DOI: 10.5061/dryad.6q573n62f
title: Hybrids between Rubus caesius and Rubus sect. Corylifolii (Rosaceae) and their relation to R. cyclomorphus, R. tiliaster, R. glauciformis, R. slesvicensis and R. firmus

description: Using flow cytometry and microsatellite DNA analysis (MSDA), we studied
 hybrids between Rubus caesius and various other Rubus species, with an
 emphasis on Sweden. We show that hybrids between Rubus caesius and Rubus
 sect. Corylifolii arise easily. They show a large variation in morphology,
 but can normally be recognised by a number of characters. They are
 typically hexaploids, but ~13% of the hybrids are pentaploids and ~8%
 tetraploids. With MSDA, they are harder to identify than hybrids with R.
 idaeus, partly because all Corylifolii species have themselves arisen from
 R. caesius hybrids and therefore share many alleles with R. caesius,
 partly because hybridisation with R. caesius seems to give rise to some
 variation in microsatellite regions. Hybrids with species of R. sect.
 Rubus are much rarer in Sweden and we have only identified one such case.
 We show that R. cyclomorphus and R. tiliaster are not proper apomictic
 species, at least not in Sweden, but rather a collection of genetically
 unrelated hybrids between R. caesius and R. raduloides or R. tiliaster.
 MSDA analysis of R. slesvicensis and R. firmus shows that these species
 are problematic. We identify two distinct taxa, one from Skåne in Sweden,
 which we describe as a new species, R. ruboculus, and another from the
 neighbourhood of Viborg and Schleswig, although the latter is probably not
 identical with R. slesvicensis s.s. All the other samples form a diverse
 group of putative R. caesius hybrids at three different ploidy levels.
 Rubus glauciformis is uniform in Småland, Öland and Blekinge, but becomes
 harder to distinguish from various hybrids in Skåne. We conclude that once
 R. caesius and R. idaeus hybrids are properly identified, along with a
 small number of new apomictic species (often with a local distribution),
 the genus Rubus does not pose any serious taxonomical problems in Sweden.
description: Five hundred seventy-six samples (young leaves from the tip of
 the annual shoot) of <em>R. caesius</em>, several
 <em>Corylifolii </em>species and putative <em>R.
 caesius</em> hybrids were collected, mainly from Sweden, but also
 from Denmark, Norway and Germany. The localities 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 they were used. 
 DNA was extracted with the CTAB method (Doyle and Doyle 1987).
 The concentration of the DNA was determined by a fluorometer and the
 samples were diluted with ddH2O 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). 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 ddH2O, 0.66 µL 10´ reaction buffer (100 mM Tris-HCl, pH 8.3, 500 mM
 KCl and 15 mM MgCl2), 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 altogether. 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).
description: Data can be accessed using Ms Excel.