Molecular profiling of 35 Phacelia species using SCoT molecular markers and genome size estimation

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Despite its diversity, the genetic relationships and genome size variation within Phacelia have remained poorly characterized. In this study, the nuclear DNA content (genome size) of 40 accessions representing 35 Phacelia species was estimated using flow cytometry (FCM), and genetic diversity was assessed using Start Codon Targeted (SCoT) molecular markers. The 2C nuclear DNA content ranged from 1.00 pg ( P. ciliata ) to 3.61 pg ( P. egena ), with notable inter- and intra-specific variation observed. SCoT marker analysis generated clear and reproducible banding patterns, enabling the construction of a UPGMA dendrogram that resolved the accessions into distinct groups, largely consistent with morphological and taxonomic relationships. Some closely related taxa, such as P. capitata and P. brachyloba , exhibited both high genetic similarity and comparable genome sizes, while significant divergence was observed among others. Although several well-supported clades identified in the SCoT-based phylogeny corresponded closely to established sections and subsections, certain taxa traditionally classified together were found in separate branches, suggesting complex evolutionary histories and potential morphological convergence. The results demonstrate that combining SCoT markers and genome size estimation provides valuable insights into the genetic diversity and evolutionary relationships within Phacelia , underscoring the importance of integrative molecular approaches for taxonomic and conservation studies in this genus. Boraginaceae DNA content flow cytometry genetic diversity molecular identification Figures Figure 1 Figure 2 Figure 3 1. Introduction The genus Phacelia Juss. has historically been placed in the family Hydrophyllaceae; however, recent molecular studies have led to its reclassification. According to current taxonomic consensus (Luebert et al., 2016 ), Phacelia is now included in the family Boraginaceae sensu lato . This change is supported by molecular analyses, such as the ndhF sequence study conducted by Ferguson ( 1998 ), which provided evidence for a closer relationship between Hydrophyllaceae and Boraginaceae, justifying the inclusion of Phacelia within Boraginaceae in the modern systematics. Phacelia represents the largest and most taxonomically diverse genus in the family, with approximately 150–200 of annual and perennial herbaceous species (Constance and Chuang, 1982 ; Gilbert et al., 2005 ; Hofmann et al., 2016 ), with the majority distributed in the western regions of the United States and northern Mexico (Walden et al., 2014 ; Riser et al., 2017 ). California (USA) is the center of the Phacelia diversity, with more than 90 recognized taxa, with about 40 of which being endemic to the state (Raven and Axelrod, 1978 ; Wilken et al., 1993 ). Some of the Phacelia species have also been introduced and naturalized in other regions, including Southern Europe, Australia and New Zealand (Smither-Kopperl, 2018 ). Several species within the Phacelia genus are considered especially important due to their ecological, agricultural and ornamental value. Phacelia tanacetifolia is widely cultivated as a cover crop because of its rapid growth, high biomass production, and ability to suppress weeds and retain soil nitrogen. Additionally, it is recognized as an important nectar and pollen source for honey bees and other pollinators, contributing to both apiculture and the maintenance of biodiversity in agroecosystems (Thrasyvoulou and Tsirakoglou, 1998 ; Petanidou, 2003 ; Smither-Kopperl, 2018 ). Some species are used as ornamental plants in gardens (e.g. P. campanularia , P. grandiflora , P. bipinnatifida , P.distans ), while others are an important source of nectar and pollen for bees and honey production. The phacelia honey is rich in antioxidants and exhibits antimicrobial, anti-inflammatory, and anticancer properties, making it valuable for overall health, medical nutrition, and therapeutic purposes (Williams and Christian, 1991 ; Sakač et al., 2022 ; Kunat-Budzyńska et al., 2023 ). Many Phacelia species are adapted to arid and semi-arid environments, exhibiting drought tolerance and the ability to colonize disturbed or marginal soils (Hickman, 1993 ; Baldwin et al., 2011 ). The plants can improve soil structure, enhance nutrient cycling, and support beneficial soil microorganisms (Kilian, 2016 ). Additionally, several taxa (e.g. P. argentea, P. insularis var. insularis , P. cookei, P. ramosissima ) are considered rare or threatened, particularly in the USA and Canada, underscoring the importance of conservation measures (California Native Plant Society, 2024 https://rareplants.cnps.org ; https://www.fs.usda.gov/ ). The genus Phacelia is genetically and morphologically diverse, with complex evolutionary relationships among species (Glass, 2007 ). Species divergence within the genus is caused by ecological adaptation, geographic isolation, and reproductive barriers, which explain why some species appear morphologically similar but genetically distinct, while others differ morphologically but are genetically close (Hansen et al., 2009 ; Vasile et al., 2024 ). The genus has been subdivided into three subgenera ( Cosmanthus, Howellanthus and Phacelia ), 11 sections and 12 subsections, and exhibits notable genetic diversity, particularly reflected in its chromosome numbers, polyploidy and chromosomal rearrangements, which have important implications for its evolution and speciation (Constance, 1963 ; Walden and Patterson, 2012 ). Constance ( 1963 ) reviewed cytological data for 145 species within the genus Phacelia and reported that 75 species exhibit a haploid chromosome number of n = 11, which suggested that is the base chromosome number for the genus. Chromosome counts in other species, ranging from n = 5 to 33, are considered to have originated from this base number through mechanisms such as aneuploidy, involving the gain or loss of individual chromosomes, and polyploidy, characterized by whole-genome duplications. Notably, substantial morphological and ecological variation frequently occurs among groups sharing the same chromosome number, most commonly n = 11, suggesting that factors beyond chromosome number contribute significantly to the genus’s diversity (Constance, 1963 ; Gillett, 1968 ; Munz and Keck, 1973 ; Gilbert et al., 2005 ; Walden et al., 2014 ; Welsh et al., 2016 ; Levy at al., 2021). The taxonomic and systematic relationships in Phacelia were inferred primarily through comparative morphology and cytological studies. The traits such as leaf shape, inflorescence architecture, seed morphology, and floral characteristics to delimit species and higher taxonomic groupings were examined (Constance and Chuang, 1982 ; Lee, 1986 ; Levy et al., 2021 ). Cytological investigations, focusing on chromosome counts and karyotype analysis, provided additional insight into genetic variation and evolutionary relationships. Moreover, aneuploidy and polyploidy were recognized as important mechanisms generating chromosomal diversity and, by extension, contributing to speciation and morphological differentiation (Constance, 1963 ; Gillet, 1968; Walden et al., 2014 ; Ozkan and Benlioglu, 2015 ). Furthermore, the molecular phylogenetic analyses, including nuclear ribosomal DNA (ITS), chloroplast DNA ( rpl 16 intron) sequences, SSR markers, and ddRADseq were employed to reveal relationships between and within species of the Phacelia genus as well as the other genera within the Boraginaceae (Hydrophyllaceae) family (Levy et al., 1996 ; Gilbert et al., 2005 ; Riser et al., 2017 , Vasile et al., 2024 ; Torres-Diaz et al., 2025), although some of them can be insufficient in the analysis of phylogenetic relationships. Application of additional molecular markers, including ISSR (Inter-Simple Sequence Repeats, Kurane et al., 2009 ) or SCoT (Start Codon Targeted; Collard and Mackill, 2009 ), may be useful for better understanding the genetic relationships in the Phacelia genus as well as within the Boraginaceae family. Moreover, nuclear markers combined with the flow cytometry method (FCM) seem to be promising in phylogenetic assessment of Phacelia and species identification in this genus. Recent studies provide no information on the application of such a combination of methods in the genus Phacelia . Additionally, there is no information regarding phylogenetic investigations involving such a large number of species and varieties. The combination of genome size estimation and molecular markers has been successfully applied to other medicinal plants, including Ocimum (Rewers and Jedrzejczyk, 2016 ), Mentha (Jedrzejczyk and Rewers, 2018 ), Malva (Jedrzejczyk and Rewers, 2020 ), and Echinacea (Jedrzejczyk, 2018 ). Given the significant potential of the Phacelia genus, which contains biologically active compounds with pharmaceutical and cosmetic properties, the use of combined analytical approaches allows a comprehensive characterization of Phacelia ’s complex and therefore optimalization of its use in sustainable agriculture and medicinal research. This study aimed to apply the nuclear DNA content (genome size) and SCoT molecular markers to identify species and determine the inter- and intra-specific variation within the Phacelia genus. 