New estimates and synthesis of chromosome number, ploidy level and genome size variation in Allium sect. Codonoprasum: a step towards understanding the hitherto unresolved diversification and evolution of the section

doi:10.21203/rs.3.rs-4972960/v1

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New estimates and synthesis of chromosome number, ploidy level and genome size variation in Allium sect. Codonoprasum: a step towards understanding the hitherto unresolved diversification and evolution of the section

https://doi.org/10.21203/rs.3.rs-4972960/v1

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Background

The genus Allium is known for its high chromosomal variability, but most chromosome counts are based on a few individuals and genome size (GS) reports are limited in certain taxonomic groups. This is evident in the Allium sect. Codonoprasum, a species-rich (> 150 species) and taxonomically complex section with weak morphological differences between taxa, the presence of polyploidy and frequent misidentification of taxa. Consequently, a significant proportion of older karyological reports may be unreliable and GS data are lacking for the majority of species within the section. This study, using chromosome counting and flow cytometry (FCM), provides the first complex and detailed insight into variation in chromosome number, polyploid frequency and distribution, and GS in section members, a step towards understanding the section's unresolved diversification and evolution.

Results

We analysed 1,582 individuals from 311 populations of 25 taxa and reported DNA ploidy levels and their GS, with calibration from chromosome counts in 21 taxa. Five taxa had multiple ploidy levels. GS estimates for 16 taxa are primary estimates. A comprehensive review of chromosome number and DNA-ploidy levels in 128 taxa of the section revealed that all taxa had x = 8, except A. rupestre with two polyploid series (x = 8, descending dysploidy x = 7), unique for this section. Diploid taxa dominated (71.1%), while di-/polyploid (12.5%) and pure polyploid (16.4%) taxa were less common. Ploidy diversity showed that diploid taxa were dominant in the eastern Mediterranean (> 85%), decreasing towards the west and north, with only polyploid taxa present in northern and northwestern Europe. A 4.1-fold variation in GS was observed across 33 taxa (2C = 22.3–92.1 pg), mainly due to polyploidy, with GS downsizing observed in taxa with multiple ploidy levels. Intra-sectional GS variation suggests evolutionary relationships, and intraspecific GS variation within some taxa may indicate taxonomic heterogeneity and/or historical migration patterns.

Conclusions

Our study showed advantages of FCM as an effective tool for detecting ploidy levels and determining GS within the section. GS could be an additional character in understanding evolution and phylogenetic relationships within the section.

cytogeography

DNA ploidy level

chromosome number

flow cytometry

genome size

polyploidy

As the evolutionary history of organisms is inscribed in their chromosomes, chromosome number is a fundamental genomic attribute of an organism (Mayrose and Lysák 2020). Information on chromosome number (Rice et al., 2015) and nuclear DNA content (Greilhuber et al. 2005; Pellicer and Leitch 2020; Siljak-Yakovlev et al. 2020) highlights the role of numerical variation (Van de Peer et al., 2017) and, together with the study of karyotypic differentiation, helps to understand the role of structural changes in evolutionary processes (e.g. Schubert and Lysák 2011; Mandáková and Lysák 2018). Chromosome number and genome size (hereafter GS) are important species-specific traits (Stebbins 1971; Guerra 2008, 2012; Goldblatt and Lowry 2011; Carta et al. 2020; Pellicer and Leitch 2020), which are useful tools for discriminating between taxa and resolving taxonomy within groups that are critical, for example, due to morphological reduction, phenotypic plasticity, mating systems and reticulate evolution (e.g. Castro et al. 2012; Hajrudinović et al. 2015; Prančl et al. 2018; Popelka et al. 2019; Afonso et al. 2021). GS and its intraspecific variation may also help to understand the evolutionary forces shaping genomic features (Šmarda and Bureš 2010; Prančl et al. 2014, 2018; Becher et al. 2021) and functional diversity of plants (e.g. Šímová and Herben 2012; Roddy et al. 2020; Bitomský et al. 2022, 2023).

Reports on the chromosomes of species are usually based on small numbers of individuals. Such an approach may underestimate the variability in cytotype composition within and between populations, which is usually the result of genome duplication (Stuessy 2009). From a practical perspective, flow cytometry has proven valuable in plant biosystematics over the last two decades (Bourge et al. 2018; Sliwinska et al. 2022; Loureiro et al., 2023). This technique allows rapid and non-destructive estimation of DNA-ploidy levels and nuclear DNA content in a large number of samples (e.g. Trávníček et al. 2012; Čertner et al. 2017, 2022; Rejlová et al. 2019). Its application has led to the discovery of diverse cytotypes in various plant taxa, providing a better understanding of the mechanisms underlying cytotype origin and coexistence (reviewed in Kolář et al. 2017), cytogeographic diversity across species ranges (e.g. Mráz et al. 2008; Duchoslav et al. 2010, 2020; Šafářová and Duchoslav 2010; Šafářová et al. 2011; Kobrlová et al. 2016, 2022; Taraška et al. 2021, 2024; Horák et al. 2023; Vejvodová et al. 2024) or the identification of patterns of ecological diversification or habitat shifts (e.g. Duchoslav et al. 2020; Kobrlová et al. 2022r et al. 2023), which can provide insight into the evolutionary history of species (Blommaert 2020; Cang et al. 2024).

The genus Allium L. (Amaryllidaceae, Allieae) is one of the largest monocotyledonous genera (Chase et al. 2009; Costa et al. 2020), with over a thousand accepted species (POWO 2024), represented by perennial rhizomatous or bulbiferous herbs that combine sexual and asexual reproduction (Rabinowitch and Currah 2002), and is widely distributed throughout the Northern Hemisphere (Fritsch and Friesen 2002; Friesen et al. 2006; Li et al. 2010; Hauenschild et al. 2017). The genus exhibits multiple basic chromosome numbers, including x = 7, 8, 9, 10, 11 (Hanelt et al. 1992; Friesen et al. 2006; Li et al. 2016; Peruzzi et al. 2017; Han et al. 2020). The genus also displays high levels of polyploidy (Friesen 1992; Hanelt et al. 1992; Han et al. 2020) and occasional B chromosome appearance (Speta 1984; Holmes and Bougourd 1989; Vujošević et al. 2013). Polyploidisation is considered the main driver of adaptations and speciation across diverse environments within the genus (Han et al. 2020).

A number of studies have been conducted on the diversity of chromosome number (for a comprehensive survey see Peruzzi et al. 2017; Han et al. 2020) and GS (Leitch et al. 2019) in the genus Allium. However, despite the genus's taxonomic richness, certain groups within the genus are underrepresented in databases of chromosome count and GS. This is particularly evident in the Allium sect. Codonoprasum Rchnb. This section is one of the largest and taxonomically most complicated within the genus (Hanelt 1996; Salmeri et al. 2014, 2016; Özhatay and Koçyiğit 2019), exhibiting minor morphological differences between taxa and presence of polyploid species/species groups (Tzanoudakis and Vosa 1988; Peruzzi et al. 2017; Duchoslav et al. 2020; Han et al. 2020). This often leads to misidentification of taxa (Salmeri et al. 2016; Vojtěchová et al. 2023), and thus a significant proportion of older karyological reports for certain species may not be reliable due to uncertain identification of the studied individuals. In addition, GS data are lacking for the majority of species within the section, with only a small subset of taxa having been studied (Ohri et al. 1998; Baranyi and Greilhuber 1999; Duchoslav et al. 2013; Šmarda et al. 2019; Vojtěchová et al. 2023, 2024). Furthermore, more detailed analyses focusing on population-level differences in GS (i.e. global and local distribution patterns of cytotypes) are almost absent for section members (Duchoslav et al. 2010, 2013; 2020; Šafářová and Duchoslav 2010; Šafářová et al. 2011).

To address these issues in a broader context, we collected population samples of 25 taxa (species, subspecies) of A. sect. Codonoprasum across Europe and neighbouring regions. Using classical karyology, flow cytometry and a comprehensive review of the available literature, our aims were to (i) determine the diversity of DNA ploidy levels (sensu Suda et al. 2006) within and between populations for each taxon studied, (ii) validate the detected DNA ploidy levels by chromosome counting, (iii) estimate the GS of the detected ploidy levels and evaluate its variation and spatial pattern, and (iv) critically compare the new data with those from the literature. Finally, we aimed to discuss the patterns obtained in more general content. Therefore, we extracted all available data on chromosome number and GS for the remaining section members not covered by our present research and synthesised the current knowledge on cytogenetic diversity within the section. Our goal is to highlight problematic groups and to stimulate further studies aimed primarily at understanding the taxonomic relationships and phylogeny of this evolutionarily young group.

Plant material and species identification

Determination of the studied taxa was based on the descriptions in the original species/subspecies descriptions, regional floras or studies dealing with their taxonomy. Specifically, we mostly accepted the recent treatments dealing with the respective species. Plant individuals were collected between 2004 and 2023 from natural populations across Europe, Caucasus and Israel, with emphasis to cover as much of the range of the taxa studied as possible (Table S1). Plants were transported and cultivated in the experimental garden of Palacký University in Olomouc, Czech Republic. All analyses were done with the cultivated plants. The voucher specimens were deposited in the Herbarium of Palacký University in Olomouc (OL).

