Genome Insights into Autopolyploid Evolution: A Case Study in Senecio doronicum (Asteraceae) from the Southern Alps

Citation: Fernández, P.; Hidalgo, O.; Juan, A.; Leitch, I.J.; Leitch, A.R.; Palazzesi, L.; Pegoraro, L.; Viruel, J.; Pellicer, J. Genome Insights into Autopolyploid Evolution: A Case Study in Senecio doronicum (Asteraceae) from the Southern Alps. Plants 2022,11, 1235. https:// doi.org/10.3390/plants11091235 Academic Editor: Alex Troitsky Received: 9 April 2022 Accepted: 29 April 2022 Published: 2 May 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). plants Article Genome Insights into Autopolyploid Evolution: A Case Study in Senecio doronicum (Asteraceae) from the Southern Alps Pol Fernández 1,* , Oriane Hidalgo 1,2 , Ana Juan 3, Ilia J. Leitch 2, Andrew R. Leitch 4, Luis Palazzesi 5, Luca Pegoraro 6, Juan Viruel 2and Jaume Pellicer 1,2,* 1 Institut Botànic de Barcelona (IBB, CSIC-Ajuntament de Barcelona), Passeig del Migdia s.n., Parc de Montjuïc, 08038 Barcelona, Spain; [email protected] 2Royal Botanic Gardens, Kew, Kew Green, Richmond TW9 3AE, UK; [email protected] (I.J.L.); [email protected] (J.V.) 3 Departamento de Ciencias Ambientales y Recursos Naturales, Universidad de Alicante, 03080 Alicante, Spain; [email protected] 4School of Biological and Chemical Sciences, Queen Mary University of London, London E1 4NS, UK; a.r[email protected] 5Museo Argentino de Ciencias Naturales, CONICET, División Paleobotánica, Buenos Aires C1405DJR, Argentina; [email protected].ar 6 Biodiversity and Conservation Biology Research Unit, Swiss Federal Institute for Forest, Snow and Landscape Research WSL, 8903 Bimensdorf, Switzerland; luca.pegorar[email protected] * Correspondence: [email protected] (P.F.); [email protected] (J.P.); Tel.: +34-932890611 (P.F. & J.P.) Abstract: Polyploidy is a widespread phenomenon across angiosperms, and one of the main drivers of diversification. Whilst it frequently involves hybridisation, autopolyploidy is also an important feature of plant evolution. Minority cytotypes are frequently overlooked due to their lower frequency in populations, but the development of techniques such as flow cytometry, which enable the rapid screening of cytotype diversity across large numbers of individuals, is now providing a more comprehensive understanding of cytotype diversity within species. Senecio doronicum is a relatively common daisy found throughout European mountain grasslands from subalpine to almost nival elevations. We have carried out a population-level cytotype screening of 500 individuals from Tête Grosse (Alpes-de-Haute-Provence, France), confirming the coexistence of tetraploid (28.2%) and octoploid cytotypes (71.2%), but also uncovering a small number of hexaploid individuals (0.6%). The analysis of repetitive elements from short-read genome-skimming data combined with nuclear (ITS) and whole plastid DNA sequences support an autopolyploid origin of the polyploid S. doronicum individuals and provide molecular evidence regarding the sole contribution of tetraploids in the formation of hexaploid individuals. The evolutionary impact and resilience of the new cytotype have yet to be determined, although the coexistence of different cytotypes may indicate nascent speciation. Keywords: Asteraceae; cytotype; genome size; repetitive DNA; polyploidy; transposable elements 1. Introduction Polyploidy (or whole-genome multiplication—WGM) refers to the coexistence of more than two copies of the genome in a nucleus. It arises from dysfunctional meiotic or mitotic division that results in the formation of unreduced gametes [ 1 ], generating polyploid individuals. Polyploidy is widespread across plants, and it is considered to be a major driver of evolutionary change in angiosperms [ 2 , 3 ]. Through the analysis of chromosome data, it is estimated that about 15% of speciation events in angiosperms involve polyploidy [ 4 ], generating similar frequencies of autopolyploid and allopolyploid species [ 5 ]. Chromosome counts and flow cytometry can efficiently identify recent polyploidisation events; however, they are limited to detect ancient polyploidisation events whose chromosomal signature might have been eroded over time by diploidising processes [ 6 ]. Nevertheless, the advent of new sequencing technologies has resulted in significant progress in our understanding Plants 2022,11, 1235. https://doi.org/10.3390/plants11091235 https://www.mdpi.com/journal/plants Plants 2022,11, 1235 2 of 15 of ancient polyploidy and diploidisation, revealing that WGMs have been more frequent than previously thought. For example, we currently know that the common ancestor of all angiosperms has undergone at least one ancient WGM event, predating their origin and subsequent diversification [ 7 , 8 ]. Since then, multiple episodes of WGM have been estimated along the major plant taxonomic lineages, supporting the role of polyploidy as one of the main engines of plant evolution [9]. Certainly, autopolyploidy results in changes in gene dosage and in levels of gene expression, which can in turn influence tolerance to environmental