2. Materials and methods 2.1. Plant material Forty Phacelia accessions representing 35 species were used in the investigation. Seeds were obtained from GRIN-ARS-USDA gene bank (Table 1 ). All seeds were sown in pots containing a mixture of sand and commercial humus (1:2, w/w), and placed in a growth chamber, with a day/night temperature of 26/18°C, and with a 16/8-hour photoperiod. Table 1 Nuclear DNA content of analyzed the Phacelia genotypes. No. Species Taxon Origin DNA content CV sample Internal standard Genome size (pg/2C) ± SD Mbp/2C 1 P. adenophora USA 1.99 ± 0.08 g* 1946 4.18 PH VS 2 P. bicolor USA 1.90 ± 0.04 hij 1858 4.11 PH VS 3 P. brachyloba USA 1.86 ± 0.04 j 1819 3.24 RS VS 4 P. californica USA 3.32 ± 0.02 c 3247 2.68 SL S 5 P. campanularia Poland 1.25 ± 0.02 s 1223 2.99 SL VS 6 P. campanularia ssp. vasiformis USA 1.50 ± 0.04 n 1467 2.85 SL VS 7 P. capitata USA 1.87 ± 0.03 j 1829 3.12 RS VS 8 P. cicutaria USA 3.39 ± 0.02 b 3315 2.39 SL S 9 P. cicutaria var. hispida USA 3.42 ± 0.03 b 3345 2.32 SL S 10 P. ciliata USA 1.00 ± 0.02 v 978 3.67 SL VS 11 P. coerulea USA 1.64 ± 0.02 lm 1604 4.70 RS VS 12 P. congesta USA 1.65 ± 0.02 lm 1614 2.79 SL VS 13 P. corymbosa USA 1.78 ± 0.06 k 1741 4.17 RS VS 14 P. crenulata USA 1.36 ± 0.01 pr 1330 2.77 RS VS 15 P. crenulata var. ambigua USA 1.70 ± 0.01 l 1663 2.92 RS VS 16 P. crenulata var. corrugata USA 1.96 ± 0.06 gh 1917 2.84 RS VS 17 P. distans USA 1.36 ± 0.03 pr 1330 3.83 RS VS 18 P. douglasii USA 1.95 ± 0.01 ghi 1907 3.97 PH VS 19 P. egena USA 3.61 ± 0.03 a 3531 2.68 SL S 20 P. fremontii USA 1.99 ± 0.04 g 1946 4.16 PH VS 21 P. glandulosa var. glandulosa USA 2.25 ± 0.01 e 2001 3.03 RS VS 22 P. grisea USA 1.19 ± 0.02 t 1164 4.97 SL VS 23 P. hastata USA 1.86 ± 0.07 j 1819 3.58 RS VS 24 P. heterophylla USA 1.90 ± 0.06 ij 1858 3.06 RS VS 25 P. humilis var. humilis USA 1.61 ± 0.02 m 1575 3.98 SL VS 26 P. integrifolia USA 2.12 ± 0.07 f 2073 2.78 RS VS 27 P. inundata USA 1.96 ± 0.06 g 1917 4.92 PH VS 28 P. linearis USA 1.08 ± 0.02 u 1056 3.99 SL VS 29 P. lutea USA 2.00 ± 0.08 g 1956 4.24 RS VS 30 P. malvifolia USA 1.34 ± 0.01 r 1311 2.77 SL VS 31 P. nemoralis ssp. nemoralis USA 3.12 ± 0.02 d 3051 3.61 SL S 32 P. pachyphylla USA 1.05 ± 0.02 uv 1027 4.65 SL VS 33 P. palmeri USA 1.43 ± 0.02 o 1399 2.76 SL VS 34 P. parryi USA 1.35 ± 0.03 r 1320 3.55 SL VS 35 P. pulchella var. gooddingii USA 1.22 ± 0.01 st 1193 5.66 PH VS 36 P. ramosissima USA 1.35 ± 0.02 r 1329 3.55 SL VS 37 P. ramosissima var. latifolia USA 1.32 ± 0.03 r 1291 3.00 SL VS 38 P. rupestris USA 1.02 ± 0.01 v 998 3.28 SL VS 39 P. sericea USA 1.41 ± 0.02 op 1379 3.59 SL VS 40 P. tanacetifolia Czech Republic 1.26 ± 0.02 s 1232 3.16 SL VS * a-v, values (in columns) followed by the same letter are not significantly different at P = 0.05 (Duncan’s test); Internal standards: Raphanus sativus ‘Saxa’ (RS), Solanum lycopersicum ‘Stupicke’ (SL), Petunia hybrida ‘PxPc6’ (PH); Genome size: Very Small (VS), Small (S); 1pg = 978 Mbp Three internal standards: Raphanus sativus ‘Saxa’, Solanum lycopersicum ‘Stupicke’ and Petunia hybrida ‘PxPc6’ were used for FCM analysis. The plants were grown in a growth chamber, with a day/night temperature of 22/18°C, and with a 16/8-hour photoperiod. 2.2. Flow cytometry Nuclear DNA content was measured in young and freshly picked leaves of all Phacelia accessions obtained from the seeds. The samples for flow cytometric analysis were prepared according to the procedure described by Jedrzejczyk and Sliwinska ( 2010 ). Plant material was chopped with a sharp razor blade in a Petri dish with 1ml of nucleus isolation buffer (0.1 M Tris, 2.5 mM MgCl 2 x6H 2 O, 85 mM NaCl, 0.1% (v/v) Triton X-100) supplemented with propidium iodide (PI, 50 mg/ml) and ribonuclease A (RNase A, 50 µg/ml). The chopped plant material was filtered through a nylon filter with a mesh diameter of 50 µm. For each assayed sample, fluorescence was measured in 5,000–7,000 nuclei using a CyFlow SL Green (Partec GmbH, Münster, Germany) flow cytometer equipped with a high-grade solid-state laser with green light emission at 532 nm, long-pass filter RG 590 E, DM 560 A, and side (SSC) and forward (FSC) scatter. Histograms were analyzed with FloMax software (Partec GmbH, Münster, Germany). Analyses were performed in five biological replicates (five individual plants) per genotype. Three different internal standards were used for genome size estimation: Raphanus sativus ‘Saxa’ (2C = 1.10 pg; Dolezel et al., 1998), Solanum lycopersicum ‘Stupicke’ (2C = 1.15 pg; Dolezel et al., 1992) and Petunia hybrida ‘PxPc6’ (2C = 2.85 pg/2C; Marie and Brown, 1993 ). The nuclear DNA content was calculated using the linear relationship between the ratio of the 2C peak positions of Phacelia genotypes and the internal standard on the histogram of fluorescence intensities. The coefficient of variation (CV) of the G 0 /G 1 peak of Phacelia species ranged between 2.32 and 5.66%. The 2C DNA contents (pg) were transformed to megabase pairs of nucleotides using the following conversion: 1 pg = 978 Mbp (Doležel and Bartoš, 2005 ; Table 1 ). The results were estimated using a one-way analysis of variance and a Duncan’s test (P = 0.05; Statistica v. 13.3, StatSoft, Poland) 2.3. DNA extraction Genomic DNA was extracted from 100 mg of fresh leaf tissue collected from three randomly selected plants per accession using the Plant DNA GPB Mini Kit (GenoPlast Biotech S.A., Poland). The quality and concentration of the isolated DNA were evaluated spectrophotometrically and confirmed by electrophoresis on a 1% agarose gel. Only samples exhibiting high integrity and purity were selected for SCoT-PCR analyses. 2.4. SCoT-PCR SCoT-PCR amplifications were performed in a total reaction volume of 12.0 µL, containing 30 ng of genomic DNA template, 0.1 U/µL Taq DNA polymerase, 4 mM MgCl₂, 0.5 mM of each dNTP (2x PCR Master Mix Plus; A&A Biotechnology, Poland), 10 µM of SCoT primer (Genomed, Poland), and sterile deionized water. The primers were 18 nucleotides in length, with GC content ranging from 50–72%. Amplifications were conducted using a T100 Thermal Cycler (Bio-Rad, USA) under the following conditions: initial denaturation at 94°C for 5 min; 35 cycles consisting of denaturation at 94°C for 1 min, annealing at primer-specific temperatures between 49.5°C and 63.5°C for 1 min, and extension at 72°C for 2 min; followed by a final extension at 72°C for 7 min. A total of 15 SCoT primers were initially screened, of which ten generated reproducible and stable banding patterns and were subsequently selected for the analysis of Phacelia accessions (Table 2 ). Each PCR reaction with the selected primers was performed in duplicate to ensure reproducibility. Amplified products were separated by electrophoresis on 1.5% (w/v) agarose gels prepared in 1× TBE buffer and stained with ethidium bromide. Fragment sizes were estimated using a 3000 bp DNA ladder (GenoPlast Biotech S.A., Poland). Gel images were captured under UV light using the GelDoc XR + system (Bio-Rad, USA). Table 2 Features of SCoT primers used in molecular characterization of 40 Phacelia accessions. Primer code Primer sequence (5’-3’) Annealing temperature (˚C) TNB NPB PPB PIC SCoT-2 CAACAATGGCTACCACCC 51.0 21 21 100 0.340 SCoT-4 CAACAATGGCTACCACCT 49.5 24 24 100 0.375 SCoT-5 CAACAATGGCTACCACGA 50.0 24 24 100 0.361 SCoT-6 CAACAATGGCTACCACGC 51.0 23 23 100 0.331 SCoT-11 AAGCAATGGCTACCACCA 50.0 25 25 100 0.289 SCoT-12 ACGACATGGCGACCAACG 56.0 27 27 100 0.327 SCoT-32 CCATGGCTACCACCGCAC 56.0 24 24 100 0.294 SCoT-34 ACCATGGCTACCACCGCA 54.0 24 24 100 0.330 SCoT-35 CATGGCTACCACCCGCCC 63.5 20 20 100 0.354 SCoT-36 GCAACAATGGCTACCACC 51.0 28 28 100 0.381 Average 24 24 100 0.338 Note: TNB – the number of total bands; NPB – the number of polymorphic bands, PPB (%) - the percentage of polymorphism; PIC – polymorphism information content for each starter 2.5. SCoT-PCR data analysis The SCoT banding patterns were scored using a binary system, assigning a value of 1 for the presence and 0 for the absence of bands. Only distinct, bright, and well-resolved bands were considered, while faint bands were excluded to minimize scoring artefacts. For each primer, the numbers of monomorphic and polymorphic amplification products were recorded. Polymorphism Information Content (PIC) was calculated following Ghislain et al. ( 1999 ) using the formula: PIC = 1 – p 2 – q 2 , where p is the band frequency and q is band absence. Genetic distances among all accessions were estimated according to Nei and Li ( 1979 ). A dendrogram was constructed using the unweighted pair group method with arithmetic mean (UPGMA) via Treecon version 3.1 software (Van de Peer and Wachter, 1994 ). Branch support was evaluated by bootstrap analysis with 2000 replicates. 