Chromosome counts

Actively growing, young roots were harvested from the pot-cultivated plants, pre-treated with 8-hydroxyquinoline (0.002 M) in darkness at room temperature for 4 h, fixed in a cold mixture of ethanol and acetic acid (3:1) overnight and then stored at 4°C until use. Selected root tips were hydrolysed in 5 N HCl for 25 minutes, stained with Schiff reagent for 40 minutes and squashed in 45% acetic acid (Lillie 1951). Preparations were photographed and counted using an Olympus CX-31 light microscope. Usually, at least five metaphases were counted for each individual studied.

Flow cytometry

Flow cytometry (FCM) was used to estimate the DNA ploidy level (relative genome size, RGS, i.e. ratio of the 2C-peak of the sample to the 2C-peak of the internal standard; Suda et al. 2006) and to determine nuclear DNA content, i.e. the holoploid genome size (absolute genome size, AGS; 2C value sensu Greilhuber et al. 2005) of samples. The methodical recommendations of Sliwinska et al. (2022) were followed. Chromosome counts for selected individuals analysed by FCM served as reference material for the estimates obtained using FCM. The monoploid genome size (1Cx value sensu Greilhuber et al. 2005) was calculated as the 2C value of the sample divided by its ploidy level. Samples were prepared according to the protocol described by Duchoslav et al. (2010) and stained with propidium iodide (PI) with addition of RNAse (both 50 µg·ml–¹). The analyses were performed on a Partec PAS (Partec GmbH, Münster, Germany) or BD Accuri C6 (BD Biosciences, San Jose, USA) cytometer. Secale cereale L. ‘Daňkovské’ (2C = 16.19 pg; Doležel et al. 1998); Triticum aestivum ‘Saxana’ (2C = 34.24 pg; Šafářová and Duchoslav 2010); Pisum sativum ‘Ctirad’ (2C = 8.75 pg; Vojtěchová et al. 2023); Vicia faba 'Inovec' (2C = 26.81 pg, the value recalculated to the primary standard S. cereale) served as internal standards. Where multiple internal standards were used for FCM within a given taxon, the observed values based on the less frequently used standards were recalculated to the dominant standard and these values are presented.

Separate plants or mixed samples of up to four plants per population were measured for the RGS estimation. For each (mixed) sample, fluorescence intensity of usually 3,000 particles were recorded for the RGS estimations. If more peaks were identified in the mixed sample, measurements were repeated with each individual measured separately. The following measurement strategy was chosen to ensure validity of the AGS estimation: (i) all measurements were made over period when the plants were in an identical phenological phase of development, with young fresh leaves without any symptoms of senescence or pathogen attack (March to May), (ii) at least 5000 nuclei per sample were recorded, (iii) only CV for the G₀/G₁ peaks of the standard and Allium samples below 5% were accepted, (iv) each sample was measured by the same operator at least three different times on different days and mean AGS value was calculated from these three measurements (Doležel et al. 2007).

Bibliographic review on chromosome counts and GS

An extensive bibliographic review was performed using the metadatabase CCDB (Rice et al. 2015), the plant C-value database (Leitch et al. 2019) and additional own searches, providing a detailed list of chromosome numbers and nuclear DNA contents (2C values) estimated by different methods (FCM, Feulgen microdensitometry (FEM), Vickers M86 scanning microdensitometry (VIM), Hardie et al. 2002; Doležel et al. 2007) of the taxa of A. sect. Codonoprasum published to date. All records were critically reviewed from a taxonomic point of view to ensure correct determination and nomenclature, according to the original publications and recent taxonomic concepts, as well as by direct contact with the authors of the original descriptions, if possible. The assignment of the studied taxa to A. sect. Codonoprasum usually followed the original description or later taxonomic revisions. Recently, Özhatay and Koçyiğit (2019) transferred 23 species, mostly described from Turkey and originally assigned to A. sect. Codonoprasum, to A. sect. Scorodon K. Koch. We present both taxonomic treatments in the review, i.e. with and without acceptance of the above-mentioned section change.

The majority of extracted chromosome data sets of the studied taxa (those with a given locality) were georeferenced. Distribution maps of the different ploidy levels were constructed for selected di-&polyploid and polyploid taxa with a large number of records. Additionally, all available GS estimates for Allium (Leitch et al. 2019) were extracted and used as a background GS data to describe the known variation in GS of the genus.

Data analyses

Frequency of different cytotype compositions of populations was estimated for each studied taxon based on the presence of cytotypes within populations. Frequency of each cytotype within each taxon was based on the total number of FCM-analysed individuals, ignoring their population assignment. Summary statistics of GS parameters were calculated for each studied taxon, based on population-level data. Relationship between genome size (AGS, RGS) and geographic coordinates (latitude, longitude) for selected ploidy levels of the taxa studied was assessed by Spearman correlation coefficient. Data were analysed in NCSS 9 (www.ncss.com). The maps were created in QGIS 3.28 (www.qgis.org), using the Terrain Elevation Above Sea Level map provided by the Global Solar Atlas 2.0 (https://globalsolaratlas.info) as a background.

Chromosome number, DNA-ploidy level and GS assessment for 25 studied taxa

The new data on the diversity and frequency of cytotypes in their populations and the RGS and AGS for each ploidy level in each taxon are summarised in Table 1, while Table S1 gives these data for the individual populations studied. Detailed bibliographic reviews of published karyological and GS data for the taxa studied are given in Tables S2 and S3, respectively. A total of 25 taxa were analysed by FCM (311 populations/1 582 individuals), with first AGS estimates provided for 16 taxa (Table 1). For nine taxa, several reports on nuclear DNA content have been published previously (Tables S3), whereas for two of them we detected the occurrence of new cytotypes. In five taxa more than one ploidy level was found. Chromosome numbers were counted for 21 taxa, with a new report for A. rupestre Steven. Multiple ploidy levels were confirmed in four taxa. A commentary on the data obtained for each taxon studied is given below, together with critical assessment of the available literature.

Allium aetnense Brullo, Pavone & Salmeri. – The species is a regional endemic of Mt. Etna in Sicily (Brullo et al. 2013). The FCM of six plants from a population on the northern slopes of Mt. Etna revealed a single cytotype identified as diploid (2n = 16, Fig. S1A), consistent with the only previous report (Brullo et al. 2013). Our AGS estimate is the first for the species (Table 1).

Allium carinatum subsp. carinatum. – The subspecies is the most common member of an informal A. carinatum complex (sensu Levan 1933; Stearn 1980), although in some taxonomic treatments (e.g. Jauzein and Tison 2001) more complex treatment is applied, where A. flexum Waldst. & Kit. and A. consimile Jordan ex Gren. are separated from it as independent species. It is native to northern Turkey and most parts of Europe except southwestern and northwestern Europe, Finland, Belarus and Russia (POWO 2024). The FCM of 464 plants from 77 populations revealed the presence of three cytotypes (Table 1): dominant triploids (2n = 3x = 24), less frequent diploids (2n = 2x = 16) and a very rare cytotype with the RGS corresponding to DNA-tetraploids, detected for the first time in this subspecies. The RGS data in di- and triploids were confirmed by chromosome counts (Fig. S1B,C), whereas this was not possible in the inferred DNA-tetraploids due to poor growth of the plants. Previously, two ploidy levels (i.e. 2x, 3x) have been reported for this taxon in numerous studies (e.g. Levan 1933; Geitler and Tschermak-Woess 1962; Table S2). Jauzein and Tison (2001) report tetraploid count for related A. consimile from France. Since some authorities (POWO 2024) consider this species to be a synonym of A. carinatum (= A. carinatum subsp. carinatum), this count might be confirmation of supposed DNA-4x we measured by FCM. However, we agree with Jauzein and Tison (2001) that its taxonomic status requires further study.

In addition, aneuploids (2n = 25, 26) have rarely been documented in several Austrian populations (Tschermak-Woess 1947; Geitler and Tschermak-Woess 1962). However, later authors (e.g. Speta 1984; Wetschnig 1992), who have also detected supernumerary chromosomes in triploid Austrian populations, have presented them as B chromosomes. Similarly, Cheshmedzhiev (1973) has reported the presence of a B chromosome in one triploid Bulgarian population (Table S2).

Pure triploid populations (79.2%) followed by pure diploid populations (14.3%) were the most frequent in our data set. Pure DNA-4x and mixed 2x + 3x and 2x + DNA-4x populations were rare, accounting for a total of five populations (Table 1). Previously, only pure cytotype populations had been reported, with triploids being more common than diploids (Table S2). In agreement with published data, diploids and triploids had a similar geographical distribution and occurred throughout the range of the subspecies (Fig. 1A). Several new national records were found for both di- and triploids (Table S1). Putative DNA-4x were found in three countries (Bulgaria, Italy, Slovakia).

