Hybridogenic Activity of Invasive Species of Asteraceae

We studied taxa from genus Bidens , Solidago , and Erigeron , sect. Conyza (Asteraceae). By analyzing the nucleotide sequences of the internal transcribed spacer (ITS1)-ITS2 site, the hybrid origin of the Bidens (cid:1) decipiens , previously attributed to the North American alien species B. connata , was confirmed. The analysis of trnL-trnF sequences showed that the native B. cernua is the maternal species and the invasive B. frondosa is the paternal species in all probabilities. Diagnostic morphological features of the three Solidago taxa growing together in the vicinity of Pskov have been studied: a native S. virgaurea , an invasive species of North American origin S. canadensis , and their hybrid S. (cid:1) niederederi . S. (cid:1) niederederi has an intermediate position between S. virgaurea and S. canadensis . The hybrid origin of S. (cid:1) niederederi is proven by molecular analysis of nuclear DNA nucleotide sequences (ITS1-ITS2 site). It is not yet possible to unambiguously answer the question which parent species is maternal and which is paternal. We also studied invasive species of the genus Erigeron sect. Conyza in the Mediterranean. Occasionally occurring in Southern Europe, individuals of E. canadensis (cid:1) E. sumatrensis with intermediate morphological features, described as “ Conyza (cid:1) rouyana , ” are likely unstable and soon “ absorbed ” by the parent species E. sumatrensis . Contrary to the hypothesis by C. Elton explaining the success of plant invasion in a new homeland by strengthening hybridization processes in the secondary distribution range.


Introduction
There is a hypothesis that the strengthening of hybridization processes in the secondary distribution range contributes to more successful existence of plants in their new homeland [1,2]. Under unusual conditions, alien species can form hybrids with closely related native species, as well as with other alien plants inhabiting a given area. Often, hybrids are better adapted to secondary distribution range conditions than parent taxa [3][4][5], resulting in landscapes in a new home area. Successful recombination of genetic traits of parent species reduces the lag phase (a period of adaptation of an alien taxon to new conditions during which there is not yet active introduction into natural phytocoenosis and expansion of the secondary distribution range) and leads to the formation of new active "species transformers." Cross-pollinated plant species are the most predisposed to hybridization. Sympatric species are less likely to be cross-species hybridized than allopatric species or populations [6]. The share of hybrid taxa among invasive species of Middle Russia reaches 10% [7].
For a long time, the most important factor in limiting hybridization was geographical isolation, but nowadays closely related taxa come into contact with each other through a multitude of anthropogenic "corridors" [8]. Thus, inter-regional immigration occurs by means of the introduction of plants, which can be frequent and repetitive, and therefore it significantly increases immigration flow [9]. If we consider the situation where conditions for the hybridization of closely related taxa already exist, there may be several possible developments that coexist: (a) New hybrid taxon may appear, and (b) native taxon disappears. During hybridization, genetic assimilation occurs, and new genes are injected into one or both parent species. Hybrids, even being fertile, can, however, be reproductively isolated from parent plants due to the effect of the selection on reproductive traits (allopolyploidy, heterozygous translocations, recombination, mitochondrial DNA-specific differences) and/or due to factors that predetermine crossing (flowering phenology, separation of ecological niches). Interspecific hybridization may also facilitate the naturalization of rare genotypes and cause an increase in their proportion by inverse crossbreeding with alien parent taxa or hybridization between the hybrids themselves. Greater selection advantages for alien alleles should lead to faster replacement of natural alleles through hybridization and slower replacement without hybridization [10]. The period of displacement (substitution) decreases significantly with increasing immigration flow and selective differentiation. Immigration and selection operate in a variety of ways: increasing immigration levels result in the substitution of native species by suppressing them, while increasing selective differentiation in favor of an alien species results in the substitution of an alien species by genetic assimilation without leaving "pure" native species. At moderate and high immigration rates, the loss of native species can be rapid with or without hybridization. Given the high number of species introduced by humans, the loss of native species can increase only as a result of hybridization [11].
Hybridization increases the threat of extinction of many species due to introgression [12,13]. High degree of introgression is often manifested by windpollinated species such as oaks. Hybridization and introgression can lead to a hybrid complex consisting of many hybrids due to a large number of loci. Thus, multiloci seem to increase the number of hybrid types and genetic complexes and accelerate the reduction of "pure" natural species. In addition, the large number of loci essentially reduces the probability of having a "pure" individual of any parent origin [11]. Without introgression, hybrids, being reproductively isolated, can quickly form a new species. With introgression, speciation slows down as inverse crosses with parent lines occur. The impact of hybridization and introgression on the rate of substitution of native species by closely related ones has been addressed by a mathematical model involving a one-loop bipartite inheritance scheme with different levels of cross-species hybridization [11]. Although the model did not take into account vegetatively propagating hybrids, the results showed that the substitution of natural taxa by alien ones could occur very quickly (in less than five generations). According to the results, hybridization and introgression can increase the degree of substitution of native species by non-native ones. Introgression increases species substitution with low immigration, but prevents substitution when an indigenous species has a significant advantage in selection as well as with higher immigration levels. However, as introductions are associated with increased frequency of hybrids, the impact on the indigenous taxon remains high, and the likelihood of extinction increases significantly [11].
It is known that the highest invasive activity is exhibited by species of Asteraceae family [14], so we have focused our attention on representatives of this group of alien species. It is often impossible to say with certainty whether plants with intermediate morphological features are hybrids (between two species of the same genus). This may also be the case for new ecological forms, resulting from microevolution of species. To confirm or disprove the hypothesis about hybrid origin of certain taxon, it is best to use molecular genetic methods.