3. Results 3.1. Genome size Significant statistical differences in genome size were observed among the studied Phacelia accessions. The 2C nuclear DNA content ranged from 1.00 pg in P. ciliata to 3.61 pg in P. egena , corresponding to 978 Mbp and 3531 Mbp, respectively (Table 1 and Fig. 1 ). Notably, intraspecific genome size variation was detected, as exemplified by three accessions of P. crenulata : the 2C DNA content was 1.36 pg in P. crenulata , 1.70 pg in P. crenulata var. ambigua , and 1.96 pg in P. crenulata var. corrugata . Similarly, genome size differences between P. campanularia (1.25 pg/2C) and P. campanularia subsp. vasiformis (1.50 pg/2C) enabled clear discrimination of these genotypes. The majority of accessions (78%) exhibited genome sizes within the range of 1.02 to 1.99 pg/2C, reflecting significant variation both within and between species. According to Soltis et al. ( 2003 ) classification, 35 accessions possessed a very small genome, whereas five displayed a small genome. The coefficient of variation (CV) was low (< 5), indicating high reliability and reproducibility of the flow cytometric measurements. 3.2. SCoT markers Ten SCoT primers were screened to study the genetic relationships among Phacelia species. All primers produced reproducible polymorphic bands across all 40 Phacelia accessions. An image of the SCoT amplification generated by one primer of all studied accessions is shown in Fig. 2 . A total of 212 polymorphic bands were amplified across the studied species (Table 2 ). The sizes of the amplified fragments ranged from approximetely100 to 3300 bp. All tested primers revealed 100% polymorphism. The lowest and highest numbers of polymorphic bands were 20 for SCoT-35 and 28 for SCoT-36, respectively, with an average of 24 polymorphic bands per primer. The polymorphism information content (PIC) values for the 10 SCoT primers ranged from 0.289 (SCoT-11) to 0.381 (SCoT-36), with an average PIC of 0.338 per primer. Primers SCoT-5, SCoT-12, SCoT-32, and SCoT-55 proved to be particularly effective, allowing for the discrimination of even closely related genotypes. The genetic distance analysis among the studied accessions revealed distinguished variation in genetic similarity across the examined Phacelia species. The lowest genetic distance (0.28) was observed between P. capitata and P. brachyloba . In contrast, the highest genetic distance values, reaching 0.85, were recorded between P. rupestris and P. coerulea , as well as between P. rupestris and P. pachyphylla (Fig. 3 ; Table S1 ). Using the binary matrix derived from SCoT marker data, a dendrogram was constructed to visualize the genetic relationships among the accessions. The dendrogram effectively grouped the studied species into distinct clusters, each containing two, three or four accessions. Notably, four species - P. grisea , P. integrifolia , P. pachyphylla , and P. rupestris – were not grouped into any of the clades (Fig. 3 ). Furthermore, the analysis demonstrated strong clustering of species represented by two or three accessions, reflecting their genetic correlation. For instance, P. ramosissima clustered tightly with its variety P. ramosissima var. latifolia , underscoring their close genetic affinity. Similarly, P. cicutaria grouped closely with its variety P. cicutaria var. hispida . The species P. campanularia and its subspecies P. campanularia ssp. vasiformis also clustered together, highlighting their taxonomic similarity. Additionally, the three accessions of P. crenulata were grouped within a single cluster, further supporting the genetic consistency within this species (Fig. 3 ). 4. Discussion The genus Phacelia exhibits considerable morphological variability and ecological adaptability, which complicates accurate taxonomic classification. Understanding the genetic diversity within and among Phacelia species is essential for effective species preservation and conservation strategies. Flow cytometry, as a rapid and reliable method for estimating genome size, serves as an essential preliminary step in species identification, providing valuable cytogenetic data that can distinguish closely related taxa. Using flow cytometry, genome size screening was performed on 40 Phacelia accessions. To the best of our knowledge, this study represents the first report of 2C DNA content determination for 39 of these accessions. The measured 2C DNA content across all accessions ranged from 1.00 to 3.61 pg/2C, with approximately 88% of accessions exhibiting very small genome sizes (< 2.25 pg/2C). Until now, the 2C DNA content had only been reported for P. tanacetifolia by Zonneveld ( 2019 ), with a value of 0.22 pg higher than that obtained in the present study (1.26 pg/2C). Variations in genome size enabled the discrimination of nearly 18% of the examined Phacelia accessions. Notably, intraspecific differences in 2C DNA content allowed clear differentiation between P. campanularia (1.25 pg) and P. campanularia ssp. campanularia (1.50 pg), as well as among three accessions of P. crenulata (1.36 pg), P. crenulata var. ambigua (1.70 pg), and P. crenulata var. corrugata (1.96 pg). Conversely, nuclear DNA content did not permit distinction between P. cicuriata and P. cicuriata var. hispida , nor between P. ramosissima and P. ramosissima var. latifolia , where genome size differences were minimal (0.03 pg). The literature contains numerous reports demonstrating the successful application of flow cytometry as a reliable and efficient method for species identification, particularly in herbaceous plants where morphological differentiation is minimal or ambiguous (e.g. Jedrzejczyk, 2018 ; Jedrzejczyk, 2020 ; Jedrzejczyk and Rewers, 2018 ). In cases where flow cytometry is insufficient to discriminate closely related species or varieties due to overlapping 2C DNA content, the incorporation of molecular markers serves as a complementary tool that provides a more precise and reliable means of genotype differentiation. The application of molecular markers such as SCoT markers offers a powerful approach for resolving taxonomic ambiguities, particularly among morphologically similar accessions (Jedrzejczyk, 2020 ; Liu et al., 2021 ; Rewers et al., 2024 ). In the present study, 10 SCoT primers were successfully employed, which amplify conserved genomic regions flanking the ATG start codon, thereby generating gene-targeted polymorphisms with potential functional relevance (Collard and Mackill, 2009 ; Tabasi et al., 2020 ; Liu et al., 2021 ). This marker system facilitates the detection of genetic variation at coding sequences, providing higher discriminatory power for intraspecific differentiation compared to genome size estimation applied alone. To the best of our knowledge, this is the first report of the application of SCoT molecular markers for genetic diversity assessment within the genus Phacelia or even in the Boraginaceae family. In our study, all primers used in SCoT-PCRs revealed 100% polymorphism between all accessions, highlighting the high efficiency and discriminatory power of SCoT markers in capturing genetic variation within the Phacelia genus. Using these primers, it was possible to identify all tested species and varieties. The highest genetic similarity observed between P. capitata and P. brachyloba indicated a close genetic relationship between these accessions, which was also confirmed by similar genome size, 1.87 pg/2C and 1.86 pg/2C, respectively. This genetic similarity could reflect retained ancestral states, relatively recent divergence, or gene flow within the genus, despite these ecological distinctions. Conversely, the lowest similarity was detected between P. rupestris and both P. coerulea and P. pachyphylla , reflecting significant genetic divergence within these accessions, probably due to geographic isolation or ecological specialization. These findings correspond with prior observations of genetic subdivision and limited gene flow within Phacelia populations (Castillo, 1994 ; Glass, 2007 ). The mapping of genome size onto the molecular tree highlights both parallel and divergent evolutionary trends within Phacelia . In the majority of clades, relative genome size shifts were observed, suggesting the occurrence of polyploidy or chromosomal rearrangements. Even closely related accessions, in some clades (e.g. P. campanularia, P. crenulata ), differ substantially in genome size, indicating independent events of genome expansion or contraction. Although genome size generally aligns with SCoT-based clustering, additional evolutionary and ecological factors probably influenced genome size variation within the genus. The phylogenetic tree constructed using SCoT molecular markers provides clear insights into the genetic relationships among the examined Phacelia species. The topology reveals several well-supported clades, grouping taxa that are congruent with previously proposed sectional or infrageneric classifications based on morphological or molecular evidence (Ferguson, 1998 ; Luebert et al., 2016 ). The clustering of species such as P. crenulata , P. crenulata var. corrugata , and P. crenulata var. ambigua further aligns with their recognized taxonomic treatment as closely related taxa (Wilken, 2012 ), supporting the