stress and therefore promote adaptation (e.g., [ 10 – 12 ]). Examples of autopolyploid speciation have been reported in several species, such as Tolmiea menziesii (Pursh) Torr. & A.Gray [ 13 ], Heuchera micrantha Douglas [ 14 ] and Centaurea tentudaica (Rivas Goday) Rivas Goday & Rivas Mart. [ 15 ] (see also a review on the topic by Parisod et al. [ 16 ] for further examples). One of the consequences of autopolyploidy is the reduction, or loss, of gene flow between ploidy levels, especially from higher to lower ploidy levels, although recurrent polyploidy formation may facilitate gene flow to higher ploidy levels. Nevertheless, the barriers to gene flow generated by polyploidy facilitate divergence within ploidy levels, increasing the potential for speciation. Current understanding of the occurrence of autopolyploid complexes has made unprecedented progress through the application of fast and cost-effective tools such as flow cytometry, which enables reliable screening of hundreds of individuals (e.g., [ 17 – 20 ]) in a relatively short period of time. Indeed, large-scale analyses have facilitated the discovery of hidden minority cytotypes (e.g., Elymus L. [ 21 ]), which might have otherwise remained hidden using solely chromosome-based approaches. In parallel, High-Throughput Sequencing (HTS) technologies have contributed significantly to our understanding of polyploid evolution, polyploid complexes and to polyploid genome dynamics [ 22 , 23 ]. For example, repetitive DNA sequences, including transposable elements (TE) and tandemly arranged elements (e.g., satellite DNA), which form a substantial component of plant genomes [ 24 , 25 ], have been shown to diverge independently in polyploid lineages compared with their diploid progenitors [ 26 , 27 ]. Repeat dynamics is not only of interest to study genome organisation, function and evolution [ 28 , 29 ], but is shown to harbour phylogenetic signal among closely related species, based on their abundance and sequence similarity recovered from HTS analysis [ 30 , 31 ]. The question now arises as to whether intraspecific (and intrapopulation) repeat variation can also be identified, and how is it influenced by mechanisms such as polyploidy. Senecio doronicum L. (Asteraceae) is a relatively common species of herbaceous perennial found throughout European mountain ranges between 1000–2400 m of elevation, exceptionally up to 3000 m [ 32 ]. Its habitat spans from alpine meadows to rocky screes (Figure 1). Even though diploids (2n = 20) have been described in the genus, chromosome numbers for the species have been frequently reported as 2n = 40, 80 (i.e., 2n = 4x and 8x). Additionally, higher ploidy levels (e.g., 12x) have also been found anecdotally on gametophytic records according to the Chromosome Counts Database (CCD) [ 33 ]. Aiming at evaluating and testing the existence of minority cytotypes in this species, we conducted a cytotype-screening analysis focusing on a population of the southern Alps (Tête Grosse, Alpes-de-Haute-Provence, France, Figure 2A). This analysis was combined with genome skimming approaches to characterise repetitive DNA across multiple individuals and cytotypes. Specifically, our goals were to assess: (i) the impact of polyploidy on the composition of the repetitive fraction of the genome in S. doronicum, (ii) investigate if there is intraand intercytotype variation in the repetitive DNA content, and (iii) determine to what extent TEs can be used to unravel evolutionary pathways leading to polyploid complexes at the population level, beyond what can be interpreted from traditional nuclear and chloroplast markers. Plants 2022,11, 1235 3 of 15 Plants 2022, 11, x FOR PEER REVIEW 3 of 15 complexes at the population level, beyond what can be interpreted from traditional nuclear and chloroplast markers. Figure 1. Habitat preferences and morphology of tetraploid (A,B,C) and octoploid (D,E,F) cytotypes observed in Senecio doronicum from the population of Tête Grosse (Alpes-de-Haute-Provence, France). Figure 2. (A) Geographical distribution of the population of Senecio doronicum from Tête Grosse ( ). Additional sites sampled in the Southwestern French Alps are also indicated in the map and the cytotypes recovered indicated (4x = , 8x = ). (B) Distribution of individuals in the population of Tête Grosse, colored according to their cytotype. (C) Boxplots depicting the DNA ploidy levels assigned on the basis of relative fluorescence ratios of nuclei. Data Maps from Google Earth: Google Landsat/Copernicus Data SIO, NOAA, U.S. Navy, NGA, GEBCO. GeoBasis-DE/BKG (©2009) Inst. Geogr. Nacional. Figure 1. Habitat preferences and morphology of tetraploid ( A – C ) and octoploid ( D – F ) cytotypes observed in Senecio doronicum from the population of Tête Grosse (Alpes-de-Haute-Provence, France). Plants 2022, 11, x FOR PEER REVIEW 3 of 15 complexes at the population level, beyond what can be interpreted from traditional nuclear and chloroplast markers. Figure 1. Habitat preferences and morphology of tetraploid (A,B,C) and octoploid (D,E,F) cytotypes observed in Senecio