Faruk Bogunić
email: f.bogunic@sfsa.unsa.ba

Sonja Siljak-Yakovl

Irma Mahmutović-Dizdarević
email: irma.m@pmf.unsa.ba

Alma Hajrudinović-Bogunić

Mickaël Bourge
email: mickael.bourge@i2bc.paris-saclay.fr

Spencer C Brown
email: spencer.brown@sfr.fr

Edina Muratović
email: edina.muratovic@pmf.unsa.ba

Genome Size, Cytotype Diversity and Reproductive Mode Variation of Cotoneaster integerrimus (Rosaceae) from the Balkans

Keywords: apomixis, Balkans, clonality, Cotoneaster integerrimus, cytotype, genome size, polyploidy

Cotoneaster integerrimus represents a multiploid and facultative apomictic system of widely distributed mountain populations. We used flow cytometry to determine genome size, ploidy level, and reproduction mode variation of the Balkan populations, supplemented by analysis of nuclear microsatellites in order to address: (i) geographic distribution and variation of cytotypes among the populations; (ii) variation of reproduction mode and the frequency of sexuality; (iii) pathways of endosperm formation among the sampled polyploids and their endosperm balance requirements; (iv) genotypic diversity and geographic distribution of clonal lineages of polyploids. The prevalence of apomictic tetraploid cytotype followed by sexual diploids and extremely rare triploids was demonstrated. This prevalence of tetraploids affected the populations' structure composed from clonal genotypes with varying proportions. The co-occurrence of diploids and tetraploids generated higher cytotype, reproductive mode, and genotypic diversity, but mixed-ploidy sites were extremely rare. The endosperm imbalance facilitates the development and the occurrence of intermediate triploids in mixed-ploidy populations, but also different tetraploid lineages elsewhere with unbalanced endosperm. All these results showed that the South European populations of C. integerrimus have higher levels of cytotype and reproductive diversity compared to the Central European ones. Therefore, the South European populations can be considered as a potential reservoir of regional and global diversity for this species.

Introduction

Genome size, as a fundamental biological characteristic of evolutionary significance, has implications on overall biodiversity [START_REF] Pellicer | Genome size diversity and its impact on the evolution of land plants[END_REF]. The causes of genome size variation in plants are varied, but the most striking is polyploidization. The evolutionary significance of polyploidy has tremendous and far-reaching implications for plant diversity [START_REF] Van De Peer | The evolutionary significance of polyploidy[END_REF]. It is assumed that almost 50% of plant groups have been affected by at least a single polyploidization event throughout their evolutionary history [START_REF] Soltis | The polyploidy revolution then and now: Stebbins revisited[END_REF]. Polyploidy recomposes genome structure, alters gene expression, induces phenotypic and physiological changes, and provide adaptive potential to the polyploid plant [START_REF] Ďurkovič | Leaf traits in parental and hybrid species of Sorbus (Rosaceae)[END_REF][START_REF] Soltis | Polyploidy and angiosperm diversification[END_REF][START_REF] Hegarty | Genomic clues to the evolutionary success of polyploid plants[END_REF]. Allopolyploidy is considered to be more frequent than autopolyploidy [START_REF] Parisod | Evolutionary consequences of autopolyploidy[END_REF], but both processes profoundly shape plant diversity, either independently or in concert ( [START_REF] Barker | On the relative abundance of autopolyploids and allopolyploids[END_REF], and references therein).

One of the consequences of being a polyploid is breakdown of self-incompatibility, allowing a shift towards asexual reproductive mode [START_REF] Dobeš | Asymmetric reproductive interference: The consequences of cross-pollination on reproductive success in sexual-apomictic populations of Potentilla puberula (Rosaceae)[END_REF][START_REF] Hörandl | The evolution of self-fertility in apomictic plants[END_REF][START_REF] Barrett | The evolution, maintenance, and loss of self-incompatibility systems[END_REF]. Apomixis (asexual seed formation) represents an effective life strategy by which apomicts preserve and maintain hybrid and heterozygous genetic lineages, cytotypes with unbalanced chromosomes, enabling their long-term persistence and dispersal [START_REF] Hörandl | The complex causality of geographical parthenogenesis[END_REF]. Apomixis is highly correlated with polyploidy and hybridity. However, it is still unclear as to which of these factors precedes and what exact genetic and developmental mechanisms regulate the emergence of apomixis [START_REF] Hojsgaard | The rise of apomixis in natural plant populations[END_REF].

Gametophytic apomixis, the most common type of asexual reproduction, represents the formation and development of unreduced megagametophyte (parthenogenesis) coupled with the fertilization of unreduced central cell (pseudogamy) [START_REF] Hörandl | Understanding the geographic distributions of apomictic plants: A case for a pluralistic approach[END_REF][START_REF] Ozias-Akins | Mendelian genetics of apomixis in plants[END_REF]. As a consequence of bypassing meiosis and lack of genome recombination, apomicts have a genetic structure identical to the maternal plant, thus creating clonal populations. Apomictic polyploids show a greater colonization ability by occupying more extreme ecological niches and persisting in larger distribution areas (geographic parthenogenesis, [START_REF] Hörandl | Understanding the geographic distributions of apomictic plants: A case for a pluralistic approach[END_REF]) than their diploid sexual relatives [START_REF] Mráz | Greater reproductive assurance of asexual plant compared to sexual relative in a low density sympatric population-experimental evidence for pollen limitation[END_REF][START_REF] Alonso-Marcos | Difference in reproductive mode rather than ploidy explains niche differentiation in sympatric sexual and apomictic populations of Potentilla puberula[END_REF][START_REF] Coughlan | Widespread generalist clones are associated with range and niche expansion in allopolyploids of Pacific Northwest Hawthorns (Crataegus L.)[END_REF]. Although genetically uniform, apomicts acquire genetic variation over time via accumulation of spontaneous mutations and via residual sexuality fostering diversity within clonal populations [START_REF] Hojsgaard | A little bit of sex matters for genome evolution in asexual plants[END_REF][START_REF] Majeský | The pattern of genetic variability in apomictic clones of Taraxacum officinale indicates the alternation of asexual and sexual histories of apomicts[END_REF][START_REF] Paun | The role of hybridization, polyploidization and glaciation in the origin and evolution of Ranunculus cassubicus complex[END_REF]. One of the mechanisms enhancing genetic diversity of apomictic populations comes from crosses between asexual and related sexual lineages, resulting in offspring that have a predominant apomictic mode of reproduction [START_REF] Alonso-Marcos | Difference in reproductive mode rather than ploidy explains niche differentiation in sympatric sexual and apomictic populations of Potentilla puberula[END_REF][START_REF] Lepší | Versatility of reproductive modes and ploidy level interactions in Sorbus s.l. (Malinae, Rosaceae)[END_REF][START_REF] Hajrudinović | When sexual meets apomictgenome size, ploidy level and variation of reproductive mode in Sorbus aria s.l. and S. austriaca (Rosaceae) in Bosnia and Herzegovina[END_REF]. Given the long-life cycle of woody species, apomictic reproduction and independence of mates is extremely important in the early stages of the establishment of new populations and colonization of new environments.

Genus Cotoneaster Medik. (Rosaceae, Spiroaeoideae, Pyreae, [START_REF] Potter | Phylogeny and classification of Rosaceae[END_REF]) includes shrubs and some small trees that are distributed in Europe, North Africa, and temperate areas of Asia (except Japan) [START_REF] Fryer | Cotoneasters: A Comprehensive Guide to Shrubs for Flowers, Fruit, and Foliage[END_REF]. The species number varies greatly among authors and ranges from 50-70 [START_REF] Dickoré | Species of Cotoneaster (Rosaceae, Maloideae) indigenous to, naturalising or commonly cultivated in Central Europe[END_REF], to ≈90 [START_REF] Lu | Flora of China[END_REF], to ≈400 [START_REF] Fryer | Cotoneasters: A Comprehensive Guide to Shrubs for Flowers, Fruit, and Foliage[END_REF]. Evolutionary history and diversification of Cotoneaster have been affected by homoploid and heteroploid hybridization, as well as autopolyploidy and apomixis [START_REF] Fryer | Cotoneasters: A Comprehensive Guide to Shrubs for Flowers, Fruit, and Foliage[END_REF][START_REF] Meng | Phylogenomic analyses based on genome-skimming data reveal cyto-nuclear discordance in the evolutionary history of Cotoneaster (Rosaceae)[END_REF][START_REF] Bourge | Cytotypes and reproductive modes of Cotoneaster integerrimus (Rosaceae) from Bosnia and Herzegovina: Preliminary results[END_REF][START_REF] Macková | Insights into the cytotype and reproductive puzzle of Cotoneaster integerrimus in the Western Carpathians[END_REF][START_REF] Li | Molecular evidence for natural hybridization between Cotoneaster dielsianus and C. glaucophyllus[END_REF][START_REF] Rothleutner | Ploidy levels, relative genome sizes, and base pair composition in Cotoneaster[END_REF][START_REF] Nybom | DNA markers and morphometry reveal multiclonal and poorly defined taxa in an apomictic Cotoneaster species complex[END_REF][START_REF] Kroon | Polyploidy in Cotoneaster II[END_REF][START_REF] Sax | Polyploidy and apomixis in Cotoneaster[END_REF]. Both classical chromosome countings and cytometric analyses confirmed a series of ploidy levels in the genus ranging from diploids to hexaploids, with a prevalence of tetraploids.

Apomixis has been proven to be the most frequent reproductive mode in Cotoneaster that was primarily inferred from embryological data [START_REF] Hjelmquist | Embryo sac development of Cotoneaster[END_REF][START_REF] Mandryk | Seed reproduction features in Cotoneaster melanocarpus Fisch. ex Blytt. (Rosaceae) from Ukrainian Carpathian flora. Ukr. Botan[END_REF][START_REF] Mandryk | Embryologic investigation of Cotoneaster melanocarpus Fisch. ex Blytt (Rosaceae)[END_REF], breeding experiments [START_REF] Flinck | Cotoneaster harrysmithii, a new species from western China[END_REF], and later by molecular analyses [START_REF] Li | Molecular evidence for natural hybridization between Cotoneaster dielsianus and C. glaucophyllus[END_REF][START_REF] Nybom | DNA markers and morphometry reveal multiclonal and poorly defined taxa in an apomictic Cotoneaster species complex[END_REF]. Recently, apomixis has been confirmed using flow cytometry and different reproductive pathways of seed formation in tetraploids of C. integerrimus Medik. [START_REF] Bourge | Cytotypes and reproductive modes of Cotoneaster integerrimus (Rosaceae) from Bosnia and Herzegovina: Preliminary results[END_REF][START_REF] Macková | Insights into the cytotype and reproductive puzzle of Cotoneaster integerrimus in the Western Carpathians[END_REF].