resolution power of SCoT markers for both species-level and infra-species differentiation. Furthermore, the grouping of P. campanularia with its subspecies P. campanularia ssp. vasiformis , and the close placement of species pairs like P. fremontii and P. douglasii , suggested a robust relationship between morphologically similar or geographically proximate taxa. Notably, some clusters contain species that are morphologically diverse or distributed across different sections in traditional taxonomy, which supports previous molecular studies indicating that morphological convergence or plasticity is relatively common in Phacelia (Hileman et al., 2006 ). For instance, taxa such as P. californica , P. cicutaria and its variety P. cicutaria var. hispida appear in a distinct and well-separated branch, reflecting their unique evolutionary lineage, as noted in earlier phylogenetic reconstructions based on ITS and chloroplast sequences (Ferguson, 1998 ). The observed arrangement of P. integrifolia and P. glandulosa var. glandulosa in separate branches despite some morphological similarities echoes earlier findings pointing to complex evolutionary patterns within the genus (Ferguson, 1998 ; Luebert et al., 2016 ). Overall, the SCoT-based phylogeny corroborates and refines previous hypotheses regarding the genetic relatedness and taxonomic boundaries within Phacelia . These findings are consistent with the conclusions drawn from earlier analyses employing nuclear and chloroplast markers (Ferguson, 1998 ; Luebert et al., 2016 ), underscoring the value of SCoT markers for resolving fine-scale phylogenetic relationships within the genus. Furthermore, the results emphasize the importance of integrating multiple molecular marker systems to fully capture the evolutionary complexity of Phacelia . Several well-supported clades in the SCoT-based phylogenetic tree correspond closely to sections and subsections recognized in classical taxonomy. However, certain taxa traditionally assigned to the same sectional or subsectoral groups are dispersed across different branches of the tree. This incongruence indicates that the relationships inferred from SCoT markers do not always fully align with morphological classification schemes, pointing to a more complex evolutionary history for the genus. Possible explanations for such discrepancies include convergent evolution of morphological traits, incomplete lineage sorting, or differences in the resolution power among molecular markers. Similar patterns of discordance have been reported in other molecular phylogenetic studies of Phacelia (Ferguson, 1998 ; Luebert et al., 2016 ), highlighting the necessity of integrative approaches that combine diverse molecular datasets with comprehensive morphological analyses to achieve a more robust and accurate understanding of phylogenetic relationships within the genus. 5. Conclusions This study provides a comprehensive assessment of genetic diversity and genome size variation within the genus Phacelia , combining flow cytometry and SCoT molecular markers for the first time in this taxon. The integration of flow cytometry and molecular marker analyses enables precise assessment of genetic relationships and species delimitation, complementing traditional morphological methods. This combined strategy enhances the accuracy of phylogenetic studies, germplasm characterization, and breeding programs, ultimately facilitating the optimized utilization of Phacelia species in ecological restoration, sustainable agriculture, and pharmaceutical research. Declarations Author contribution IJ: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing – Original Draft, Writing – Review & Editing, Visualization, Resources, Supervision, Project administration. AL : Investigation. MR: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing – Original Draft, Writing – Review & Editing, Visualization. Acknowledgements This work was financially supported by the Minister of Science under the program “Regional Initiative of Excellence” (RID/SP/0017/2024/01). The authors are gratefully thankful to the GRIN-ARS-USDA gene bank for supplying Phacelia seeds. Appendix A. 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Cite Share Download PDF Status: Published Journal Publication published 31 Mar, 2026 Read the published version in Journal of Applied Genetics → Version 1 posted Editorial decision: Major Revisions Needed 26 Jan, 2026 Reviewers agreed at journal 29 Sep, 2025 Reviewers invited by journal 15 Sep, 2025 Editor assigned by journal 10 Sep, 2025 First submitted to journal 05 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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06:09:49","extension":"html","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":213788,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7542683/v1/f8e0fabe1b0000313836cbe3.html"},{"id":92052127,"identity":"cbe5b710-c594-46c1-90e1-0c3c17dfa2c8","added_by":"auto","created_at":"2025-09-24 06:09:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":219133,"visible":true,"origin":"","legend":"\u003cp\u003eSelected histograms of 2C DNA content of \u003cem\u003ePhacelia \u003c/em\u003especies.\u003c/p\u003e","description":"","filename":"Fig.1.Selectedhistogramsof2CDNAcontent.png","url":"https://assets-eu.researchsquare.com/files/rs-7542683/v1/ef9e975955ca3414497e46a0.png"},{"id":92052542,"identity":"92337bec-4a44-4292-896c-2278cac34952","added_by":"auto","created_at":"2025-09-24 06:17:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3996315,"visible":true,"origin":"","legend":"\u003cp\u003eBanding profiles generated by\u003cstrong\u003e \u003c/strong\u003eSCoT-PCR using SCoT-12 primer. M, 3000 bp DNA ladder; C, negative control; 1, \u003cem\u003eP. adenophora\u003c/em\u003e; 2, \u003cem\u003eP. bicolor\u003c/em\u003e; 3, \u003cem\u003eP. brachyloba\u003c/em\u003e; 4, \u003cem\u003eP. californica\u003c/em\u003e; 5, \u003cem\u003eP. campanularia\u003c/em\u003e; 6, \u003cem\u003eP. campanularia\u003c/em\u003e ssp. \u003cem\u003evasiformis\u003c/em\u003e; 7, \u003cem\u003eP. capitata\u003c/em\u003e; 8, \u003cem\u003eP. cicutaria\u003c/em\u003e; 9, \u003cem\u003eP. cicutaria\u003c/em\u003e var. \u003cem\u003ehispida\u003c/em\u003e; 10, \u003cem\u003eP. ciliata\u003c/em\u003e; 11, \u003cem\u003eP. coerulea\u003c/em\u003e; 12, \u003cem\u003eP. corymbosa\u003c/em\u003e; 13, \u003cem\u003eP. crenulata\u003c/em\u003e; 14, \u003cem\u003eP. crenulata\u003c/em\u003e var. \u003cem\u003eambigua\u003c/em\u003e; 15, \u003cem\u003eP. crenulata\u003c/em\u003evar. \u003cem\u003ecorrugata\u003c/em\u003e; 16, \u003cem\u003eP. congesta\u003c/em\u003e; 17, \u003cem\u003eP. distans\u003c/em\u003e; 18, \u003cem\u003eP. douglasii\u003c/em\u003e; 19, \u003cem\u003eP. egena\u003c/em\u003e; 20, \u003cem\u003eP. fremontii\u003c/em\u003e; 21, \u003cem\u003eP. glandulosa\u003c/em\u003e var. \u003cem\u003eglandulosa\u003c/em\u003e; 22, \u003cem\u003eP. grisea\u003c/em\u003e; 23, \u003cem\u003eP. hastata\u003c/em\u003e; 24, \u003cem\u003eP. heterophylla\u003c/em\u003e; 25, \u003cem\u003eP. humilis\u003c/em\u003e var. \u003cem\u003ehumilis\u003c/em\u003e; 26, \u003cem\u003eP. inundata\u003c/em\u003e; 27, \u003cem\u003eP. integrifolia\u003c/em\u003e; 28, \u003cem\u003eP. linearis\u003c/em\u003e; 29, \u003cem\u003eP. lutea\u003c/em\u003e; 30, \u003cem\u003eP. nemoralis\u003c/em\u003e ssp. \u003cem\u003enemoralis\u003c/em\u003e; 31, \u003cem\u003eP. malvifolia\u003c/em\u003e; 32, \u003cem\u003eP. pachyphylla\u003c/em\u003e; 33, \u003cem\u003eP. palmeri\u003c/em\u003e; 34, \u003cem\u003eP. parryi\u003c/em\u003e; 35, \u003cem\u003eP. pulchella\u003c/em\u003e var. \u003cem\u003egooddingii\u003c/em\u003e; 36, \u003cem\u003eP. ramosissima\u003c/em\u003e; 37, \u003cem\u003eP. ramosissima\u003c/em\u003e var. \u003cem\u003elatifolia\u003c/em\u003e, 38, \u003cem\u003eP. rupestris\u003c/em\u003e; 39, \u003cem\u003eP. sericea\u003c/em\u003e; 40, \u003cem\u003eP. tanacetifolia\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Fig.2.BandingprofilesgeneratedbySCoTprimer.png","url":"https://assets-eu.researchsquare.com/files/rs-7542683/v1/395ff91fe59b7068bbc04f0d.png"},{"id":92052132,"identity":"386ece09-4e27-480a-8d95-7921416dd882","added_by":"auto","created_at":"2025-09-24 06:09:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":464873,"visible":true,"origin":"","legend":"\u003cp\u003eThe UPGMA dendrogram computed using genetic distance matrix based on SCoT markers. Only bootstrap values \u0026gt;50% are indicated; scale indicates genetic distance.\u003c/p\u003e","description":"","filename":"Fig.3.TheUPGMAdendrogram.png","url":"https://assets-eu.researchsquare.com/files/rs-7542683/v1/3f7e28ee19b063434c8afc27.png"},{"id":106344305,"identity":"77493f1d-071f-4df4-81b7-8fef032c1de0","added_by":"auto","created_at":"2026-04-07 16:13:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4739859,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7542683/v1/561fd196-8c2a-44f6-b346-9fa7c3c43ad9.pdf"},{"id":92052125,"identity":"a502d3d9-665f-4da0-8fa7-2acbaa366038","added_by":"auto","created_at":"2025-09-24 06:09:49","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":24852,"visible":true,"origin":"","legend":"\u003cp\u003eTable S1. Genetic distances between 40 \u003cem\u003ePhacelia \u003c/em\u003eaccessions.