doronicum from the population of Tête Grosse (Alpes-de-Haute-Provence, France). Figure 2. (A) Geographical distribution of the population of Senecio doronicum from Tête Grosse ( ). Additional sites sampled in the Southwestern French Alps are also indicated in the map and the cytotypes recovered indicated (4x = , 8x = ). (B) Distribution of individuals in the population of Tête Grosse, colored according to their cytotype. (C) Boxplots depicting the DNA ploidy levels assigned on the basis of relative fluorescence ratios of nuclei. Data Maps from Google Earth: Google Landsat/Copernicus Data SIO, NOAA, U.S. Navy, NGA, GEBCO. GeoBasis-DE/BKG (©2009) Inst. Geogr. Nacional. Figure 2. ( A ) Geographical distribution of the population of Senecio doronicum from Tête Grosse ( Plants 2022, 11, x FOR PEER REVIEW 3 of 15 complexes at the population level, beyond what can be interpreted from traditional nuclear and chloroplast markers. Figure 1. Habitat preferences and morphology of tetraploid (A,B,C) and octoploid (D,E,F) cytotypes observed in Senecio doronicum from the population of Tête Grosse (Alpes-de-Haute-Provence, France). Figure 2. (A) Geographical distribution of the population of Senecio doronicum from Tête Grosse ( ). Additional sites sampled in the Southwestern French Alps are also indicated in the map and the cytotypes recovered indicated (4x = , 8x = ). (B) Distribution of individuals in the population of Tête Grosse, colored according to their cytotype. (C) Boxplots depicting the DNA ploidy levels assigned on the basis of relative fluorescence ratios of nuclei. Data Maps from Google Earth: Google Landsat/Copernicus Data SIO, NOAA, U.S. Navy, NGA, GEBCO. GeoBasis-DE/BKG (©2009) Inst. Geogr. Nacional. ). Additional sites sampled in the Southwestern French Alps are also indicated in the map and the cytotypes recovered indicated (4x = Plants 2022, 11, x FOR PEER REVIEW 3 of 15 complexes at the population level, beyond what can be interpreted from traditional nuclear and chloroplast markers. Figure 1. Habitat preferences and morphology of tetraploid (A,B,C) and octoploid (D,E,F) cytotypes observed in Senecio doronicum from the population of Tête Grosse (Alpes-de-Haute-Provence, France). Figure 2. (A) Geographical distribution of the population of Senecio doronicum from Tête Grosse ( ). Additional sites sampled in the Southwestern French Alps are also indicated in the map and the cytotypes recovered indicated (4x = , 8x = ). (B) Distribution of individuals in the population of Tête Grosse, colored according to their cytotype. (C) Boxplots depicting the DNA ploidy levels assigned on the basis of relative fluorescence ratios of nuclei. Data Maps from Google Earth: Google Landsat/Copernicus Data SIO, NOAA, U.S. Navy, NGA, GEBCO. GeoBasis-DE/BKG (©2009) Inst. Geogr. Nacional. , 8x = Plants 2022, 11, x FOR PEER REVIEW 3 of 15 complexes at the population level, beyond what can be interpreted from traditional nuclear and chloroplast markers. Figure 1. Habitat preferences and morphology of tetraploid (A,B,C) and octoploid (D,E,F) cytotypes observed in Senecio doronicum from the population of Tête Grosse (Alpes-de-Haute-Provence, France). Figure 2. (A) Geographical distribution of the population of Senecio doronicum from Tête Grosse ( ). Additional sites sampled in the Southwestern French Alps are also indicated in the map and the cytotypes recovered indicated (4x = , 8x = ). (B) Distribution of individuals in the population of Tête Grosse, colored according to their cytotype. (C) Boxplots depicting the DNA ploidy levels assigned on the basis of relative fluorescence ratios of nuclei. Data Maps from Google Earth: Google Landsat/Copernicus Data SIO, NOAA, U.S. Navy, NGA, GEBCO. GeoBasis-DE/BKG (©2009) Inst. Geogr. Nacional. ). ( B ) Distribution of individuals in the population of Tête Grosse, colored according to their cytotype. ( C ) Boxplots depicting the DNA ploidy levels assigned on the basis of relative fluorescence ratios of nuclei. Data Maps from Google Earth: Google Landsat/Copernicus Data SIO, NOAA, U.S. Navy, NGA, GEBCO. GeoBasis-DE/BKG ( © 2009) Inst. Geogr. Nacional. Plants 2022,11, 1235 4 of 15 2. Results 2.1. Cytotype Screening of Senecio doronicum Identifies Three Ploidies in Tête Grosse Our flow-cytometry DNA ploidy screening confirmed the coexistence of tetraploid and octoploid cytotypes in the population (Supplementary Table S1), with the latter being over 2.5x more abundant (i.e., 141 tetraploid individuals versus 356 octoploid individuals), and with very little overlap in the distribution of each cytotype across the population (Figure 2B,C). Additionally, the analyses uncovered three hexaploid individuals growing close together (<5 cm apart), which displayed intermediate DNA ratios (i.e., genome sizes) compared to those of tetraploids and octoploids (Figure 2C). Genome sizes (Mb/1C) estimated for each ploidy level were as follows: 4x (4205.4 ± 19.56), 6x (6357 ± 29.34), 8x (8097.84 ± 48.9). The survey conducted on 36 accessions from outside Tête Grosse, in neighbouring mountain valleys, also reported a high incidence of octoploid individuals (32 out of 36; Supplementary Table S2). 