Interspecific Differences in Phenolics

Phenolic compounds, namely, chlorogenic acid, rutin, isoquercitrin, hyperoside, and quercitrin, were detected in leaves and inflorescences of S. virgaurea, S. canadensis, S. gigantea, and S.×niederederi. Leaves showed considerably higher mean values for chlorogenic acid compared with inflorescences, whereas inflorescences showed significant accumulation of isoquercitrin. All analyzed Solidago species shared the same common phenolic constituents.

ANOVA revealed high significant differences (P≤0.001) for the mean quantities of all phenolic compounds in leaves and inflorescences among the investigated species. Post hoc multiple comparisons of mean quantities specified that S. gigantea significantly differed for all compounds among all species. The highest values of phenolics were found in the leaves and inflorescences of S. gigantea compared with other species, except for rutin. Meanwhile, the rutin content in both the leaves and inflorescences of other species greatly exceeded that of S. gigantea, showing the highest accumulation in S.×niederederi leaves and in S. canadensis inflorescences. Furthermore, the differences for phenolics were analyzed between S.×niederederi and putative parental species S. virgaurea and S. canadensis. Solidago gigantea was excluded from the multiple-comparison test because the remaining taxa were not different for most phenolic compounds when S. gigantea was included. Significant differences among the three taxa were observed for chlorogenic acid and quercitrin in the leaves and for chlorogenic acid, rutin, and isoquercitrin in the inflorescences (Tables 1 and 2). The leaves of S.×niederederi significantly accumulated the highest (P<0.01) level of chlorogenic acid, whereas the flowers of S. canadensis showed significant accumulation of this compound in comparison with the other two species. However, the amount of chlorogenic acid in the flowers did not differ significantly (P>0.05) between S.×niederederi and S. virgaurea. The rutin content in the inflorescences of S. canadensis greatly exceeded that in the flowers of S.×niederederi and S. virgaurea. The highest amount of quercitrin was accumulated in the leaves and inflorescences of S. canadensis, and the mean content of this compound was found in leaves and inflorescences of S.×niederederi. Meanwhile, quercitrin was detected in minor quantities or not found at all in the flowers of S. virgaurea. Species significantly varied for content of isoquercitrin in the inflorescences, the highest levels of which were found in S. virgaurea followed by S.×niederederi and S. canadensis. However, the amount of hyperoside did not differ significantly among the species for both plant parts.

Table 1 Differences of phenolic compound accumulation in leaves among four Solidago species.Footnote ^a

^a Differences among four species for all compounds were significant at P≤0.001. n=the number of accessions analyzed. Values (mean±SD) followed by different letters among first three species are significantly (P≤0.05) different according to Scheffe’s test. Solidago gigantea was excluded from the multiple-comparison test because the remaining taxa were not different for most phenolic compounds when S. gigantea was included.

^b Abbreviation: DM, dry mass.

Table 2 Differences of phenolic compound accumulation in inflorescences among four Solidago species.Footnote ^a

^a Differences among four species for all compounds were significant at P≤0.001. n=the number of accessions analyzed. Values (mean±SD) followed by different letters among three first species are significantly (P≤0.05) different according to Scheffe’s test. Differences among four species for all compounds were significant at P≤0.001. Solidago gigantea was excluded from the multiple-comparison test because the remaining taxa were not different for most phenolic compounds when S. gigantea was included.

^b Abbreviation: DM, dry mass.

Consequently, S. virgaurea accumulated the lowest quantities of phenolic compounds compared with S. canadensis and S.×niederederi, with the exception of isoquercitrin in leaves. In contrast, among the three Solidago species investigated, for the most part S. canadensis contained the highest amounts of the phenolic compounds. Solidago×niederederi showed average values of phenolics except for having the highest concentration of chlorogenic acid in leaves.