\u003c/p\u003e","description":"","filename":"TableS1.Geneticdistances.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7542683/v1/da19d38979f775b89435dba8.xlsx"}],"financialInterests":"","formattedTitle":"Molecular profiling of 35 Phacelia species using SCoT molecular markers and genome size estimation","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe genus \u003cem\u003ePhacelia\u003c/em\u003e Juss. has historically been placed in the family Hydrophyllaceae; however, recent molecular studies have led to its reclassification. According to current taxonomic consensus (Luebert et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), \u003cem\u003ePhacelia\u003c/em\u003e is now included in the family Boraginaceae \u003cem\u003esensu lato\u003c/em\u003e. This change is supported by molecular analyses, such as the ndhF sequence study conducted by Ferguson (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1998\u003c/span\u003e), which provided evidence for a closer relationship between Hydrophyllaceae and Boraginaceae, justifying the inclusion of \u003cem\u003ePhacelia\u003c/em\u003e within Boraginaceae in the modern systematics. \u003cem\u003ePhacelia\u003c/em\u003e represents the largest and most taxonomically diverse genus in the family, with approximately 150\u0026ndash;200 of annual and perennial herbaceous species (Constance and Chuang, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1982\u003c/span\u003e; Gilbert et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Hofmann et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), with the majority distributed in the western regions of the United States and northern Mexico (Walden et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Riser et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). California (USA) is the center of the \u003cem\u003ePhacelia\u003c/em\u003e diversity, with more than 90 recognized taxa, with about 40 of which being endemic to the state (Raven and Axelrod, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1978\u003c/span\u003e; Wilken et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). Some of the \u003cem\u003ePhacelia\u003c/em\u003e species have also been introduced and naturalized in other regions, including Southern Europe, Australia and New Zealand (Smither-Kopperl, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSeveral species within the \u003cem\u003ePhacelia\u003c/em\u003e genus are considered especially important due to their ecological, agricultural and ornamental value. \u003cem\u003ePhacelia tanacetifolia\u003c/em\u003e is widely cultivated as a cover crop because of its rapid growth, high biomass production, and ability to suppress weeds and retain soil nitrogen. Additionally, it is recognized as an important nectar and pollen source for honey bees and other pollinators, contributing to both apiculture and the maintenance of biodiversity in agroecosystems (Thrasyvoulou and Tsirakoglou, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Petanidou, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Smither-Kopperl, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Some species are used as ornamental plants in gardens (e.g. \u003cem\u003eP. campanularia\u003c/em\u003e, \u003cem\u003eP. grandiflora\u003c/em\u003e, \u003cem\u003eP. bipinnatifida\u003c/em\u003e, \u003cem\u003eP.distans\u003c/em\u003e), while others are an important source of nectar and pollen for bees and honey production. The phacelia honey is rich in antioxidants and exhibits antimicrobial, anti-inflammatory, and anticancer properties, making it valuable for overall health, medical nutrition, and therapeutic purposes (Williams and Christian, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Sakač et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Kunat-Budzyńska et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Many \u003cem\u003ePhacelia\u003c/em\u003e species are adapted to arid and semi-arid environments, exhibiting drought tolerance and the ability to colonize disturbed or marginal soils (Hickman, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Baldwin et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The plants can improve soil structure, enhance nutrient cycling, and support beneficial soil microorganisms (Kilian, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Additionally, several taxa (e.g. \u003cem\u003eP. argentea, P. insularis\u003c/em\u003e var. \u003cem\u003einsularis\u003c/em\u003e, \u003cem\u003eP. cookei, P. ramosissima\u003c/em\u003e) are considered rare or threatened, particularly in the USA and Canada, underscoring the importance of conservation measures (California Native Plant Society, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://rareplants.cnps.org\u003c/span\u003e\u003cspan address=\"https://rareplants.cnps.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.fs.usda.gov/\u003c/span\u003e\u003cspan address=\"https://www.fs.usda.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe genus \u003cem\u003ePhacelia\u003c/em\u003e is genetically and morphologically diverse, with complex evolutionary relationships among species (Glass, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Species divergence within the genus is caused by ecological adaptation, geographic isolation, and reproductive barriers, which explain why some species appear morphologically similar but genetically distinct, while others differ morphologically but are genetically close (Hansen et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Vasile et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The genus has been subdivided into three subgenera (\u003cem\u003eCosmanthus, Howellanthus\u003c/em\u003e and \u003cem\u003ePhacelia\u003c/em\u003e), 11 sections and 12 subsections, and exhibits notable genetic diversity, particularly reflected in its chromosome numbers, polyploidy and chromosomal rearrangements, which have important implications for its evolution and speciation (Constance, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1963\u003c/span\u003e; Walden and Patterson, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Constance (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1963\u003c/span\u003e) reviewed cytological data for 145 species within the genus \u003cem\u003ePhacelia\u003c/em\u003e and reported that 75 species exhibit a haploid chromosome number of \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;11, which suggested that is the base chromosome number for the genus. Chromosome counts in other species, ranging from \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5 to 33, are considered to have originated from this base number through mechanisms such as aneuploidy, involving the gain or loss of individual chromosomes, and polyploidy, characterized by whole-genome duplications. Notably, substantial morphological and ecological variation frequently occurs among groups sharing the same chromosome number, most commonly \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;11, suggesting that factors beyond chromosome number contribute significantly to the genus\u0026rsquo;s diversity (Constance, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1963\u003c/span\u003e; Gillett, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1968\u003c/span\u003e; Munz and Keck, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1973\u003c/span\u003e; Gilbert et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Walden et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Welsh et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Levy at al., 2021).