2.2. Repetitive DNA Content in S. doronicum A representative summary of the results of one individual per ploidy level is illustrated in Figure 3and given in Table 1. The proportion of the genome containing repetitive elements was almost identical for the three cytotypes (i.e., mean: 85.54% ( ± 0.32%)). Around 19–20% of the repetitive genome were unclassified elements. Ty3/Gypsy-like Long Terminal Repeats (LTR) elements dominate the repetitive landscape of each ploidy level, with genomic proportions (GP) of 46.86% in 4x, 42.02% in 8x and 44.06% in 6x, followed by Ty1/Copia LTRs (see Table 1for detailed composition of the different repeat lineages comprising these LTR repeat superfamilies). Among four lineages of Ty3/Gypsy LTR elements identified, the chromovirus-type Tekay element was by far the most abundant (ranging between 39.08% to 43.85%). The Ty1/Copia superfamily was represented by seven lineages, with SIRE elements being the most abundant (GP: 13.08–13.60%, Table 1). Plants 2022, 11, x FOR PEER REVIEW 5 of 15 Figure 3. Genomic composition of Senecio doronicum representative of each cytotype (4x, 6x and 8x). Estimates of the genomic abundances (in Mb/1C) of different repeats are indicated and colored by repeat class [GP = Genome proportion (%), LTR = Long Terminal Repeat]. Table 1. Repetitive DNA composition estimated in individuals TG422 (4x), TG507 (6x) and TG84 (8x) as illustrative of the overall dynamics reported across cytotypes. Genome Proportion (GP) 4x 6x 8x Repeat Type Lineage [%] [Mb] [%] [Mb] [%] [Mb] Ty1/Copia 14.51 610.40 14.86 944.78 15.28 1,237.56 SIRE 13.08 550.07 13.34 848.01 13.60 1,101.70 Angela 0.97 40.96 1.11 70.64 0.95 76.83 TAR 0.11 4.64 0.10 6.43 0.14 11.51 Bianca 0.06 2.44 0.06 3.99 0.31 24.73 Ale 0.03 1.24 0.01 0.82 0.03 2.81 Tork 0.06 2.37 0.04 2.48 0.06 4.60 Ikeros 0.21 8.68 0.20 12.42 0.19 15.39 Ty3/Gypsy 46.86 1,970.85 44.02 2,798.58 42.06 3,406.09 Tekay 43.85 1,843.88 40.61 2,581.82 39.08 3,164.91 Athila 1.67 70.09 1.60 101.55 1.56 126.28 CRM 0.39 16.41 0.42 26.46 0.36 29.44 Retand 0.96 40.47 1.40 88.76 1.06 85.47 LTR-unclassified 3.39 142.61 5.49 348.91 6.42 519.65 Other repeats Pararetrovirus 0.01 0.46 0.22 13.93 0.22 17.71 DNA transposons 0.61 25.54 0.71 44.93 0.56 45.12 TIR/Enspm-CACTA 0.00 0.00 0.00 0.00 0.00 0.00 TIR/MuDR-Mutator 0.40 16.71 0.49 31.33 0.34 27.15 TIR/haT 0.18 7.59 0.16 10.00 0.17 13.62 TIR/PIF-Harbinger 0.03 1.24 0.06 3.60 0.05 4.35 Tandem repeats Ribosomal DNA 0.28 11.76 0.40 25.35 0.25 19.97 Satellite 0.37 15.64 0.62 39.16 0.45 36.71 Unclassified repeat clusters (GP ≥ 0.01%) 5.92 249.17 5.64 358.81 5.94 480.84 Small unclassified clusters (GP < 0.01%) 13.49 567.21 13.95 886.71 14.10 1141.67 Total repeats 85.45 3,593.65 85.91 5,461.17 85.27 6,905.31 Single copy 14.55 611.75 14.09 895.83 14.73 1,192.53 Figure 3. Genomic composition of Senecio doronicum representative of each cytotype (4x, 6x and 8x). Estimates of the genomic abundances (in Mb/1C) of different repeats are indicated and colored by repeat class [GP = Genome proportion (%), LTR = Long Terminal Repeat]. Non-LTR retrotransposons were only represented by Pararetrovirus repeats, occurring in very low abundance (i.e., ≤ 0.22% GP). DNA transposons included Mutator, haT and Harbinger lineages, with Mutator being the most prevalent in all three cytotype accessions (GP: 0.34–0.49%, Table 1). (N.B. Details on the number of reads analysed for the 12 individuals comprising five tetraploids, three hexaploids and four octoploids and the classification and GP of the highly repetitive elements identified in their genomes are available in Tables S3 and S4 and Figure S1). Despite some variation observed in the GP of DNA repeats between the individuals analysed (Table S4), we did not find significant Plants 2022,11, 1235 5 of 15 differences in the composition of the repetitive genomes between the three cytotypes (permanova p= 0.41). Moreover, linear regressions of the number of reads of each repeat type between all cytotype combinations, had an R 2 around 0.99, with a high level of statistical significance (Figure 4and Table 2, see also Figure S2 for regressions focusing on Ty1/Copia and Ty3/Gypsy elements). Table 1. Repetitive DNA composition estimated in individuals TG422 (4x), TG507 (6x) and TG84 (8x) as illustrative of the overall dynamics reported across cytotypes. Genome Proportion (GP) 4x 6x 8x Repeat Type Lineage [%] [Mb] [%] [Mb] [%] [Mb] Ty1/Copia 14.51 610.40 14.86 944.78 15.28 1237.56 SIRE 13.08 550.07 13.34 848.01 13.60 1101.70 Angela 0.97 40.96 1.11 70.64 0.95 76.83 TAR 0.11 4.64 0.10 6.43 0.14 11.51 Bianca 0.06 2.44 0.06 3.99 0.31 24.73 Ale 0.03 1.24 0.01 0.82 0.03 2.81 Tork 0.06 2.37 0.04 2.48 0.06 4.60 Ikeros 0.21 8.68 0.20 12.42 0.19 15.39 Ty3/Gypsy 46.86 1970.85 44.02 2798.58 42.06 3406.09 Tekay 43.85 1843.88 40.61 2581.82 39.08 3164.91 Athila 1.67 70.09 1.60 101.55 1.56 126.28 CRM 0.39 16.41 0.42 26.46 0.36 29.44 Retand 0.96 40.47 1.40 88.76 1.06 85.47 LTR-unclassified 3.39 142.61 5.49 348.91 6.42 519.65 Other repeats Pararetrovirus 0.01 0.46 