**3.** *Bidens 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. 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. 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. decipiens* in Russia and found that features of this species are intermediate between the North American invasive


*B. frondosa* L. and the native *B. cernua* L. *B. decipiens* are covered with two types of hairs—duplex, from two cells (as in *B. frondosa*), and simple multicellular (as in *B. cernua*). In addition, the seeds of *B. decipiens* are quadrilateral and have four axes (as in *B. cernua*) and are covered with warts (as in *B. frondosa*). Heads of *B. decipiens* are similar in size and shape to heads of *B. frondosa*, and the leaves are whole, as in *B. cernua*. On the basis of these data, we hypothesize the hybrid origin

Nucleotide sequence analysis shows that not all individuals defined as *B. decipiens* can be called hybrids. The point is that in cases of nucleotide substitutions

of *B. decipiens* [25].

22 MK397990/ / –

*Samples of the studied taxa of Asteraceae.*

**Table 1.**

**99**

**Sample no.**

**Number of ITS/rpl32– trnL/trnL–trnF sequence**

*Hybridogenic Activity of Invasive Species of Asteraceae DOI: http://dx.doi.org/10.5772/intechopen.91370*

c\_3a MK491854/MK474085/ – *S. canadensis*

3 MK397980/ / – *Erigeron*

8a MK397986/ / – *E. sumatrensis*

20 MK397995/ / – *E. canadensis*

*Note: "E. sumatrensis E. canadensis (?)" – putative hybrids.*

**Taxon Date and place of collection, notes**

near the village of Chudinovo, idle field, 2018

National Park, Pine forest, 2014

Italy, Pompeii, 2016 40.7° N, 14.5° E

40.8° N, 14.4° E

38.7° N, 9.1° W

Italy, Naples, 2016

v\_4 – /MK474088/ – *S. virgaurea* Russia, Moscow region, Chekhov district,

v\_6a MK491858/ / – *S. virgaurea* Russia, Moscow region, "Losiny Ostrov"

*sumatrensis*

5a MK397981/ / – Italy, the Island of Ischia, 2016

6 MK397984/ / – Italy, Herculaneum, 2016

*E. canadensis* (?)

13a MK397991/ / – *E. canadensis* Italy, the Island of Ischia, 2016

55.1° N, 37.5° E c\_5a MK491857/MK474090/ – *S. canadensis*

55.89° N, 37.77° E v\_6b MK491859/ / –

40.7° N, 13.9° E 5b MK397982/ / –

40.8° N, 14.2° E 8b MK397987/ / – 10a MK397988/ / – Italy, Pompeii, 2016 40.7° N, 14.5° E 10b MK397989/ / –

40.7° N, 13.9° E 13b MK397992/ / –

40.4° N, 3.7° W 19 MK397994/ / – *Erigeron* sp.

16 MK397985/ / – Portugal, Lisbon, 2017

18 MK397993/ / – Spain, Madrid, park, 2017

**in GeneBank**

n\_2b MK491853/MK474083/ –

c\_3b MK491855/MK474086/ – c\_3c MK491856/MK474087/ –

n\_2c – /MK474084/ –

c\_5b – /MK474089/ –

5c MK397983/ / –

**Sample no.**

**Number of ITS/rpl32– trnL/trnL–trnF sequence**

de\_1a МК559763/ – /MK575566 *Bidens*

*decipiens* (= "*B. connata*")

*Invasive Species - Introduction Pathways, Economic Impact, and Possible Management Options*

2013 54.4914° N, 34.3393° E de \_1b МК559764/ – /MK575567

de \_2a МК559767/ – /MK575570 Russia, Kaliningrad region, 2013

de \_3a МК559771/ – /MK575573 Russia, Vladimir region, near Tasinsky

de \_4a МК559774/ – /MK575575 Russia, Vladimir region, near Tasinsky

fr\_5a МК559780/ – /MK575581 *B. frondosa* Russia, Vladimir region, near Tasinsky

fr\_5b МК559781/ – /MK575582 Russia, Vladimir region, near Tasinsky

cr\_6a МК559755/ – /MK575559 *B. cernua* Russia, Moscow region, near Zvenigorod

t\_7 МК559754/ – /MK575558 *B. tripartita* Russia, Vladimir region, near Tasinsky

de \_13 МК559779/ – /MK575580 Russia, Moscow, park near Sviblovo estate,

*virgaurea*

57.80° N, 28.25° E v\_1b MK491850/MK474080/ –

village, 2014 55.567° N, 40.172° E

village, 2018

town, 2014

village, 2018

2018 55.8639° N, 37.6396° E

of Pskov, idle field, 2018

Russia, Pskov region, Pskov district, vicinity

leaves 55.567° N, 40.172° E de \_3b МК559772/ – / –

55.567° N, 40.172° E de \_4b МК559775/ – /MK575576

55.567° N, 40.172° E fr\_5c МК559782/ – /MK575583

55.69° N, 36.74° E cr\_6b МК559756/ – /MK575560

55.567° N, 40.172° E cr\_8a МК559757/ – /MK575561 *B. cernua*

cr\_9a МК559760/ – /MK575563 Belarus, Dzerzhinsk, 2018 53.693° N, 27.165° E <sup>с</sup>r\_9b МК559761/ – /MK575564