Materials and methods
DNA was extracted from silica gel dried leaves of Bidens, Solidago, and Erigeron taxa according to the method by Rogers and Bendich [15]. The herbarium specimens are stored in the herbarium of the Tsitsin Main Botanical Garden (MHA). Polymerase chain reaction (PCR) was carried out in a DNA Engine Dyad Peltier Thermal Cycler amplifier (Bio-Rad, United States). For the nuclear ribosomal internal transcribed spacer (ITS) 1-2 (ITS1-ITS2), nnc18s10 (forward) and c26A (reverse) primers with an annealing temperature of 50°С were used. For the chloroplast loci (rpl32-trnL and trnL-trnF intergenic spacers), primers were used at the annealing temperature from 0.3 to 65°С [16]. For the chloroplast locus rpl32-trnL, we used primers rpl32F (forward) and trnL UAG (reverse), and for the other chloroplast locus trnL-trnF, we used primers c (forward) and f (reverse). Purification of the PCR product for sequencing was carried out in a mixture of ammonium acetate with ethanol. The nucleotide DNA sequences were determined on an automatic sequencer (Syntol). Further processing of the nucleotide sequences was carried out in the BioEdit program. The data were sent to GenBank (2019), in which these nucleotide sequences can be found by the additional numbers assigned to them ( Table 1). Phylogenetic trees were constructed using SplitsTree4.

Bidens Â decipiens
Bidens connata Muhl. ex Willd. is a North American species whose natural area extends from Alaska in the north to Mexico in the south [17]. The species has high polymorphism within native area, and several varieties have been described: B. connata var. ambiversa Fassett, var. anomala Farwell, var. fallax (Warnstorf) Sherff, var. gracilipes Fernald, var. inundata Fernald, var. petiolata (Nuttall) Farwell, var. pinnata S. Watson, and var. submutica Fassett [18,19]. In the second half of the twentieth century, American botanists made suggestions about the hybrid nature of B. connata based on morphological features [20]. This species was indicated as an alien for Europe [21]. However, European plants called "B. connata" are morphologically different from American samples. Their outer leaves are clearly leafshaped, well-developed, and 3-6 cm long, with no reedy flowers, and the first real leaves are less narrow and with more pronounced petioles than those of B. connata and fewer denticles on the leaves, and the denticles, in turn, are usually larger and less regularly located [22]. Plants from European populations have been described as B. Â decipiens Warnst. in 1895. The typical excrement material collected by Carl Warnstorf is stored in the herbaria of Edinburgh (E), Frankfurt (FR), and Charles University in Prague (PRC) [23]. In Europe, the locations of B. Â decipiens are few and far between. A map of the gradual eastward expansion of this species was previously compiled by the authors of this paper [24] and is shown in Figure 1. Previously, we studied morphological features of B. Â decipiens in Russia and found that features of this species are intermediate between the North American invasive Sample no.