Principal Component Analysis

The first PCA1 model that represented the two-dimensional scatter plots showed differentiation between the sets of chemical data of the four Solidago species studied. The score plots for the first two PCs explained 85% of the total variance and represented enough visualization for any possible patterns of the data. PC1 accounted for 69.51% of the observed total variability and was strongly associated with negative loadings of quercitrin (Quer F, Quer L) and isoquercitrin (Isoquer F, Isoquer L) in flowers and leaves and chlorogenic acid (Chlora L) in leaves as well as the positive variable of rutin (Rut L) in leaves. The PC2 model explained 15.58% of the total variance and was characterized by negative loadings of rutin (Rut F) and chlorogenic acid (Chlora F) in flowers (Figure 2A). The PCA1 score plots with 95% confidence limit ellipses for the four species showed an arrangement of accessions into two distinct groups (Figure 2B). Within the right-hand plots, one group was formed by three overlapping ellipses along PC2 covering all accessions of S. canadensis, S. virgaurea and S.×niederederi. The location of accessions on the score plots can be explained by the position of variables on the loading plots. Rut F, Rut L, and Chlora F, with higher negative loading on PC2, were found to have high to moderate values in corresponding samples, while Quer F, Quer L, Isoquer F, and Isoquer L, with high negative loading on PC1, were accumulated in low quantities in all accessions of this group. The second group of accessions within the left-hand plots brought together all S. gigantea samples, which were closely clustered along negative PC1. In contrast to the first group within the right-hand plots, Quer L, Quer F, Isoquer L, and Isoquer F, scoring high in PC1, were detected as having the highest contents in those accessions; conversely, Rut L and Rut F, scoring low in PC1, were found in minor quantities. Chlora L and Chlora F, loading high to moderately in both PCs, were associated with higher levels of chlorogenic acid, especially in flowers. The results clearly suggested that accessions of S. gigantea were highly different with respect to phenolic content compared with the first group, which was formed by accessions of the other three Solidago species. The PCA1 model did not clearly separate morphologically different taxa as S.×niederederi, S. virgaurea, and S. canadensis. Consequently, the PCA2 model for the three species was used, in order to focus more specifically on differentiation among S.×niederederi, S. virgaurea, and S. canadensis accessions.

Figure 2 PCA1 representing four Solidago taxa for accumulation of phenolic compounds. (A) Loading plots for compound variables contributing to PC1 and PC2. (B) Score plots for the testing accessions with 95% confidence ellipses limit for each species.

In the new PCA2, the first two PCs explained more than 56% of the total variance in the data set and presented much better separation between accessions of corresponding species. PC1 accounted for 37% of the observed variance and was best characterized by positive loadings of Chlora F, Rut F, and Quer L variables and negative Isoquer F. PC2 was highly associated with negative loadings of Chlora L and Rut L and moderate with positive Quer F (Figure 3A). Graphical representation of the score plots showed separation of all S. canadensis accessions in a separate cluster on the right-hand plot (Figure 3B). The positioning of S. canadensis samples coincided with higher loadings of Chlora F, Rut F, Quer L, and Quer F, indicating the greater influence of those variables on the colocated scores. The opposing location of Isoquer F to Chlora F, Rut F Quer L, and Quer F displayed negative correlations, which reflect opposite values of corresponding compounds in the accessions. One sample in the lower right-hand plot was separated from all of the others within the ellipses, suggesting that its composition differs significantly from the other accessions. Indeed, this sample contained the highest values of rutin and chlorogenic acid in both leaves and inflorescences. Meanwhile, the scores of S.×niederederi and S. virgaurea represented two overlapping ellipses. Additionally, S.×niederederi samples were much more scattered on PC1 versus PC2 along with Rut L and Chlora L loadings, displaying high variation in rutin and chlorogenic acid content in leaves. For these compounds, S.×niederederi was similar to S. canadensis. Some of S.×niederederi accessions exposed a tight position with S. virgaurea, indicating a great similarity in chemical content. Some hybrid samples demonstrated a remote position from all the others, scoring a high negative in PC2 and a low positive in PC1, which suggested higher amounts of chlorogenic acid and rutin in their leaves. The opposing location of some accessions on the upper left-hand score plot associated with low values of Chlora L and Rut L in those samples. Meanwhile, Quer F, scoring moderately in PC2, was found in mean quantities in previous samples. Isoquer F, scoring high in PC1 and moderately in PC2, had the greatest influence on colocated accessions and associated mean content of isoquercitrin in flowers of both species. Moreover, negative correlation of Isoquer F with Chlora F, Rut F, and Quer L suggested low values of these compounds in the corresponding samples. Consequently, the PCA2 model demonstrated that S. canadensis accessions formed a separate cluster, indicating a quantitative composition of phenolics highly different from that of from S.×niederederi and S. virgaurea. Conversely, S.×niederederi and S. virgaurea accessions did not reveal the separation of two well-defined groups, with high overlapping on the score plots.

