed with the NTSYSpc v.2.1q
 software (Rohlf, 1994), using a pair-wise comparison of Jaccard
 coefficients (Jaccard, 1908) based on the occurrence of alleles for all
 four loci.
description: # Rubus wahlbergii and related species in Sweden
 [https://doi.org/10.5061/dryad.3bk3j9ktf](https://doi.org/10.5061/dryad.3bk3j9ktf) Mingyue Zhou, Melanie S. Montes, Tore Mattsson, Tomas Burén, Mikael Hedrén & Ulf Ryde (2024) Nordic J. Bot., in press; DOI: 10.1111/njb.04373 **Explanation of the columns:** A Suggested species according to the investigation (text) B DNA bank number - Unique extraction number (integer) C Collection code - Unique identification code (text) D Genus - Always "Rubus" (text) E Collected as - species name at collection (text) F Habitat (text) G Province, mainly in Sweden (text) H Parish (text) I Locality (text) J x coordinate (N/S) in Swedish RT90 (integer) K y coordinate (E/W) in Swedish RT90 (integer) L Date I - date of first observation or collection (year-month-day) M Voucher no - unique for each date (integer) - only if a herbarium voucher is collected, otherwise empty N Date II - date of collection, if different from Data I (year-month-day) O Collector (text) P Chromosome number Other columns are the results from the microsatellite DNA analysis Columns Q -BW represent probe 105b Columns BY-ET represent probe 117b Columns EV-IF represent probe 2A8 Columns IH-MC represent probe 275a The first row shows the band length in base pairs The other rows show the results for each sample as a number between 1 and 5, representing the relative intensity (1 weak, 5 strongest) Empty cells mean that no band of that length was observed.