Key information on embryo sac development of several tri-and tetraploid Cotoneaster species (C. rosea Edgew., C. nitens Rehd. & Wils., C. bullata Bois, C. obscura Rehd. & Wils., C. acutifolia var. villosula Rehd. & Wils., C. racemiflora var. soongorica Schneid.) was provided in 1962 [START_REF] Hjelmquist | Embryo sac development of Cotoneaster[END_REF], and for C. melanocarpus Fisch. ex Blytt much later [START_REF] Mandryk | Seed reproduction features in Cotoneaster melanocarpus Fisch. ex Blytt. (Rosaceae) from Ukrainian Carpathian flora. Ukr. Botan[END_REF][START_REF] Mandryk | Embryologic investigation of Cotoneaster melanocarpus Fisch. ex Blytt (Rosaceae)[END_REF]. These species showed a strong tendency towards apomictic reproduction, namely, apospory and rarely diplospory, and the early development of their embryo sacs had general similarities with the other apomictic rosaceous genera. The most frequent development of unreduced embryo sac implied degeneration of the primary megaspore mother cell in the earliest developmental stages of the ovule and its replacement with unreduced embryo sac originating from nucellar cells (apospory). In several cases, the unreduced embryo sac developed from archesporial cells following the degeneration of the primary megaspore mother cell (diplospory). The secondary mother megaspore cell also occurred and was developed from unreduced embryo sac or via meiosis [START_REF] Hjelmquist | Embryo sac development of Cotoneaster[END_REF]. The reduced embryo sacs were rarely observed in the studied species [START_REF] Hjelmquist | Embryo sac development of Cotoneaster[END_REF][START_REF] Mandryk | Seed reproduction features in Cotoneaster melanocarpus Fisch. ex Blytt. (Rosaceae) from Ukrainian Carpathian flora. Ukr. Botan[END_REF][START_REF] Mandryk | Embryologic investigation of Cotoneaster melanocarpus Fisch. ex Blytt (Rosaceae)[END_REF]. The authors did not provide data on the polar nuclei. Conclusion of those findings is that most polyploid species of Cotoneaster are apomictic [START_REF] Fryer | Cotoneasters: A Comprehensive Guide to Shrubs for Flowers, Fruit, and Foliage[END_REF][START_REF] Rothleutner | Ploidy levels, relative genome sizes, and base pair composition in Cotoneaster[END_REF]. Sexual reproduction is less represented in Cotoneaster and follows a common pattern of development of Polygonum type of embryo sac: seeds of diploid mothers have diploid embryo and triploid endosperm; seeds of tetraploid mothers have tetraploid embryo and hexaploid endosperm [START_REF] Bourge | Cytotypes and reproductive modes of Cotoneaster integerrimus (Rosaceae) from Bosnia and Herzegovina: Preliminary results[END_REF][START_REF] Macková | Insights into the cytotype and reproductive puzzle of Cotoneaster integerrimus in the Western Carpathians[END_REF].

In recent studies, only a tetraploid cytotype in C. integerrimus populations from Central Europe was found [START_REF] Macková | Insights into the cytotype and reproductive puzzle of Cotoneaster integerrimus in the Western Carpathians[END_REF][START_REF] Kši Ňan | Genome size estimation of Cotoneaster species (Rosaceae) from the Western Carpathians[END_REF]. The same authors [START_REF] Macková | Insights into the cytotype and reproductive puzzle of Cotoneaster integerrimus in the Western Carpathians[END_REF] proved that facultative apomixis was the main reproductive mode, followed by autonomous apomixis and haploid parthenogenesis, while the proportion of sexuality was 10% in the studied sample. The authors showed that different reproductive pathways involved the interaction of reduced and unreduced gametes documented in both sexuals and asexuals.

In this study, we analyzed the variation of genome size, ploidy level, and the diversity of reproductive modes of the C. integerrimus populations from the Balkans with additional samples from several other European regions. In addition, molecular analyses based on nuclear microsatellite markers were performed on the studied cytotypes. Our questions aimed to assess: (i) the geographic distribution and variation of cytotypes within populations in the sampled area; (ii) the most prevalent mode of reproduction in polyploid cytotypes and the frequency of sexuality; (iii) the pathways of endosperm formation among the sampled polyploids and their endosperm balance requirements; (iv) genotypic diversity and geographic distribution of clonal lineages of polyploids. Finally, we discuss the importance of South European populations of C. integerrimus within the context of cytotype diversity and variation in reproduction mode.

Results

Genome Size and Ploidy Level Variation

The flow cytometry (FCM) of 208 Cotoneaster integerrimus individuals from Bosnia and Herzegovina resulted in three distinct groups whose holoploid genome sizes (2C) corresponded to three different ploidy levels, namely, di-, tri-and tetraploid cytotypes (Table 1, Figure 1A). The two singular values corresponded to pentaploid (2C = 2.99 pg, 1Cx = 0.598 pg) and hexaploid cytotype (2C = 3.35 pg, 1Cx = 0.558 pg); thus, both values were excluded from further analyses. The 2C genome size mean values were 1.28 pg in diploids, 1.856 pg in triploids, and 2.481 pg in tetraploids. The significantly (F 2, 207 = 10.98, p > 0.001) highest monoploid (1Cx) genome size value was recorded in diploid, and the lowest in triploid cytotype (Table 1). Geographic distribution of cytotypes showed a clear prevalence of tetraploids (89% in the total sample) in each population except the Umoljani (Um) in Bosnia and Herzegovina (Table 2, Figure 1B). The sample for Figure 1B encompassed the ploidies obtained from flow cytometry as well as the estimated ploidy levels from microsatellite data for 65 individuals (explained in Table 2 and the Material and Methods section). Diploids (7.3% in total sample) were recorded in only three populations with a single individual in Borova glava (Bg) and Devečani (De), and 18 individuals in Umoljani (Um) (Table 2, Figure 1B). Triploids (3.7% in total sample) were observed in seven populations and were represented with just one individual in six populations (Vrbe-Vr, Rujište-Ru, Sovićka vrata-So, Vošac-Vo, Premužićeva staza-Pr, Rtanj-Rt) and four individuals in the population Umoljani (Table 2). 2.

Flow Cytometric Seed Screening

Only unambiguous results of flow cytometric seed screening (FCSS) are presented. Approximately one quarter of analyzed seeds had minor additional peaks corresponding to doubled nuclear DNA levels mirroring endoreplicated embryo and/or endosperm nuclei. In total, the reproductive mode was characterized for 591 seeds from three C. integerrimus cytotypes (Table 2).

In general, inferred pathways of sexual and asexual seed formation showed great diversity and included both reduced and unreduced gametes within each cytotype (Figure 2, Table 3). The tetraploid cytotype was the most abundant, and it consequently resulted in the highest number of reproductive pathways, predominantly apomictic. Diploids involved only sexual reproduction (Table 3). The triploid cytotype was represented with only three seeds, one of sexual and two of apomictic origin (Table 3). In any case, sexual and asexual seed formation was clearly distinguished.

Flow Cytometric Seed Screening

Only unambiguous results of flow cytometric seed screening (FCSS) are presented. Approximately one quarter of analyzed seeds had minor additional peaks corresponding to doubled nuclear DNA levels mirroring endoreplicated embryo and/or endosperm nuclei. In total, the reproductive mode was characterized for 591 seeds from three C. integerrimus cytotypes (Table 2).

In general, inferred pathways of sexual and asexual seed formation showed great diversity and included both reduced and unreduced gametes within each cytotype (Figure 2, Table 3). The tetraploid cytotype was the most abundant, and it consequently resulted in the highest number of reproductive pathways, predominantly apomictic. Diploids involved only sexual reproduction (Table 3). The triploid cytotype was represented with only three seeds, one of sexual and two of apomictic origin (Table 3). In any case, sexual and asexual seed formation was clearly distinguished.

Seed Origin in Diploids

Diploids yielded exclusively sexually originated seeds having the same or increased ploidy level compared to the mother plant, which depended on sperm cell ploidies (Figure 2(S1,S2), Table 3). The profile 2x emb.:3x end. was as expected the most represented (103 seeds) and included both reduced female and male gametes. The profile 3x emb.:4x end. (eight seeds) was a result of interploid crosses between diploids and tetraploids. Those seeds originated from joint of reduced gametes (2x) from tetraploid males and a reduced egg cell (1x) of diploid mother.

Seed Origin in Triploids

All three analyzed triploid seeds had different profiles (Figure 2(S3,A1,A2), Table 3). The only sexual profile 3x emb.:4x end. from one seed included 2x male gamete and reduced (1x) egg cell (Table 3). The other two seeds were of apomictic origin. One included unreduced egg cell fecundated with reduced (1x) sperm cell creating 3x emb.:7x end. Profile, and the other included unreduced (3x) sperm cell creating 3x emb.:9x end. profile (Figure 2(A1,A2), Table 3).

Seed Origin in Tetraploids

Three pathways of sexual seed formation were observed in tetraploids (Figure 2(S4-S6), Table 3). The most frequent profile 4x emb.:6x end. (92 seeds) was formed by fertilization of a meiotically reduced egg cell (2x) with reduced 2x sperm cell (Figure 2(S4)). Fecundation of unreduced female gamete (4x) with reduced 2x male gamete resulted in the formation of B iii hybrids (6x emb.:10x end.) and was detected in 22 seeds (Figure 2(S5)). Two cases of gamete exchange between cytotypes were registered. The fecundation of a reduced egg cell (2x) of tetraploid female with reduced gamete (1x) of diploid male resulted in a 3x emb.:5x end. profile (Figure 2(S6), Table 3).

Pseudogamous seed formation had various pathways. The two most frequent apomictic profiles were 4x emb.:10x end. (148 seeds, Figure 2(A3)) and 4x emb.:12x end. (137 seeds, Figure 2(A4)). In the first case, unreduced polar nuclei were fertilized with reduced 2x male gametes (or, less likely, two 1x gametes). The second case included fecundation of unreduced polar nuclei with two 2x male gametes (Table 3). The following profiles were observed with lower frequencies (Table 3): 4x emb.:11x end. (22 seeds), 4x emb.:9x end. (11 seeds), 4x emb.:7x end. (9 seeds). The 4x emb.:11x end. profile included the fecundation of polar nuclei with 3x pollen or simultaneously with 2x and 1x sperm cells (Figure 2(A5), Table 3). The 4x emb.:9x end. (Figure 2(A6)) profile resulted from the fecundation of polar nuclei with 1x sperm cell. Exact pathway of origin for the 4x emb.:7x end. profile (Figure 2(A8)) remained unresolved, as well as several profiles with oddly high ploidies of endosperm (14x, 15x, 16x, 20x, data not shown).

Moreover, profiles that corresponded to autonomous apomixis (4x emb.:8x end.) have been documented in 32 seeds (Figure 2(A7)). Finally, two seeds originated via haploid parthenogenesis (2x emb.:6x end.) were observed. These cases involved reduced (2x) sperm cells fecundating solely central nuclei (Figure 2(A9), Table 3).

Endosperm Balance

All sexually derived seeds that involved the exchange of reduced gametes within diploid and tetraploid cytotypes had a balanced endosperm with a maternal to paternal genome ratio of 2m:1p (Table 3). Unbalanced endosperms with a ratio of 1m:1p and 4m:1p were observed in sexually produced seeds originating from interploid crosses between diploids and tetraploids (Table 3). On the other hand, within the sample of apomictically originated seeds (354 seeds), the apomictic profile 4x emb.:12x end. was the only one with the balanced endosperm (137 seeds, 38.7%). The remaining seeds of apomictic origin (217 seeds, 61.3%) had unbalanced endosperms (Table 3). Interestingly, the same tetraploid mother plants produced sexual and apomictic seeds having both balanced and unbalanced endosperm (Table S1).

Genotypic Variation in Cotoneaster integerrimus Populations

Five microsatellite loci were successfully amplified in the analyzed C. integerrimus populations and yielded a total of 99 alleles from 254 individuals (Table S2).

A total of 92 multilocus genotypes (MLGs) were obtained from five loci (Ng, Table 4). Each of the studied diploids (N = 13) had a unique genotype (Table S2). The clonal genotypes (N = 32) were found within a polyploid pool of 241 individuals (Table S2).

Each population contained at least one clonal genotype shared by a different number of individuals within a population, indicating clonal reproduction (Ni = 194, Table 4). The proportion of the detected clones (Ng/N) ranged from 0.1 to 0.91 (Table 4), reflecting the different level of clonal structure of populations. In each population, at least two individuals shared the same genotype (Table 4). Moreover, certain populations were completely clonal (Pu-Mt. Trebević, Si-Sinjajevina, He-Austria; Ng = 1, Table 4). In the majority of populations, the effective number of genotypes was higher than one, indicating the presence of unique genotypes in populations (Table 4). Different values of genotypic diversity of populations (Table 4) showed their diverse structure of clonal and unique genotypes, suggesting different ratios of sexuality and asexuality in populations.

