Seeds were obtained from various suppliers, with different seed stocks indicated by numbers for anonymity. All information regarding the original indication of the species and the country of origin of the ordered seed samples were documented ( Table 1 ). The seeds were sown and grown in a mixture of sand and soil (Einheitserde Classic Profi, Balster Einheitserdewerk GmbH, Fröndenberg, Germany) in a greenhouse under controlled conditions as previously described (Turcios et al., 2023). Given the highly variable germination rates among seed samples, approximately 200 seeds per seed stock were sown (June 10, 2024), and once seedlings of at least 5 cm height were established in all growth containers, plants of similar size per seed stock were selected for DNA extraction (with five plants per supplier labeled with a-e). Two additional plants per supplier were transferred to separate pots to monitor development and morphology during the vegetative phase. A further three plants per supplier were initially isolated in pots and transferred to short-day conditions (9 h photoperiod) for 3 months to induce flowering. The remaining plants were left to grow for 3–6 months to obtain fresh shoot tips for flow cytometry.

To identify potential species-specific morphological characteristics, a detailed examination of the habitus and floral structures of mature plants was conducted for each supplier, following previously published methods (Kadereit et al., 2012). As the published dichotomous key (Piirainen et al., 2017) relies on floral traits and flower induction was not possible for suppliers 5 and 7, morphological comparisons of these accessions were limited to photographic documentation of magnified shoots and branching points. These images are presented alongside floral images obtained for the other flowering samples, with all floral photographs captured at a comparable developmental stage. Habitus assessments, however, encompassed plants of all eleven suppliers, with two individual plants per stock photographed at three distinct developmental stages: early (August 27, 2024), middle (October 10, 2024), and late (December 6, 2024). This analysis facilitated the observation of ontogenetic changes in plant architecture and overall morphology across the different suppliers.

A previously described method (Singh et al., 2014) was used for DNA extraction with modifications: Young stems of plants from all suppliers ( Table 1 ) were homogenized in liquid nitrogen using a mortar and pestle. Subsequently, genomic DNA was extracted from 100 mg of the resulting fine powder via the Plant Nucleospin II Kit (Macherey & Nagel, Düren, Germany) following the manufacturer’s instructions for plants via the CTAB lysis buffer system. The concentration of the extracted DNA was determined photometrically via a SynergyMx plate reader (BioTek Instruments, Winooski, USA) with a TAKE-3 microvolume plate (BioTek Instruments). Its quality was assessed via gel electrophoresis via 1.5% agarose stained with MIDORI Green Advance (NIPPON Genetics EUROPE GmbH, Düren, Germany). The ratio of the absorbance at 260 nm to that at 230 nm (ideally between 2.0 and 2.2) and the ratio at 260 nm to that at 280 nm (ideally ≥ 1.7) were also controlled (Heptinstall and Rapley, 2000). Only DNA isolates demonstrating these required purity ratios and exhibiting nonfragmented bands on agarose gel electrophoresis were selected for subsequent analyses.

All PCR amplifications for phylogenetic barcoding markers were performed in a lid-heated thermocycler (peqSTAR XS, VWR International GmbH, Darmstadt, Germany) with 25 µl reaction volumes, including 0.2 mM dNTPs (dNTP-Set, Life Technologies GmbH, Darmstadt, Germany), 1 U Taq polymerase and 10× standard Taq reaction buffer (both New England Biolabs, Ipswich, MA, USA), 30–100 ng template DNA and 0.2 µM of each primer (Eurofins Genomics GmbH, Ebersberg, Germany). The nuclear ETS region was amplified via the primer pair “18S-II-rev” (Ochsmann, 1998) and the slightly modified “ETS-Salicornia” (Kadereit et al., 2007). The nuclear ITS region was amplified via the primer pair “ITS-28S” and “ITS-18S” (Kadereit et al., 2006). The nuclear matK–trnK region of the cpDNA was amplified via the primer pair “MatK–1RKIM–f” and “MatK–3FKIM-r” (Kuzmina et al., 2012). The atpB-rbcL region of the cpDNA was amplified via the primer pair “atpB-rbcL-F” and “atpB-rbcL-R” (Xu et al., 2000). The primer sequences and individual PCR conditions are summarized in an additional file ( Supplementary Table S1 ).