The RGS of diploids showed a tendency for bimodal distribution and a significant geographical pattern of increasing RGS westwards (latitude: r_s = 0.341, P = 0.195; longitude: r_s = -0.785, P < 0.001, Fig. 2A, Fig. S2A). In triploids, RGS also increased westwards (latitude: r_s = 0.305, P = 0.019; longitude: r_s = -0.485, P < 0.001; Fig. 2A), but the pattern was more complex, with occurrences of mosaic parapatry or mosaic sympatry of populations with high and low RGS (Fig. S2B). The AGS of diploids and triploids were variable, i.e. 31.2–36.9 pg (mean 32.9 ± 1.8 pg) and 45.0–51.7 pg (mean 47.9 ± 1.5 pg), respectively. The AGS of likely DNA-tetraploids was 56.7 pg (Fig. S2C). The 1Cx values decreased with increasing ploidy level (Table 1). Previous estimates of nuclear DNA content based on various techniques for both di- and triploids (Nagl and Fusenig 1979; Labani and Elkington 1987; Ohri et al. 1998; Baranyi and Greilhuber 1999; Šmarda et al. 2019) were within the range of the AGS we measured and followed the spatial patterns we observed (although sometimes reported with incorrect ploidy, as the authors did not count chromosomes), considering the geographical origin of the measured plants (Table S3). Only one of the previously published DNA amounts (2C = 22.4 pg, Bösen and Nagl 1978) was completely different, most probably belonging to another species. Divergent AGS values we measured might suggest the existence of several lineages within the taxon and require additional study employing molecular markers.

Allium carinatum subsp. pulchellum (Regel) Bonnier & Layens. – The subspecies is distributed as native in southeastern France, southern parts of Central Europe, Italy, and southeastern Europe to western Romania (POWO 2024) and northeastern Turkey (Marmara region, Kollmann 1984). The FCM of 120 plants from 21 populations (Table 1, Table S1) revealed a single cytotype, identified as diploid by multiple chromosome counts (2n = 16; Fig. S1D). This agrees with numerous previous records of diploids from several European countries, the only exceptions being a triploid count (2n = 24 + 1B; Cheshmedzhiev 1973) and several diploid counts with the presence of a B chromosome (2n = 16 + 1B; Cheshmedzhiev 1973, 1975a) from Bulgaria (Fig. 1B, Table S2).

The RGS showed substantial variation (Table 1), with a significant increase towards the northwest (latitude: r_s = 0.620, P = 0.003; longitude: r_s = -0.788, P < 0.001; Fig. 2B). The AGS followed the spatial pattern observed in the RGS, ranging from 28.1 to 38.8 pg with a mean of 33.6 ± 3.60 pg (Table 1). The Bulgarian and Serbian populations had considerably lower RGS and AGS (-38%) than the other populations (Fig. S2D). So far, measurements of the nuclear DNA amount using FEM and FCM have been provided by Labani and Elkington (1987) and Veselý et al. (2012) for samples of unknown origin and SE France, respectively (Table S3). Both agree well with the majority of our estimates. As the rather divergent AGS values were confirmed by chromosome counting, there are at least two diploid lineages within the taxon: one with lower AGS typical of the eastern localities and the other with higher AGS common to the more western localities.

Allium daninianum Brullo, Pavone & Salmeri. – Widespread representative of the A. stamineum species group in the Middle East (Brullo et al. 1996a, 2007). FCM of 10 plants from three Israeli populations (Table S1) revealed a single cytotype identified as diploid (2n = 16, Fig. S1G). This is consistent with previous records of diploids from Israel and Lebanon (Table S2), with the occurrence of 1–2 B chromosomes reported by Brullo et al. (1996a) from the Coastal Galilee. Our AGS estimate is the first for the species (Table 1).

Allium dentiferum Webb and Berthel. – The species is considered taxonomically critical due to confusion with A. longispathum Redouté. Some authorities (Bartolucci et al. 2024; POWO 2024) and French authors (Jauzein and Tison 2001; Dobignard and Chatelain 2010; Tison and de Foucault 2014) treat A. dentiferum as a synonym of A. longispathum, considering some diagnostic characters of the former species (especially the presence of interstaminal teeth) to be variable and not distinctive, whereas Brullo (Brullo et al. 1991, 2008; Brullo and Guarino 2017) considers A. dentiferum to be distinct from A. longispathum. The taxonomic confusion is related to the fact that both the protologue and the original illustration of A. longispathum (Redoute 1811) and the lectotype designated by Wilde-Duyfjes (1976) do not allow an unequivocal morphological differentiation from some related species. The treatment on the origin and morphological variation and habitat of the type material of A. longispathum by Jauzein and Tison (2001) and the recent photographs of plants of the species from the vicinity of the type locality (Bordeaux, France) on iNaturalist (https://www.inaturalist.org/observations/14161096) suggest that both taxa might be closely related, if not identical. If identical, the name A. longispathum has priority over A. dentiferum (Jauzein and Tison 2001).

During our research (Vojtěchová et al., unpubl. results) we observed two groups of plants: (i) plants that closely resembled the description of A. dentiferum (sensu Brullo et al. 2008), (ii) plants that differed from “typical” A. dentiferum by shorter (4–5.5 mm), apiculate, truncate or subobtuse petals with a slightly different colour (dirty white with pinkish to purplish strips or strikes), ovary cylindrical-elliptical, smooth or slightly papillose in the upper part, stamens exerted from the perigon and often inconspicuous or absent interstaminal teeths (provisionally labelled as “A. dentiferum-pallens”). The second group of plants mix characters of several species, i.e. they partly resemble the description of A. longispathum (sensu Jauzein and Tison 2001), but also A. pallens L. p. p. (sensu Brullo et al. 2003). Genetic analysis is urgently needed to clarify the relationships within this complex, as the morphological variation observed could be partly due to hybridisation between A. dentiferum and A. pallens, or introgression of A. pallens into A. dentiferum, as speculated by Jauzein and Tison (2001).

The FCM of 199 plants from 39 populations of “typical” A. dentiferum (Table S1) revealed two cytotypes, confirmed by several chromosome counts (Table 1, Fig. S1E,F): tetraploid (2n = 32) and pentaploid (2n = 40), both also reported in previous reports, which were directly referred to as A. dentiferum (Brullo et al. 1991, 2008). However, in contrast to previous reports (Table S2) where tetraploids dominated (69%) over pentaploids (41%), our data suggest the opposite (4x: 12.8%, 5x: 87.2%, Table 1). We confirmed previous records of tetraploids from Spain, Italy and Greece (Crete). Brullo et al. (1991, 2008) also recorded tetraploids in Malta, Cyprus and Turkey (Table S2). Pentaploids were documented from Italy and France (Table S2), and we recorded them as new records for Croatia, Slovenia, Greece and Spain (Fig. 1C, Table S1). Only pure-cytotype populations were found (Table 1). Several chromosome records attributed to the Bulgarian A. pallens var. pallens by Cheshmedziev (1970, 1975b) may actually belong to A. dentiferum, considering the photographs of the analysed plants and the notes on their morphology made by the author in his later paper (Cheshmedziev 1975b). On the other hand, some chromosome reports (Cheshmedziev 1975c, 1977) of Bulgarian plants considered as A. longispathum probably represent other, presently unidentified, species (Table S2). Koçyiğit and Özhatay (2011) reported diploids of A. dentiferum for plants sampled in Turkey, but we consider this number to belong to other species.

The RGS in tetraploids were almost constant (latitude: r_s = 0.500, P = 0.667; longitude: r_s = 0.500, P = 0.667; Fig. 2C, Fig. S2E) while it showed intermediate variation in pentaploids, with nearly significant increase towards the east (latitude: r_s = -0.062, P = 0.728; longitude: r_s = 0.340, P = 0.053; Fig. 2C, Fig. S2F). The AGS of pentaploids followed the spatial pattern observed in the RGS, with the easternmost populations having about 5 pg (7%) higher AGS than the westermost populations (Table S1). Our AGS estimates are the first for the both ploidies, with the 1Cx values decreasing with increasing ploidy level (Table 1).

Twenty-six plants of the nine populations assigned to the second group (“A. dentiferum-pallens”) were all found to be tetraploid (Table 1, Fig. S1H), which agrees with data from France for A. longispathum (Jauzein and Tison 2001). In contrast to tetraploid A. dentiferum, however, their RGS were more variable (Fig. S2G) and mean AGS was slightly shifted to lower values (Table 1, Table S1). These populations were found in Spain, Italy, Croatia, Bulgaria and Greece (Fig. 1D, Table S1) and require additional taxonomic study.

Allium flavum L. – The taxonomy and cytogenetics of this species complex are still poorly understood. The most recent taxonomic treatment (POWO 2024) recognizes four infraspecific taxa: subsp. flavum, subsp. ionochlorum Maire, subsp. tauricum (Besser ex Rchb.) K. Richt., and var. pilosum (Kollmann & Koyuncu) Koçyiğit & Özhatay. In addition, several other taxa very similar in morphology to members of the A. flavum complex have been described from the Eastern Mediterranean and neighbouring regions, most of them being assigned into the informal A. stamineum species group (see Brullo et al. 2007). In the Balkan Peninsula this is the case for A. guicciardii Heldr. (see below) or A. croaticum Bogdanović, Brullo, Mitić and Salmeri (Brullo et al. 2007; Bogdanović et al. 2008). Many karyological reports of A. flavum remain unclear, as they refer to A. flavum s.l., without further differentiation at the intraspecific level (Liveri et al. 2019). However, in some cases, knowing the sampling location and the range of the taxon or taxonomic concept used in the respective study, it is possible to infer the infraspecific taxon for which the corresponding chromosome number was published (Table S2).

