Low genetic variation of invasive Fallopia spp. in their northernmost European distribution range

Abstract Knowledge about the reproduction strategies of invasive species is fundamental for effective control. The invasive Fallopia taxa (Japanese knotweed s.l.) reproduce mainly clonally in Europe, and preventing spread of vegetative fragments is the most important control measure. However, high levels of genetic variation within the hybrid F. × bohemica indicate that hybridization and seed dispersal could be important. In Norway in northern Europe, it is assumed that these taxa do not reproduce sexually due to low temperatures in the autumn when the plants are flowering. The main objective of this study was to examine the genetic variation of invasive Fallopia taxa in selected areas in Norway in order to evaluate whether the taxa may reproduce by seeds in their most northerly distribution range in Europe. Fallopia stands from different localities in Norway were analyzed with respect to prevalence of taxa, and genetic variation within and between taxa was studied using amplified fragment length polymorphism (AFLP). Taxonomic identification based on morphology corresponded with identification based on simple sequence repeats (SSR) and DNA ploidy levels (8× F. japonica, 6× F. × bohemica and 4× F. sachalinensis). No genetic variation within F. japonica was detected. All F. × bohemica samples belonged to a single AFLP genotype, but one sample had a different SSR genotype. Two SSR genotypes of F. sachalinensis were also detected. Extremely low genetic variation within the invasive Fallopia taxa indicates that these taxa do not reproduce sexually in the region, suggesting that control efforts can be focused on preventing clonal spread. Climate warming may increase sexual reproduction of invasive Fallopia taxa in northern regions. The hermaphrodite F. × bohemica is a potential pollen source for the male‐sterile parental species. Targeted eradication of the hybrid can therefore reduce the risk of increased sexual reproduction under future warmer climate.


| INTRODUCTION
The invasive Fallopia taxa, belonging to the Polygonaceae family, are among the most problematic invasive alien plants in Europe and North America (recently summarized by Robinson, Inger, Crowley, and Gaston (2017)). These taxa develop extensive rhizome systems and form large monocultures that displace native species, reduce biodiversity, and degrade habitats (Aguilera, Alpert, Dukes, & Harrington, 2010) (Figure 1). Spread of rhizome and stem fragments by humans and water streams are the main means of spread in Europe (Bailey, Bímová, & Mandák, 2009). However, hybridization between the taxa is common, and reproduction by seeds may be more important than previously assumed (Bzdęga et al., 2016;Hollingsworth & Bailey, 2000b;Krebs et al., 2010;Mandák, Bímová, Pyšek, Štěpánek, & Plačková, 2005;Tiébré, Bizoux, Hardy, Bailey, & Mahy, 2007). Sexual reproduction increases genetic diversity and produces new genotypes that may be better adapted to the environment and more tolerant to chemical or biological control treatments. Furthermore, seed dispersal can also increase spread and consequently complicate management.
Three Fallopia taxa are invasive in Europe: Fallopia japonica (Houtt.) Ronse de Craene var. japonica (here referred to as F. japonica), F. sachalinensis (F. Schmidt ex Maxim) Ronse de Craene, and their hybrid, F. × bohemica (Chrtek and Chrtková) J. P. Bailey. All three taxa are rhizomatous perennials that are polyploid, gynodioecious, and generally self-incompatible. Fallopia japonica and F. sachalinensis are native to East Asia and were introduced to Europe as ornamental and fodder plants in the mid-1800s (Bailey, 1994). The two species escaped cultivation and have hybridized in the introduced range to produce F. × bohemica (Bailey, 1994). In Norway, Schübeler reported in 1883 that F. japonica had been cultivated as an ornamental plant for several years and had been distributed north to Bodø (67°N) (Schübeler, 1883). The earliest herbarium records of F. sachalinensis and F. × bohemica in Norway are dated 1935 and 1964, respectively (Fremstad & Elven, 1997). Fallopia × bohemica is sometimes misidentified as either of the parental species, and its distribution is likely underestimated (Bailey & Wisskirchen, 2004). The hybrid can be more invasive (Parepa, Fischer, Krebs, & Bossdorf, 2014) and difficult to control (Bímová, Mandák, & Pyšek, 2001) than the parental species.
Fallopia sachalinensis and F. × bohemica are both present as malesterile and hermaphrodite individuals in Europe. Fallopia × bohemica can be partially to fully fertile. The two taxa can reproduce sexually within taxa, as well as by hybridization and backcrossing, and genetic variation is found within both taxa. They have various ploidy levels, of which tetraploid F. sachalinensis and hexaploid F. × bohemica are the most common Tiébré, Bizoux, et al., 2007).
Particularly high levels of genetic and cytological variation within F. × bohemica indicate that hybridization and sexual reproduction play important roles in the development and spread of this species (Bzdęga et al., 2016;Gaskin et al., 2014;Mandák et al., 2005).
Although the seeds of invasive Fallopia taxa germinate and grow into viable plants when grown under optimal conditions in experiments, observations of seedlings are not very common in nature in Europe (Funkenberg, Roderus, & Buhk, 2012;Mandák et al., 2005;Tiébré, Vanderhoeven, et al., 2007). This indicates that seedling establishment in the field requires certain environmental conditions. Seedling establishment is negatively affected by dry conditions, late spring frost, and competition for light (Forman & Kesseli, 2003; F I G U R E 1 The invasive Fallopia taxa form large monocultures that displace native species, reduce biodiversity, and degrade habitats. The picture shows a stand of Fallopia × bohemica, photographed in July in Ås, Norway Funkenberg et al., 2012). In addition, seeds are susceptible to fungal attacks and predation by birds (Bailey & Spencer, 2003;Engler, Abt, & Buhk, 2011).
Information about the reproduction modes of the invasive Fallopia taxa in more northern regions of Europe is lacking. In Norway in northern Europe, it is assumed that late flowering (end of August to October) combined with a cold autumn climate and early frosts limits the production of mature seeds (Fremstad & Elven, 1997). However, seed production has been observed in these taxa in Norway (personal observation), and one record of F. × conollyana from Stavanger in southwest Norway (Fremstad & Elven, 1997) indicates that occasional establishment from seed may occur.
The objective of this study was to examine the genetic diversity of F. japonica, F. sachalinensis, and F. × bohemica in selected areas of Norway, in order to evaluate whether the taxa reproduce sexually in their northern distribution range in Europe. This is the first genetic diversity study of these taxa in Norway and the hitherto northernmost study on this subject.