\u003c/p\u003e\u003cp\u003eThe taxonomic and systematic relationships in \u003cem\u003ePhacelia\u003c/em\u003e were inferred primarily through comparative morphology and cytological studies. The traits such as leaf shape, inflorescence architecture, seed morphology, and floral characteristics to delimit species and higher taxonomic groupings were examined (Constance and Chuang, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1982\u003c/span\u003e; Lee, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1986\u003c/span\u003e; Levy et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Cytological investigations, focusing on chromosome counts and karyotype analysis, provided additional insight into genetic variation and evolutionary relationships. Moreover, aneuploidy and polyploidy were recognized as important mechanisms generating chromosomal diversity and, by extension, contributing to speciation and morphological differentiation (Constance, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1963\u003c/span\u003e; Gillet, 1968; Walden et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Ozkan and Benlioglu, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Furthermore, the molecular phylogenetic analyses, including nuclear ribosomal DNA (ITS), chloroplast DNA (\u003cem\u003erpl\u003c/em\u003e16 intron) sequences, SSR markers, and ddRADseq were employed to reveal relationships between and within species of the \u003cem\u003ePhacelia\u003c/em\u003e genus as well as the other genera within the Boraginaceae (Hydrophyllaceae) family (Levy et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Gilbert et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Riser et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Vasile et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Torres-Diaz et al., 2025), although some of them can be insufficient in the analysis of phylogenetic relationships. Application of additional molecular markers, including ISSR (Inter-Simple Sequence Repeats, Kurane et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) or SCoT (Start Codon Targeted; Collard and Mackill, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), may be useful for better understanding the genetic relationships in the \u003cem\u003ePhacelia\u003c/em\u003e genus as well as within the Boraginaceae family. Moreover, nuclear markers combined with the flow cytometry method (FCM) seem to be promising in phylogenetic assessment of \u003cem\u003ePhacelia\u003c/em\u003e and species identification in this genus. Recent studies provide no information on the application of such a combination of methods in the genus \u003cem\u003ePhacelia\u003c/em\u003e. Additionally, there is no information regarding phylogenetic investigations involving such a large number of species and varieties. The combination of genome size estimation and molecular markers has been successfully applied to other medicinal plants, including \u003cem\u003eOcimum\u003c/em\u003e (Rewers and Jedrzejczyk, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), \u003cem\u003eMentha\u003c/em\u003e (Jedrzejczyk and Rewers, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), \u003cem\u003eMalva\u003c/em\u003e (Jedrzejczyk and Rewers, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and \u003cem\u003eEchinacea\u003c/em\u003e (Jedrzejczyk, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Given the significant potential of the \u003cem\u003ePhacelia\u003c/em\u003e genus, which contains biologically active compounds with pharmaceutical and cosmetic properties, the use of combined analytical approaches allows a comprehensive characterization of \u003cem\u003ePhacelia\u003c/em\u003e\u0026rsquo;s complex and therefore optimalization of its use in sustainable agriculture and medicinal research.\u003c/p\u003e\u003cp\u003eThis study aimed to apply the nuclear DNA content (genome size) and SCoT molecular markers to identify species and determine the inter- and intra-specific variation within the \u003cem\u003ePhacelia\u003c/em\u003e genus.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Plant material\u003c/h2\u003e\u003cp\u003eForty \u003cem\u003ePhacelia\u003c/em\u003e accessions representing 35 species were used in the investigation. Seeds were obtained from GRIN-ARS-USDA gene bank (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). All seeds were sown in pots containing a mixture of sand and commercial humus (1:2, w/w), and placed in a growth chamber, with a day/night temperature of 26/18\u0026deg;C, and with a 16/8-hour photoperiod.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eNuclear DNA content of analyzed the \u003cem\u003ePhacelia\u003c/em\u003e genotypes.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"10\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eNo.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSpecies\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eTaxon\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eOrigin\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e\u003cp\u003eDNA content\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eCV sample\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eInternal standard\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eGenome size\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e(pg/2C)\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eMbp/2C\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. adenophora\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eg*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1946\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e4.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003ePH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eVS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. bicolor\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ehij\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1858\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e4.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003ePH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eVS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. brachyloba\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ej\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1819\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e3.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eRS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eVS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. californica\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e3.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ec\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e3247\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eSL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. campanularia\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePoland\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003es\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1223\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eSL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eVS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. campanularia\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003essp. \u003cem\u003evasiformis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003en\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1467\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eSL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eVS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. capitata\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ej\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1829\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e3.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eRS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eVS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. cicutaria\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e3.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e3315\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eSL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. cicutaria\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003evar. \u003cem\u003ehispida\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e3.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e3345\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eSL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. ciliata\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ev\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e978\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e3.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eSL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eVS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. coerulea\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003elm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1604\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e4.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eRS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eVS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. congesta\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003elm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1614\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eSL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eVS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. corymbosa\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ek\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1741\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e4.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eRS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eVS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. crenulata\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003epr\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1330\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eRS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eVS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. crenulata\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003evar. \u003cem\u003eambigua\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003el\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1663\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eRS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eVS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. crenulata\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003evar. \u003cem\u003ecorrugata\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003egh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1917\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eRS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eVS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. distans\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003epr\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1330\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e3.