0.22 13.93 0.22 17.71 DNA transposons 0.61 25.54 0.71 44.93 0.56 45.12 TIR/Enspm-CACTA 0.00 0.00 0.00 0.00 0.00 0.00 TIR/MuDR-Mutator 0.40 16.71 0.49 31.33 0.34 27.15 TIR/haT 0.18 7.59 0.16 10.00 0.17 13.62 TIR/PIF-Harbinger 0.03 1.24 0.06 3.60 0.05 4.35 Tandem repeats Ribosomal DNA 0.28 11.76 0.40 25.35 0.25 19.97 Satellite 0.37 15.64 0.62 39.16 0.45 36.71 Unclassified repeat clusters (GP ≥0.01%) 5.92 249.17 5.64 358.81 5.94 480.84 Small unclassified clusters (GP < 0.01%) 13.49 567.21 13.95 886.71 14.10 1141.67 Total repeats 85.45 3593.65 85.91 5461.17 85.27 6905.31 Single copy 14.55 611.75 14.09 895.83 14.73 1192.53 The variation in TE composition between individuals with the same cytotype were also small; in all cases differences were not significant (paired Wilcoxon tests p= 0.3–0.89) (Table S5), confirming high levels of similarity in the repetitive DNA content regardless of the ploidy level. The same trend was observed when comparing samples from Tête Grosse population with those analysed from outside the population (Table S5). Plants 2022,11, 1235 6 of 15 Plants 2022, 11, x FOR PEER REVIEW 6 of 15 Non-LTR retrotransposons were only represented by Pararetrovirus repeats, occurring in very low abundance (i.e., ≤0.22% GP). DNA transposons included Mutator, haT and Harbinger lineages, with Mutator being the most prevalent in all three cytotype accessions (GP: 0.34–0.49%, Table 1). (N.B. Details on the number of reads analysed for the 12 individuals comprising five tetraploids, three hexaploids and four octoploids and the classification and GP of the highly repetitive elements identified in their genomes are available in Tables S3 and S4, and Figure S1). Despite some variation observed in the GP of DNA repeats between the individuals analysed (Table S4), we did not find significant differences in the composition of the repetitive genomes between the three cytotypes (permanova p = 0.41). Moreover, linear regressions of the number of reads of each repeat type between all cytotype combinations, had an R2 around 0.99, with a high level of statistical significance (Figure 4 and Table 2, see also Figure S2 for regressions focusing on Ty1/Copia and Ty3/Gypsy elements). Figure 4. Pairwise scatterplot comparisons of the number of reads included in repeat clusters from each cytotype. The slope indicates the genome size ratio between each cytotype. (A) 4x vs. 6x. (B) 4x vs. 8x. (C) 8x vs. 6x. Table 2. Statistics for the linear regression analyses carried out between cytotype pairs based on an analysis of the number of reads of Ty1/Copia-like elements, Ty3/Gypsy-like elements, and all repetitive elements (All). (SE: Standard error, Sig.: *** p-value < 0.0001). 8x–6x 6x–4x 8x–4x Slope SE R2 Sig. Slope SE R2 Sig. Slope SE R2 Sig. Ty1/Copia 1.37 0.01 0.996 *** 1.5 0.01 0.999 *** 2.06 0.02 0.996 *** Ty3/Gypsy 1.3 0.01 0.999 *** 1.5 0 0.999 *** 1.95 0.01 0.998 *** All 1.31 0 0.997 *** 1.5 0 0.999 *** 1.97 0.01 0.997 *** The variation in TE composition between individuals with the same cytotype were also small; in all cases differences were not significant (paired Wilcoxon tests p = 0.3–0.89) (Table S5), confirming high levels of similarity in the repetitive DNA content regardless of the ploidy level. The same trend was observed when comparing samples from Tête Grosse population with those analysed from outside the population (Table S5). 2.3. Phylogenetic Implications of Cytotype Diversity in S. doronicum To confirm that all recovered cytotypes belong to the same species, and to discard any individuals which showed evidence of hybridisation with closely related species, we conducted a phylogenetic analysis using the internal transcribed spacer (ITS) on a dataset comprising all 4x, 6x and 8x individuals analysed here and the extended sampling of the European clade of Senecio section Crociseris (Rchb.) Boiss. by Calvo et al. [34]. Both Neighbour-Net (NN) and Neighbour-Joining (NJ) trees confirmed that all analysed samples fell within S. doronicum (Figure 5 A,B), but were embedded in different subclades, supporting an autopolyploid scenario, whilst indicating that octoploids most likely arose from Figure 4. Pairwise scatterplot comparisons of the number of reads included in repeat clusters from each cytotype. The slope indicates the genome size ratio between each cytotype. ( A ) 4x vs. 6x. ( B ) 4x vs. 8x. (C) 8x vs. 6x. Table 2. Statistics for the linear regression analyses carried out between cytotype pairs based on an analysis of the number of reads of Ty1/Copia-like elements, Ty3/Gypsy-like elements, and all repetitive elements (All). (SE: Standard error, Sig.: *** p-value < 0.0001). 