| Sample collection
Sampling localities were situated in lowland coast and fjord regions of Norway. The southernmost locality had a latitude of 58.0°N, and the northernmost locality had a latitude of 69.6°N (Figure 2).
According to the Köppen-Geiger climate classification system (Peel, Finlayson, & McMahon, 2007), the Norwegian sampling area has a temperate to cold climate, with no dry seasons, and with warm to cold summers. Seven meters is the length the rhizome system can extend from a parent plant (Child & Wade, 2000).
The number of samples collected from each locality varied (Table 1), which reflects variation in sampling intensity and not necessarily the abundance of invasive Fallopia plants.
For morphological identification, some of the largest leaves from the mid to lower part of the main stems were used. For molecular and flow cytometric analysis, young leaves without signs of fungal infections or herbivory were collected from a single shoot in each stand.
Leaves were transported fresh in plastic bags to the laboratory. Each sample was divided into two parts; one part was flash frozen using liquid nitrogen and stored at −20°C, the other part was dried in silica gel and stored at room temperature. For 35 samples collected in west Norway, the complete samples were silica-dried directly after collection due to longer transportation time from the field to the laboratory.
A silica-dried sample of octoploid F. japonica from the UK was provided by John Bailey, University of Leicester. This sample was used for comparison with Norwegian samples in all analyses.

| Morphological identification
Each sample was assigned to a species based on the diagnostic characters described by Bailey and Wisskirchen (2004). If samples contained flowers, the flowers were examined under a stereomicroscope (Leica MZ125), and sex expression was determined according to Bailey (1994).

| Flow cytometric analysis
Flow cytometry allows for rapid determination of the relative DNA content by measuring the intensity of fluorescent light emitted from isolated nuclei that have been stained with a DNA-specific fluorochrome (Galbraith et al., 1983). Silica-dried leaves from 124 samples, including the octoploid control sample from the UK, were subjected to flow cytometric analysis for determination of DNA ploidy level. The remaining 22 samples were not analyzed due to lack of silica-dried leaves. The analysis was performed by Plant Cytometry Services, the Netherlands (http://plantcytometry.nl). The samples were chopped together with the internal standard, Pachysandra terminalis, and collected in a DNA buffer with DAPI as the fluorochrome. After sieving, the nuclei were analyzed with CyFlow Space (Partec GmbH, Germany) with a UV High power LED (365).