The nominate subspecies (A. flavum subsp. flavum, including var. minus Boiss.) is distributed as native to southwestern and southern Europe, the southern part of Central Europe and southeastern Europe from southern France to Romania, and Turkey (POWO 2024). The FCM of 337 plants from 76 populations revealed two cytotypes, confirmed by several chromosome counts (Table 1, Fig. S1I,J): diploid (2n = 16, 89.9%) and tetraploid (2n = 32, 10.1%), both also reported in previous reports (Table S2), rarely with the presence of additional 1–3 B chromosomes (see Vujošević et al. 2013 for survey). Cheshmedzhiev (1970) also reported an aneuploid plant (2n = 33) from Bulgaria. In addition, triploids (2n = 24) have rarely been reported from Bulgaria and Greece (Ved Brat 1965; Cheshmedzhiev 1994), but solely for var. minus (= A. webbii G. C. Clementi). Consistent with previous reports (Table S2), pure diploid populations were the most common (89.5%), followed by pure tetraploid populations (9.2%). Diploids were found throughout the range of the species, as previously reported (Fig. 1E). The occurrence of tetraploids has been reported from the Balkan Peninsula (Table S2) and from Granada, Spain (Ruiz Rejón and Sañudo 1976). However, A. flavum is not considered to be present in Spain (Aedo 2013) and the count mentioned therefore belongs to a different species. The range of tetraploids increased towards the north (Fig. 1E), with new records for Romania, Hungary and Slovakia. We also found a 2x + 4x mixed population in Slovakia, which is the first report of a mixed-ploidy population for the species (Table 1).

The RGS of diploids showed considerable variation (Table 1), with a significant increase towards the south-east (latitude: r_s = -0.285, P = 0.020; longitude: r_s = 0.380, P = 0.002; Fig. 2E, Fig. S2I). In tetraploids, the RGS showed much less variation (Table 1), but the RGS showed the opposite spatial pattern to the diploids, increasing towards the northwest (latitude: r_s = 0.714, P = 0.047; longitude: r_s = -0.690, P = 0.058; Fig. 2F, Fig. S2J). Except for one measurement, the AGS of diploids had a unimodal pattern with a mean of 2C = 27.9 pg (Table 1), which is in good agreement with previous reports based on both FEM and FCM (Table S3; Baranyi and Greilhuber 1999; Veselý et al. 2012). An extremely low AGS (i.e. 2C = 21.4 pg) was detected for the population sampled near Minerve in southern France, far from the rest of the analysed populations (Table S1). We interpret this value as a result of processes acting on isolated and/or marginal populations at the edge of the species' range (Šmarda and Bureš 2010). However, more samples from southwestern Europe are needed to draw a firm conclusion on the observed pattern.

The AGS of the tetraploids showed a bimodal pattern, with one population group originating from Bulgaria having AGS values between 2C = 38 and 41 pg and the second group (Eastern Central Europe, Serbia) having AGS values between 2C = 42 and 46 pg (Fig. S2J). These divergent AGS values were confirmed by chromosome counting (Table S1) and fit well with previous estimates of DNA content using various methods (Labani and Elkington 1987; Ohri et al. 1998; Baranyi and Greilhuber 1999; Ohri and Pistrick 2001), although some of them (Labani and Elkington 1987; Ohri et al. 1998; Ohri and Pistrick 2001) erroneously present them as diploid (Table S3). The 1Cx values decreased with increasing ploidy level (Table 1).

The subspecies Allium flavum subsp. tauricum (Besser ex Rchb.) K. Richt. is native to southeastern Europe, Ukraine, European Russia, Kazakhstan, Caucasian countries, Turkey and Iran (POWO 2024). Plants show rather variable size, shape and colouring of perigon, filaments and anthers (e.g. Vvedensky 1935; Stearn 1980; Ciocârlan 2000; Cheshmedzhiev 2011), which has probably led to the description of several taxa of low taxonomic rank (e.g. Zahariadi 1966; Özhatay and Koçyiğit 2019), but also to species misidentifications (Bogdanović et al. 2011; Tzanoudakis et al. 2019). Recently, Özhatay and Koçyiğit (2019) questioned A. paczoskianum as a synonym of A. flavum subsp. tauricum and consider both taxa as separate, different species. Furthermore, the possibility of confusion with morphologically very similar and rarely reported species of the A. stamineum group, e.g. A. guicciardii (Brullo et al. 2007)d croaticum (Bogdanović et al. 2008), cannot be excluded in published records. The FCM of 62 plants from 19 populations revealed two cytotypes: karyologically confirmed tetraploids (2n = 32, Fig. S1K) and a cytotype with the RGS approximately 75% of that measured in tetraploids (Table 1). Despite the lack of chromosome counts for these lower RGS plants, we classify them as DNA-diploids, based on a similar pattern of RGS differences between diploids and tetraploids in the closely related A. flavum subsp. flavum. However, to be sure, additional chromosome counts are desirable.

Four ploidy levels and two aneuploid counts were previously reported for this taxon (Table S2). Tetraploids were the most frequently reported ploidy, followed by diploids. This is consistent with our data, with pure tetraploid populations being the most common (73.7%), followed by pure DNA-diploid populations (26.3%), and no records of cytotype-mixed populations (Table 1). Cheshmedzhiev (1975a, 1982) reported several additional cytotypes, mostly as single records from the Rhodopi Mts and the Thracian Lowlands in Bulgaria, i.e. penta- (2n = 40) and hexaploid (2n = 48) as well as tetraploid with 0–3 B chromosomes and aneuploids with 2n = 33 and 34.

Previous records suggested partially different distribution patterns of diploids and tetraploids, with diploids reported from the Caucasus (Pogosian 1983; Magulaev 1992), Turkey (Tanker and Kurucu 1979 sub A. amphipulchellum Zahar.; Johnson and Brandham 1997), Northern and Southern Greece (Strid and Franzén 1981; Tzanoudakis and Vosa 1988 sub A. flavum) and the island of Lesvos (Karavokyrou and Tzanoudakis 1991 sub A. flavum), and tetraploids from Bulgaria (Cheshmedzhiev 1970, 1975a, 1982) and Turkey (Özhatay 1990, 1993). We recorded diploids as a new cytotype for Bulgaria and North Macedonia, and tetraploids as a new cytotype for Ukraine and North Macedonia (Table S1). Bulgaria represents the most cytotype-diverse region (Fig. 1F, Table S2).

The range of RGS in DNA-diploids was relatively narrow (Table 1), with an almost significant increase towards the east (latitude: r_s = -0.154, P = 0.804; longitude: r_s = 0.872, P = 0.054; Fig. 2F, Fig. S2K). In tetraploids, RGS was variable with a significant increase towards the southwest (latitude: r_s = -0.688, P = 0.007; longitude: r_s = -0.842, P < 0.001; Fig. 2F, Fig. S2L), i.e. opposite to that found in DNA-diploids. The pattern of AGS followed that of RGS. Outlier RGS/AGS values were recorded from two Bulgarian populations, which were approximately 2–4 pg higher than those of the remaining tetraploid populations (Table S1). Whether this could be an indication of taxonomic heterogeneity should be the subject of future investigation. There is only one nuclear DNA estimate using FEM, which was erroneously reported as diploid (2C = 39.6 pg, Vakhtina et al. 1977), although according to our measurements it is more likely to be tetraploid. The mean 1Cx of the tetraploids was lower than that of the diploids (Table 1).

Allium garbarii Peruzzi. – The species is endemic to the Calabrian coast in Italy (Peruzzi 2007). The FCM of four plants from the locus classicus revealed a single cytotype identified as diploid (2n = 16, Fig. S1L), confirming previous results (Peruzzi 2007). Our AGS estimate is the first for this species (Table 1).

Allium guicciardii Heldr. – The species of the A. stamineum group is only very rarely reported in the literature, although it is reported to occur in Central and Northern Greece and Romania (Brullo et al. 2007) and is considered to be a Greek endemic with a limited range (Dimopoulos et al. 2023). We observed a northern Greek population that clearly belonged to the A. flavum/A. stamineum groups, but differed from typical nominate A. flavum by yellow-greenish perigon and stamens white below and violet above. Though literature is not consistent in diagnostic characters to unequivocally distinguish this species from closely related taxa (Brullo et al. 2007; Bogdanović et al. 2008), we provisionally assign this population to A. guicciardii. The FCM of eight plants revealed a single cytotype (Table 1), which was not confirmed by chromosome counting (material not available). However, considering the RGS/AGS of plants analysed and those of closely related A. flavum s. str., a diploid ploidy level is suggested for these plants. Previously, diploids (2n = 16; Alden 1976) and tetraploids (2n = 32; Brullo et al. 2007) have been reported for A. guicciardii, with both ploidy levels occurring in Greece and tetraploids occurring also in Romania (Brullo et al. 2007). Our AGS estimate is the first for this species (Table 1).