8x–6x 6x–4x 8x–4x Slope SE R2Sig. Slope SE R2Sig. Slope SE R2Sig. Ty1/Copia 1.37 0.01 0.996 *** 1.5 0.01 0.999 *** 2.06 0.02 0.996 *** Ty3/Gypsy 1.3 0.01 0.999 *** 1.5 0 0.999 *** 1.95 0.01 0.998 *** All 1.31 0 0.997 *** 1.5 0 0.999 *** 1.97 0.01 0.997 *** 2.3. Phylogenetic Implications of Cytotype Diversity in S. doronicum To confirm that all recovered cytotypes belong to the same species, and to discard any individuals which showed evidence of hybridisation with closely related species, we conducted a phylogenetic analysis using the internal transcribed spacer (ITS) on a dataset comprising all 4x, 6x and 8x individuals analysed here and the extended sampling of the European clade of Senecio section Crociseris (Rchb.) Boiss. by Calvo et al. [ 34 ]. Both Neighbour-Net (NN) and Neighbour-Joining (NJ) trees confirmed that all analysed samples fell within S. doronicum (Figure 5A,B), but were embedded in different subclades, supporting an autopolyploid scenario, whilst indicating that octoploids most likely arose from tetraploids outside Tête Grosse. In contrast, we found that tetraploid and hexaploid individuals were grouped together in the same subclade, mixed with closely related accessions from the Alpes Maritimes. Octoploid individuals were clustered together in a separate subclade with accessions of Jacobaea kirghisica (DC.) E.Wiebe and S. doronicum from other populations of the Alps and Jura. The splitstree NN also supported these relationships between Tête Grosse individuals (i.e., octoploids belonging to a separate clade from the tetraploids and hexaploids, Figure 5B). Additionally, nuclear evidence was complemented by the analysis of conserved regions in a satellite DNA repeat (207 bp) shared between all analysed individuals from Tête Grosse. From the alignment matrix, we found that all tetraand hexaploid individuals shared exactly the same sequence while octoploids shared five unique SNPs. Plastid DNA reconstruction was carried out on all 12 individuals, generating plastid genome sequences ranging between 151,196 and 151,222 bp. The comparison of plastid sequences between tetraploid and hexaploid individuals from Tête Grosse revealed 0–15 SNPs between the eight individuals analysed, with individual TG370 being the most divergent, although it still grouped with the other seven individuals analysed. In contrast, among octoploid individuals, 14–30 SNPs were found. Further, when comparing the plastid Plants 2022,11, 1235 7 of 15 sequences of hexaploids and tetraploids, to octoploids from Tête Grosse, 66–87 variants were found. The plastid sequences of individuals outside Tête Grosse [FR626 and FR627 (4x), FR475 (8x)] were more similar to each other, regardless of ploidy level, than to the individuals of the same cytotype from the Tête Grosse site (Figure 5C). The NN reconstruction also supported the very close relationship between tetraploid and hexaploid individuals from Tête Grosse, while octoploids from this population appeared clustered in a different lineage (Figure 5C). Phylogenetic analyses carried out using TE outputs from RepeatExplorer2 failed to produce any systematic signal at the population level (data not shown), given the high similarity of the repetitive DNA elements between individuals and ploidy levels. Plants 2022, 11, x FOR PEER REVIEW 7 of 15 tetraploids outside Tête Grosse. In contrast, we found that tetraploid and hexaploid individuals were grouped together in the same subclade, mixed with closely related accessions from the Alpes Maritimes. Octoploid individuals were clustered together in a separate subclade with accessions of Jacobaea kirghisica (DC.) E.Wiebe and S. doronicum from other populations of the Alps and Jura. The splitstree NN also supported these relationships between Tête Grosse individuals (i.e., octoploids belonging to a separate clade from the tetraploids and hexaploids, Figure 5B). Additionally, nuclear evidence was complemented by the analysis of conserved regions in a satellite DNA repeat (207 bp) shared between all analysed individuals from Tête Grosse. From the alignment matrix, we found that all tetraand hexaploid individuals shared exactly the same sequence while octoploids shared five unique SNPs. Figure 5. Neighbour joining tree (A) and Neighbour net analysis (B) based on ITS sequences of European clade of Senecio sect. Crociseris from Calvo et al. [34], and including 4x, 6x and 8x individuals of Senecio doronicum from the present study. (C) Neighbour net analysis of whole plastid sequences among those same individuals sequenced in our study. Dashed line separates individuals of Tête Grosse from other populations in the area. Capital letters indicate genetic clusters. Plastid DNA reconstruction was carried out on all 12 individuals, generating plastid genome sequences ranging between 151,196 and 151,222 bp. The comparison of plastid sequences between tetraploid and hexaploid individuals from Tête Grosse revealed 0–15 SNPs between the eight individuals analysed, with individual TG370 being the most divergent, although it still grouped with the other seven individuals analysed. In contrast, among octoploid individuals, 14–30 SNPs were found. Further, when comparing the plastid sequences of hexaploids and tetraploids, to octoploids from Tête Grosse, 66–87 variants were found. The plastid sequences of individuals outside Tête Grosse [FR626 and FR627 (4x), FR475 (8x)] were more similar to each other, regardless of ploidy level, than to the individuals of the same cytotype from the Tête Grosse site (Figure 5C). The NN reconstruction also supported the very close relationship between tetraploid and Figure 5. Neighbour joining tree ( A ) and Neighbour net analysis ( B ) based on ITS sequences of European clade of Senecio sect. Crociseris from Calvo et al. [ 34 ], and including 4x, 6x and 8x individuals of Senecio doronicum from the present study. ( C ) Neighbour net analysis of whole plastid sequences among those same individuals sequenced in our study. Dashed line separates individuals of Tête Grosse from other populations in the area. Capital letters indicate genetic clusters. 