| DNA extraction
DNA was extracted from frozen or silica-dried leaves using Qiagen's Plant DNeasy Mini Kit (Qiagen Inc., Hilden, Germany) according to the manufacturer's protocol.

| Simple sequence repeats analysis
To aid taxonomic identification, all 145 samples were analyzed with the simple sequence repeats (SSR) markers KW2 and KW6, developed by Grimsby et al. (2007). KW6 amplifies a fragment in F. sachalinensis and F. × bohemica, but not in F. japonica (Grimsby et al., 2007). KW2 amplifies a fragment in all three taxa (Grimsby et al., 2007) and was included in the analysis as a control to verify that the DNA quality was appropriate for PCR amplification and that the samples were from Fallopia. PCR mixture and cycling conditions were essentially as described by Grimsby et al. (2007). Amplification result was visualized by running 10 μl PCR product on agarose (1.5%) gel electrophoresis at 90-110 V for approximately 2-3 hr. Approximate fragment size was determined by comparing with a 100 bp DNA ladder (New England Biolabs).

| Amplified fragment length polymorphism analysis
The genetic variation within and between the Fallopia taxa was examined by use of AFLP analysis (Vos et al., 1995). This robust, reproducible method allows for simultaneous analysis of many polymorphic loci with relatively few primers (Costa, Pereira, Garrido, Tavares-de-Sousa, & Espinosa, 2016;Jones et al., 1997). AFLP analysis has previously been used successfully to detect genetic diversity within the invasive Fallopia taxa (Bzdęga et al., 2012(Bzdęga et al., , 2016Gaskin et al., 2014). AFLP analysis was performed as previously described (Elameen et al., 2008),

| Genetic analysis
The genetic similarity (GS) was estimated using the Dice coefficient,  (Dice, 1945). The genetic similarity among the individual plants, based on the presence or absence of amplified fragments, was also calculated by Jaccard's coefficient (Jaccard, 1908).
The similarity matrices obtained were analyzed using the unweighted pair group method with arithmetic mean (UPGMA), as suggested by Sneath and Sokal (1973). The similarity matrices were also used to perform principal coordinate analyses (PCO) to classify and detect potential structure within and among the taxa. The analyses and the dendrogram constructions were performed using NTSYS-pc software (Rohlf, 2000).
The genetic structure of the Fallopia samples was also investigated using the model-based Bayesian clustering approach of genetic mixture analysis (Structure 2.3.4 software) (Falush, Stephens, & Pritchard, 2003). These analyses assume that the clusters (K) are at Hardy-Weinberg equilibrium and in linkage equilibrium, but the approach has proven to be robust also for deviations from these assumptions (Falush et al., 2003). Simulations were performed with a data set from K = 1 to K = 6. Six independent runs were conducted to assess the consistency of the results across runs, and all runs were based on 500,000 iterations after a burn-in period of 100,000 iterations.
The method developed by Evanno, Regnaut, and Goudet (2005) was used to identify the number of genetically homogeneous clusters (K).
For the parameter set in our study, we selected admixture as ancestry model, and the allele frequencies were assumed to be correlated, as it is more reasonable to assume common ancestry of such closely related species. We used a burn-in period length of 100,000 runs and 500,000 MCMC runs to compute the probability of the data for estimating K. Among the six independent runs, the one with the highest Ln Pr (X|K) value (log probability) was chosen and represented as bar plots. Bar plots of likelihoods and ΔKs were made with STRUCTURE HARVESTER (Earl & vonHoldt, 2012). while the third sample did not contain flowers. Only hermaphrodite flowers were observed on F. × bohemica, but one of these had flowers that fit the description of female-sterile flowers (Bailey, 1994). These flowers had long filaments with well-filled anthers and poor stigma development. In addition, the inflorescence panicles of the same F. × bohemica were less compact and with longer branches than the panicles of other F. × bohemica. Furthermore, the leaves of this plant were more narrowly oblong.

| DNA ploidy levels
The ratios between the fluorescence peaks of samples and the internal standard were used to deduce different ploidy levels.