To evaluate the length, specificity, and quantity of each PCR product, all reactions were separated on 1.5% agarose gels stained with MIDORI Green Advance. Direct sequencing of the PCR product was performed by Microsynth (Microsynth AG, Göttingen, Germany).

All subsequent analyses of the sequence data were conducted within the CLC Main Workbench (version 23.0.4, Qiagen, Venlo, Netherlands) environment, except where noted. Consensus sequences for both our generated and NCBI reference sequences were obtained, if needed, through reverse complementation. For sequences generated by Microsynth, initial quality assessment of the.ab1 files was performed, and all ambiguous terminal regions were trimmed. Following alignment with subsets of reference sequences [gap cost settings: gap opening cost = 10; gap extension cost = 1; free end gap cost], a minimal consensus sequence length was determined on the basis of a trade-off between the number of sufficiently long reference sequences and the maximum overall length of each sequence. For Salicornia spp. ETS marker references, only the previously annotated reference sequences from Kadereit et al. (2012) were used. The reference dataset was expanded for determining suppliers 5 and 7, including ETS sequences from Sarcocornia sp. and Arthrocnemum sp., as well as closely related species, namely, Microcnemum coralloides subsp. coralloides, Tecticornia australasica, and Halosarcia indica subsp. bidens reference sequences. To simplify the phylogenetic tree, redundant sequences of the same ribotype were subsequently hidden. To identify redundant, too short and inaccurate sequences of all available data for ETS, ITS, matK and atpB-rbcL, reference data were subjected to sequence alignments and comparisons. Sequences were considered redundant if they were 100% identical to another reference sequence, including the species name. However, redundant sequences from NCBI with different species names were retained, taking a lack of genetic variation, synonyms or imprecisely determined plant samples into account. All reference sequences for ETS, ITS, matK, and atpB-rcL used in the final trees and partial RNA structure predictions, including GenBank-IDs, vouchers, and species names, are documented in an additional file ( Supplementary Table S2 ).

The alignments of our generated sequences and the processed reference datasets were analyzed for the best nucleotide substitution model in the maximum likelihood reconstruction. The analyses involved the application of the hLRT, BIC, AIC, and AICc model testing algorithms. The best fitting algorithm for phylogenetic reconstruction was determined on the basis of the results of these analyses. Phylogenetic analyses were conducted via maximum likelihood with UPGMA tree construction. The general time reversible (GTR) model with rate and topology variation was employed for ETS and ITS, whereas the Felsenstein 81 model with rate variation was used for matK and atpB-rbcL. To control the reliability of the phylogenetic trees, a bootstrap analysis with 1,000 replicates was conducted. Clades containing the species of interest were identified, assuming that samples within the same region or connected by the same branch likely belong to the same species. To simplify the main figures, we reduced the number of reference sequences by eliminating irrelevant species or species only distantly related to our target species while preserving clades of key species as well as contradictory connections of reference sequences for comparison and quality assessment. However, for the initial identification of the species, a broader perspective, considering all available and reliable reference sequences for a specific barcode, was taken.

SNP analysis was applied exclusively to the ETS sequences because of the absence of clear references for the other markers. A previously published SNP comparison of the ETS region (Kadereit et al., 2012) provided an alignment of representative core sequences of S. europaea, S. perennans, S. procumbens, and S. persica, along with the outgroup Salicornia sp. (S. “crassa” group), including variable and diagnostic positions. Following this method, the generated ETS sequences were analyzed for homologies at these verified species-specific locations. Representative sequences were selected from the consensus of the five sequences obtained from each supplier. However, owing to substantial heterogeneity among the five samples of supplier 3, all individual sequences from this supplier were presented.