54.95° N, 20.49° E de \_2b МК559768/ – /MK575571

**Taxon Date and place of collection, notes**

Russia, Kaluga region, Milyatinsky Reservoir,

village, 2014 Formed with dissected lower

village, 2014 Formed with whole lower leaves

**in GeneBank**

de \_1c МК559765/ – / MK575568 de \_1d МК559766/ – /MK575569

de \_2c МК559769/ – /MK575572

de \_3c МК559773/ – / MK575574

cr\_8b МК559758/ – /MK575562

cr\_9c МК559762/ – /MK575565

fr\_10b МК559784/ – /MK575585

de \_11c МК559778/ – /MK575579

v\_1c MK491851/MK474081/ –

**98**

fr\_10a МК559783/ – /MK575584 *B. frondosa*

de \_11a МК559776/ – /MK575577 *B. decipiens* (= "*B. connata*") de \_11b МК559777/ – /MK575578

v\_1a MK491849/MK474079/ – *Solidago*

n\_2a MK491852/MK474082/ – *S. niederederi*

cr\_8c МК559759/ – /

de \_2d МК559770/ – / –


#### **Table 1.**

*Samples of the studied taxa of Asteraceae.*

*B. frondosa* L. and the native *B. cernua* L. *B. decipiens* are covered with two types of hairs—duplex, from two cells (as in *B. frondosa*), and simple multicellular (as in *B. cernua*). In addition, the seeds of *B. decipiens* are quadrilateral and have four axes (as in *B. cernua*) and are covered with warts (as in *B. frondosa*). Heads of *B. decipiens* are similar in size and shape to heads of *B. frondosa*, and the leaves are whole, as in *B. cernua*. On the basis of these data, we hypothesize the hybrid origin of *B. decipiens* [25].

Nucleotide sequence analysis shows that not all individuals defined as *B. decipiens* can be called hybrids. The point is that in cases of nucleotide substitutions

#### **Figure 1.**

*Map of the secondary distribution range of* Bidens decipiens*.*

at the ITS1-ITS2 site differentiating *B. frondosa* and *B. cernua*, we encounter heterozygosity of samples of *B. decipiens* (and, accordingly, ambiguity of reading of the sequence) in many cases, but still not in all (as it should be expected for the hybrid F). At the same time, each of our samples of *B. decipiens* is characterized by at least some such ambiguous readings of nucleotides (C or T, A or T, A or C) (**Figure 2**) in the case of substitutions, which, of course, confirms the hypothesis of the hybrid origin of this taxon. Populations of *B. decipiens* from different parts of the range differ in the number of substitutions. Thus, samples from the banks of the Milyatinskoye reservoir in the Kaluga region demonstrate in most cases the presence of ambiguous readings in the case of nucleotide substitutions, with the exception of sample de\_1a, in which heterozygosity is not observed in all cases of substitutions. It means that this population is a hybrid one. In addition, both parent species grow on the banks of the Milyatinskoye reservoir in close proximity to the population of *B. decipiens*, which indirectly supports this view [26]. Probably, the differences in the sample de\_1a are due to the presence of introgression, i.e., this sample is a backcross resulting from crossing of *B. decipiens* with *B. cernua*, because ITS1-ITS2 of this sample has a stronger DNA area similar to *B. cernua* than others. The same situation is observed with individuals of *B. decipiens* from Belarus. *B. decipiens* specimens from the Kaliningrad region, by contrast, in most cases show similarities with *B. cernua* in the case of substitutions rather than heterozygosity, except for sample de\_2a. In this case, there are two possible variants in the first case, we collected samples of backcrosses and see the result of introgression; in the second case, the parent form is another form of *B. frondosa*, not the widespread *B. frondosa* var. *frondosa.* The second variant is less probable. However, other forms of *B. frondosa* have been recently found [27]. Among plants of *B. decipiens* collected in the Vladimir region, two forms distinct on lower leaves—with a dissected sheet plate (samples de\_3a, de\_3b, and de\_3c) and with a whole sheet plate (de\_4a, de\_4b)—are clearly distinguished. As it turned out, these forms have genetic differences, but samples 4a and 4b are also not identical in the ITS1-ITS2 section sequences. In this case, it is only possible to estimate which form is closer to *B. cernua* and which one to *B. frondosa* using statistical methods. The plant collected on the territory of Sviblovo Estate in Moscow and based on a set of morphological features defined as *B. decipiens*, in the section ITS1-ITS2, has a very high

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. decipiens B. cernua* but also backcrosses are formed (*B. decipiens 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

*Fragment of ITS1-ITS2 site of nuclear DNA of various taxa of* Bidens *genus. The nucleotides are coded using*

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.*

For trnL-trnF site of chloroplast DNA, samples of *B. 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. decipiens*. This means that the aboriginal *B. cernua i*s the maternal species and *B. frondosa* is the paternal

by the high polymorphism of this species in its natural area.