In the current research, the main phenolic compounds in Solidago spp. plant materials, the quercetin-3-O-glycosides: quercitrin, isoquercitrin, rutin, and hyperoside, together with chlorogenic acid, were used to ascertain relationships and differences between S.×niederederi and other Solidago species growing in mixed populations. In general, the phenolic compounds are the most widely used of all secondary constituents in chemotaxonomic studies, mainly due to their ubiquitous occurrence in plants and their structural variability and chemical stability (Braunberger et al. Reference Braunberger, Zehl, Conrad, Wawrosch, Strohbach, Beifuss and Krenn2015; Clark et al. Reference Clark, Bliss, Suzuki and Borris2014; Švehlíková et al. Reference Švehlíková, Mráz, Piacente and Marhold2002). Furthermore, the phenolics listed above are considered principal bioactive compounds in Solidago spp. and are used for plant-derived medical preparations (Melzig Reference Melzig2004; Sabir et al. Reference Sabir, Ahmad, Hamid, Khan, Athayde, Santos and Boligon2012). The present research on the comparison of four taxa demonstrated that all analyzed Solidago spp. exposed the same investigated phenolic constituents, which seems to be characteristic of the whole Solidago genus. On the other hand, the data obtained showed the significant quantitative differences in phenolics among corresponding taxa. The highest values of all studied phenolic compounds, with the only exception being rutin content in leaves and flowers, were found in S. gigantea. Further, chlorogenic acid was the only compound whose content in the leaves of S.×niederederi significantly exceeded that found in parental species, while the content of other compounds was intermediate or did not differ significantly. The accumulation of high amounts of phenolics may provide a competitive advantage via suppression of neighboring plants (Kim and Lee Reference Kim and Lee2011). Moreover, phenolic compounds are known to be particularly important for plant interactions with abiotic and biotic environments and constitute one of the most common defense groups against herbivores (Mallikarjuna et al. Reference Mallikarjuna, Kranthi, Jadhav, Kranthi and Chandra2004; Treutter Reference Treutter2006). Furthermore, chlorogenic acid is known to be associated with plant resistance to insects, fungi, bacteria, and viruses (Leiss et al. Reference Leiss, Maltese, Choi, Verpoorte and Klinkhamer2009; Niggeweg et al. Reference Niggeweg, Michael and Martin2004). It should be noted that chlorogenic acid and rutin in the leaves of S.×niederederi was highly variable. Moreover, chemical variability provides abundant material for environment-mediated selection and may play a role in superior resistance to herbivores and pathogens. Therefore, this variability may contribute to a higher hybrid fitness and persistence in a different environment (Oberprieler et al. Reference Oberprieler, Eder, Meister and Vogt2011).

Figure 3 PCA2 representing three Solidago taxa for accumulation of phenolic compounds. (A) Loading plots for compound variables contributing to PC1 and PC2. (B) Score plots for the testing accessions with 95% confidence ellipses limit for each species.