3. Discussion 3.1. Flow Cytometry Uncovers the Existence of Minority Cytotypes in S. doronicum Our flow-cytometry-based cytotype screening not only confirmed the coexistence of tetraploids and octoploids in Tête Grosse, but also uncovered the existence of a novel minority hexaploid cytotype, illustrating the utility of this kind of approach in detecting rare cytotypes. Both autoand allopolyploid speciation in the genus Senecio has been frequently reported, and the consequences at chromosome and gene levels investigated (see review [ 35 ]). Whilst diploid species are reported to occur in the genus, S. doronicum has so far only been reported to exist at higher ploidy levels [ 33 ]. To a large extent, and based on our field observations across the southwestern Alps, the results reported here in Plants 2022,11, 1235 8 of 15 Tête Grosse mimic the dynamics observed more broadly for the species, where octoploids are more abundant and widely distributed than tetraploids (Figure 2A,B). Indeed, 70% of the individuals surveyed in the Tête Grosse population were octoploids. Each cytotype showed evidence of distinct habitat preference in the population studied (Figure 1), which is illustrated by the little overlap in distribution of tetraploids and octoploids across the site (Figure 2B). Whether tetraploids are, in general, being outcompeted by octoploids is still an open question, and requires future extensive samplings across the Alps. One possible explanation for such a scenario could be that octoploids have more efficient dispersal mechanisms than tetraploids. Increased rates of self-pollination and efficient local dispersal of polyploid Ranunculus adoneus A. Gray have been key for increased persistence and long-term expansion compared to diploids [ 36 ]. Despite both cytotypes being reported to reproduce sexually by seeds [ 37 ], clonal reproduction cannot be ruled out given the high frequency of individuals that we observed growing together (in clumps). Based on such observations, it is possible that differential rates of sexual versus asexual reproduction between cytotypes may be an important factor contributing to the contrasting evolutionary successes of the cytotypes, which in the long term may contribute to the higher frequency of the octoploids over tetraploids in the genus. To our knowledge, the summit of Tête Grosse is one of the very few sites where both ploidy levels coexist, with other populations consisting mostly of scattered and isolated tetraploid individuals among octoploids (Figure 2A). The higher abundance of tetraploids at Tête Grosse means that opportunities for inter-cytotype hybridisation are more likely. Indeed, we observed that despite each cytotype having relatively different flowering periods, there was some overlap (c. 10 days) in flowering time between them (Pegoraro et al., unpublished), thus a window of opportunity for potential pollen exchange between cytotypes exists. Based on these observations, two main scenarios for the rise of the hexaploid individuals in the population can be considered, including a single cytotype origin or intercytotype crossing (discussed below). The very low number of hexaploid individuals found (3 out of 500 in the population) could represent the early stages of the establishment of hexaploids, but future surveys will be needed to confirm whether this is the case or just an isolated event, and potentially an evolutionary dead end. 3.2. Correlation of TE Amounts among Ploidy Levels Supports the Autopolyploid Evolution in S. doronicum Among the repeat clusters identified using RepeatExplorer2, the majority were classified as Ty3/Gypsy and Ty1/Copia superfamilies, supporting the dominance of LTR retrotransposons across land-plant genomes. An overall dominance of Ty3/Gypsy-like elements has been reported in many plant genomes, including species of Asteraceae [ 38 ], but there are exceptions such as in Urospermum Scop. (in a different tribe from Senecio), where Ty1/Copia elements are the major contributors [ 39 ]. In S. doronicum, by far the most abundant repeats were Ty3/Gypsy-Tekay elements, with a GP of c. 43%. Indeed, these repeats comprised more than double the GP of the second most abundant repeat lineage which was the Ty1/Copia SIRE elements (GP c. 13%, Table 1). Such analyses highlight the important role of Tekay repeats in shaping the genome of S. doronicum. At the population scale, the repetitive landscape of the individuals analysed was highly conserved, both within cytotypes and between accessions of different ploidy levels ( Figures 3and S1 , Tables S4 and S5 ). This was evidenced by the significant correlations observed, either when ‘all repeats’ were analysed (Figure 4, Table 2) or when the Ty3/Gypsy and Ty1/Copia repeat classes were analysed separately (Table 2, Figure S2). Together, these data provide support for an autopolyploid origin of both hexaand octoploid S. doronicum. One of the many outcomes of autopolyploidy is an increase in the amount of TEs due to the increased number of genome copies within the cell. However, mechanisms driving TE dynamics following WGM and their impact at evolutionary scales are diverse, and vary between autoand allopolyploids and between species (reviewed in [ 16 , 40 ]). For example, whilst here we observed the above-mentioned significant—and almost linear—correlation Plants 2022,11, 1235 9 of 15 between DNA repeats across ploidy levels, this is not necessarily the case in other plant species following autopolyploidy (although studies are limited). For example, a study of 300 autopolyploid Arabidopsis arenosa (L.) Lawalrée showed evidence of differential accumulation of TEs in different individuals, potentially influencing patters of gene expression and providing opportunities for local adaptations [ 41 ]. However, no such changes in repeat dynamics associated with cytotype was observed in S. doronicum, perhaps because they are recently formed cytotype variants. Although most studies of how repeat dynamics change in response to WGM come from the study of allopolyploids [ 40 ], which have increased complexity due to the associated interspecific hybridisation, the data are highlighting that genome upsizing and downsizing associated with changes in repetitive DNA activity can change over time [ 41 ]. These results therefore emphasise the importance of considering how the tempo of polyploidisation will impact the overall composition and organisation of a polyploid genome, rather than the actual polyploidisation process itself [ 42 ]. Evidence from population-wide comparative approaches is now needed to more fully evaluate the changes in the composition and organisation of repetitive elements following polyploidy over time, particularly in autopolyploids, which are very limited. 3.3. Unreduced Gamete Formation in Tetraploid Individuals as the Most Likely Origin of the Hexaploid Cytotype As indicated above, two potential yet exclusive evolutionary scenarios can be invoked to explain the origin of hexaploid individuals of S. doronicum in Tête Grosse: Scenario 1-they have arisen from an inter-cytotype cross involving reduced gametes from both the tetraploid and octoploid, and Scenario 2-their origin solely involves tetraploid individuals, arising from a cross between reduced and unreduced gametes (which have been reported in CCD). To distinguish between these two pathways we note the following: (i) The NN splitstree analysis of the whole plastid sequence data (Figure 5C) and the SNP counting from both the satellite and plastid DNA suggest an extremely close relationship between tetraploid and hexaploid individuals; (ii) Bearing in mind that the plastid genome is maternally inherited without meiosis [ 43 ], we assume that at least, the maternal contribution to the hexaploids came from the original tetraploid gene pool at Tête Grosse; (iii) The nuclear data (ITS) also support a close relationship between tetraploids and hexaploids based on the reduced number of SNPs from the satellite analysis, and from the phylogenetic inferences depicted in Figure 5A where no ITS variants were found in hexaploids. In summary, and taking all these observations together, we propose that hexaploid individuals on Tete Grôsse most likely arose via Scenario 2. Given the small number of SNPs, together with the low number of hexaploid individuals found in this population (just three out of 500 analysed), the data also suggest that these hexaploid individuals may have arisen very recently. Many of the examples of autopolyploid evolution reported in the literature involve diploid individuals that produce unreduced gametes leading to polyploid cytotypes (see review [ 16 ]). In contrast, our analysis suggests an origin arising from the tetraploid level. Minority cytotypes must overcome competition disadvantage and stochastic effects prior to becoming established, a process that can be favoured if recurrent WGM takes place. Certainly, the very low number of hexaploid individuals observed suggest that their longterm survival may be limited unless the production of unreduced gametes is high and/or there is the potential for vegetative growth or reproduction via apomictic pathways [ 44 ], although the latter does not seem to be present in the species [ 37 ]. In contrast, phylogenetic analyses (Figure 5A–C) indicate that octoploids have arisen from different populations of S. doronicum . Thus, their occurrence in sympatry suggests that long-distance dispersal has brought the populations together. The linear boundary between tetraploids and octoploids at Tête Grosse (Figure 2B) suggests expansion of one cytotype, perhaps to the detriment of the other.