While the majority of clonal genotypes (N = 29) were geographically restricted within a single or two populations, the two clonal genotypes occurred at multiple sites (Table S2). The more widespread clonal genotype occurred in populations Bo-Borova glava (N = 20), Go-Mt. Igman (N = 9), Ru-Rujište (N = 9), So-Sovićka vrata (N = 2), and Um-Umoljani (N = 1), which was equivalent to 17% of all polyploids. The second clonal genotype was less widespread and was shared by plants from three populations: So-Sovićka vrata (N = 4), Um-Umoljani (N = 1), and Vr-Vrbe (N = 3). One hundred and thirty-seven (56.8%) individuals belonged to 25 spatially limited clonal genotypes, while other individuals possessed unique genotypes.

Principal coordinate analysis (PCoA) based on Jaccard distances was applied on C. integerrimus polyploid MLGs (Figure 3). PCoA revealed the pattern of grouping of MLGs dispersed along the first (PCo1) and the second (PCo2) coordinates (Figure 3). A weak regional geographic pattern of separation among MLGs groups was evident. The MLGs from western Balkans formed three groups along the PCo1: the first group consisted of MLGs from Bosnia and Herzegovina (Bg, Bo, Ru, Go, So, Um, Vr) and Croatia (Vo); the second consisted of several Bosnian MLGs (Go and Pu); and the third one was the most heterogeneous and consisted of MLGs originating from Bosnia and Herzegovina (Ca, De, Pr, Um, Vr), Croatia (Pr), and Montenegro (Si). MLGs from Italy (Mo), Austria (He), and Germany (Ro) were intermingled with Balkan MLGs. The southern Balkan MLGs from Greece (Ol and Pa) and North Macedonia (Su) clustered into a separate group along the second coordinate (Figure 3). Serbian MLGs (Mu and Rt) formed a group neighboring the southern Balkan cluster. Certain MLGs from De and Um populations were interspersed within the southern Balkan cluster. Greece (Ol and Pa) and North Macedonia (Su) clustered into a separate group along the second coordinate (Figure 3). Serbian MLGs (Mu and Rt) formed a group neighboring the southern Balkan cluster. Certain MLGs from De and Um populations were interspersed within the southern Balkan cluster.

Discussion

Genome Size Variation and Geographic Distribution of Cotoneaster integerrimus Cytotypes

Cotoneaster integerrimus showed stability of the monoploid genome size of three cytotypes, despite asymmetric sample size and significant differences between them. Mon-

Discussion

Genome Size Variation and Geographic Distribution of Cotoneaster integerrimus Cytotypes

Cotoneaster integerrimus showed stability of the monoploid genome size of three cytotypes, despite asymmetric sample size and significant differences between them. Monoploid values were quite similar to those obtained by Macková et al. [START_REF] Macková | Insights into the cytotype and reproductive puzzle of Cotoneaster integerrimus in the Western Carpathians[END_REF] and Kši ňan et al. [START_REF] Kši Ňan | Genome size estimation of Cotoneaster species (Rosaceae) from the Western Carpathians[END_REF]. A slight decrease of monoploid genome size of polyploids compared to diploids was evident. Genome downsizing is a common phenomenon during polyploidization and inter-cytotype hybridization [START_REF] Leitch | Genome downsizing in polyploid plants[END_REF][START_REF] Liu | Polyploidy does not control all: Lineage-specific average chromosome length constrains genome size evolution in ferns[END_REF][START_REF] Zenil-Ferguson | Evaluating the role of genome downsizing and size thresholds from genome size distributions in angiosperms[END_REF]. The obtained genome sizes and ploidies of seed embryos were consistent with leaf data. However, certain inconsistencies were present in precise determination of endosperm ploidy, which was calculated using the monoploid genome size of embryo. This issue has been often reported in studies dealing with flow cytometric seed screen [START_REF] Lepší | Versatility of reproductive modes and ploidy level interactions in Sorbus s.l. (Malinae, Rosaceae)[END_REF][START_REF] Macková | Insights into the cytotype and reproductive puzzle of Cotoneaster integerrimus in the Western Carpathians[END_REF]. The source of endosperm variation lies in possibility of the central cell's fertilization by unbalanced and aneuploid gametes of variable genome size and in different number of nuclei in the central cells [START_REF] Lepší | Versatility of reproductive modes and ploidy level interactions in Sorbus s.l. (Malinae, Rosaceae)[END_REF][START_REF] Hajrudinović | When sexual meets apomictgenome size, ploidy level and variation of reproductive mode in Sorbus aria s.l. and S. austriaca (Rosaceae) in Bosnia and Herzegovina[END_REF][START_REF] Dobeš | Prospects and limits of the flow cytometric seed screen-insights from Potentilla sensu lato (Potentilleae, Rosaceae)[END_REF][START_REF] Talent | The potential for ploidy level increases and decreases in Crataegus (Rosaceae, Spiraeoideae, tribe Pyreae)[END_REF]. However, the observed variation in endosperm ploidy did not hinder the discrimination between the sexual and asexual origin of seeds but, in some cases, rather made it difficult to accurately infer the number and ploidy of sperm cells involved in the fertilization of central cell's nuclei.

Cotoneaster integerrimus tetraploids are the dominant cytotype in the studied Balkan populations (Figure 1B), as also documented in Central Europe [START_REF] Macková | Insights into the cytotype and reproductive puzzle of Cotoneaster integerrimus in the Western Carpathians[END_REF][START_REF] Kši Ňan | Genome size estimation of Cotoneaster species (Rosaceae) from the Western Carpathians[END_REF]. Populations from Central Europe were homogeneously tetraploid. Sexual diploids are detected only in the Western Alps [START_REF] Macková | Insights into the cytotype and reproductive puzzle of Cotoneaster integerrimus in the Western Carpathians[END_REF]. In comparison, our study showed the greater presence of sexual diploids in the Balkans but stressed their rare occurrence as well. They were registered at the three populations in Bosnia and Herzegovina, of which two contained only one diploid individual. In contrast, it was the prevailing cytotype at the most numerous Umoljani population, outnumbering the polyploids (Figure 1B, Table 2). The observed co-occurrence of tetraploids and triploids in seven populations (Figure 1B) implicate the exchange of reduced gametes between tetraploid and diploid cytotypes. However, the sympatry of diploids, triploids, and tetraploids was documented only in the population Umoljani. We assume that diploids in other populations simply were not covered by sampling due to their overall rarity. Hence, a more extensive sampling strategy should be applied to test this assumption. If we consider the present results jointly with Macková et al. [START_REF] Macková | Insights into the cytotype and reproductive puzzle of Cotoneaster integerrimus in the Western Carpathians[END_REF] and Kši ňan et al. [START_REF] Kši Ňan | Genome size estimation of Cotoneaster species (Rosaceae) from the Western Carpathians[END_REF], as a comprehensive dataset on C. integerrimus ploidy variation, it is apparent that diploids are a minority cytotype in the overall cytotype structure of the species. Diploids of C. integerrimus as a rare cytotype never form monoploid populations [START_REF] Kolář | Mixed-ploidy species: Progress and opportunities in polyploid research[END_REF]. The coexistence of diploid and tetraploid cytotypes is also extremely rare in C. integerrimus. Diploid cytotype at the Umoljani site contributes markedly to ploidy richness, reproductive mode variation, and genotypic diversity. Namely, diploids affect cytotype dynamics through reproductive interactions with sexual and asexual tetraploids, generating intermediate triploid cytotype (Table 3). Furthermore, interploid crossings contribute to greater diversity of reproductive pathways, which was confirmed at the Umoljani site (Table S1). Finally, gene flow between di-and tetraploid cytotypes contributes to the higher genotypic diversity (Table 4).

The question remains as to why C. integerrimus diploids are so rare. Geographic distribution of C. integerrimus diploids in our sample confirmed a restricted and smaller geographic range relative to asexual tetraploids, which is a common pattern in different agamic groups [START_REF] Kolář | Mixed-ploidy species: Progress and opportunities in polyploid research[END_REF][START_REF] Goad | Hybridization, polyploidy and clonality influence geographic patterns of diversity and salt tolerance in the model halophyte seashore paspalum (Paspalum vaginatum)[END_REF][START_REF] Dickinson | Niche shifts, hybridization, polyploidy and geographic parthenogenesis in western North American hawthorns (Crataegus subg. Sanguineae, Rosaceae)[END_REF][START_REF] Cosendai | Cytotype stability, facultative apomixis and geographical parthenogenesis in Ranunculus kuepferi (Ranunculaceae)[END_REF]. These areas inhabited with diploids are considered as relict habitats in glacial refugia during the Pleistocene climate changes [START_REF] Hörandl | The evolution of self-fertility in apomictic plants[END_REF][START_REF] Kolář | Towards resolving the Knautia arvensis agg. (Dipsacaceae) puzzle: Primary and secondary contact zones and ploidy segregation at landscape and microgeographic scales[END_REF]. This assumes that the site Umoljani served as refugium for the persistence of diploids during glacial cycles. Such a scenario seems plausible for Umoljani as it is situated just above the huge Rakitnica gorge within the high mountains in Bosnia and Herzegovina, which has been confirmed as a refugium for many plant species [START_REF] Ritter-Studnička | Reliktgesellschaften auf Dolomitböden in Bosnien und der Hercegovina[END_REF]. Moreover, the Umoljani site has been identified as one of the hotspots of Sorbus cytotype diversity in the Balkans [START_REF] Hajrudinović | When sexual meets apomictgenome size, ploidy level and variation of reproductive mode in Sorbus aria s.l. and S. austriaca (Rosaceae) in Bosnia and Herzegovina[END_REF]. The current coexistence of the C. integerrimus diploids and tetraploids reflects the primary contact zone of cytotypes that involved in situ ancient formation of tetraploids via autopolyploidy or postglacial cytotype overlap of the two cytotypes (secondary contact zone). Nuclear microsatellites showed genetic divergence between diploids and tetraploids (Figure S1) and rather support the secondary contact zone. However, due to the small sample size and occurrence of clonal genotypes (six out of eight individuals) at the Umoljani site, it remains uncertain as to whether the current coexistence resulted from the primary or secondary contact. Such sites, where different sexual and asexual cytotypes coexist and interact, encouraging the formation of novel biodiversity, represent areas of particular conservation concern [START_REF] Ennos | Process-based species action plans: An approach to conserve contemporary evolutionary processes that sustain diversity in taxonomically complex groups[END_REF].