Allium hermoneum (Kollmann & Shmida) Brullo, Guglielmo, Pavone & Salmeri. – The species is reported to occur in the alpine belt of the Anti-Lebanon Mts in Syria, Israel and Lebanon (Kollmann and Shmida 1977; Brullo et al. 2007; Danin and Fragman-Sapir 2016+). FCM analysis and chromosome counting of two plants from two micropopulations between the peaks of Mt. Habushic and Mt. Hermon (Israel) revealed a single cytotype, identified as tetraploid (2n = 32, Fig. S1M). Previously, Shmida and Kollmann (1977) and Kollmann (1985) reported both diploid and tetraploid chromosome numbers for samples collected at the type locality (Mt. Hermon). Our AGS estimate is the first for this species (Table 1).

Allium karsianum Fomin. – The range of this species is restricted to NE Turkey and the Transcaucasus (POWO 2024). It is closely related to A. kunthianum Vved. from which it is problematic to distinguish due to contradictions in diagnostic characters in regional floras and frequently reported intermediates (e.g. Vvedensky 1935; Oganesian and Agababian 2001). The FCM of five plants from three populations from Georgia and Armenia revealed a single cytotype (Table 1), identified as diploid (2n = 16, Fig. S1N), confirming previous diploid records from Armenia (Pogosian 1983) and Turkey (Özhatay 1993). Our AGS estimate for three populations of diploids was very narrow, around 2C = 35.4 pg (Table 1). There is only one previous AGS report by FEM (2C = 28.5 pg) for diploids of unknown origin by Vakhtina et al. (1977), which is, however, quite distant from our data.

Allium kunthianum Vved. – This species, which is closely related to A. karsianum (see above), is reported to occur in Iran, North Caucasus, Transcaucasus and Turkey (POWO 2024). The FCM of ten plants from three populations from Georgia revealed a single cytotype, identified as diploid (2n = 16, Fig. S1O), confirming the previous diploid records from Georgia (Gagnidze et al. 2015) and Armenia (e.g. Pogosian 1983, Table S2). Vakhtina and Kudryashova (1985) also report a tetraploid count from Armenia. Our AGS estimate for three populations of diploids was very narrow, around 2C = 32.4 pg (Table 1). There is only one previous AGS report by FEM (2C = 35.1 pg) for diploids of unknown origin by Vakhtina et al. (1977), which is rather closer to our AGS of A. karsianum.

Allium macedonicum Zahar. – This rare species is reported to occur in the Pangaion Hills (NE Greece), from where it was described (Zahariadi 1975), and NC Greece (Dimopoulos et al. 2023). A specimen from a locality in the southern part of North Macedonia, close to known distribution range, has 2C = 28.9 pg, which could be considered as DNA-diploid, based on the only diploid (2n = 16) chromosome report (Papanicolaou 1984) and the similarity of the AGS value with that of the related species of the A. paniculatum L. group (Table 1).

Allium melanantherum Pančić. – This rare Balkan endemic species occurs in Bulgaria, Serbia, Kosovo, North Macedonia and northern Greece (Anderson 1991; Anačkov 2009; Assyov et al. 2012; Teofilovski 2021; Dimopoulos et al. 2023; POWO 2024). The FCM of 14 plants from two nearby populations in the Rila Mts (Bulgaria) revealed a single cytotype, identified as triploid (2n = 24, Fig. S1P). Our chromosome count confirms previous triploid records (some of which indicate the presence of 0–1 B chromosomes) from Bulgaria, from which diploids (2n = 16) and tetraploids (2n = 32) have also been reported (Cheshmedzhiev 1970, 1971, 1976, 1979, 1992). Triploids have also been reported from northern Greece (Tzanoudakis and Vosa 1988, Table S2). Our AGS estimate is the first for triploids (Table 1). Previously, AGS of tetraploids (2C = 47.5 pg) was estimated using FEM (Ohri et al. 1998; Ohri and Pistrick 2001). The 1Cx value of tetraploids were found to be lower than that of triploids (Fig. 4, Table).

Allium oporinanthum Brullo, Pavone & Salmeri. – The species is considered a northwestern Mediterranean species, occurring in Spain, France (Brullo et al. 1997a) and the Aosta Valley in the northwestern Italy (Rey et al. 2015). The FCM of 65 plants from 11 populations revealed a single cytotype, which was identified as tetraploid (2n = 32, Table 1, Fig. S1Q), confirming previous tetraploid records from several localities in Spain and France (Brullo et al. 1997a; Jauzein and Tison 2001). The range of RGS and AGS values were relatively narrow, and our AGS estimate is the first for this species (Table 1).

Allium orestis Kalpoutz., Trigas & Constantin. – The species was described from the Parnon Mt and Taigetos Mt of the southern Peloponnese, Greece (Kalpoutzakis et al. 2012). The FCM of nine plants from the locus classicus resulted in a single cytotype, which was identified as diploid (2n = 16) by the authors of the species description (Kalpoutzakis et al. 2012). Our AGS estimate is the first for this species (Table 1).

Allium pallens L. – Widespread semi-ruderal species in the Mediterranean (Brullo et al. 2003). FCM of 121 plants from 19 populations, clearly matching the species description in Brullo et al. (2003), yielded a single cytotype identified as tetraploid (2n = 32, Table 1, Fig. S1R). Our chromosome counts confirm previous tetraploid records from several European, Turkish and north-African localities (e.g. Jauzein and Tison 2001; Brullo et al. 2003; Fig. 1F), frequently referred under the names A. coppoleri Tineo or A. stearnii Pastor & Valdés (Table S2). Reported diploid records (2n = 16, Table S2), e.g. from Spain (e.g. Pastor 1982; Ruíz Rejón et al. 1980, 1986), Greece (Karavokyrou and Tzanoudakis 1991) and Turkey (Ved Brat 1965) likely belong to other, presently unidentifiable, species and require further study, owing to frequent misidentification or misinterpretation of the species (Brullo et al. 2003), and are not presented in the map (Fig. 1F). The range of RGS and AGS values of tetraploids was relatively wide, with a clear increase towards the northwest (RGS: latitude: r_s = 0.515, P = 0.029; longitude: r_s = -0.492, P = 0.038; Fig. 2D, Fig. S2H). The AGS estimate is the first for this species (Table 1).

Allium praescissum Rchb. – The species is distributed from east of the Dnieper River in Ukraine to western Siberia and typically occurs on saline soils (Dobrochaeva et al. 1999; Seregin 2007; Sinitsina 2019). The FCM of eight plants from a Russian population revealed a single cytotype. Despite the lack of chromosome counts for these plants, we classify them as DNA-diploids, based on the similarity of our AGS estimates (2C = 31.6 pg, Table 1) to the karyologically verified nuclear DNA content using FEM (2n = 16, 2C = 28.5 pg) by Vakhtina et al. (1977). Zakirova and Nafanailova (1988) reported diploids also from Kazakhstan (Table S2).

Allium pseudostamineum Kollmann & Shmida. – This endemic species is native to Israel, Syria and Lebanon (Brullo et al. 2007; POWO 2024) and was described from Mt. Hermon in the Anti-Lebanon Mts (Kollmann and Shmida 1977). FCM analysis of a single plant from the population between the peaks of Mt. Habushic and Mt. Hermon (Israel) revealed a single cytotype, identified as a DNA-diploid, consistent with the previous report of 2n = 16 from the type locality (Shmida and Kollmann 1977). Our AGS estimate is the first for this species (Table 1).

Allium rhodopeum Velen. – This rare species is native to Bulgaria, Serbia, North Macedonia, Albania, Greece and Turkey (Brullo et al. 1998; Barina and Pifko 2011; Nikolov 2021; POWO 2024). The FCM of 31 plants from five populations sampled in Bulgaria and Greece revealed a single cytotype identified as diploid (2n = 16, Table 1, Fig. S1S). This confirms previous diploid records from other regions of Bulgaria (Cheshmedzhiev 1970, 1973), Greece (Brullo et al. 1998) and Turkey (Özhatay 1990, 1993). Ricci (1965) published tetraploid count (2n = 32) in a plant of unknown origin but we omit this count due to uncertainty of the species determination. Our AGS estimate is the first for this species (Table 1).

Allium rupestre Steven – The species occurs in Crimea, Turkey and Caucasus (Vvedensky 1935; POWO 2024). The FCM of 23 plants from six populations sampled in Georgia and Crimea revealed four distinct RGS/AGS groups. Most of our AGS estimates are the first for the species (Table 1). Three groups with increasing AGS mean of 2C = 22.3, 29.0 and 42.9 pg probably represent a polyploid series with basic chromosome number x = 7 and chromosome numbers 2n = 2x = 14, 2n = 3x = 21 and 2n = 4x = 28, respectively (Fig. S1T, U, V). The 2n = 14 is most likely the first such count for the species. However, Vakhtina and Kudryashova (1985) commented on the possibility that a count of 2n = 14 for A. kunthianum by Vakhtina (1964) might actually belong to A. rupestre. Chromosome counts of 2n = 21 and 2n = 28 have already been published for individuals sampled in the Crimea (Ukraine) and the foothills of the Caucasus (Georgia), respectively (Vakhtina and Kudryashova 1985).