The chemistry of hybrids varied not only qualitatively, but also quantitatively (Orians Reference Orians2000). Generally, hybrids express parental chemicals, but parental compounds are sometimes missing, or novel compounds are present. The survey by Cheng et al. (Reference Cheng, Vrieling and Klinkhamer2011), based on 1,112 secondary metabolites of different hybrids, showed that the frequency of metabolite novelty accounted for 5.5% of all studied compounds. The probability of occurrence of novel compounds increases in polyploids, especially in F₂ and later-generation hybrids (Orians Reference Orians2000). Accordingly, we have not detected any novel chemicals in S.×niederederi, which is diploid and apparently only a first-generation hybrid (Karpavičienė and Radušienė Reference Karpavičienė and Radušienė2016).

PCA has been used here as an exploratory method to describe data sets without a priori knowledge of the data structure and to allow the visualization of the similarities and differences within analyzed data to indicate the compounds most responsible for separation of different plant groups. In this regard, the position of S. gigantea accessions on PC1 versus PC2 plots at a distance from others represented a greater difference in chemical composition as determined by the study of all phenolic compounds. Meanwhile, the other three taxa formed a separate cluster, indicating similarities in their quantitative accumulation of phenolics. It can be assumed that interspecific chemical variability among Solidago species should be genetically based. However, the mode of qualitative inheritance of most chemical compounds is Mendelian with dominance; if both or one of the parents produce a chemical, the hybrids almost always produce it as well (Orians Reference Orians2000). It can be assumed that the genes responsible for the synthesis of phenolics are constitutively, but differently expressed in the analyzed species (Cheng et al. Reference Cheng, Vrieling and Klinkhamer2011). Although morphologically and phylogenetically S. canadensis is closer to S. gigantea than to S. virgaurea (Semple Reference Semple2016), invasive Solidago spp. differ in their ploidy level. S. gigantea is tetraploid (2n=36), whereas S. canadensis is diploid (2n=18), as are S. virgaurea and S.×niederederi (Karpavičienė and Radušienė Reference Karpavičienė and Radušienė2016). The ploidy level is known to have a significant effect on the intraspecific variation in concentrations of metabolites in S. gigantea (Hull-Sanders et al. Reference Hull-Sanders, Johnson, Owen and Meyer2009). In this context, tetraploid S. gigantea showed high mean phenolic content, while diploid Solidago spp. exhibited comparatively lower content for the compounds analyzed.

The additional PCA for the three species studied, with the exception of S. gigantea, demonstrated that the parental species S. virgaurea and S. canadensis were well distinguished from each other based on the analyzed phenolics. Although interspecific hybrids can often be clearly distinguished from parental species based on their biochemical phenotypes (Kirk et al. Reference Kirk, Cheng, Choi, Vrieling and Klinkhamer2012), S.×niederederi accessions did not show itself to be a clearly defined group compared with the parental species, being in close position with S. virgaurea. They clustered less tightly and displayed greater variation in chemical composition, with some of the samples falling outside the range of the parental species. These results are similar to those reported previously by Kirk et al. (Reference Kirk, Choi, Kim, Verpoorte and Van Der Meijden2005) for chemical composition of two Senecio species and their F₁ hybrids, which did not cluster intermediately to parents on the basis of PCA. According to Orians (Reference Orians2000), parental chemicals in hybrids are mostly either expressed at concentrations similar to one of the parents or at intermediate concentrations. However, S.×niederederi significantly exceeded the parental species in mean chlorogenic acid content in leaves, but the range of variation of this compound in the hybrid was similar to that of S. canadensis. The results agree with other studies showing that hybrid plants have much more genetic variation than the parental species (Ellstrand and Schierenbeck Reference Ellstrand and Schierenbeck2006; Zalapa et al. Reference Zalapa, Brunet and Guries2010), which leads to higher phenotypic diversity. Overall, in our research, S.×niederederi was characterized by the accumulation of phenolic compound content intermediate or similar to that of S. virgaurea, suggesting its hybrid origin.