Asexual Seed Formation in Monoploid and Mixed-Ploidy Populations

Flow cytometric seed screening unequivocally confirmed pseudogamous apomixis as the most prevalent mode of reproduction in the C. integerrimus complex along the sampled area. While diploids are exclusively sexual, tetraploids combine asexual and sexual reproduction. Our results for tetraploids are fully consistent with those of Macková et al. [START_REF] Macková | Insights into the cytotype and reproductive puzzle of Cotoneaster integerrimus in the Western Carpathians[END_REF], as well as with other related genera: Amelanchier [START_REF] Burgess | Effects of apomixis and polyploidy on diversification and geographic distribution in Amelanchier (Rosaceae)[END_REF], Crataegus [START_REF] Talent | Endosperm formation in aposporous Crataegus (Rosaceae, Spiraeoideae, tribe Pyreae): Parallels to Ranunculaceae and Poaceae[END_REF], Potentilla [START_REF] Dobeš | Prospects and limits of the flow cytometric seed screen-insights from Potentilla sensu lato (Potentilleae, Rosaceae)[END_REF], Rubus [START_REF] Šarhanová | New insights into the variability of reproduction modes in European populations of Rubus subgen. Rubus: How sexual are polyploid brambles? Sex[END_REF], and Sorbus [START_REF] Lepší | Versatility of reproductive modes and ploidy level interactions in Sorbus s.l. (Malinae, Rosaceae)[END_REF][START_REF] Hajrudinović | When sexual meets apomictgenome size, ploidy level and variation of reproductive mode in Sorbus aria s.l. and S. austriaca (Rosaceae) in Bosnia and Herzegovina[END_REF]. All C. integerrimus tetraploids in monoploid populations showed similar patterns of seed formation involving both reduced and unreduced gametes (Table 3). Seed formation in tetraploids mainly involved central cell fertilization with one or two 2x sperm cells (two dominant profiles: 4x emb.:10x end. and 4x emb.:12x end., Table 3). The latter profile suggested dispermy of the central cell during endosperm formation [START_REF] Burgess | Effects of apomixis and polyploidy on diversification and geographic distribution in Amelanchier (Rosaceae)[END_REF][START_REF] Talent | Endosperm formation in aposporous Crataegus (Rosaceae, Spiraeoideae, tribe Pyreae): Parallels to Ranunculaceae and Poaceae[END_REF][START_REF] Šarhanová | New insights into the variability of reproduction modes in European populations of Rubus subgen. Rubus: How sexual are polyploid brambles? Sex[END_REF], rather than fertilization with unreduced sperm cells. Namely, we did not notice the occurrence of male unreduced gametes in any of the supposed reproductive pathway of tetraploids (Table 3). The 10x endosperm was prevalent in Crataegus and Rubus but quite rare in Amelanchier and Sorbus, having 12x endosperm. Both 10x and 12x endosperm had almost equal frequency in C. integerrimus tetraploids, as confirmed by Macková et al. [START_REF] Macková | Insights into the cytotype and reproductive puzzle of Cotoneaster integerrimus in the Western Carpathians[END_REF]. In addition, both ploidy levels of endosperm were present in seeds produced by the same plants, even yielding sexually originated seeds with balanced endosperm at the same time (Table S1). The capability of a single plant to simultaneously produce pseudogamous seeds with balanced/unbalanced endosperm, as well as seeds of sexual origin with a balanced endosperm, represents an advanced reproductive strategy. The potential to use the full benefits from both reproduction modes, accompanied with self-compatibility and self-pollination [START_REF] Hörandl | The evolution of self-fertility in apomictic plants[END_REF][START_REF] Hörandl | The complex causality of geographical parthenogenesis[END_REF], makes the Cotoneaster integerrimus tetraploid apomicts ecologically superior for rapid adaptation, colonization, and long-term persistence of populations in dynamic environments. The prevalence of tetraploid apomictic populations over diploid sexual ones goes in favor of geographic parthenogenesis along the sampled area [START_REF] Hörandl | Understanding the geographic distributions of apomictic plants: A case for a pluralistic approach[END_REF].

The frequency of endosperm with odd ploidy levels was low (8.8%) in tetraploids of C. integerrimus, although this is common in related genera [START_REF] Lepší | Versatility of reproductive modes and ploidy level interactions in Sorbus s.l. (Malinae, Rosaceae)[END_REF][START_REF] Hajrudinović | When sexual meets apomictgenome size, ploidy level and variation of reproductive mode in Sorbus aria s.l. and S. austriaca (Rosaceae) in Bosnia and Herzegovina[END_REF][START_REF] Macková | Insights into the cytotype and reproductive puzzle of Cotoneaster integerrimus in the Western Carpathians[END_REF][START_REF] Ennos | Process-based species action plans: An approach to conserve contemporary evolutionary processes that sustain diversity in taxonomically complex groups[END_REF][START_REF] Talent | Endosperm formation in aposporous Crataegus (Rosaceae, Spiraeoideae, tribe Pyreae): Parallels to Ranunculaceae and Poaceae[END_REF][START_REF] Šarhanová | New insights into the variability of reproduction modes in European populations of Rubus subgen. Rubus: How sexual are polyploid brambles? Sex[END_REF]. Such endosperm in our case would require the participation of triploid and haploid pollen that was not observed in the field, except for the population Umoljani. Lepší et al. [START_REF] Lepší | Versatility of reproductive modes and ploidy level interactions in Sorbus s.l. (Malinae, Rosaceae)[END_REF] showed that the exact embryo and endosperm genome sizes primarily depend on the combination of actual gamete genome sizes involved in the fertilization and may markedly deviate from expected mean values, making profile interpretation difficult. We assume that these profiles with an unbalanced endosperm (4x emb.:7x end., 4x emb.:9x end. and 4x emb.:11x end.) represent pseudogamy that most likely included aneuploid sperm cells with variable genome size [START_REF] Talent | Endosperm formation in aposporous Crataegus (Rosaceae, Spiraeoideae, tribe Pyreae): Parallels to Ranunculaceae and Poaceae[END_REF][START_REF] Šarhanová | New insights into the variability of reproduction modes in European populations of Rubus subgen. Rubus: How sexual are polyploid brambles? Sex[END_REF] or products of an irregular pseudogamous process [START_REF] Dobeš | Prospects and limits of the flow cytometric seed screen-insights from Potentilla sensu lato (Potentilleae, Rosaceae)[END_REF]. However, our data showed that tetraploid cytotype apparently tolerate endosperm imbalance, but the relationship between endosperm imbalance tolerance and seed viability and germination remains uninvestigated.

In addition to pseudogamous development, we also observed a profile corresponding to autonomous apomixis (4x emb.:8x end.) (Table 3). Unlike Macková et al. [START_REF] Macková | Insights into the cytotype and reproductive puzzle of Cotoneaster integerrimus in the Western Carpathians[END_REF], in our dataset, we detected some cases of G2 peaks of tetraploid embryos (Figure 2(S4)), and therefore we cannot precisely distinguish endosperm with endoreduplicated embryos. Finally, several cases of haploid parthenogenesis have been found in tetraploid seeds as a distinct rare pathway of asexual seed formation yielding unviable seeds and anormal development of the plant.

Sexuality in Cotoneaster integerrimus Populations

The fertilization via reduced (2x) gametes, resulting in seeds with a balanced endosperm (2m:1p), represents the main (19.4%, N = 92, Table 3) sexual reproductive pathway of tetraploid C. integerrimus, which is noticeably higher than in Central European populations (6.3%, [START_REF] Macková | Insights into the cytotype and reproductive puzzle of Cotoneaster integerrimus in the Western Carpathians[END_REF]). As mentioned earlier, different seed formation pathways (apomictic and sexual) involving different combinations of gamete number can operate in one plant. On the other hand, the rate of sexuality within a single plant/population greatly varies, as evidenced by the proportion of clonal genotypes and complete clonality in some populations (Table 4).

The frequency (N = 22, 4.2%, Table 3) of unreduced female gametes (4x) in crosses with reduced male gametes (2x) resulted in B iii hybrid seeds, with a similar proportion found in Central European populations. Our screening revealed only one fruitless hexaploid individual in the field, and we assume that this cytotype is extremely rare. The viability of B iii hybrid seeds and seedlings is most likely low due to endosperm imbalance [START_REF] Lepší | Versatility of reproductive modes and ploidy level interactions in Sorbus s.l. (Malinae, Rosaceae)[END_REF].

Sexuality in C. integerrimus also included bidirectional intercytotype crosses, resulting in 12 seeds with triploid embryos that accounted for ≈9% of all analyzed seeds from the Umoljani population. We expected that the number of such crosses would be much higher in a larger sample. Singular occurrence of triploid individuals in other populations (So, Ru, and Vr) also requires more extensive sampling to depict cytotype structure in populations. Our results provide evidence that gene flow occurs between diploid and tetraploid cytotypes in their sympatry, but the small number of triploids that have a weak or no fruit yield suggest a lower fitness than either parent (only three fertile pyrenes from two individuals). The occurrence of triploid cytotype in mixed-ploidy populations indicates the existing weak reproductive barriers between diploid and tetraploid cytotype, which nevertheless allow the formation of triploids but with a low frequency.

The key reproductive isolation mechanism in the formation of triploids is considered to be the triploid block, which operates in different polyploid systems [START_REF] Lepší | Versatility of reproductive modes and ploidy level interactions in Sorbus s.l. (Malinae, Rosaceae)[END_REF][START_REF] Sutherland | Variation in heteroploid reproduction and gene flow across a polyploid complex: One size does not fit all[END_REF][START_REF] Köhler | The impact of the triploid block on the origin and evolution of polyploid plants[END_REF][START_REF] Husband | The role of triploid hybrids in the evolutionary dynamics of mixed-ploidy populations[END_REF][START_REF] Scott | Parent-of-origin effects on seed development in Arabidopsis thaliana[END_REF]. The triploid block is caused by malfunction of the endosperm as a result of an imbalance between paternally and maternally imprinted genes during the endosperm development, and often leads to seed abortion [START_REF] Vinkenoog | Genomic imprinting and endosperm development in flowering plants[END_REF]. The observed endosperm imbalance in all triploid seeds originated from di-, tri-, and tetraploid mother plants in the present dataset (Table 3), suggesting that a small portion of seeds are insensitive to the triploid block. These seeds, originated via intercytotype crosses in C. integerrimus, represent novel polyploid forms of genomic variation with potential for divergence. The frequency of repeated crosses between diploids and tetraploids accompanied by the relaxed endosperm balance would directly affect the frequency of triploids in mixed-ploidy populations. Although triploids are considered unstable and less fertile relative to diploids and tetraploids in Rosaceae [START_REF] Talent | Endosperm formation in aposporous Crataegus (Rosaceae, Spiraeoideae, tribe Pyreae): Parallels to Ranunculaceae and Poaceae[END_REF][START_REF] Ludwig | Breeding systems, hybridization and continuing evolution in Avon Gorge[END_REF], they play an important role as intermediates in the formation of tetraploids via 'triploid bridge' [START_REF] Hajrudinović | When sexual meets apomictgenome size, ploidy level and variation of reproductive mode in Sorbus aria s.l. and S. austriaca (Rosaceae) in Bosnia and Herzegovina[END_REF][START_REF] Husband | The role of triploid hybrids in the evolutionary dynamics of mixed-ploidy populations[END_REF][START_REF] Hamston | Apomixis and hybridization drives reticulate evolution and phyletic differentiation in Sorbus L.: Implications for conservation[END_REF]. Our scenario involves bidirectional crosses between a sexual diploid cytotype and a predominantly apomictic tetraploid cytotype that produces a mostly apomictic triploid progeny of C. integerrimus. The reproductive success and persistence of the triploid cytotypes will primarily depend on the capabilities provided by their mating system [START_REF] Hörandl | The evolution of self-fertility in apomictic plants[END_REF]. Certainly, a future sampling design will encompass a larger geographic distribution of C. integerrimus populations and a larger number of individual plants/progenies to answer the question on the occurrence, dynamics, and distribution of triploids and their potential role in evolution of this complex.

Patterns of Genotypic Diversity in Cotoneaster integerrimus

The use of nuclear microsatellite markers confirmed cytometric results and shed a new light on the breeding system of C. integerrimus. The patterns of genotypic diversity within and among populations primarily reflected on the breeding system of C. integerrimus that favors pseudogamous reproduction. Favored asexuality is evident in the low level of genotypic diversity and the high proportion of clonal genotypes in most populations. While the studied diploids have unique MLGs, a trait of exclusive out-crossers [START_REF] Hamston | Apomixis and hybridization drives reticulate evolution and phyletic differentiation in Sorbus L.: Implications for conservation[END_REF][START_REF] Robertson | Hybridization and polyploidy as drivers of continuing evolution and speciation in Sorbus[END_REF], polyploids share the same genotypes in each population. Heteroploid populations showed higher levels of genotypic diversity. These patterns may reflect ancient hybridization between cytotypes that has taken place over time or current processes in which younger apomictic lineages tend to prevail via intercytotypic crosses [START_REF] Coughlan | Widespread generalist clones are associated with range and niche expansion in allopolyploids of Pacific Northwest Hawthorns (Crataegus L.)[END_REF][START_REF] Šarhanová | Hybridization drives evolution of apomicts in Rubus subgenus Rubus: Evidence from microsatellite markers[END_REF]. The proportion of sexuality of tetraploids significantly differs among the populations, but the factor that governs the sexual reproduction in C. integerrimus remains unclear. On the other hand, the sexual reproduction of facultative apomicts may depend on changes in environmental conditions [START_REF] Šarhanová | New insights into the variability of reproduction modes in European populations of Rubus subgen. Rubus: How sexual are polyploid brambles? Sex[END_REF]. The interaction between the breeding system and environmental conditions allows for the facultative apomict to increase the rate of sexuality at a given time, reshuffling the overall genetic variability of populations in dynamic environments [START_REF] Karunarathne | Sexual modulation in a polyploid grass: A reproductive contest between environmentally inducible sexual and genetically dominant apomictic pathways[END_REF]. In any case, sexuality is an important and propulsive agent of genetic variability in monoploid and mixed-ploidy C. integerrimus populations. Additional factors contributing to genetic variability in different apomictic systems are mutations and residual recombination of the female gametophyte [START_REF] Majeský | The pattern of genetic variability in apomictic clones of Taraxacum officinale indicates the alternation of asexual and sexual histories of apomicts[END_REF][START_REF] Hamston | Apomixis and hybridization drives reticulate evolution and phyletic differentiation in Sorbus L.: Implications for conservation[END_REF][START_REF] Paule | Apomictic and sexual lineages of the Potentilla argentea L. group (Rosaceae): Cytotype and molecular genetic differentiation[END_REF][START_REF] Paun | Patterns, sources and ecological implications of clonal diversity in apomictic Ranunculus carpaticola (Ranunculus auricomus complex, Ranunculaceae)[END_REF], but these aspects are beyond the scope of this study.