To elucidate the phylogenetic relationships among Salicornia species, an in-depth analysis of RNA topology was performed following the methods of previous studies (Keller et al., 2008; Schultz and Wolf, 2009). The underlying hypothesis is that SNPs can induce variations in the predicted RNA secondary structure, thereby creating distinct topological profiles for each species. For this purpose, the sequences and reference sequence sets for the markers ETS, ITS, matK, and atpB-rbcL, which were already generated in 2.3 - 2.4, were used.

The primary objective of this computational approach using the CLC Main Workbench was to enhance the visualization and interpretation of SNPs, including gaps and ambiguous base pairs. For each supplier, RNA secondary structures of reference sequences from various species exhibiting congruence were grouped in vertically aligned clusters alongside the corresponding supplier sequences. This arrangement facilitated the assessment of species discrimination and the potential of the associated marker. To ensure clarity in individual analyses, a single, representative RNA secondary structure was chosen for each supplier. This selection was based on the fact that it was overwhelmingly supported by other predictions for the same supplier. Where different suppliers yielded identical structures, these structures were consolidated into a unified representation.

To facilitate comprehension of the RNA secondary structure, a color-coded system was implemented. Specific RNA motifs are represented as follows:

The youngest stem segments (or leaves for reference plants) were sampled from mature specimens. The total nuclear DNA content of the stem tissue was determined via flow cytometry via either propidium iodide (PI) or 4’,6-diamidino-2-phenylindole (DAPI) staining, following established protocols (Bohanec, 2003; Dolezel et al., 2007). Propidium iodide staining was performed with Lycopersicon esculentum Mill. convar. infiniens Lehm. var. flammatum Lehm. ‘Stupicke Rane’ (Gene Bank Gatersleben acc. No. LYC 418) [1.96 pg/2C] as the internal standard, was exclusively employed for precise DNA content estimation of Trifolium pratense ‘Bingenheimer’. For Salicornia species, T. pratense served as the internal standard for DAPI staining, while Glycine max (L.) Merr. convar. max var. max ‘Cina 5202’ (Gene Bank Gatersleben acc. No. SOJA 392) [2.23 pg/2C] was used for Sarcocornia fruticosa and Arthrocnemum macrostachyum. Nuclei were initially extracted separately for reference peak profiling and subsequently coextracted by chopping with a razor blade in the manufacturer’s recommended nuclei extraction buffer (CyStain^® PI Absolute P or CyStain UV Precise P Automate, Sysmex Deutschland GmbH, Norderstedt, Germany). The resulting suspension was filtered through a 30 μm nylon mesh, and 2 ml of the respective staining solution was added. Flow cytometric measurements were performed on a Sysmex XF-1600™ flow cytometer (Sysmex Deutschland GmbH, Norderstedt, Germany) (linear scale), which acquired data for ten thousand nuclei from the primary peak per sample. The G0/G1 peak positions of the standard and target accessions were calculated via CyView and CyBatch software (Sysmex Deutschland GmbH).

The extracted DNA of the five samples from the 11 suppliers was amplified via three previously reported promising nuclear microsatellite loci markers (Vanderpoorten et al., 2009). All PCR amplifications were performed in a lid-heated thermocycler (peqSTAR XS) with 100 µl reaction volumes including 0.2 mM dNTPs (dNTP-Set, Life Technologies GmbH), 1 U Taq polymerase and 10× Standard Taq reaction buffer (New England Biolabs), 100–400 ng template DNA and 0.4 µM of each primer (Eurofins Genomics GmbH). The microsatellite regions were amplified with primers for the previously published S2, S5, and S19 loci (Vanderpoorten et al., 2009), where each forward marker was labeled with a fluorophore suitable for multiplexing ( Supplementary Table S1 ). Amplification was carried out under slightly modified conditions ( Supplementary Table S1 ). Gel electrophoresis of the amplicons revealed no specific fragments for samples from suppliers 5 and 7, which were subsequently excluded from further processing. The PCR products were purified via the NucleoSpin™ Gel and PCR Clean-up Kit (Macherey & Nagel), which employs 50% diluted NTI buffer to remove small fragments such as primers and excess dNTPs. The DNA concentration of the purified PCR products was subsequently photometrically determined to combine all three markers at the same concentration in 50 µl volumes per sample, according to Eurofins’ specifications. Five samples from each of the suppliers where clear amplicons could be generated were subsequently forwarded to Eurofins for analysis for fragment length analysis, and the internal lane standard (ILS-600) was added.