Another group with an AGS of 2C = 38.6 pg, intermediate between the AGS of the tri- and tetraploids (see above) is represented by plants from a population originating from Georgia (Table 1), for which we counted 2n = 24 (Fig. S1W). A very similar nuclear DNA amount (2C = 37.8 pg) was also measured for plants originating from the other Georgian locality (Borjomi), from which a chromosome number 2n = 24 was previously reported (Ohri et al. 1998; Ohri and Pistrick 2001). The authors consider this chromosome count to be triploid, based on x = 8 and two diploid reports (2n = 16) from Armenia (Pogosian 1990) and Turkey (Özhatay 1993). These rare records suggest a rather complicated evolution within the species and require more detailed research over the whole Caucasian region.

Allium telmatum Bogdanović, Brullo, Giusso & Salmeri. – The species is endemic to the northwestern Croatian coast (Bogdanović et al. 2009). The FCM of ten plants from one population revealed a single cytotype identified as tetraploid (2n = 32, Fig. S1S). Our chromosome count confirms previous records from two localities (Bogdanović et al. 2009) close to newly sampled locality. Our AGS estimate is the first for this species (Table 1).

Allium tenuiflorum Ten. – This Mediterranean species is reported to occur in southern Europe from southeastern France to Bulgaria and in Libya (POWO 2024). However, the species is critical because of its morphological similarity to some other species such as A. pallens and A. dentiferum (Jauzein and Tison 2001; Brullo et al. 2003). The taxonomic status of the eastern Balkan populations (e.g. Bulgaria) also requires further study. FCM of 24 plants from five populations sampled at the southern edge of its range in Italy (cf. Brullo et al. 2003) and 12 plants from two localities in Croatia (Istria, N Dalmatia) yielded a single cytotype identified as diploid (2n = 16, Table 1, Fig. S1Y), confirming previous diploid records (rarely with 1–6 B chromosomes) from Italy (e.g. Marcucci and Tornadore 1994; Jauzein and Tison 2001; Brullo et al. 2003; Peruzzi 2003; Tornadore and Marcucci 2005), Croatia (Puizina et al. 1997) and Bulgaria (Cheshmedzhiev 1975c). In addition, Fernandes and Queiros (1971) also reported diploid count, but for a plant of unknown origin, so this count must be considered doubtful. Apart from diploids, there are two records of triploids, one from Hyères in southern France (Jauzein and Tison 2001) and one from Apulia in southern Italy (Tornadore 1981). The pentaploids (2n = 40) reported by Vosa (1976) from central Italy clearly belong to another species, most probably A. dentiferum. The ranges of RGS and AGS of the diploids we analysed were wide, with a weak tendency of Croatian populations towards slightly higher GS compared to Italian populations (Table S1). Our AGS estimate is the first for this species (Table 1).

Allium valdesianum Brullo, Pavone & Salmeri. – The species is endemic to the alpine belt of Sierra Nevada Mts in Spain (Brullo et al. 1996b). The FCM of five plants from a population in the northwestern part of the Sierra Nevada Mts revealed a single cytotype identified as diploid (2n = 16, Fig. S1Z), in agreement with the only previous diploid record from the locus classicus (Brullo et al. 1996b), which is almost identical to our locality. Our AGS estimate is the first for this species (Table 1).

Ploidy variation and frequency in Allium sect. Codonoprasum: overview

The issue of high chromosomal variation in the genus Allium has long been of interest since the benchmarking studies of Levan (1931, 1933). A recent review found that only 3.2% of Allium species are pure polyploids (exclusively 4x), but almost 30.2% of species within the genus show intraspecific variation in ploidy levels, up to eight ploidies (2x-10x; Han et al. 2020). However, only a subset of species of A. sect. Codonoprasum, sometimes even with inaccurate cytotype composition, was included in Han’s review. Almost half of the taxa (i.e. 44%) we screened using FCM were polyploid or di-&polyploid, with tetraploids/DNA-tetraploids being the most common polyploid cytotype (Table 1). The synthesis of our new data (Table S1) and the published chromosomal/DNA-ploidy data for 160 recognised taxa of A. sect. Codonoprasum (Tables S2, S4, all taxa summarised in Table S5) showed that 32 species (20.0% of total) have no data on their ploidy. Moreover, many older chromosome number records in specimens assigned by the given authors especially to members of the A. paniculatum complex (e.g. A. paniculatum L., A. fuscum Waldst. & Kit.) should be considered with caution or even excluded due to species misconception/misidentification (Salmeri et al. 2016; Vojtěchová et al. 2023; see Table S4 for details). Considering only reliable records of taxa with at least one chromosome record (128 taxa), diploid taxa are dominant (71.1%), while di-&polyploid and pure polyploid taxa are less common, occurring at similar frequencies of 12.5% and 16.4%, respectively (Fig. 3A). After excluding taxa that were recently transferred to another section according to Özhatay and Koçyiğit (2019), 80 out of 116 taxa with at least one chromosome count are diploid (69.0%), 16 di-&polyploid (13.8%) and 20 polyploid only (17.2%, Fig. 3B). While proportion of diploid species is consistent with the overall pattern observed in the entire genus Allium (Han et al. 2020), proportion of di-&polyploid and polyploid taxa within the A. sect. Codonoprasum is apparently different from those found by Han et al. (2020). We explain these differences by using a narrower and more critical species concept and more detailed survey in our study compared to Han et al. (2020).

Detailed insight into ploidy composition in di-&polyploid and polyploid taxa shows that 4x, 3x, 2x + 3x and 2x + 3x + 4x taxa are dominant, while frequencies of other ploidies and/or their combinations are rare (Fig. 3C, Table S5), e.g. pentaploid A. pseudotelmatum Duchoslav & Jandová, hexaploid A. exaltatum (Meikle) Brullo, Pavone, Salmeri & Venora, and up to hexaploid A. flavum subsp. tauricum. Allium oleraceum L. shows the most intriguing ploidy composition ranging from tri- to octoploids (for details Duchoslav et al. 2020). This contrasts with Han et al. (2020), who found the 2x + 4x combination to be the most common (46.3%) within di-&polyploid and polyploid Allium species. In addition, our synthesis adds some new cytotype combinations not covered by Han et al. (2020), suggesting a high diversity of cytotype composition within A. sect. Codonoprasum. It also shows that the true proportion of mixed ploidy species may be underestimated due to the sample-size limitations of classical chromosome counting (Duchoslav et al. 2020), and mixed populations may not be detected at all, although reports of different cytotypes coexisting in the same regions in several taxa (Tables S1, S2, S4) might suggest this. In this respect, the use of more convenient and efficient FCM is beneficial (Siljak-Yakovlev et al. 2020; Sliwinska et al. 2022), although its use within the genus is still scarce (but see Duchoslav et al. 2010, 2013, 2020; Vojtěchová et al. 2023, 2024).

However, contrary to our expectations (Fig. 3), we recorded only four mixed-ploidy populations, representing 1.3% of the populations analysed, and in only two taxa, A. carinatum subsp. carinatum and A. flavum subsp. flavum (Table S1). Adding previously published population-level ploidy data (Table S4), only three other taxa in the section (A. oleraceum, A. paniculatum, A. marginatum Janka) had reported ploidy-mixed populations. Apart from the unique A. oleraceum with complex ploidy structure of populations (Duchoslav et al. 2010, 2020), only 2x + 3x and 2x + 4x populations were rarely found in the above four mentioned taxa (Table S4), which is far below the average percentage (16.1%) of populations with multiple cytotypes found in 39 mixed-ploidy species (Kolář et al. 2017). However, in contrast to our study, the majority of studies reviewed by Kolář et al. (2017) were specifically designed to detect within-population ploidy diversity by sampling a high number of individuals per population (16 vs. 5 on average, Kolář et al. 2017 vs. our study) and extensively covering the entire area of the population, which has been shown to increase the probability of detecting multiple ploidies (Duchoslav et al. 2020). Therefore, the combination of intensive within-population sampling and FCM may increase the frequency of mixed-ploidy populations, especially within the taxa with increased vegetative reproduction (bulbils, bulblets), which may help different cytotypes to overcome the process of minority cytotype exclusion in local sympatry (Herben et al. 2017; Kolář et al. 2017).

The spatial pattern of ploidy diversity of taxa from A. sect. Codonoprasum across Europe and adjacent regions is shown in Fig. 3D. Diploid taxa dominate in the eastern Mediterranean, where they represent more than 85% of all taxa with counted chromosome numbers. The proportion of diploid taxa gradually decreases both westwards and northwards, with only polyploid taxa present in northern and northwestern Europe. The dominance and diversity of diploid taxa in the Eastern Mediterranean and the adjacent Irano-Turanian region are explained by the fact that these regions are considered as genus/section evolutionary centres (Hanelt 1996; Fritsch and Friesen 2002; Friesen et al. 2006), from which the ancestors of extant species migrated westward in the past, e.g. across newly opened dry environments during the Messinian salinity crisis (Krijgsman et al. 1999; Trájer et al. 2021), followed by evolution in isolation of newly colonised regions after the marine transgression at the end of the Tertiary (Garcia-Castellanos et al. 2009). This east-west phylogeographical break has frequently been inferred, and sometimes dated in various plant groups (Nieto Feliner 2014). Several authors (e.g., Bogdanović et al. 2009; Salmeri et al. 2016) hypothesised this scenario as very likely in autumn-flowering taxa of A. sect. Codonoprasum, where all but one (A. apolloniensis Biel et al.) extant taxa in Eastern Mediterranean are diploid while those occurring westward are (paleo-)polyploids (Brullo et al. 1997b; Bogdanović et al. 2009; Özhatay et al. 2018), with both groups forming different subclades in the concatenated nr-ITS and cp-DNA tree (Salmeri et al. 2016).