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eRS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eVS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. douglasii\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eghi\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1907\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e3.97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003ePH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eVS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. egena\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e3.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e3531\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eSL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. fremontii\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1946\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e4.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003ePH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eVS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. glandulosa\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003evar. \u003cem\u003eglandulosa\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e2.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ee\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e2001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e3.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eRS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eVS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. grisea\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003et\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1164\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e4.97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eSL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eVS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. hastata\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ej\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1819\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e3.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eRS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eVS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. heterophylla\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eij\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1858\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e3.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eRS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eVS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. humilis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003evar. \u003cem\u003ehumilis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003em\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1575\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e3.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eSL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eVS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. integrifolia\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e2.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ef\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e2073\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2.78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eRS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eVS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. inundata\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1917\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e4.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003ePH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eVS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. linearis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eu\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1056\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e3.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eSL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eVS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. lutea\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e2.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1956\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e4.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eRS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eVS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. malvifolia\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003er\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1311\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eSL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eVS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. nemoralis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003essp. \u003cem\u003enemoralis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e3.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ed\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e3051\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e3.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eSL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. pachyphylla\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003euv\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1027\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e4.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eSL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eVS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. palmeri\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1399\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eSL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eVS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. parryi\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003er\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1320\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e3.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eSL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eVS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. pulchella\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003evar. gooddingii\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003est\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1193\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e5.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003ePH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eVS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. ramosissima\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003er\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1329\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e3.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eSL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eVS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. ramosissima\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003evar. \u003cem\u003elatifolia\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003er\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1291\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e3.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eSL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eVS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. rupestris\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ev\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e998\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e3.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eSL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eVS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. sericea\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eop\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1379\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e3.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eSL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eVS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. tanacetifolia\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCzech Republic\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003es\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1232\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e3.