Morphological examination of the habitus ( Figure 1 ) and flowers ( Figure 2 ) from suppliers 1, 2, 3, 6, 8, 9, and 10 revealed only a few discernible differences. All habitus images, depicting two plants per supplier throughout the three growth stages, are summarized in an additional file ( Supplementary Table S3 ). Another additional file compiles all images showing flower details at various magnifications of the plants from all suppliers, contingent on successful flowering induction ( Supplementary Table S4 ). On the basis of vegetative and flower characteristics, tentative species assignments of suppliers 1, 2, 3, 6, 8, 9, and 10 to Salicornia europaea subsp. europaea or Salicornia perennans subsp. perennans were made utilizing a previously published identification key (Kadereit et al., 2012). Classification on the basis of the species identification key was limited to two primary characteristics: “Flowers consistently 3 per cyme, with the visible portion of lateral flowers smaller than that of the central flower” ( Figure 2 ) and “fertile spikes usually shorter than the rest of the plant.” These criteria are applicable to S. persica, S. europaea, and S. perennans.

Independent of the identification key application, differences in growth habits were noted among these groups. Suppliers 1 and 8 exhibited denser growth than did suppliers 2, 3, and 11 ( Figure 1 ). Furthermore, all the plants of these suppliers (1, 2, 3, 8, 11) presented a more upright growth form with a greater overall height than did the plants of suppliers 6, 9, and 10, the latter of which presented predominantly narrow and bushy development ( Figure 1 ). A somewhat strict arborescent or pyramidal growth form with a clear main stem and distinctly decussated lateral branches further characterized the habitus of the plants from supplier 3 ( Figure 1 ).

Plants from supplier 4 were characterized by an inflorescence axis that was consistently and significantly longer than those observed in the previously described suppliers, often accompanied by predominantly irregular growth forms ( Figure 1 ). These traits align with the characteristics of Salicornia procumbens subsp. procumbens (Kadereit et al., 2012). Confirming features included the perianth tube of all flowers being sunken but clearly separate from the fleshy inflorescence axis, and flowers arranged “3 per cyme, visible part of lateral and central flowers subequal in size”. Furthermore, another morphological feature of supplier 4 was the comparatively larger size of its flowers ( Figure 2 ).

Morphological classification of suppliers 5 and 7 via a previously published identification key (Piirainen et al., 2017) was not feasible because of the absence of flowering. Alternatively, growth habits were evaluated to distinguish these samples. Both supplier 5 and supplier 7 exhibited a strong upright growth habit with a distinct main stem and strictly ascending, long lateral branches, which contrasts with the narrow-growth types (suppliers 6, 9, and 10) and shows a more strictly ascending habit than the more numerous and widely spreading lateral branches observed in supplier 4 ( Figure 1 ). Furthermore, variations in stem coloration were observed. Supplier 5 presented a darker, more intense green hue, whereas supplier 7 presented a glaucous (blue–green) appearance ( Figure 1 ). The remaining suppliers generally displayed lighter green tones. Overall, while distinctions in growth form were observed between suppliers 1, 2, 3, 8, and 11 compared with suppliers 6, 9, and 10 and further from suppliers 5 and 7, a definitive taxonomic assignment for any of these groups using identification keys was not possible. Only the distinct characteristics of Supplier 4 strongly suggested an assignment to S. procumbens subsp. procumbens.