Taxon
Date and place of collection, notes  similarity with B. cernua. However, in one case this specimen still has heterozygosity in nucleotide substitutions differentiating B. cernua and B. frondosa, so we cannot say that this specimen is a form of B. cernua; most likely, it is the result of introgressive hybridization (Figure 2). It is possible that in the case of introgressive hybridization, not only B. Â decipiens Â B. cernua but also backcrosses are formed (B. Â decipiens Â B. frondosa). It is interesting that ambiguous readings of a certain nucleotide are also observed for all B. frondosa samples in the same position, but they are not related to nucleotide substitutions in other taxa (Figure 2). It is not excluded that B. frondosa itself is a species of hybrid origin. This is indirectly proved by the high polymorphism of this species in its natural area. Based on the nucleotide sequences of the ITS1-ITS2 site in the SplitsTree program, the dendrogram is built using the UPGMA method (Figure 3). With high probability (with 100% bootstrap support), two clades were separated-sample t_7 (B. tripartita). One clade was separated by species, specimens B. frondosa (fr_5a, fr_5b, fr_5b, fr_10a, fr_10b), and the other clade included all samples of B. Â decipiens and B. cernua, indicating a high similarity.
For trnL-trnF site of chloroplast DNA, samples of B. Â decipiens and B. cernua have no differences (this applies to all plants, including those collected in different regions), while B. frondosa differs from these taxa by six substitutions of one to two nucleotides and deletion of seven nucleotides (Figure 4). B. tripartita has another deletion (Figure 4), which is absent in other taxa, which once again indirectly confirms its non-participation to the hybrid origin of B. Â decipiens. This means that the aboriginal B. cernua is the maternal species and B. frondosa is the paternal species.

Solidago Â niederederi
We have set ourselves the task to study the features of Solidago hybrids in northwest Russia, because in contemporary publications the most numerous references to S. Â niederederi are in the northeastern part of Europe and the Pskov region is the closest region of Russia to it. Previously, on the basis of the analysis of the highly variable noncoding chloroplast region rpl32-trnL by Polish botanists, it was found that hybridization between S. canadensis and S. virgaurea can occur in both  directions and both species can be both mother and father plants [28]. We aimed to determine the situation with respect to parental taxa in the Pskov populations of these species.
The main difference between the hybrid S. Â niederederi and its parents is the structure of shoot systems (mainly the inflorescence structure, Figure 5). In S. canadensis numerous heads are collected in a compound raceme, and in S. Â niederederi the number of heads is smaller and is collected in a compressed compound raceme, whereas in S. virgaurea the number of heads is smaller, and the branches of the compound raceme are so short that the inflorescence is more like a spike.
With regard to the length of the head, S. Â niederederi plants in Pskov cannot be clearly distinguished from S. canadensis due to the high variability of this indicator in S. canadensis. However, in terms of average head lengths, the hybrid also occupies an intermediate position between parent species (Figure 6). S. canadensis and S. Â niederederi shoots are pubescent, while S. virgaurea shoots are glabrous, glossy, and sometimes reddish. The leaves of S. Â niederederi in the middle part of the shoot are linear-lanceolate and dentate along the edge, with three distinct veins (as in S. canadensis), while in the basal part of the shoot large, ovate, with reticulate veins (as in S. virgaurea). To confirm hybrid origin of S. Â niederederi population in the vicinity of Pskov, nucleotide sequences of nuclear and chloroplast DNA of Pskov individuals (both parent and hybrid species) as well as individuals of parent species from Moscow region were analyzed. The analysis of the ITS1-ITS2 site showed that in all cases of nucleotide substitutions differentiating S. virgaurea and S. canadensis, S. Â niederederi has ambiguous readings ( Table 2), indicating heterozygosity, which confirms the hybrid origin of individuals from this population. One sample of S. canadensis (c_3c) showed heterozygosity in three cases of nucleotide substitutions out of four, although morphologically this sample did not differ from other individuals of S. canadensis, which indicates the presence of introgressive hybridization within the Solidago genus. It is likely that this sample is a backcross (result of crossbreeding S. Â niederederi with the parent species S. canadensis).
The analysis of the rpl32-trnL high-variable intergenic spacer made it impossible to give an unambiguous answer which species is maternal to S. Â niederederi and which is paternal. In contrast to the results obtained by A. Plizhko and J. Zalevska-Galosh [30], our samples of S. canadensis have a higher variability of this section of chloroplast DNA. For example, a sample of s_3a from the Pskov region has a DNA fragment that is absent in other plants of S. canadensis not only in the Pskov region but also near Moscow (242-264 nucleotides, Table 3). The area occupying positions 271-306 in the alignment of our sequences (292-330 for sample c_3a, Table 3) and differentiating parental species in Polish populations [30] may differ not only in S. Â niederederi but also in both parental species. The analysis of another noncoding site of chloroplast DNA, trnL-trnF, also failed to answer this question because all the samples examined were identical in this site. Based on the data obtained, we can only v_1a The nucleotides are coded using IUPAC nomenclature.  The nucleotides are coded using IUPAC nomenclature. assume that hybridization occurs in both directions in the Pskov population, but there is also a possibility that only one species may be maternal and the other paternal, and it is necessary to search for other, more variable sites of chloroplast DNA.