Although the most clonal MLGs were spatially restricted within the populations, few were dispersed among the populations. Moreover, the most widespread clonal MLG constituted the complete structure of the Bg population, and significantly in the Go and Ru sites. Geographical distances between the sites/populations with the clonal MLGs ranged between 40 and 70 km. The wide geographic occurrence of a certain clonal lineage confirmed its high abilities to colonize and spread in new geographic areas. This pattern might fit the idea of generalist clonal genotypes that are geographically widespread and occur in different habitats [START_REF] Coughlan | Widespread generalist clones are associated with range and niche expansion in allopolyploids of Pacific Northwest Hawthorns (Crataegus L.)[END_REF]. The widely distributed clonal MLGs may represent old C. integerrimus lineages that had the chance for multiple long-distance dispersal events, and spatially restricted ones may represent relatively new clonal genotypes that have not had time to disperse across environments [START_REF] Šarhanová | Hybridization drives evolution of apomicts in Rubus subgenus Rubus: Evidence from microsatellite markers[END_REF]. In that context, the lack of clear geographic structure among the polyploid MLGs, as revealed by PCoA, might lie in their multiple origins via recurrent mating between widely distributed lineages and local genotypes, as well as multiple colonization of sites by different clones [START_REF] Coughlan | Widespread generalist clones are associated with range and niche expansion in allopolyploids of Pacific Northwest Hawthorns (Crataegus L.)[END_REF][START_REF] Paun | Patterns, sources and ecological implications of clonal diversity in apomictic Ranunculus carpaticola (Ranunculus auricomus complex, Ranunculaceae)[END_REF]. In such a scenario, recurrent admixture of divergent genotypes and their interaction will blur the geographic structuring of populations.

Materials and Methods

Plant Material

We sampled a total of 18 natural populations of C. integerrimus originating from the Balkans (covering Bosnia and Herzegovina and the neighboring regions of Serbia, Montenegro, Croatia, North Macedonia, and Greece (Figure 1A, Table 2)). Our sample was complemented with three populations from Italy, Austria, and Germany. More detailed sampling was performed in 10 Bosnia and Herzegovina populations encompassing 17-30 individuals per site, depending on the population size (Table 2). The distance between adult plants at all sites was at least 20 m to avoid collecting ramets. Altogether, 254 adult individuals from 21 populations were used for genome size, ploidy level determination, and molecular analysis. To determine the reproductive mode variation, we studied 591 seeds from the 63 mother individuals. The vouchers were deposited in the Herbarium of the National Museum of Bosnia and Herzegovina (SARA herbarium, voucher numbers 51809-51818).

Genome Size and Ploidy Level Determination

Absolute genome size and ploidy level determination was performed by flow cytometry (FCM) for 208 individuals from Bosnia and Herzegovina. Genome size determination followed the protocol previously used for Sorbus spp. [START_REF] Hajrudinović | When sexual meets apomictgenome size, ploidy level and variation of reproductive mode in Sorbus aria s.l. and S. austriaca (Rosaceae) in Bosnia and Herzegovina[END_REF]. Briefly, parts (around 1 cm 2 ) of C. integerrimus fresh leaves and leaf material of internal standard (Solanum lycopersicum cv. Montfavet 63-5, 2C = 1.99 pg, [START_REF] Lepers-Andrzejewski | Diversity and dynamics of plant genome size: An example of polysomaty from a cytogenetic study of Tahitian vanilla (Vanilla × tahitensis, Orchidaceae)[END_REF]) were chopped together using a razor blade in cold Gif Nuclear Buffer [START_REF] Bourge | Flow cytometry as tool in plant sciences, with emphasis on genome size and ploidy level assessment[END_REF]. The suspension was filtered through a 50 µm nylon mesh (CellTrics from Partec-Sysmex, Goertliz, Germany), and RNAse (Roche CustomBiotech, Mannheim, Germany) was added to 2.5 U mL -1 . The nuclei were stained with propidium iodide (Sigma-Aldrich, Saint-Louis, United States of America) to a final concentration 50 µg mL -1 and incubated on ice for at least five minutes. The fluorescence of 5000 nuclei was recorded for each sample using a Partec CyFlow SL3 (Partec, Münster, Germany) 532 nm laser cytometer. Fluorescence histograms were analyzed using FloMax ver. 2.8 (Partec, Münster, Germany). The absolute 2C DNA values of Cotoneaster individuals were obtained by calculation of linear relationship between the fluorescence signals of unknown sample and known internal standard. Individual DNA ploidy levels [START_REF] Suda | Ploidy level versus DNA ploidy level: An appeal for consistent terminology[END_REF] were inferred following earlier chromosome counts on Cotoneaster species and compared with obtained 2C DNA values. Each individual was assigned into particular cytotype: di-, tri-, and tetraploid. The total 2C DNA value/ploidy level ratio was used to obtain monoploid genome size (1Cx, [START_REF] Greilhuber | The origin, evolution and proposed stabilization of the terms 'genome size' and 'C-value' to describe nuclear DNA contents[END_REF]) of each cytotypes. The mean values of monoploid genome size of cytotype groups were tested using one-way ANOVA followed by Tukey's HSD test. Prior to ANOVA, homogeneity of groups' variances using Levene's test and data distribution using Kolmogorov-Smirnov test were checked. Analyses were conducted using PAST 3.17 [START_REF] Hammer | Paleontological statistics software package for education and data analysis[END_REF].

For individuals sampled outside Bosnia and Herzegovina (N = 65), the ploidy level was inferred from microsatellite data. The maximum number of alleles per locus was used to determine the ploidy level for those samples. This procedure was successfully applicable in certain groups [START_REF] Greilhuber | The origin, evolution and proposed stabilization of the terms 'genome size' and 'C-value' to describe nuclear DNA contents[END_REF][START_REF] Feulner | Central European polyploids of Sorbus subgenus Aria (Rosaceae) recurrently evolved from diploids of central and south-eastern Europe: Evidence from microsatellite data[END_REF][START_REF] Uhrinová | Origin and genetic differentiation of pinkflowered Sorbus hybrids in the Western Carpathians[END_REF][START_REF] Beck | Does hybridization drive the transition to asexuality in diploid Boechera?[END_REF], but this criterion was reliable for tetraploids only.

Flow Cytometric Seed Screening

Flow cytometric seed screening was successfully conducted on 591 seeds following the procedure described by Hajrudinović et al. [START_REF] Hajrudinović | When sexual meets apomictgenome size, ploidy level and variation of reproductive mode in Sorbus aria s.l. and S. austriaca (Rosaceae) in Bosnia and Herzegovina[END_REF]. Seeds (pyrenes) were collected from attached fruits on 14 diploid, 3 triploid, and 63 tetraploid mother individuals, previously cytotyped (Table S1). Only well-formed seeds, dried for 48 h at room temperature and kept in paper bags at 4 • C prior to analysis, were used. Each seed was analyzed separately. Only three healthy seeds were recovered from triploid plants, which indicates a high level of sterility of this cytotype. Entire seeds were co-chopped with part of fresh leaf (around 0.5 cm 2 ) of internal standard Oryza sativa ssp. japonica cv. Nipponbare (2C = 0.9 pg, [START_REF] Uozu | Repetitive sequences: Cause for variation in genome size and chromosome morphology in the genus Oryza[END_REF]) in cold Gif Nuclear Buffer following the steps in procedure described for genome size and ploidy level determination. Both linear and log histograms were recorded after the fluorescence was divided between two photomultipliers with a 50/50 mirror. Endosperm ploidy was calculated using the inferred monoploid genome size of embryo of the same seed. Estimated DNA ploidies of embryo and endosperm were compared to distinguish between sexual and apomictic origin of each analyzed seed and to deduce fertilization pathways according to [START_REF] Hajrudinović | When sexual meets apomictgenome size, ploidy level and variation of reproductive mode in Sorbus aria s.l. and S. austriaca (Rosaceae) in Bosnia and Herzegovina[END_REF][START_REF] Talent | Endosperm formation in aposporous Crataegus (Rosaceae, Spiraeoideae, tribe Pyreae): Parallels to Ranunculaceae and Poaceae[END_REF][START_REF] Matzk | An efficient screen for reproductive pathways using mature seeds of monocots and dicots[END_REF].

Analysis of Nuclear Microsatellites

A modified CTAB-procedure [START_REF] Tel-Zur | Modified CTAB procedure for DNA isolation from epiphytic cacti of genera Hylocereus and Selenicereus (Cactaceae)[END_REF] was used to extract total genomic DNA from around 20 mg of silica-dried leaf material. Amplification of five nuclear microsatellite markers was performed for 254 individuals (Table 2) following Robertson et al. [START_REF] Robertson | Hybridization and polyploidy as drivers of continuing evolution and speciation in Sorbus[END_REF]. The applied primers are primarily designed for Malus × domestica (CH01H10, CHO1FO2, and CHO2D11, [START_REF] Gianfranceschi | Simple sequence repeats for the genetic analysis of apple[END_REF]) and S. torminalis (MSS5 and MSS16, [START_REF] Oddou-Muratorio | Microsatellite primers for Sorbus torminalis and related species[END_REF]) but were successfully applied in different rosaceous genera. An ABI PRISM 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) was used for electrophoretic separation of PCR products. Alleles were sized relative to the internal size standard TAMRA 500 (Applied Biosystems, Warrington, United Kingdom). Electropherograms were analyzed using GeneMapper v. 5 (Applied Biosystems).

To study genetic diversity in polyploid C. integerrimus populations, we determined the multilocus genetic genotypes (MLGs or simply genotypes) for each individual on the basis of microsatellite allele data for each of five loci in the software program GenoType v. 1.2 [START_REF] Meirmans | GENODIVE: Two programs for the analysis of genetic diversity of asexual organisms[END_REF]. Assignment of individuals into a particular clone was performed using the Meirmans and Van Tienderen [START_REF] Meirmans | GENODIVE: Two programs for the analysis of genetic diversity of asexual organisms[END_REF] algorithm according to the calculation of a genetic distance matrix and a clonal threshold (set to two for the study, after testing different threshold values upon recommendations by the authors of the programs) under the stepwise mutation model option. In order to analyze clonal diversity of C. integerrimus, we included following measures: the total number of genotypes (Ng) and effective genotypes (Eff), the number of unique genotypes (Un), and Simpson's diversity index (D) calculated in the software GenoDive v. 1.2 [START_REF] Meirmans | GENODIVE: Two programs for the analysis of genetic diversity of asexual organisms[END_REF]. The proportion of distinguishable genotypes (Ng⁄ N) [START_REF] Ellstrand | Patterns of genotypic diversity in clonal plant species[END_REF] was calculated as well. Relationships among multilocus genotypes were visualized by principal coordinate analysis (PCoA) based on Jaccard distances using PAST 3.17 [START_REF] Hammer | Paleontological statistics software package for education and data analysis[END_REF].

The maximum number of alleles per loci corresponded to ploidy levels obtained using cytometry. For 65 individuals with unknown genome size, these data were used to infer the ploidy level (Tables 2 andS2).