The sparse chemical diversity of invasive species can be explained by the assumption that invasive species undergo one to several bottlenecks when they are introduced to new areas, which leads to genetic uniformity in plant populations (Müller-Schärer and Schaffner Reference Müller-Schärer and Schaffner2008). In addition, there exist some data about the lack of differentiation in DNA content observed in native S. virgaurea (Szymura et al. Reference Szymura, Szymura and Kreitschitz2015) that may be associated with low chemical diversity. However, in contrast to low chemical diversity, high intraspecific morphological variations have been observed in the analyzed Solidago species (Karpavičienė and Radušienė Reference Karpavičienė and Radušienė2016; Kiełtyk and Mirek Reference Kiełtyk and Mirek2014; Semple et al. Reference Semple, Rahman, Bzovsky, Sorour, Kornobis, Laphitz and Tong2015). Based on the hypothesis of Bossdorf et al. (Reference Bossdorf, Auge, Lafuma, Rogers, Siemann and Prati2005), high plasticity of morphotype allows introduced species to naturalize across different environments, revealing the potential for their invasiveness. Therefore, the use of only morphological descriptions to examine taxa with large intraspecific variation may result in wrong conclusions and taxonomic confusion, as has been the case with Solidago taxa. Consequently, the phytochemical pattern in the present study complemented the evidence of hybrid S.×niederederi origin between native S. virgaurea and invasive S. canadensis. However, it can be assumed that S.×niederederi is continuously formed in mixed Solidago spp. populations, because the phytochemical intermediate of the hybrid is not fixed, suggesting that it is not a completely stabilized hybrid derivative leading to the creation of new genotypes and evolutionary novelty. Solidago×niederederi has occurred rather frequently in mixed Solidago spp. populations; therefore, it has the potential to spread and increase due to viable seed production (Migdałek et al. Reference Migdałek, Kolczyk, Pliszko, Kościńska-Pająk and Słomka2014) as well as through vegetative propagation by clonal growth (Pliszko and Kostrakiewicz-Gierałt Reference Pliszko and Kostrakiewicz-Gierałt2017). However, accessions exhibiting an intermediate chemical composition between spontaneous S. virgaurea and invasive S. gigantea were not detected. This can be explained by the differing number of chromosomes of these species, which leads to more complicated hybridization. On the other hand, S.×snarskisii Gudž. & Žaln., the spontaneous putative hybrid between S. virgaurea and S. gigantea, has been recently described (Gudžinskas and Žalneravičius Reference Gudžinskas and Žalneravičius2016). Furthermore, the appearance of a new hybrid indicated the growing impact of invasive Solidago spp. on local flora.

Although statistical analysis revealed the presence of phenolic compounds such as the chlorogenic acid and quercitrin in leaves and rutin with isoquercitrin in inflorescences of S.×niederederi at significantly different quantities from its parental species, high variability of these components and their overlapping ranges between species impact clear identification of hybrids in the wild. Nevertheless, the present study provided the first examination of expression of phenolic compounds in S.×niederederi. It is important to further assess its inheritance of phenolic compound expression in the second and later generations of the hybrids, which is very likely, because according to Pliszko and Kostrakiewicz-Gierałt (Reference Pliszko and Kostrakiewicz-Gierałt2017), hybrids are able to generate their own offspring by sexual reproduction. On the other hand, the implication of phenolic compounds does not preclude the importance of other secondary metabolites in species differentiation. Additional analysis of these species could show some differences for minor components, which could expose their importance in chemotaxonomy and the evolutionary dynamics of invasion. Overall, Solidago is a suitable model genus for the study of the plant invasion process, and the present study provides a basis for further research on the role of genetic diversity and expression of secondary metabolites during hybridization on invasive potential.