Erigeron sect. Conyza
Earlier we studied in detail morphological differences between species of the genus Erigeron that grow in Eurasia [30], and they are shown in Table 4.
Previously a hybrid of Erigeron canadensis and E. sumatrensis-Conyza Â rouyana Sennen-was described. A typical specimen of this taxon (P04315552), collected by F. Sennen in Catalonia in 1904, is kept in the herbarium of the Museum of Natural History in Paris [P] [7]. Nevertheless, some botanists did not recognize this hybrid and referred C. Â royana to E. floribundus [31], which is now treated as synonymous with E. sumatrensis. However, it cannot be excluded that morphological differences in several individuals could have been caused not by hybrid processes but by adverse environmental conditions.
The analysis of the ITS1-ITS2 site of 16 samples of Erigeron sect. Conyza (supposed hybrids and parent taxa) confirmed our conclusions about the higher polymorphism of E. sumatrensis than E. canadensis: ITS1-ITS2 sites of E. canadensis samples were identical, while E. sumatrensis has substitutions and ambiguous readings ( Table 5). As for the supposed hybrids (samples 8a, 8b, 10a, 10b, and 22), only in one case an ambiguous reading of the nucleotides coincides with the substitution differentiating E. canadensis and E. sumatrensis, which indicates that the hybridization has taken place, but since in other cases the substitutions are identical ( Table 5) and the supposed hybrids have no ambiguous readings, most likely, the reason for their morphological differences is the high polymorphism of E. sumatrensis taxon, to which they can be classified.

Conclusions
Thus, the obtained data on hybrid activity among the Asteraceae family of invasive species are ambiguous.
Hybrid B. Â decipiens has a low polymorphism. B. cernua is the most polymorphic taxon, and we can assume the presence of introgressive hybridization of B. Â decipiens with a maternal species. The analysis of ITS1-ITS2 and trnL-trnF sequences showed that B. Â decipiens is of hybrid origin and its maternal form is an aboriginal sequence of B. cernua and the paternal one is probably invasive B. frondosa. It is possible that B. Â decipiens in its present form has already appeared by introgression, but no morphological differences between supposed hybrids (Belarusian and Kaluga plants) and supposed backcrosses (plants from Moscow and Kaliningrad region) have been revealed. It should be noted that B. frondosa itself may be of hybrid origin.
In the northwest of Russia, the populations of three taxa of Solidago genus-an invasive species of North American origin of S. canadensis, an indigenous species of S. virgaurea, and their hybrid S. Â niederederi-grow together in the vicinity of the city of Pskov, which was confirmed by the sequence analysis of the ITS1-ITS2 site. Since both parents, especially S. canadensis, are quite polymorphic taxa, it is impossible to answer unambiguously which of the two species is maternal and which is paternal.
In Southern Europe, the hybriogenic activity of representatives of the genus Erigeron is close to zero. The low hybriogenic activity can also be explained by differences in the chromosome set: in E. canadensis 2n = 18 and in E. sumatrensis 2n = 54 [32].
Our data on the rare occurrence of hybrids in comparison with parental species in the Asteraceae family contradict the hypothesis explaining the success of plant growth in the new homeland by strengthening hybridization processes in the secondary distribution range [1,2], but this situation may change in the coming decades, so the hybriogenic activity of invasive species requires attention of the scientific community.