Variable and diagnostic nucleotide positions of the ETS marker, which characterize the different Salicornia taxa, have been reported (Kadereit et al., 2012) and evaluated for congruence and clarity. Diagnostic positions 76, 111, and 303, characteristic of S. europaea, were found in the sequences of suppliers 1, 2, 8, and 11 and partially in those of supplier 3 ( Figure 4 ). S. procumbens is characterized by five variable (134, 144, 170, 270, and 428) and nine diagnostic (122, 221, 260, 333, 374, 395, 402, 432, and 444) nucleotides (Kadereit et al., 2012). The sequences from supplier 4 uniquely exhibited this combination, whereas they were not found for any other supplier ( Figure 4 ). For S. perennans, while 18 variable positions have been reported, no synapomorphic mutation has been identified (Kadereit et al., 2012). A synapomorphy is a shared, derived character that is common between an ancestor and its descendants. Consistent with their assignment to S. perennans, suppliers 6, 9, and 10 presented no contradictions at these variable positions or in the remaining sequence data ( Figure 4 ). Furthermore, these suppliers were clearly assigned to S. perennans by distinguishing diagnostic positions from other species, including S. europaea (positions 76, 111, 303) and S. procumbens (positions 122, 221, 260, 333, 374, 395, 402, 432, 444). Differentiation from S. persica relies on a verified diagnostic nucleotide at position 404 and differences in variable, nonmatching nucleotides at positions 369 and 458 ( Figure 4 ; (Kadereit et al., 2012). Further differentiation from S. sp. (“Crassa” group) utilized three diagnostic nucleotides (positions 286, 287, 473) (Kadereit et al., 2012), which were exclusive to the “Crassa” group, confirming that there was no homology with sequences from suppliers 6, 9, and 10 ( Figure 4 ).

The samples from supplier 3 presented mixed and ambiguous homology to S. europaea and S. perennans sequences. Samples 3b and 3c were homologous to S. europaea on the basis of their diagnostic nucleotides and could not be differentiated from S. europaea by other diagnostic nucleotides ( Figure 4 ). Sample 3d displayed three variable nucleotides ( Figure 4 ) intermediate between those of S. europaea and other Salicornia taxa at diagnostic positions for S. procumbens, S. persica, and Salicornia sp. (“Crassa” group). Samples 3a and 3e differed from S. europaea, S. procumbens, S. persica, and Salicornia sp. (“Crassa” group) at the aforementioned diagnostic positions. Consequently, samples 3a, 3d, and 3e were more likely homologous to S. perennans sequences ( Figure 4 ).

Taken together, the analysis revealed distinct taxonomic assignments for most suppliers. Suppliers 1, 2, 8, and 11 were largely assigned to the S. europaea taxon, whereas supplier 4 was unambiguously identified as belonging to the S. procumbens taxon. Suppliers 6, 9, and 10 were attributed to the S. perennans taxon. Notably, supplier 3 could not be definitively assigned to either the S. europaea or S. perennans taxon. These clear assignments to the five main taxa were supported by diagnostic positions from the SNP analysis.

An analysis of the predictions for RNA secondary structures of the ETS region revealed that the structures from suppliers 1, 2, 3, 8, and 11 presented identical features and were homologous to reference sequences of S. europaea subsp. europaea (ribotypes 17 and 18), summarized as group 1 ( Figure 5A ). The ETS RNA secondary structures of supplier 4 were homologous to each other and to reference sequences of S. procumbens subsp. procumbens (ribotype 1), forming group 2 ( Figure 5A ). Supplier 5 displayed ETS RNA secondary structures corresponding to those of reference sequences from Sarcocornia fruticosa, represented as group 3 ( Figure 5A ). In contrast, the ETS RNA secondary structures of suppliers 6, 9, and 10 showed sufficient topological differences to warrant separate categorization and matched references of S. perennans subsp. perennans (ribotypes 1 and 8), summarized as group 4 ( Figure 5A ). Furthermore, the ETS RNA secondary structure predictions for supplier 7 formed a distinct group (group 5), matching a reference of Arthrocnemum macrostachyum ( Figure 5A ).