| Genetic diversity
Amplified fragment length polymorphism analyses of the three taxa Structure analysis showed the maximum likelihood distribution L (K) at the real number of three groups (K = 3). This value was obtained using the value of ad hoc quantity (∆K) rather than maximum likelihood value L (K) as described by Evanno et al. (2005). Structure analysis clustered the Fallopia taxa into three main clusters ( Figure 5) as for the UPGMA and PCO groupings (data not shown).
The analysis of pairwise distances showed that the greatest genetic distance was as expected between F. japonica and F. sachalinensis (0.71), while the shortest genetic distance was between F. japonica and F. × bohemica (0.36) ( Table 2).

| DISCUSSION
Fallopia japonica was the most frequent of the three Fallopia taxa sampled in Norway, followed by F. × bohemica and F. sachalinensis. F. japonica is generally the most common of the three taxa in Europe, but the relative frequencies of the taxa vary between regions (Krebs et al., may reflect a lower frequency of this species compared to the other two taxa in Norway. Also in other parts of Europe, F. sachalinensis is less frequent and seems to be less invasive, than F. japonica and F. × bohemica (Krebs et al., 2010;Mandák et al., 2005;Tiébré, Bizoux, et al., 2007). This may be due to the lower potential of F. sachalinensis to regenerate from rhizome fragments .
The taxonomic identification based on morphology corresponded with the results of the molecular analyses (AFLP and SSR) as well as with the results of the flow cytometric analysis. AFLP analysis clearly clustered the samples corresponding to morphological identification.
The diagnostic SSR marker, KW6, was detected in all samples identified as F. × bohemica or F. sachalinensis, but not in samples identified as F. japonica. The ploidy levels found for the taxa in the present study are the most common ploidy levels for these taxa in Europe (Krebs et al., 2010;Mandák et al., 2003). This indicates that morphological characters can be used to distinguish between the taxa in Norway.
However, morphological variations within the taxa could confuse identification, especially when experience with the different taxa is lacking. Distinguishing F. japonica and F. × bohemica by morphological means can be even more challenging in regions where hybridization and backcrossing occur (Tiébré, Bizoux, et al., 2007).
This is the first genetic diversity study of the invasive Fallopia taxa in Norway. Extremely low levels of genetic variation were uncovered within the taxa in the present study. In terms of F. japonica, the present results corresponded with most previous studies in Europe (Hollingsworth & Bailey, 2000a;Krebs et al., 2010;Mandák et al., 2005;Tiébré, Bizoux, et al., 2007) (Krebs et al., 2010;Pashley et al., 2007).
While other European studies have detected high levels of genetic variation within F. × bohemica by use of methods such as AFLP (Bzdęga et al., 2012(Bzdęga et al., , 2016, RAPD (Hollingsworth & Bailey, 2000b;Krebs et al., 2010;Tiébré, Bizoux, et al., 2007) and isoenzyme variation (Mandák et al., 2005), only one AFLP genotype was detected in the present study, indicating no genetic variation within F. × bohemica in Norway. However, as for F. sachalinensis, the SSR analysis indicated that one F. × bohemica sample was a different genotype than the rest of the F. × bohemica samples. Differences in the leaf and flower morphology between the one stand and the other F. × bohemica stands supported this result. Unique genotypes could result from rare, sexual reproduction in Norway or may have been introduced to Norway as seeds or vegetative propagules. It is not uncommon to find more genetic diversity when using SSR analysis compared to AFLP (Medini, Hamza, Rebai, & Baum, 2005;Powell et al., 1996). SSR regions are highly variable due to the high frequency of slipped strand mispairing mutation during DNA replication and repair, which is the main mechanism creating SSRs (Tautz, 1989).
Moreover, low genetic diversity may limit sexual reproduction of these taxa due to general self-incompatibility (Bailey, 1994).
Climate warming may affect the reproductive strategies of invasive Fallopia taxa. Climate scenario projections for Norway indicate that temperature will increase for all seasons and that the length of the growing season will increase with 1-3 months during this century (Official Norwegian Reports 2010). These changes can increase the opportunities for invasive Fallopia taxa to reproduce sexually in northern regions. Extension of the frost-free period in the autumn may have increased germination of F. × bohemica in the Quebec region in Canada (Groeneveld et al., 2014) and may also have contributed to increased seed production in F. japonica in the UK (Bailey et al., 2009). Hermaphrodite F. × bohemica is a potential pollen source for the male-sterile parental species and could play an important role for whether new, invasive genotypes can be produced under future warmer climate in Norway. Targeted eradication of F. × bohemica can therefore be a means to reduce this potential future effect of climate warming.

ACKNOWLEDGMENTS
We thank the Norwegian Public Roads Administration and the Regional