Conclusions

From genome size, ploidy level, mode of reproduction, and genotypic variation, we demonstrated that South European C. integerrimus populations from the Balkans represent a dynamic polyploid aggregate with complex breeding. The prevalence of apomictic tetraploids determines the overall cytotype structure of populations, confirming the pattern of geographic parthenogenesis along the sampled area. Genotypic variability of populations mainly results from the interaction of predominant facultative apomixis and residual sexual reproduction in tetraploids on a large geographical scale. Sexual diploids, due to their rarity, also contribute to the cytotype, reproductive diversity, and genotypic diversity of polyploid populations but at the local scale. The endosperm imbalance facilitates the development and the occurrence of intermediate triploids in mixed-ploidy populations, but also different tetraploid lineages elsewhere with unbalanced endosperm. South European populations of C. integerrimus showed higher levels of cytotype and reproductive diversity compared to the Central European ones, representing a potential reservoir of regional and global diversity for the species. Future efforts should be focused on more extensive sampling strategy of C. integerrimus populations to reveal the underlying processes shaping the diversity of this multiploid species.

Supplementary Materials:

The following are available online at https://www.mdpi.com/article/10 .3390/plants10122798/s1, Table S1: Individual FCSS profiles. Table S2: Genome composition of the studied Cotoneaster integerrimus individuals at five microsatellite loci. Funding: Not applicable.

Figure 1 .

 1 Figure 1. Absolute nuclear genome size (2C) of diploid, triploid, and tetraploid cytotypes (A) (boxes define 95th percentile interval; horizontal lines indicate mean value; whiskers indicate standard deviation: outliers are shown as circles), and geographical distribution of Cotoneaster integerrimus cytotypes and their frequency (%) per site/population (B). Site abbreviations (Site IDs) correspond to Table2.

Figure 1 .

 1 Figure 1. Absolute nuclear genome size (2C) of diploid, triploid, and tetraploid cytotypes (A) (boxes define 95th percentile interval; horizontal lines indicate mean value; whiskers indicate standard deviation: outliers are shown as circles), and geographical distribution of Cotoneaster integerrimus cytotypes and their frequency (%) per site/population (B). Site abbreviations (Site IDs) correspond to Table2.

Figure 2 .

 2 Figure 2. Flow cytometric histograms (log abscissa) of Cotoneaster integerrimus seed formation from sexual (S1-S6) or asexual (A1-A9) origin. The first fluorescence peaks correspond to the internal standard (ST, Oryza sativa L. ssp. japonica 'Nipponbare'), the second ones to the embryo, and the third ones to the endosperm (blue: 2x mother, red: 3x mother, green: 4x mother).

Figure 2 .

 2 Figure 2. Flow cytometric histograms (log abscissa) of Cotoneaster integerrimus seed formation from sexual (S1-S6) or asexual (A1-A9) origin. The first fluorescence peaks correspond to the internal standard (ST, Oryza sativa L. ssp. japonica 'Nipponbare'), the second ones to the embryo, and the third ones to the endosperm (blue: 2x mother, red: 3x mother, green: 4x mother).

Figure 3 .

 3 Figure 3. Principal coordinate analysis of the Jaccard distance matrix of multilocus genotypes of Cotoneaster integerrimus polyploids according to nuclear microsatellite data. Population abbreviations correspond to those in the Table 4. The numbers in the brackets denote the number of clones.

Figure 3 .

 3 Figure 3. Principal coordinate analysis of the Jaccard distance matrix of multilocus genotypes of Cotoneaster integerrimus polyploids according to nuclear microsatellite data. Population abbreviations correspond to those in the Table 4. The numbers in the brackets denote the number of clones.

  Figure S1: Principal coordinate analysis of multilocus genotypes from the Umoljani site. Author Contributions: Conceptualization, F.B. and E.M.; methodology, F.B., S.S.-Y., E.M. and A.H.-B.; validation, M.B. and S.S.-Y.; formal analysis, F.B. and E.M.; investigation, I.M.-D. and A.H.-B.; resources, F.B. and S.S.-Y.; writing-original draft preparation, F.B.; writing-review and editing, E.M., A.H.-B., S.S.-Y., I.M.-D., M.B., S.C.B. and F.B.; visualization, F.B. and A.H.-B.; supervision, S.S.-Y.; funding acquisition, F.B. All authors have read and agreed to the published version of the manuscript.

Table 1 .

 1 Absolute genome size values and ploidy levels of Cotoneaster integerrimus cytotypes.

	DNA Ploidy	N	2C DNA	CV %

1Cx DNA Mean ± SD (pg) Min-Max (pg) Mean ± SD (pg) * Mean (Mbp **)

	2x	20	1.280 ± 0.046	1.23-1.36	2.47	0.636 ± 0.015 a	622.00
	3x	7	1.856 ± 0.091	1.63-1.90	5.04	0.608 ± 0.030 b	594.62
	4x	181	2.481 ± 0.060	2.36-2.73	2.45	0.621 ± 0.015 b	607.73
	TOTAL	208

Table 2 .

 2 Geographic origin of Cotoneaster mother individuals covering three cytotypes and corresponding sample sizes (N) for genome size/ploidy level, reproductive mode, and microsatellite analysis.

	Origin

of Sample Number of Samples (N)

	Site No.	Site ID	Locality	East	North	Altitude (m)	Diploids	Triploids	Tetraploids	Total N per Site
	1	Go	Gornja Grkarica, Mt. Igman, Bosnia and Herzegovina (B&H)	18.28833	43.74528	1350	-, -, -	-, -, -	20, 98, 19	20, 98, 19
	2	Um	Umoljani, Mt. Bjelašnica, B&H	18.22167	43.66194	1300	18, 108, 12	4, 3, 4	8, 27, 8	30, 138, 24
	3	Vr	Vrbe, Čemerno, B&H	18.57167	43.17861	1090	-, -, -	1, -, -	19, -, 18	20, -, 18
	4	Bo	Bosiljna, Mt. Čvrsnica, B&H	17.48806	43.51194	1319	-, -, -	-, -, -	20, 7, 16	20, 7, 16
	5	Ru	Rujište, Mt. Prenj, B&H	17.95472	43.46194	1010	-, -, -	1, -, 1	17, 14, 18	18,14, 19
	6	Bg	Borova glava, Livno, B&H	17.12139	43.77861	1183	1, 3, -	-, -, -	22, 28, 21	23, 31, 21
	7	So	Sovićka vrata, Blidinje, B&H	17.50472	43.59528	1220	-, -, -	1, -, 1	16, 4, 13	17, 4, 14
	8	Pu	Puhova ravan, Mt. Trebević, B&H	18.43833	43.82861	1450	-, -, -	-, -, -	20, 55, 20	20, 55, 20
	9	Ca	Čavljak, Mt. Ozren, B&H	18.43833	43.91194	1237	-, -, -	-, -, -	21, 75, 19	21, 75, 19
	10	De	Devečanske stijene, Mt. Vlašić, B&H	17.62139	44.27889	1752	1, 2, 1	-, -, -	18, 57, 18	19, 59, 19
	11	Rt	Mt. Rtanj, Serbia	21.88917	43.77861	1282	-, -, -	1 *, -, 1	8 *, -, 8	9, -, 9
	12	Mu	Mt. Mučanj, Serbia	20.03889	43.54528	1530	-, -, -	-, -, -	7 *, 36, 7	7, 36, 7

Table 2 .

 2 Cont.

	Origin

of Sample Number of Samples (N)

	Site No.	Site ID	Locality	East	North	Altitude (m)	Diploids	Triploids	Tetraploids	Total N per Site
	13	Pr	Premužićeva staza, Mt. Velebit, Croatia	14.98722	44.76222	1613	-, -, -	1 *, -, 1	3 *, 2, 3	4, 2, 4
	14	Vo	Vošac, Mt. Biokovo, Croatia	17.05472	43.31194	1320	-, -, -	1 *, -, 1	8 *, 58, 8	9, 58, 9
	15	Si	Mt. Sinjajevina, Monte Negro	19.33861	42.96167	1773	-, -, -	-, -, -	5 *, -, 5	5, -, 5
	16	He	Hechenberg-Ostgipfel von Kranebitten, Innsbruck, Austria	11.31972	47.27972	1390	-, -, -	-, -, -	5 *, -, 5	5, -, 5
	17	Ro	Rotenfels, Rheinland-Pfalz, Germany	7.818611	49.83028	232	-, -, -	-, -, -	5 *, 6, 5	5, 6, 5
	18	Mo	Monte Ventosola, Monte Sibillini, Umbria, Italy	13.17028	42.77833	1493	-, -, -	-, -, -	4 *, 8, 4	4, 8, 4
	19	Su	Mt. Suva Gora, Northern Macedonia	21.25583	41.84472	1184	-, -, -	-, -, -	7 *, -, 7	7, -, 7
	20	Ol	Mt. Olympus, Greece	22.37278	40.07778	1935	-, -, -	-, -, -	5 *, -, 5	5, -, 5
	21	Pa	Mt. Pangaion, Greece	24.07333	40.92778	1662	-, -, -	-, -, -	5 *, -, 5	5, -, 5
			Total N per cytotype			20, 113, 13	10, 3, 9	243, 475, 232	273, 591, 254

* Ploidy level was inferred from microsatellite data.

Table 3 .

 3 Flow cytometric seed screening results and hypothesized pathways of seed formation and reproduction modes in analyzed Cotoneaster integerrimus populations.

			Cytometric Results					Hypothesized Pathways of Seed Formation
	Maternal ploidy a	Embryopg ± SD	Endosperm pg ± SD	Embryo ploidy	Endosperm ploidy	Number of seeds	Egg ploidy	Polar nuclei ploidy/ number	Sperm ploidy	Number of sperms fe-cundating the en-dosperm	Endosperm maternal: paternal ratio genome	Endosperm/ embryo ratio) (mean fluores-cence	Seed origin
	2x	1.28 ± 0.03	1.95 ± 0.05	2x	3x	103	1x	1x/2	1x	1	2m:1p	1.52	Sexual
	2x	1.91 ± 0.05	2.59 ± 0.06	3x	4x	8 b	1x	1x/2	2x	1	1m:1p	1.35	Sexual
	3x	1.94	2.64	3x	4x	1	1x	1x/2	2x	1	1m:1p	1.5	Sexual
	3x	1.85	3.93	3x	7x	1	3x	3x/2	1x	1	6m:1p	1.35	Pseudogamous Apomixis
	3x	1.94	5.72	3x	9x	1	3x	3x/2	3x	1	2m:1p	2.12	Pseudogamous Apomixis
	4x	2.54 ± 0.07	3.84 ± 0.11	4x	6x	92	2x	2x/2	2x	1	2m:1p	1.53	Sexual
	4x	3.80 ± 0.10	6.43 ± 0.21	6x	10x	22 c	4x	4x/2	2x	2	4m:1p	2.5	Sexual
	4x	1.90 ± 0.01	3.19 ± 0.05	3x	5x	2 d	2x	2x/1	1x	1	4m:1p	1.7	Sexual
	4x	2.56 ± 0.1	6.38 ± 0.15	4x	10x	148	4x	4x/2	1x or 2x	2 or 1	4m:1p	2.5	Pseudogamous Apomixis
	4x	2.54 ± 0.01	7.51 ± 0.37	4x	12x	137	4x	4x/2	2x	2	2m:1p	2.98	Pseudogamous Apomixis
	4x	2.55 ± 0.04	7.04 ± 0.25	4x	11x	22	4x	4x/2	3x	1	2.7m:1p	2.75	Pseudogamous Apomixis
	4x	2.58 ± 0.12	5.83 ± 0.28	4x	9x	11	4x	4x/2	1x	1	8m:1p	2.17	Pseudogamous Apomixis
	4x	2.53 ± 0.05	5.08 ± 0.17	4x	8x	32	4x	4x/2	-	0	4m:0p	4:1	Autonomous Apomixis
	4x	2.55 ± 0.08	4.46 ± 0.17	4x	7x	9	4x						Not resolved
													Haploid
	4x	1.28 ± 0.00	3.84 ± 0.04	2x	6x	2	2x	2x/2	1x or 2x	2 or 1			Parthenogen-
													esis
						591

a Maternal ploidy inferred from cytometric leaf measurements. b Embryo ploidy was increased compared to mother ploidy. c Biii hybrids (explanation Section 2.2.3). d Embryo ploidy was decreased compared to mother ploidy.