The ITS RNA secondary structure predictions resulted in the same number of groups as those for the ETS region ( Figure 5B ). Group 1 comprised suppliers 1, 2, 8, and 11, along with references from S. disarticulata, S. obscura, S. ramosissima, and S. x marshallii, but no sequences from supplier 3. In contrast, group 2 contained the RNA secondary structures of supplier 3, as well as those of suppliers 6, 9, and 10, together with references from S. europaea, S. herbacea, S. patula, S. perennans, S. persica, and S. prostata. Group 3 included the predictions for supplier 4 and references from S. dolichostachya, S. emerici, and S. fragilis. The ITS RNA secondary structure of supplier 5 (group 4) again matched references from Sarcocornia fruticosa and Sarcocornia pruinosa. Finally, the RNA secondary structure prediction for supplier 7 (group 5) was congruent with references from Arthrocnemum macrostachyum and Arthrocnemum glaucum.

Furthermore, the RNA secondary structure predictions for matK did not allow for a subdivision of the Salicornia species ( Figure 5C ). All sequences from suppliers 1, 2, 3, 4, 6, 8, 9, 10, and 11 resulted in homogeneous secondary structures, which matched references from S. brachiata, S. dolichostachya, S. emerici, S. europaea, S. obscura, S. patula, S. persica, and S. veneta (group 1). For the reference sequences of Sarcocornia fruticosa and Sarcocornia perennis, secondary structures that were homologous to those of supplier 5, summarized as group 2, were predicted ( Figure 5 ). The analysis of the matK RNA secondary structures for supplier 7 (group 3) revealed clear differences from all other sequence predictions ( Figure 5C ). The highest visual congruence was observed with a reference from Arthrocnemum macrostachyum, whereas the structure prediction for Arthrocnemum glaucum was distinctly different.

For the RNA structure predictions of the atpB-rbcL intergenic spacer ( Figure 5D ), the sequences from suppliers 1 and 8 were identified as identical to the prediction for a reference of S. europaea (group 1). The RNA structures of suppliers 2, 3, and 11 formed a distinct group (group 2), which clearly differed from those of group 1 but contained no homologous references. References from S. iranica subsp. iranica, S. persica, S. perspolitana, and S. x tashkensis, along with the secondary structure predictions for suppliers 4, 6, 9, and 10, formed a common homologous group (group 3). The secondary structure prediction for supplier 5 was, in turn, homologous to references from Sarcocornia alpini and Sarcocornia fruticosa (group 4). For supplier 7, an RNA secondary structure emerged (group 5) that was homologous to a reference sequence of Arthrocnemum macrostachyum and differed from that of an Arthrocnemum subterminale reference.

The ETS marker provided clear references within the five identified groups, unequivocally classifying the respective suppliers. While the ITS marker also delineated five groups with the same supplier assignments, it could not make a clear species assignment for the second group. The matK marker distinguished only three groups and could confirm the assignment of supplier 5 to Sarcocornia fruticosa or Sarcocornia perennis within the second group. The atpB-rbcL marker differentiated suppliers 1 and 8 from suppliers 2, 3, and 11 on the basis of marginal differences, but it failed to unequivocally assign the latter group to S. europaea. Furthermore, no clear species assignment was possible for group 3. In contrast, group 4 was clearly assigned to Sarcocornia fruticosa or a subspecies of Sarcocornia perennis, and group 5 was assigned to Arthrocnemum macrostachyum.

To evaluate their potential for species discrimination, the S2, S5, and S19 microsatellite (SSR) markers were analyzed for patterns of homozygosity/heterozygosity and the presence of specific fragment lengths, which demonstrated varying degrees of resolution in distinguishing among Salicornia species ( Figure 6 ). An additional table presents the results of the three markers for five samples from each supplier, with the exception of suppliers 5 and 7, which did not yield clear PCR amplicons ( Supplementary Table S5 ). As shown in Figure 6 , the S19 marker consistently distinguished the samples from suppliers 1 and 8 from supplier 2 and from supplier 11. Suppliers 1 and 8 in particular had a characteristic fragment length of 174 bp, whereas supplier 2 presented a clear fragment length of 167 bp, and supplier 11 presented 176 bp (with the exception of one sample with only 167 bp), whereas some samples from supplier 11 additionally presented smaller fragments 583 ( Supplementary Table S5 ). Supplier 11 was further delimited by the fact that a second fragment of unique length was found in the majority of the samples in both the S2 and S5 markers, although this was also true in parts of the supplier 8 samples ( Supplementary Table S5 ). Supplier 3 showed that the S2, S5 and S19 markers were mostly the same fragment size as suppliers 1 and 2 ( Supplementary Table S5 ). Figure 6 clearly shows that supplier 4 was differentiated both by fragment size and by the presence of two alleles (S2: 119 and 121 bp; S5: 97 and 100/102 bp; S19: 176 and 183 bp). Similarly, suppliers 6, 9, and 10 presented distinct allele numbers and fragment sizes across all three markers (S2: 112 and 117 bp; S5: 100 and partly 103 bp; S19: 172/173 and 176 bp) ( Figure 6 ). Overall, only the S19 marker revealed further distinct differences, specifically separating suppliers 1 and 8 from supplier 2 and supplier 2 from supplier 11, beyond what was previously identified.