A high proportion of diploid taxa, often endemic, is also associated with regions of climatic, geological and topographical complexity with less harmful glaciation effects during the Quaternary (Hughes and Woodward 2017; Noroozi et al. 2019), e.g. Turkey and Greece but also southern Italy and Sicily (Peruzzi et al. 2015). This scenario is considered to be the most parsimonious explanation of high species diversity and endemism in many species’ complexes in the Mediterranean region (Nieto Feliner 2014).

On the other hand, more northern regions of Europe are dominated by di-&polyploid or only polyploid taxa of the section (Fig. 3D) or higher ploidies (Fig. 1C, F), and also the most widespread taxa of the section are polyploids (e.g. A. oleraceum, Duchoslav et al. 2020; A. dentiferum, A. pallens; see Fig. 1C,D). Observed increase of polyploid taxa towards north fits well with the common pattern observed worldwide (Rice et al. 2019) and reflect the strong influence of the Quaternary glacial cycles on the flora of central and northern Europe (Hewitt 1999), as well as the adaptive advantage of polyploidisation and hybridisation within the section (Duchoslav et al. 2020), which might resulted from secondary contacts during postglacial range expansions (Schmitt 2007). The high proportion of polyploid and mixed-ploidy taxa within the section is considered a sign of ongoing diversification (Tzanoudakis and Vosa 1985; Hanelt 1996), which is likely to have played a role in the ecological radiation of A. sect. Codonoprasum into newly emerging habitats, especially those with mesic climates and/or fertile conditions, as demonstrated by Han et al. (2020) for the genus Allium. Indeed, many widely distributed polyploid taxa of this section dominate in mesic and/or fertile, frequently anthropogenic, disturbed habitats (Brullo et al. 2008; Duchoslav et al. 2020; Vojtěchová et al. 2024). Also, relatively high frequency of odd ploidy taxa/levels within taxa (especially 3x) observed within the section is uncommon in polyploid plants (Kolář et al. 2017) because odd ploidy causes meiotic irregularities leading to a reduction of the seed set and thus fitness of newly emerging polyploid (Ramsey and Schemske 1998, 2002). However, polyploid members of the section either form or increase their vegetative reproduction via production of aerial bulbils or underground bulblets (e.g. triploid A. corsicum, Jauzein et al. 2002; polyploid A. oleraceum, Fialová and Duchoslav 2014), which allow them to establish, persist within a site and disperse to new areas (Hörandl 2009) and thus overcome reproductive constraints.

Robust data explaining the origin of di-&polyploid and polyploid A. sect. Codonoprasum species are limited and mostly based on interpretation of mitotic chromosome arrangements (e.g. Pastor 1982; Bogdanović et al. 2011) or GS (e.g. Vojtěchová et al. 2023, 2024). Therefore, the origin of most of these taxa is generally not clarified, but only outlined in literature, and both evolutionary pathways of polyploid formation (auto- and allopolyploidy) are briefly discussed. For example, some tetraploid species are considered to be either autotetraploid (e.g. A. occultum Tzanoud. & Trigas; Tzanoudakis and Trigas 2015) or allotetraploid (e.g. A. apergii Trigas, Iatroú & Tzanoud.; Trigas et al. 2010). For some di-&polyploid species containing tri- and/or tetraploids in addition to diploids (e.g. A. paniculatum, A. marginatum), autopolyploid origin is postulated based on both GS and molecular markers (Vojtěchová et al. 2023), whereas for polyploid 4x-5x A. dentiferum and 3x-8x A. oleraceum allopolyploid origin is most likely (Brullo et al. 2008; Duchoslav et al. 2020 and references therein).

Rare dysploidy in A. rupestre

Traditionally, several basic chromosome numbers have been distinguished within the genus Allium, ranging from x = 7 to x = 11 (Jones and Rees 1968; Friesen et al. 2006). The most common is x = 8, which is dominant in the majority of subgenera (Fritsch et al. 2010), including the subgenus Allium (Ved Brat 1965; Peruzzi et al. 2017; Han et al. 2020), and has also been inferred as the ancestral basic chromosome number by ancestral state reconstruction (Peruzzi et al. 2017). Consistent with previous knowledge, all but one of the species we analysed had x = 8. When studying A. rupestre populations, we found, besides 2n = 24, corresponding to 3x with x = 8, a polyploid series 2x–3x–4x with 2n = 14, 21 and 28 (see also Vakhtina and Kudryashova 1985). So far, these results indicate a descending dysploidy leading to x = 7, which is very unusual for members of A. sect. Codonoprasum, as well as for the subgenus Allium (Mathew 1996; Peruzzi et al. 2017; Babin and Bell 2022), where only a few dysploid (x = 7) species are known, e.g. from the A. sect. Cupanioscordum Ceschm. (Brullo et al. 2015; Trigas et al. 2017), which is sister to the A. sect. Codonoprasum (Li et al. 2010).

General patterns of GS variation within the Allium sect. Codonoprasum

Our work represents the first comprehensive study of GS in A. sect. Codonoprasum, increasing by 78% the number of taxa for which GS is now known (Leitch et al. 2019). AGS estimation revealed a 2.6-fold difference in nuclear DNA content, ranging from 2C = 22.3 pg, which is the lowest known AGS within this section, to 2C = 58.5 pg (Table 1, Fig. 4A). Previously, Jones and Rees (1968) reported an even lower 2C value (18.4 pg) than the lowest we measured within this section, but this record certainly belongs to other species, probably from another section (Vojtěchová et al. 2023). Adding previous AGS records of other measured species in this section (Leitch et al. 2019), including the highly polyploid A. oleraceum (Duchoslav et al. 2013), this difference is even larger, up to 4.1-fold, with the maximum known 2C = 92.1 pg in octoploid A. oleraceum (Fig. 4A, Table S3). This variation covers most of the known range of AGS in the whole genus (Fig. 4A inset), with the exception of several species from other sections that either have lower AGS, with the lowest AGS (2C = 15.2–16.9 pg) found in A. schoenoprasum L. (Jones and Rees 1968; Baranyi and Greilhuber 1999), or an extremely high AGS record in A. validum S. Watson (2C = 148.9 pg, Ohri et al. 1996). The AGS of the analysed section members indicate that the section belongs to the plant groups with either large or very large GS sensu Leitch et al. (1998).

The observed GS variation in A. sect. Codonoprasum is mainly due to polyploidy (Fig. 4A), which has a strong influence on the genus evolution (e.g. Friesen 1992; Hanelt et al. 1992; Gurushidze et al. 2012; Han et al. 2020; Wang et al. 2023), but is also crucial for ecological radiation in Allium (Wu et al. 2010; Han et al. 2020; Wang et al. 2023). Increased chromosome number due to polyploidy resulted in higher 2C values but lower 1Cx values in all but two cases of single ploidy in two taxa examined that were represented by multiple ploidy levels (Fig. 4A,B). As previously documented, polyploid formation is often associated with DNA loss, such that polyploids often exhibit lower 1Cx values compared to their diploid/low ploidy relatives. This genome reduction may be a mechanism that promotes the success of autopolyploid/allopolyploid speciation (e.g. Ozkan et al. 2003; Leitch and Bennett 2004; Poggio et al. 2014; Wang et al. 2021). Notable differences in the difference between 1Cx values of di- and tetraploids observed in some diploid-polyploid taxa (e.g. A. flavum versus A. carinatum) may be related to the age of the polyploids, with a small decrease in 1Cx typical of very young polyploids (neopolyploids), where processes leading to genome downsizing have not yet taken place (Ekrt et al. 2010; Bressler et al. 2017; Wang et al. 2021; Pungaršek and Frajman 2024). This may also explain why the 2C values of some (neoauto-)triploids, e.g. in A. paniculatum and A. marginatum (Fig. 4B, see Vojtěchová et al. 2023 for discussion), are similar to those of tetraploids of other species, despite the similarity of 2C values between the respective diploids (Fig. 4B). Alternatively, some of such tetraploid cytotypes might represent allopolyploids, which originated by polyploidization of hybrids between diploid species with different 2C values. In A. rupestre (x = 7), tetraploids had a higher 1Cx than triploids, but this may be related to the effect of dysploidy observed in this species (see above).