Table 4 .

 4 Genetic diversity measures for the analyzed Cotoneaster integerrimus populations derived from nuclear microsatellites.

	Locality (Site ID)	Number of Individuals Sampled (N)	Number of Multilocus Genotypes Detected (Ng)	Total Number of Individuals Belonging to a Clone (Ni)	Total Number of Unique Genotypes per Population (Un)	Effective Number of Genotypes (Eff )	Proportion of Clones Detected (Ng/N)	Genotypic Diversity (Div)
	Gornja Grkarica (Go)	19	3	18	1	2.21	0.15	0.57
	Umoljani (Um)	24	21	4	19	18	0.91	0.98
	Vrbe (Vr)	18	7	12	6	4.37	0.39	0.81
	Bosiljna (Bo)	16	5	14	5	3.55	0.31	0.76
	Rujište (Ru)	19	4	17	4	2.39	0.21	0.61
	Borova glava (Bg)	21	2	20	1	1.09	0.1	0.09
	Sovićka vrata (So)	14	6	11	4	4.07	0.42	0.81
	Puhova ravan (Pu)	20	1	20	1	1	0.05	0
	Čavljak (Ca)	19	5	16	4	1.57	0.26	0.38
	Devečanske stijene (De)	19	6	16	6	2.63	0.31	0.65
	Mt. Rtanj (Rt)	9	4	7	4	3	0.44	0.75
	Mt. Mučanj (Mu)	7	2	7	2	1.86	0.28	0.47
	Premužićeva staza (Pr)	4	3	2	3	2.66	1	0.83
	Vošac (Vo)	9	4	7	3	2.07	0.33	0.58
	Mt. Sinjajevina (Si)	5	1	5	1	1	0.20	0
	Hechenberg-Ostgipfel (He)	5	1	5	1	1	0.20	0
	Rotenfels (Ro)	5	3	3	3	2.27	0.60	0.70
	Monte Ventosola (Mo)	4	3	2	3	2.66	0.75	0.83
	Mt. Suva Gora (Su)	7	3	4	2	1.81	0.71	0.52
	Mt. Olympus (Ol)	5	4	2	4	3.57	0.8	0.90
	Mt. Pangaion (Pa)	5	4	2	4	3.57	0.8	0.90
		254	92	194	81

Plants 2021, 10, x FOR PEER REVIEW 10 of 19

Acknowledgments:

We are greatly thankful to Elma Silajdžić for her help with molecular laboratory work. Božo Frajman supported us with samples from Austria, Germany, and Italy; Nevena Kuzmanović with samples from Serbia and Greece; Danka Petrović with samples from Montenegro. We thank Dubravka Šoljan for her help during collection of samples and Mario Gomez Pacheco for help with cytometry. We thank three anonymous referees for valuable comments on the manuscript.

Conflicts of Interest:

The authors declare no conflict of interest.

Data Availability Statement: Not applicable.


Robert P Adams
email: robert_adams@baylor.edu

Sam T Johnson

John Anderson

Keith Rushforth

Nicolas Valentin

Sonja Siljak-Yakovlev

Ashill, Cullompton, Devon The Shippen

Nuclear and chloroplast DNAs reveal diverse origins and mis-identifications of Juniperus chinensis cultivars

Keywords: Juniperus chinensis cultivars, origin, nrDNA, ITS, cp DNA, DNA barcoding

come   L'archive ouverte pluridisciplinaire

It has now been shown that genome size assessment using flow cytometry (FC) can be successfully used as a proxy for ploidy level in Juniperus (Farhat et al. 2019a, b) from both fresh and silica gel dried leaves of Juniperus. Thus, the ploidy of Juniper hybrids can now be determined by FC. This is very important because it is known that several J. chinensis cultivars are triploid or tetraploid [START_REF] Hall | Chromosome numbers of cultivated Junipers[END_REF]. With the confluence of both DNA methodology and FC ploidy determination, this presents us with a great opportunity to examine the origin of J. chinensis cultivars.

As a first step in this work, we recently analyzed Juniperus xpfitzeriana cultivars, one of the most commonly cultivated junipers in the world [START_REF] Adams | The origin of Juniperus xpfitzeriana, an allo-tetraploid hybrid of J. chinensis x J. sabina[END_REF]. The origin of J. xpfitzeriana is thought to be a hybrid of J. chinensis x J. sabina. Nuclear DNA (nrDNA, ITS) and 4 chloroplast gene regions were sequenced from 14 J. xpfitzeriana cultivars from Windsor Gardens, UK, and compared with all Juniperus, sect. Sabina, smooth leaf margin species. All of the 14 cultivars were identical in their chloroplast DNA and their cp DNA was identical to that of J. sabina var. balkanensis (Table 1). In addition, 13 J. xpfitzeriana cultivars were allo-tetraploids with heterozygous bases at 5 to7 sites that distinguish J. chinensis and J. sabina var. balkanensis. These cultivars had identical nrDNA. Two of the 14 cultivars, 'Old Gold' and 'Sea Green', showed a slightly different nrDNA pattern, being homozygous at sites 410 and 1139, as found in J. s. var. balkanensis. The origin of J. xpfitzeriana is from a cross of a male, tetraploid J. sabina var. balkanensis and a female, tetraploid, J. chinensis, resulting in an allo-tetraploid, dioecious, J. xpfitzeriana (Spath) Schmidt. The purpose of the present research is to present new DNA sequencing utilizing both chloroplast and nuclear DNA in the determination of the origin of J. chinensis cultivars.

G C C C G A A chinensis chinensis 15442 Arctic 4x G/T C/G C/T C/T A/C G/T A/T A/G chin x sab balkanensis 15454 Armstrongii 4x G/T C/G C/T C/T A/C G/T A/T A/G chin x sab balkanensis 15418 Aurea, Paris-sud 4x G/T C/G C/T C/T A/C G/T A/T A/G chin x sab balkanensis 15474 Aurea 4x G/T C/G C/T C/T A/C G/T A/T A/G chin x sab balkanensis 15423 Saybrook Gold 4x G/T C/G C/T C/T A/C G/T A/T A/G chin x

METHODS

Plant materials:

Samples: Leaf samples were collected in Windsor Gardens, Windsor Great Park, Windsor, SL4 2HT UK from 24 J. chinensis cultivar accessions (see Table 2) and immediately placed in activated silica gel for DNA sequencing and Flow Cytometry -ploidy determination. Reference Species: Juniperus chinensis, J. sabina var. sabina, J. s. var. balkanensis see Adams et al. (2018a) for collection details.

DNA extraction and sequencing

One gram (fresh weight) of the foliage was placed in 20 g of activated silica gel and transported to the lab, thence stored at -20 o C until the DNA was extracted. DNA was extracted from juniper leaves by use of a Qiagen mini-plant kit (Qiagen, Valencia, CA) as per manufacturer's instructions. Amplifications were performed in 30 µl reactions using 6 ng of genomic DNA, 1.5 units Epi-Centre Fail-Safe Taq polymerase, 15 µl 2x buffer E (petN, trnD-T, trnL-F, trnS-G) or K (nrDNA) (final concentration: 50 mM KCl, 50 mM Tris-HCl (pH 8.3), 200 µM each dNTP, plus Epi-Centre proprietary enhancers with 1.5 -3.5 mM MgCl2 according to the buffer used) 1.8 µM each primer. See [START_REF] Adams | A new genus, Hesperocyparis, for the cypresses of the new world[END_REF] for the ITS and petN-psbM primers utilized. The primers for trnD-trnT, trnL-trnF and trnS-trnG regions have been previously reported [START_REF] Adams | Geographic variation in nrDNA and cp DNA of Juniperus californica, J. grandis, J. occidentalis and J. osteosperma (Cupressaceae)[END_REF]. The PCR reaction was subjected to purification by agarose gel electrophoresis. In each case, the band was excised and purified using a Qiagen QIAquick gel extraction kit (Qiagen, Valencia, CA). The gel purified DNA band with the appropriate sequencing primer was sent to McLab Inc. (San Francisco) for sequencing. Chromatograms analyzed by use of Chromas 2.31 (Technelysium Pty Ltd.).

Flow cytometric analyses for ploidy level determination

Nuclear DNA amount was assessed by flow cytometry (FC) based on the technique of [START_REF] Bourge | Flow cytometry as tool in plant sciences, with emphasis on genome size and ploidy level assessment[END_REF] on silica dried leaves of Juniperus samples and fresh leaves of Hordeum vulgare L. 'Sultan'[2C= 9.81 pg in [START_REF] Garnatje | Genome size in Echinops L. and related genera (Asteraceae, Cardueae): karyological, ecological and phylogenetic implications[END_REF]] used as an internal standard. Approximately, 30 mg of leaves of both the internal standard and Juniperus were simultaneously chopped using a razor blade in a plastic Petri dish with 500 µl of cold Gif nuclear-isolation buffer-GNB [START_REF] Bourge | Flow cytometry as tool in plant sciences, with emphasis on genome size and ploidy level assessment[END_REF]): 30 mM sodium citrate, 45 mM MgCl2, 60 mM MOPS (4-morpholine propane sulphonate, pH 7), and 1% (w/v) polyvinylpyrrolidone 10,000, pH 7.2 containing 0.1% (w/v) Triton X-100, supplemented with 10 mM sodium metabisulphite and RNase (2.5 U/ml). The nuclei suspension was filtered through 50 μm nylon mesh. The nuclei were stained with 100 μg/ml propidium iodide (PI), a specific DNA fluorochrome intercalating dye, and kept at 4°C for 5 min. DNA content of about 3,000 stained nuclei was determined for each sample using the cytometer CytoFLEX S (Beckman Coulter-Life Science United States. Excitation 488 nm, 26 mW; emission through a 610/20 nm band-pass filter). Measurements of each sample were repeated twice. The software CytExpert was used for histogram analyses. The total 2C DNA value was calculated using the linear relationship between the fluorescent signals from stained nuclei of the species and the internal standard, according to the following formula: 2C DNA sample (pg) = (Sample 2C peak mean / Standard 2C peak mean) x Standard 2C DNA (pg).

RESULTS AND DISCUSSION

Ploidy was determined for twenty four (24, only 23 were Juniperus, see below) plants labeled as J. chinensis cultivars at the Windsor Gardens, UK and analyses revealed (Tables 2,3) that of the 23 juniper plants, 16 were tetraploids [START_REF] Japan | 5833 4x unknown[END_REF], 6 diploids ( 22), and one triploid (33). Farhat et al. (2019a) discovered that about 15% of Juniperus taxa were tetraploids and one, J. foetidissima, was a hexaploid, based on analysis of samples from junipers that were naturally occurring not cultivated. In this study, we found most of these cultivated plants were tetraploids. It is worthwhile to review an interesting study by [START_REF] Zinnai | Tetraploids of Sugi (Cryptomeria japonica D. Don) discovered at Forest nursery[END_REF] who in a survey Cryptomeria japonica in seedling nurseries (2 and 3-year old seedlings) found 4 seedlings with twisted needles that were thick and bent at the tip-end. In addition, the stomatal bands tended to be larger. Chromosome counts on these plants confirmed they were tetraploids. [START_REF] Chiba | Triploids and tetraploid sugi (Cryptomeria japonica D. Don) selected in forest nursery[END_REF], later, selected 39 (putative) polyploid seedlings with twisted needles from the germination beds and found 18 were diploids, 3 triploids and 18 tetraploids. The polyploids randomly occurred in beds at a rate of 5 x 10 - 6 frequency (e.g., 0.0005%). Normally in a forest seedling nursery, abnormal appearing seedlings (such as these with twisted needles) are removed by gardeners to maintain robust seedlings for out-planting. [START_REF] Ahuja | Polyploidy in gymnosperms revisited[END_REF] noted that "sporadic polyploids and aneuploids occur at a very low frequency in nurseries in conifers, but most of them show growth abnormalities, remain dwarf, and may not reach maturity". 2 is compared with literature reports of chromosome number [START_REF] Hall | Chromosome numbers of cultivated Junipers[END_REF]. Note that several literature reports differ from the flow cytometry ploidy determination: Belvedere, litr. = tetraploid (44) vs. triploid (33); Jacobiana, litr = triploid (33) vs. tetraploid [START_REF] Japan | 5833 4x unknown[END_REF]; Blaauw, litr = tetraploid (44) vs. diploid ( 22), Fruitlandii, litr. = triploid (33) vs tetraploid [START_REF] Japan | 5833 4x unknown[END_REF]; Globosa Cinerea, litr = tetraploid (44?) vs. diploid (22). It is very likely that there have been labeling errors over the decades in transferring plants among botanic gardens and nurseries. It nearly impossible to obtain samples from the original plants for which the names originated.

