Origin and genetic variability of populations of the invasive plant Rumex alpinus L. in the Giant (Krkonoše) Mountains

Monk's rhubarb, Rumex alpinus L. (R. alpinus), is a perennial plant native to the mountains of Central and Southern Europe. Currently, the distribution of R. alpinus has been partly affected by its utilization as a vegetable and a medicinal herb. In the mountains of the Czech Republic, it is considered an invasive plant, probably introduced into the Krkonoše Mountains by colonists from the Alps.

This study's main aim was to verify whether R. alpinus was introduced into the Krkonoše Mountains by alpine colonists or whether it was anthropogenically introduced from the Carpathians. Furthermore, the genetic structure of native and introduced populations of R. alpinus was determined.

For the evaluation of genetic structure, 417 samples of R. alpinus were collected from the Alps, Carpathians, Balkan, Pyrenees, and Czech Mountains. In total, 12 simple sequence repeat (SSR) markers were applied.

The results of AMOVA showed a high 60% variation within populations, 27% variation among groups, and 13% among the population within groups. The overall unbiased gene diversity was high (ĥ = 0.55). The higher level of genetic differentiation among populations (FST = 0.35; p < 0.01) indicated restricted gene flow between populations. Compared to native populations, limited genetic variability was observed in the nonnative populations. It was concluded that local adaptation, low gene exchange, and genetic drift affected the genetic diversity of nonnative R. alpinus.

The results support a genetic link between Alpine and Czech genotypes of R. alpinus, while the Carpathians genotypes corresponded to the Balkan genotype.

Methods
Description of localities
Between 2017 and 2020, plant samples of R. alpinus were collected from different locations in Europe (Table 1, Figure 1). According to Professor Klimeš research´s focused on ancestors of the original alpine colonists and individual families, R. alpinus was collected in the exact places (Krkonoše) where the colonists lived and R. alpinus probably occurred. Equally, individual plants of R. alpinus were collected in Tyrol and Styria in Austria. As control samples, R. alpinus samples were collected from other mountain localities in the Alps (Lombardy, and Graubünden) and the Pyrenees. Finally, to determine the true origin of R. alpinus were collected samples representing plant populations in the East and West Carpathians, and then in the mountainous regions of the Balkans. Populations from other mountainous regions of the Czech Republic (Jizera and Eagle Mountains) were collected for comparison and possible exclusion of other origins of R. alpinus.  

In total, 417 individual leaf samples were collected, representing 31 populations with 9–20 individual plants per population. The 329 plant samples were collected in the European Mountains, Alps, West and East Carpathians, Pyrenees, and Balkan Peninsula, which represent areas where R. alpinus is a native species.

In the Czech Mountains, represented by Eagle Mountains, Jizera Mountains, and Krkonoše (Giant) Mountains where R. alpinus is considered a nonnative/invasive plant (Kopecký, 1973; Lokvenc, 1978; Kubát, 1990; Šťastná et al., 2010; Pyšek et al., 2012), overall88 plant samples were collected (Table 1 and Figure 1).

Population sampling and DNA extraction
At each locality, leave samples were collected in plants separated approximately 200 m apart to avoid collecting the same plant because R. alpinus also reproduces vegetatively and to ensure a representative sample of the population. Samples were dried on silica gel and stored in a collection at the Faculty of Environmental Sciences, Czech University of Life Sciences Prague.

Total genomic DNA was extracted from silica gel-dried leaves of R. alpinus in two repetitions using the protocol of Doyle & Doyle (1978) with a minor modification that involved the addition of 10 mg of polyvinylpyrrolidone (PVPP) (Carl Roth, Germany) and 5 µl of 10 mg/µl RNase A (Thermo Scientific, Czech Republic) during the initial phase prior to incubation. The quality and yield of isolated DNA were assessed on a 0.8% agarose gel in 1× TAE buffer. The concentration and quality of DNA were measured using a spectrophotometer UVS-99/UVIS Drop (Avans Biotech, Taiwan). The extracted DNA samples were diluted to the concentration of 20 ng/μl for subsequent analysis and stored at - 20°C.

Microsatellite (SSR) analysis
Based on the test, 12 polymorphic primer pairs were selected from the 15 primer pairs according to Šurinová et al. (2018) and used (Table S1). DNA amplification was performed in 5 μl reactions consisting of 2.5 μl QIAGEN Multiplex PCR Master Mix; 0.125 μl of each M13-labelled forward, reverse, and fluorolabelled (NED™, PET®, 6-FAM™, VIC® – Table S1) M13 primers (10 μM each in initial volume); 20 ng of DNA dissolved in 0.5 μl TE buffer; and 1.625 μl dH2O. The PCR protocol was performed according to Schuelke (2000). PCR was performed using an Applied Biosystem Thermal Cycler (Applied Biosystems, USA) as follows: an initial denaturation step at 95°C for 15 min followed by 25 cycles of denaturation (95°C for 20 s), annealing (59°C for 30 s) and extension (72°C for 20 s), followed by 10 cycles of denaturation (95°C for 30 s), annealing (53°C for 45 s) and extension (72°C for 45 s) and a final extension at 72°C for 15 min. During the first 25 cycles, specific PCR products are produced, and in the following 10 cycles, the fluorescent M13 tag is ligated to the M13 forward primer. The quality of PCR products has been verified on 2% agarose gels. Three multiplexes were built (Table S1).

Fragment analyses were performed using capillary electrophoresis in an ABI PRISM 3500 Genetic Analyser automated sequencer (Applied Biosystems, USA). Electropherograms were analysed and scored using GeneMarker ver. 1.8 (SoftGenetics, USA).