GS variation in Allium may also be affected by phylogenetic signal (Wang et al. 2023), suggesting that GS evolution may reflect phylogenetic relationships (e.g. Weiss-Schneeweiss et al. 2006; Chrtek et al. 2009; Hutang et al. 2023). Unfortunately, only a limited number of A. sect. Codonoprasum members have been sequenced, which, together with the limited number of GS estimates available, currently precludes full reconstruction of GS evolution along the phylogeny. Two main clades [A, B] have been identified in the limited number of accessions of A. sect. Codonoprasum analysed so far using ITS and/or the combined ITS and trnH-psbA dataset (Fig. 6 in Salmeri et al. 2016; Fig. 2 in Vojtěchová et al. 2024). Interestingly, when mapping the available 2C values of diploid members of both clades onto these phylogenetic trees, diploid species with larger genomes (2C > 27 pg, Fig. 4A) and smaller genomes (with 2C < 25 pg) are recorded within the A and B clades (sensu Salmeri et al. 2016), respectively. Considering the monoploid GS, sequenced taxa with 1Cx > 12 pg (e.g. A. flavum, A. paniculatum) are placed in the A clade, whereas those with 1Cx < 12 pg (e.g. A. dentiferum, A. pallens, A. tenuiflorum) are included in the B clade. Any further consideration of the ancestral state and possible direction of GS evolution in the two main clades will require additional sequencing, as the current trees may be limited by incomplete taxon sampling.

Intraspecific intra-ploidy variation in GS and its ecological and taxonomic consequences

Traditionally considered to be a stable trait at the plant species level (Ohri 1998), GS can actually exhibit significant intraspecific variability among individuals and populations (Šmarda and Bureš 2010). Published chromosome/karyotype reports suggest that the higher intra-ploidy GS variation found in some taxa (e.g. A. flavum, Vujošević et al. 2013) could be partly explained by changes at the chromosomal level via aneuploidy or the presence of accessory B chromosomes. However, supernumerary chromosomes have only a minor effect on individual GS, and even less on plants with higher GS (Levin et al. 2005; Chumová et al. 2016; but see Leitch et al. 2009), which is the case in Allium species.

The primary mechanism contributing to genome expansion is the proliferation of transposable elements within a given ploidy level (Feschotte and Pritham 2007; Lisch 2013). On the other hand, losses in DNA content often result from unequal homologous recombination or illegitimate recombination events (Bennetzen 2005). Such observed variation may indicate the response of GS to environmental constraints along ecogeographic gradients (Knight et al. 2005) or during the range expansion/invasion (Guo et al. 2024), accompanied by drift (Cang et al. 2024). Alternatively, intraspecific GS variation could reflect a complex evolutionary history of the taxon under study (Loureiro et al. 2010) due to unrecognised phylogenetic components (Greilhuber 1998, 2005). We recorded considerable intraspecific intra-ploidy GS variation for several taxa (e.g., A. carinatum, A. flavum, Table 1, Fig. 2, Fig. 4A,B), for which we gathered samples along substantial parts of their geographical range. Longitude, which can be considered a surrogate for the continental climate gradient in Europe (Mikolaskova 2009) and a measure of the growing season and water availability (Berg et al. 2017), appeared to be the most frequent factor correlated with GS in our dataset. However, we did not observe consistent relationships between GS variation and geography, both between taxa and between-ploidy within taxa, consistent with patterns previously observed in A. oleraceum (Duchoslav et al. 2013). It can be conducted that the observed intraspecific intra-ploidy GS variation in the studied Allium taxa is likely to be complex and idiosyncratic to each taxon, probably reflecting phylogenetic heterogeneity (Rešetnik et al. 2014), as well as past processes associated with range shifts during the Holocene, or survival in refugia where several lineages may have evolved in isolation (Nieto Feliner 2014; Horák et al. 2023). Therefore, both selection and drift may have influenced the evolution of divergent GS in topographically complex and ecologically divergent landscapes (Griffiths et al. 2004), such as the Balkan Peninsula (Španiel and Rešetnik 2022). Similar results were previously obtained, for example, by Frajman et al. (2015) for representatives of Knautia L. and Terlević et al. (2022) for Dianthus sylvestris Wulfen. Longitudinal gradients in GS observed within some widely distributed Allium taxa might also reflect westward migration in the past, as discussed above. Future studies should focus on these taxa to ascertain whether there is a strong correlation between intraspecific GS variation and phylogeny, and to determine whether any taxonomic conclusions can be drawn.

Similarly, but at the interspecific level, Wang et al. (2023) found no significant relationship between GS and 19 bioclimatic variables in the 62 Allium species from the Qinghai-Tibetan Plateau in China. Also, Veselý et al. (2012), analysing 219 European geophytic species, found no relationship between GS and Pignatti's indicators of continentality, moisture and temperature, with only a tendency for species with very high GS to avoid water-stressed environments. Indeed, in the Allium species we studied, taxa with very high GS (2C > 40 pg, polyploids only) usually inhabit less stressful, often man-made habitats with higher nutrient availability (i.e. arable fields, vineyards, road ditches, moist grasslands, secondary forests; e.g. Brullo et al. 2008; Duchoslav et al. 2020, see discussion above), representing new niches for Allium polyploids (Hanelt 1996).

GS as an extra taxonomic character for species identification?

FCM has repeatedly proven beneficial in biosystematic research (e.g. Zonneveld 2001; Castro et al. 2012; Lepší et al. 2019; Popelka et al. 2019; Kobrlová et al. 2022), and GS is considered an efficient tool for taxa discrimination, especially in morphologically challenging groups (e.g. Prančl et al. 2018; Sochor et al. 2019). We have also found GS useful in combination with classical karyology within the A. sect. Codonoprasum, despite the observed intrataxon variation in GS that we found for some species (see above). An illustrative example could be the morphologically similar triplet A. tenuiflorum/A. dentiferum/A. pallens, where A. tenuiflorum is the only diploid and the rest are polyploids, i.e. A. dentiferum: 4x, 5x and A. pallens: 4x. Despite the presence of the same ploidy level (4x), these two latter species differ in AGS/RGS with no overlap (Table 1, Fig. 4A). In our pilot screening, we also found a difference in GS between two often barely distinguishable related diploid species occurring in the same area, A. karsianum and A. kunthianum (Table 1). Moreover, FCM was also effective in distinguishing between closely related and morphologically very similar diploid species of the A. paniculatum complex (Fig. 4A, Table 1, see Vojtěchová et al. 2023 for details). Furthermore, FCM could also be potentially promising for the revision of the A. stamineum complex (sensu Brullo et al. 2007), as members measured by FCM differ in 1Cx values. As precise genetic identification is required for further taxonomic assessment of such complexes, FCM may facilitate their delimitation.

This study showed the advantage of FCM for ploidy screening and estimation of GS in the A. sect. Codonoprasum. Genome size should be used regularly as an extra taxonomic character that allow to differentiate between closely related taxa, could help to detect and/or resolve the taxonomic heterogeneity of some species groups within the genus Allium and to identify poorly developed individuals (i.e., reduce misidentifications, see Salmeri et al. 2016; Vojtěchová et al. 2023). However, our population-based FCM data also clearly showed that there is an urgent need to cover as much of the geographic range of the taxa studied as possible in order to find out if variation in ploidy and GS is occurring and what the causes are. This will be of great value for future phylogenetic assessments of this taxonomically complex group.

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Availability of data and material

All data generated or analysed during this study are included in this published article [and its supplementary information files]

Competing interests

The authors declare that they have no competing interests.

Funding

We acknowledged several projects during which we had a possibility to sample wild Allium populations (Grant Agency of the Czech Republic, grant numbers 206/04/P115, 206/09/1126). Kateřina Vojtěchová was supported by internal grants from Palacky University (IGA_PrF_2023_001, 2024_001).

Authors' contributions

The design of the research was taken care by M.D. The performance of the research – sampling was taken care by M.D. with a contribution of all authors. L.K., M.J. and L.Š. Data analysis was taken care by M.D. with a contribution of L.K. and K.V. The writing of the manuscript was taken care by M.D. and L.K. All authors read and approved the final manuscript.

Acknowledgements

We thank particularly the many sample contributors mentioned in the supplement (Table S1), namely Roman Kalous, Bohumil Trávníček, Michal Hroneš, Martin Dančák, Alena Lepší, Michal Sochor, František Krahulec, Radim Vašut, David Horák, Ivan Moysiyenko, Vojtěch Taraška, Jiří Ohryzek, Miloslav Kitner, and others. Without their enthusiasm and help, we will not be able to gather such a large collection. We would like to thank Alena Fišerová (Váňová) for providing us with data from her master's thesis. We also thank Ori Fragman-Sapir (Botanical Garden Jerusalem, Israel) for his valuable advice on sampling in Israel. Salvatore Brullo (University of Catania, Italy), Dimitris Tzanoudakis (University of Patras, Greece) and Sandro Bogdanović (University of Zagreb, Croatia) are acknowledged for their consultations concerning the taxonomic identity of some populations sampled by us. Mihai Pușkaș (Babeș-Bolyai University, Cluj-Napoca) and Adam Kantor (Institute of Botany, Slovak Academy of Sciences, Bratislava) are acknowledged for consultation on distribution of selected taxa in Romania and Slovakia, respectively.

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New estimates and synthesis of chromosome number, ploidy level and genome size variation in Allium sect. Codonoprasum: a step towards understanding the hitherto unresolved diversification and evolution of the section

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