The flow cytometry measurement histograms for all samples, along with the utilized references, exhibited clear peak patterns and were averaged over three replicates. An additional table shows representative histograms for each supplier and for the Trifolium pratense reference ( Supplementary Table S6 ). The nuclear DNA content of Trifolium pratense ‘Bingenheimer’ was determined to be 0.92 pg/2C and was used as a reference for all Salicornia species. On the basis of the size of the haploid genome and in comparison with literature data (Zonneveld, 2019), assumptions were made about the ploidy level of Salicornia species, which consistently have 9 chromosomes (basic chromosome number) (Zonneveld, 2019). The samples of all suppliers displayed endopolyploidy, a phenomenon already known for other Chenopodioideae species ( Supplementary Table S6 ) (Skaptsov et al., 2017).

Suppliers 1 and 8 were classified as diploids on the basis of their DNA content and presented very similar genome sizes of 565.4 Mbp and 579.1 Mbp, respectively. Similarly, suppliers 2 and 11 were classified as diploid with slightly larger genome sizes of 594.1 Mbp and 568.7 Mbp, respectively. The diploid genome size of supplier 3, 596.7 Mbp, also fell within the range observed for the previously mentioned suppliers ( Table 2 ).

Owing to the known larger genome size (Zonneveld, 2019) of S. perennans, suppliers 6, 9, and 10 were classified as diploid representatives of this species group, with calculated genome sizes of 1047.5 Mbp (supplier 6), 1021.3 Mbp (supplier 9), and 926.9 Mbp (supplier 10), respectively ( Table 2 ).

For supplier 4, the highest DNA content among the presumed Salicornia samples was measured. This value is typical for tetraploid species, leading to a calculated (haploid) genome size of 585.5 Mbp ( Table 2 ).

For supplier 5, the ploidy level could not be definitively resolved because the reported haploid genome sizes in the literature are not particularly precise and because of the documented occurrence of variable ploidy levels within the genus. Compared with the reference data, a hexaploid level was presumed, yielding a calculated haploid genome size of 1035.6 Mbp ( Table 2 ). However, on the basis of reference data suggesting a monoploid genome size of approximately 0.985 pg DNA content (Koce et al., 2008), an octoploid level could not be excluded. This alternative ploidy would imply a monoploid genome with approximately 0.106 pg of DNA content, corresponding to a genome size of 776.7 Mbp.

For supplier 7, a tetraploid ploidy level was expected (Shepherd and Yan, 2003), resulting in a potential haploid genome size of 1460.1 Mbp ( Table 2 ). Importantly, an as-yet unrecorded hexaploid genome for the species could also be a factor, potentially leading to a genome size of 973 Mbp. suppliers 1, 2, 3, 8, and 11 presented the smallest genome contents typical of S. europaea. In contrast, supplier 4 was differentiated by a significantly larger genome content, characteristic of tetraploid species and known for S. procumbens. This also distinguished supplier 4 from supplier 6, 9, and 10, which presented an intermediate genome content typical of diploid species within the S. perennans taxon. Supplier 5 presented a considerably greater genome content, aligning more with the genus Sarcocornia. Finally, supplier 7 could not be assigned to any species on the basis solely of this analysis owing to a lack of reference data.