*the International Union of Pure and Applied Chemistry (IUPAC) nomenclature.*

*Hybridogenic Activity of Invasive Species of Asteraceae DOI: http://dx.doi.org/10.5772/intechopen.91370*

*decipiens* and *B. cernua*, indicating a high similarity.

species.

**101**

**Figure 2.**

*Hybridogenic Activity of Invasive Species of Asteraceae DOI: http://dx.doi.org/10.5772/intechopen.91370*

#### **Figure 2.**

at the ITS1-ITS2 site differentiating *B. frondosa* and *B. cernua*, we encounter heterozygosity of samples of *B. decipiens* (and, accordingly, ambiguity of reading of the sequence) in many cases, but still not in all (as it should be expected for the hybrid F). At the same time, each of our samples of *B. decipiens* is characterized by at least some such ambiguous readings of nucleotides (C or T, A or T, A or C)

*Invasive Species - Introduction Pathways, Economic Impact, and Possible Management Options*

*Map of the secondary distribution range of* Bidens decipiens*.*

**Figure 1.**

**100**

(**Figure 2**) in the case of substitutions, which, of course, confirms the hypothesis of the hybrid origin of this taxon. Populations of *B. decipiens* from different parts of the range differ in the number of substitutions. Thus, samples from the banks of the Milyatinskoye reservoir in the Kaluga region demonstrate in most cases the presence of ambiguous readings in the case of nucleotide substitutions, with the excep-

substitutions. It means that this population is a hybrid one. In addition, both parent species grow on the banks of the Milyatinskoye reservoir in close proximity to the population of *B. decipiens*, which indirectly supports this view [26]. Probably, the differences in the sample de\_1a are due to the presence of introgression, i.e., this sample is a backcross resulting from crossing of *B. decipiens* with *B. cernua*, because ITS1-ITS2 of this sample has a stronger DNA area similar to *B. cernua* than others. The same situation is observed with individuals of *B. decipiens* from Belarus. *B. decipiens* specimens from the Kaliningrad region, by contrast, in most cases show similarities with *B. cernua* in the case of substitutions rather than heterozygosity, except for sample de\_2a. In this case, there are two possible variants in the first case, we collected samples of backcrosses and see the result of introgression; in the second case, the parent form is another form of *B. frondosa*, not the widespread *B. frondosa* var. *frondosa.* The second variant is less probable. However, other forms of *B. frondosa* have been recently found [27]. Among plants of *B. decipiens* collected in the Vladimir region, two forms distinct on lower leaves—with a dissected sheet plate (samples de\_3a, de\_3b, and de\_3c) and with a whole sheet plate (de\_4a, de\_4b)—are clearly distinguished. As it turned out, these forms have genetic differences, but samples 4a and 4b are also not identical in the ITS1-ITS2 section sequences. In this case, it is only possible to estimate which form is closer to *B. cernua* and which one to *B. frondosa* using statistical methods. The plant collected on the territory of Sviblovo Estate in Moscow and based on a set of morphological

tion of sample de\_1a, in which heterozygosity is not observed in all cases of

features defined as *B. decipiens*, in the section ITS1-ITS2, has a very high

*Fragment of ITS1-ITS2 site of nuclear DNA of various taxa of* Bidens *genus. The nucleotides are coded using the International Union of Pure and Applied Chemistry (IUPAC) nomenclature.*

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. decipiens B. cernua* but also backcrosses are formed (*B. decipiens 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. decipiens* and *B. cernua*, indicating a high similarity.

For trnL-trnF site of chloroplast DNA, samples of *B. 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. decipiens*. This means that the aboriginal *B. cernua i*s the maternal species and *B. frondosa* is the paternal species.

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

The main difference between the hybrid *S. 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. 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

The size of the heads themselves also varies (**Figure 6**). *S. niederederi* heads have an oval shape and occupy an intermediate position in diameter between parental species, 2201 45 μm (mean error average) with a maximum spread of

3132 30 μm (2874–3548 μm) and 1591 22 μm (1428–1939 μm), respectively [29]. With regard to the length of the head, *S. 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. niederederi* shoots are pubescent, while *S. virgaurea* shoots are glabrous, glossy, and sometimes reddish. The leaves of *S. 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*. 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. 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

1762 to 2728 μm, while for *S. virgaurea* and *S. canadensis*, these values are

these species.

*Hybridogenic Activity of Invasive Species of Asteraceae DOI: http://dx.doi.org/10.5772/intechopen.91370*

spike.

**Figure 5.**

**103**

*Panicles of* Solidago niederederi *and its parental species.*

**Figure 3.** *Dendrogram based on analysis of the ITS region of DNA of various* Bidens *taxa with bootstrap support data.*


**Figure 4.**

*Fragment of the trnL-trnF intergenic spacer of chloroplast DNA of various taxa of* Bidens *genus. The nucleotides are coded using IUPAC nomenclature.*