Ploidy shown in Table

Analysis of nrDNA (ITS) revealed 12-14 polymorphic sites among the 24 'J. chinensis cv' studied (Table 3). Analysis of 3 chloroplast (cp) genes: petN-psbM, trnS-trnG and trnL-trnF revealed that petN-psbM (hereafter petN), as the most informative in distinguishing J. chinensis, J. sabina, and related species, thus, trnS-trnG and trnL-trnF were not further utilized. petN sequence utilized to reveal the chloroplast source (e.g., pollen, paternal) for the J. chinensis cultivars studied.

The 24 'J. chinensis cultivars' were found to be in 8 groups (Table 3). The first group (yellow) included 'Richeson' and 'Fruitlandii, both tetraploids, which have J. sabina var. balkanensis cp, and J. xpfitzeriana ITS, as seen in the Wilhelm Pfitzer (xpfitzeriana) sample (from [START_REF] Adams | The origin of Juniperus xpfitzeriana, an allo-tetraploid hybrid of J. chinensis x J. sabina[END_REF]. So, both of these are xpfitzeriana, not J. chinensis.

Japonica, and Japonica Variegata (2 nd group, blue), tetraploids, are part of J. chinensis var. sargentii (Table 3) with J. c. var. sargentii cp and ITS.

Kuriwao Sunbeam is in the 3 rd (purple) group and is very unusual being a diploid with J. sabina var. balkanensis cp and J. chinensis var. procumbens ITS, because both of these taxa are tetraploid (Farhat et al. 2019a).

The 4 th and 5 th groups are closely related with all 7 cultivars having J. chinensis ITS DNA, but the red group 4, contains Obelisk, Iowa (=Globosa), and Plumosa Aurea which are tetraploids with J. chinensis cp. In contrast, group 5 (salmon) contains 4 diploids (chinensis var. sargentii Glauca, Pfitzer Blaauw, Pfitzer Shimpaku, and Pfitzer Globose Cinerea), all have J. chinensis ITS, but each has J. tsukusiensis (sometimes treated as J. chinensis var. tsukusiensis, Adams 2014) chloroplasts. The use of Pfitzer as part of the cultivar name is confusing, as xpfitzeriana is tetraploid and of hybrid origin from J. sabina x J. chinensis, see [START_REF] Adams | The origin of Juniperus xpfitzeriana, an allo-tetraploid hybrid of J. chinensis x J. sabina[END_REF].

The 6 th group (green) is the largest with 9 tetraploids and one triploid, all have J. chinensis hybrid ITS DNA (Table 3). Seven (Aurea, Jacobiana, Shepherdii, Keteleerii, Robusta Glauce, Lombards, Belvedere) have J. chinensis var. sargentii cp. Two (Kuriwao Mist, Mathot) have J. sabina var. balkanensis cp and one (Kek) is the only plant in these analyses with J. chinensis cp. The tremendous diversity in the hybrid nature of nrDNA (ITS) in this group indicating that the maternal parent arose by hybridization with a variety of junipers.

The 9 th group was most surprising to find that 'Savill Sentinel' was not a juniper, but a cypress, Cupressus gigantea by ITS DNA (Table 3). Interestingly, this plant is of hybrid origin (note the heterozygous ITS sites, Table 3), with a male Cupressus gigantea parent chloroplast. We not able to identify the maternal parent of the hybrid at this point. Even with 3 botanists collecting samples, none of us noted that it was a cypress. Perhaps we were too focused on the mechanics of collecting and accurately labeling the samples to observe the plant.

Group 10 produced the second surprise in that 'Spartan' had ITS and cp DNA of J. virginiana (Table 3). Juniperus chinensis and J. virginiana look very similar, especially if juvenile (decurrent) leaves are present on J. virginiana, so it is not surprising that Spartan was labeled J. chinensis as some time in history.

Five diploid cultivars have cp parents that differ from their homozygous maternal parents nrDNA: Kuriwao Sunbeam (J. sabina var. balkanensis, cp, J. chinensis var. procumbens, nrDNA); Glauca, Blaauw, Shimpaku, and Globosa Cinerea (all 4 with J. tsukusiensis cp and J. chinensis, nrDNA). These 5 cultivars with conflicting cp and nrDNA seem likely to have experienced a chloroplasts capture event as has been found often in natural populations of Juniperus (Adams et al. 2017 a,b;Adams et al. 2018 a,b;[START_REF] Adams | Allopatric hybridization and introgression between Juniperus scopulorum Sarg. and Juniperus blancoi Mart. in northern Mexico: Unidirectional gene flow[END_REF]Farhat et al. 2019 a,b;[START_REF] Hojjati | Discovery of chloroplast capture in Juniperus excelsa complex by multi-locus phylogeny[END_REF].

It is interesting that some of the aforementioned diversity was discovered Le [START_REF] Duc | Using random amplification of polymorphic DNA for a taxonomic reevaluation of Pfitzer junipers[END_REF] by the use of RAPDs (Random Amplified Polymorphic DNAs). Figure 1 shows a PCO based on 122 RAPD bands of J. chinensis, J. sabina and 9 cultivars. Notice the Pfitzer cultivars group are near the base of J. chinensis, but intermediate on axis 2, to J. sabina, giving an evidence that they are chinensis x sabina hybrids, although the synthetic (computer generated) hybrid is precisely intermediate. Fruitlandii (a xpfitzeriana, Table 3) is intermediate on axis 3. Kallay's Compact, Gold Coast and Hetzii form a group near the Pfitzers (yellow oval).

Figure 1. PCO using 122 RAPD bands of J. chinensis (natural, Japan), J. sabina (natural, Switzerland) and 9 cultivars.

Possible ploidy levels of putative parents of 'J. chinensis' cultivars in this study

It is very interesting that the ploidy of all the male parents of the 'J. chinensis' cultivars as well as the female parents have been reported (Farhat et al. 2019a) as tetraploids (4x), (Table 4: J. sabina var. balkanensis, J. chinensis, J. c. var. sargentii, J. c. var. procumbens, J. tsukusiensis, and J. xpfitzeriana). However, Kuriwao Sunbeam is diploid (2x, Table 4) suggesting that haploid (1x) gametes of J. s. var. balkanensis and J. c. var. procumbens united to form the diploid. The four male tsukusiensis x female chinensis parentages resulted in diploid (2x) chinensis var. sargentii 'Glauca', and 3 Pfitzer 'Blaauw', 'Shimpaku', and 'Globosa Cinerea' (Table 4). Although Farhat et al. (2019a) found their natural tsukusiensis to be 4x, it is very possible there are cultivars of tsukusiensis that are diploid. And, it is certainly possible that putative 'chinensis' female parents were diploids. Unfortunately, we know very little about variation in ploidy of J. chinensis in the wild. In a recent study of nearly all Juniperus species, Farhat et al. (2019a) reported that J. chinensis, J. c. var. procumbens, and J. c. var. sargentii were tetraploids in nature. However, only one plant each of J. chinensis, J. c. var. procumbens, and J. c. var. sargentii were analyzed. [START_REF] Nagano | Karyomorphological study of Juniperus. In: Cytogenetic studies of forest trees and shrubs -Review, Present Status, and Outlook on the Future[END_REF][START_REF] Nagano | Karyotype analysis of three Juniperus species using fluorescence in situ hybridization (FISH) with two ribosomal genes[END_REF] analyzed J. chinensis varieties from Japan and reported that J. chinensis var. chinensis, J. c. var. kaizuka, J. c. var. jacobiana were tetraploids (2n=44), but J. c. var. sargentii was a diploid (2n=22). In [START_REF] Nagano | Karyotype analysis of three Juniperus species using fluorescence in situ hybridization (FISH) with two ribosomal genes[END_REF], they report that their J. c. var. sargentii was obtained from Mt. Shiroiwa, Miyazaki Prefecture. Farhat et al. (2019a) obtained their J. c. var. sargentii from Mt. Kirigishi, Furano-Ashibetsu Natural Park, Hokkaido. However, [START_REF] Nagano | Karyotype analysis of three Juniperus species using fluorescence in situ hybridization (FISH) with two ribosomal genes[END_REF] strongly felt the chromosome karyomorphological differences between their J. chinensis var. chinensis and J. c. var. sargentii warranted the recognition of J. sargentii at the specific level. In contrast, [START_REF] Adams | Phylogeny of Juniperus using nrDNA and four cpDNA regions[END_REF] and [START_REF] Adams | Systematics of Juniperus chinensis and J. tsukusiensis from Japan and Taiwan: DNA sequencing and terpenoids[END_REF] found that their J. c. var. sargentii (4x) material was in a well-supported clade with J. chinensis, supporting its recognition as J. c. var. sargentii. The confusion may rest on the fact that J. c. var. chinensis and J. c. var. sargentii are difficult identify when collecting.

The final unusual case is that of Belvedere, a triploid with male chinensis v. sargentii (4x, Farhat et al. 2019a;or 2x, Nagano et al. 2007) and female chinensis hybrid (4x) (Table 4). If the var. sargentii was 2x and the female chinensis hybrid was tetraploid, then the triploid follows simply (2x + 1x = 3x). If the male parent was a tetraploid, then the explanation of triploid hybrid would be more difficult. Table 4. Analyses of ploidy of putative parents' ploidy and ploidy of the cultivars at Windsor Garden.

In this study, we found tremendous variation among nrDNA and cp parentage. The development and implementation of a DNA barcode system would greatly aid botanic gardens to screen current and incoming accessions to assign taxonomic names to junipers and other conifers.

  . v. balkanensis 4x

Table 1 .

 1 nrDNA (ITS) variable sites in J. chinensis cultivars. (Windsor Gardens), J. chinensis, and J. sabina. K=G/T; S=C/G; Y=C/T; M=A/C; W=A/T; R=A/G. chloroplast types: balkanensis = J. sabina var. balkanensis/ J. thurifera; sabina = J. sabina var. sabina; and chinensis = J. chinensis. Modified from[START_REF] Adams | The origin of Juniperus xpfitzeriana, an allo-tetraploid hybrid of J. chinensis x J. sabina[END_REF]. Site numbers modified to correspond with site numbers in Table3of this report.

	taxa: J. xpfitzeriana (=xmedia),	ploidy	212 a	410	665	986	996	1034	1073	1137	ITS	chloroplast,
	unless noted otherwise		K	S	Y	Y	M	K	W	R	classification hybrid?	ex. pollen from:
	Most probable male (pollen) parent	4x	G	C	T	T	A	T	T	G	J. sabina var.	J. sabina var.
											balkanensis	balkanensis
	Most probable female parent genotype 4x	T

Table 2 .

 2 Windsor 24 Juniperus chinensis cv and origin table < = earlier than (before).

		Adams	Windsor	ploidy	Chrom
	taxon	#	acc. #	this	number, 2n,
	Juniperus chinensis '			study	litr.

ACKNOWLEDGEMENTS

This research was supported in part with funds (0324512 to RPA) from Baylor University. Thanks to the Issy Bean and Claire Sadler at Windsor Gardens for assistance in collection.