We studied genetic diversity in 54 populations of nine species of the genus Chondrilla (C. acantholepis, C. ambigua, C. brevirostris, C. canescens, C. graminea, C. juncea, C. laticoronata, C. latifolia, and C. pauciflora) from SE European Russia and the neighboring territories of NW Kazakhstan. Analyses of the trnT–trnF region of plastid DNA and the internal transcribed spacer of ribosomal DNA (ITS1–5.8S–ITS2) using Statistical Parsimony, Maximum Likelihood and Neighbor Net methods revealed two major evolutionary lineages roughly corresponding to two subgenera traditionally recognized within Chondrilla in the region. Within the first evolutionary lineage (subgenus Brachyrhynchus) sexual diploid C. ambigua and its putatively hybrid apomictic derivatives C. brevirostris, C. laticoronata and C. pauciflora can be recognized. Their identity is confirmed by analyses of ISSR markers. The second evolutionary lineage (subgenus Chondrilla) is comprised comprises by C. juncea, C. acantholepis, C. canescens, C. graminea, and C. latifolia in European Russia. Analyses of morphological variability and the genealogy of plastid and nuclear markers genealogy favor their treatment as a single species C. juncea.

Samples were collected in 2014 to 2016 from 54 natural populations of 9 Chondrilla species (C. acantholepis, C. ambigua, C. brevirostris, C. canescens, C. graminea, C. juncea, C. laticoronata, C. latifolia and C. pauciflora Ledeb.) in the Astrakhanskaya, Volgogradskaya, Penzenskaya, Samarskaya, Saratovskaya and Ulyanovskaya Provinces of Russia, Krasnodarsky Krai, the Republic of Kalmykia, the Crimea, and Western Kazakhstan. Three plants were sampled from each population as herbarium specimens (kept at the herbarium SARBG) and silica gel dried floret ligules. From sympatric populations of C. juncea, C. graminea and C. latifolia with numerous morphologically intermediate plants, three plants were randomly chosen and designated as C. juncea / C. graminea or C. juncea / C. latifolia, respectively.

DNA was extracted from silica gel dried ligules (connate petals of ligulate florets) using commercially available NucleoSpin® Plant II DNA extraction kit (MACHEREY-NAGEL GmbH, Düren, Germany) according to the manufacturer’s protocol.

The plastid trnT−trnL and trnL−trnF intergenic spacers (IGS) and the trnL intron were amplified using standard primers a and b, c and d, e and f (Taberlet et al., 1991). The nuclear internal transcribed spacer regions of ribosomal DNA (ITS1−5.8S−ITS2) were amplified using primers ITS 1, ITS2, ITS3 and ITS 4 (White et al. 1990).

Polymerase chain reactions (PCR) were carried out in 20 µl reaction volumes in a Mastercycler gradient thermocycler (Eppendorf, Germany). The reaction mixture contained 4 µl of ready-to-load PCR mix 5X MasTaq^DDMix-2025 (200 µM of each dNTP, 2 mM MgCl₂, 1.5 U “hot-start” SmarTaq DNA-polymeraze, reaction buffer, stabilizer and two inert dyes; Dialat Ltd., Moscow, Russia), 15 µl of deionized water, 3.4 pmol of each primer and 1 µl of template DNA of unknown final concentration. The PCR conditions were as follows: preliminary denaturation for 5 minutes at 95°C; 35 cycles of 30 seconds at 95°C, 30 seconds at 55°C and 2 minutes at 72°C, followed by final elongation for 10 minutes at 72°C. PCR products were purified with a DNA Gel Extraction Miniprep Kit (BioSilica Ltd., Novosibirsk, Russia) according to the manufacturer’s instructions.
Direct sequencing was performed in both directions on the ABI PRISM 3130 XL genetic analyzer (Applied Biosystems, Foster City, CA, USA), using ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit v. 3.1 for cycle sequencing reactions at the facilities of Sintol Ltd. (Moscow, Russian Federation).