Autopolyploidization has driven the successful invasion of Solidago canadensis in East Asia. However, it was believed that only diploid S. canadensis invaded Europe, whereas polyploids never did. In this study, we aim to investigate whether polyploidy S. canadensis invaded Europe and compare the ecological niche differentiation pattern driven by ploidy in Asia and Europe and North America.Here, molecular identification (combination of ribosomal ITS and psbA-trnH intergenic spacer), ploidy level, and morphological traits of ten S. canadensis populations collected in Europe were compared with previously identified S. canadensis populations from other continents and S. altissima populations. Furthermore, the ploidy-driven geographical differentiation pattern of S. canadensis in different continents was investigated. Results showed that all ten European populations were identified as S. canadensis with five diploid and five hexaploid populations. Significant differences in morphological traits existed among diploids and polyploids (tetraploids and hexaploids), rather than between polyploids from different introduced ranges and between S. altissima and polyploidy S. canadensis populations. The invasive hexaploids and diploids had few differences in latitudinal distributions in Europe which was similar to the native range but absolutely different from a distinct climate-niche differentiation in Asia. This may be attributed to the bigger difference in climate between Asia and Europe and North America.The above morphological and molecular evidences proved the invasion of polyploid S. canadensis in Europe and suggest that S. altissima may be merged into a complex of S. canadensis species. Our study may be concluded that geographical and ecological niche differentiation of an invasive plant driven by ploidy depends on the degree of difference in the environmental factors between the introduced range and the native range, which provides new insight into the invasive mechanism.

Diploid (2n = 2x = 18), tetraploid (2n = 4x = 36), and hexaploid (2n = 6x = 54) seeds of S. canadensis were collected from the native region (North Amercia) and invasive region (Europe and Asia). The detailed descriptions of material collection and identification had been presented by Cheng et al. (2021). These seeds were stored in refrigerator at 4℃ in the Weed Research Laboratory, Nanjing Agricultural University, China. The NA2x, NA4x, NA6x, IN2x, IN4x and IN6x were used to represent the native diploid, native tetraploid, native hexaploid, introduced diploid, introduced tetraploid, and introduced hexaploid, respectively. NA represents the population of North America, CN represents the population of China, JP represents the population of Japan, EU represents the population of Europe, and RUS represents the population of Russia. 

Molecular markers and phylogenetic analysis:

To confirm the correct identification of the ten populations, we sequenced the ribosomal ITS (608bp) and trnH-psbA intergenic spacer (213bp) of three individuals from each population. To further verify the species identity of our samples, we conducted a BLAST search in the database of the National Center for Biotechnology Information (NCBI) to determine the species with the most similar identities. We built phylogenetic trees for all 152 S. canadensis populations, 7 S. altissima populations and closely related species with the ITS-psbA-trnH sequences, using the MEGA 4.1 Beta 3 software based on the maximum likelihood (ML) analyses, using a GTR substitution model as determined.

Ploidy level of S. canadensis populations in Europe:

We grew up plants from each seed family in the greenhouse. Fresh leaf material was held in self-sealing plastic bags containing silica and transported to the laboratory for ploidy level determination. We applied a modified flow cytometry method by Galbraith et al. (Galbraith et al., 1983) for the determination of ploidy level. The native diploid population (CA09) was used as an internal reference to measure the ploidy of ten populations. Glycine max Merr. 'Polanka is used as an internal reference to measure the DNA content of diploids (Dolezel et al., 2007).

Comparison of the morphological traits of different geo-cytotype populations:

After plant establishment, three individuals of each population were selected, and three replicates of each individual were used to measure the 7 vegetative traits: height, stem, leaf length, leaf width, number of epidermal hairs of the main vein (per 2 mm length), number of epidermal hairs of the lateral vein (per 2 mm length), and number of epidermal hairs of the netted venation (per 4 mm2). During the flowering period,  three individuals of each population were selected, and three replicates of each individual were used to measure the 4 floral traits: length of the ray floret, height of the involucre, number of ray florets, and number of disc florets.

Geographical distribution in Europe and global differentiation of ecological niches of different ploidy populations:

The geographical differentiation of different ploidy populations in Europe and other regions was analyzed by combining with our previous research (Cheng et al., 2020a) and literature records. Climatic factors of all occurrences were extracted from worldclim (https://www.worldclim.org/) for the analysis of differences in climatic factors between the distribution ranges of different ploidy populations. In addition, the ecological niche differentiation pattern of different ploidy populations in Europe, North America, and Asia are compared.

Methods for processing the data: 

The average and square deviation of morphological traits and climate factors for different geo-cytotypes of S. canadensis in different regions were calculated, and the significance of the differences (P<0.05) between geo-cytotypes in different regions was analyzed using using one-way ANOVA with Least Significant Difference (LSD).

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