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eSL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eVS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"10\"\u003e* a-v, values (in columns) followed by the same letter are not significantly different at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.05 (Duncan\u0026rsquo;s test); Internal standards: \u003cem\u003eRaphanus sativus\u003c/em\u003e \u0026lsquo;Saxa\u0026rsquo; (RS), \u003cem\u003eSolanum lycopersicum\u003c/em\u003e \u0026lsquo;Stupicke\u0026rsquo; (SL), \u003cem\u003ePetunia hybrida\u003c/em\u003e \u0026lsquo;PxPc6\u0026rsquo; (PH); Genome size: Very Small (VS), Small (S); 1pg\u0026thinsp;=\u0026thinsp;978 Mbp\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThree internal standards: \u003cem\u003eRaphanus sativus\u003c/em\u003e \u0026lsquo;Saxa\u0026rsquo;, \u003cem\u003eSolanum lycopersicum\u003c/em\u003e \u0026lsquo;Stupicke\u0026rsquo; and \u003cem\u003ePetunia hybrida\u003c/em\u003e \u0026lsquo;PxPc6\u0026rsquo; were used for FCM analysis. The plants were grown in a growth chamber, with a day/night temperature of 22/18\u0026deg;C, and with a 16/8-hour photoperiod.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Flow cytometry\u003c/h2\u003e\u003cp\u003eNuclear DNA content was measured in young and freshly picked leaves of all \u003cem\u003ePhacelia\u003c/em\u003e accessions obtained from the seeds. The samples for flow cytometric analysis were prepared according to the procedure described by Jedrzejczyk and Sliwinska (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Plant material was chopped with a sharp razor blade in a Petri dish with 1ml of nucleus isolation buffer (0.1 M Tris, 2.5 mM MgCl\u003csub\u003e2\u003c/sub\u003e x6H\u003csub\u003e2\u003c/sub\u003eO, 85 mM NaCl, 0.1% (v/v) Triton X-100) supplemented with propidium iodide (PI, 50 mg/ml) and ribonuclease A (RNase A, 50 \u0026micro;g/ml). The chopped plant material was filtered through a nylon filter with a mesh diameter of 50 \u0026micro;m. For each assayed sample, fluorescence was measured in 5,000\u0026ndash;7,000 nuclei using a CyFlow SL Green (Partec GmbH, M\u0026uuml;nster, Germany) flow cytometer equipped with a high-grade solid-state laser with green light emission at 532 nm, long-pass filter RG 590 E, DM 560 A, and side (SSC) and forward (FSC) scatter. Histograms were analyzed with FloMax software (Partec GmbH, M\u0026uuml;nster, Germany). Analyses were performed in five biological replicates (five individual plants) per genotype. Three different internal standards were used for genome size estimation: \u003cem\u003eRaphanus sativus\u003c/em\u003e \u0026lsquo;Saxa\u0026rsquo; (2C\u0026thinsp;=\u0026thinsp;1.10 pg; Dolezel et al., 1998), \u003cem\u003eSolanum lycopersicum\u003c/em\u003e \u0026lsquo;Stupicke\u0026rsquo; (2C\u0026thinsp;=\u0026thinsp;1.15 pg; Dolezel et al., 1992) and \u003cem\u003ePetunia hybrida\u003c/em\u003e \u0026lsquo;PxPc6\u0026rsquo; (2C\u0026thinsp;=\u0026thinsp;2.85 pg/2C; Marie and Brown, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). The nuclear DNA content was calculated using the linear relationship between the ratio of the 2C peak positions of \u003cem\u003ePhacelia\u003c/em\u003e genotypes and the internal standard on the histogram of fluorescence intensities. The coefficient of variation (CV) of the G\u003csub\u003e0\u003c/sub\u003e/G\u003csub\u003e1\u003c/sub\u003e peak of \u003cem\u003ePhacelia\u003c/em\u003e species ranged between 2.32 and 5.66%. The 2C DNA contents (pg) were transformed to megabase pairs of nucleotides using the following conversion: 1 pg\u0026thinsp;=\u0026thinsp;978 Mbp (Doležel and Bartoš, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The results were estimated using a one-way analysis of variance and a Duncan\u0026rsquo;s test (P\u0026thinsp;=\u0026thinsp;0.05; Statistica v. 13.3, StatSoft, Poland)\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. DNA extraction\u003c/h2\u003e\u003cp\u003eGenomic DNA was extracted from 100 mg of fresh leaf tissue collected from three randomly selected plants per accession using the Plant DNA GPB Mini Kit (GenoPlast Biotech S.A., Poland). The quality and concentration of the isolated DNA were evaluated spectrophotometrically and confirmed by electrophoresis on a 1% agarose gel. Only samples exhibiting high integrity and purity were selected for SCoT-PCR analyses.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. SCoT-PCR\u003c/h2\u003e\u003cp\u003eSCoT-PCR amplifications were performed in a total reaction volume of 12.0 \u0026micro;L, containing 30 ng of genomic DNA template, 0.1 U/\u0026micro;L Taq DNA polymerase, 4 mM MgCl₂, 0.5 mM of each dNTP (2x PCR Master Mix Plus; A\u0026amp;A Biotechnology, Poland), 10 \u0026micro;M of SCoT primer (Genomed, Poland), and sterile deionized water. The primers were 18 nucleotides in length, with GC content ranging from 50\u0026ndash;72%. Amplifications were conducted using a T100 Thermal Cycler (Bio-Rad, USA) under the following conditions: initial denaturation at 94\u0026deg;C for 5 min; 35 cycles consisting of denaturation at 94\u0026deg;C for 1 min, annealing at primer-specific temperatures between 49.5\u0026deg;C and 63.5\u0026deg;C for 1 min, and extension at 72\u0026deg;C for 2 min; followed by a final extension at 72\u0026deg;C for 7 min. A total of 15 SCoT primers were initially screened, of which ten generated reproducible and stable banding patterns and were subsequently selected for the analysis of \u003cem\u003ePhacelia\u003c/em\u003e accessions (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Each PCR reaction with the selected primers was performed in duplicate to ensure reproducibility. Amplified products were separated by electrophoresis on 1.5% (w/v) agarose gels prepared in 1\u0026times; TBE buffer and stained with ethidium bromide. Fragment sizes were estimated using a 3000 bp DNA ladder (GenoPlast Biotech S.A., Poland). Gel images were captured under UV light using the GelDoc XR\u0026thinsp;+\u0026thinsp;system (Bio-Rad, USA).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eFeatures of SCoT primers used in molecular characterization of 40 \u003cem\u003ePhacelia\u003c/em\u003e accessions.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePrimer code\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePrimer sequence (5\u0026rsquo;-3\u0026rsquo;)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAnnealing temperature (˚C)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTNB\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNPB\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePPB\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePIC\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSCoT-2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCAACAATGGCTACCACCC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e51.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.340\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSCoT-4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCAACAATGGCTACCACCT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e49.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.375\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSCoT-5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCAACAATGGCTACCACGA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e50.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.361\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSCoT-6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCAACAATGGCTACCACGC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e51.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.331\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSCoT-11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAAGCAATGGCTACCACCA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e50.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.289\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSCoT-12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eACGACATGGCGACCAACG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e56.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.327\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSCoT-32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCCATGGCTACCACCGCAC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e56.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.294\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSCoT-34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eACCATGGCTACCACCGCA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e54.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.330\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSCoT-35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCATGGCTACCACCCGCCC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e63.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.354\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSCoT-36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGCAACAATGGCTACCACC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e51.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.381\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eAverage\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e24\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e24\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e100\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e0.338\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003eNote: TNB \u0026ndash; the number of total bands; NPB \u0026ndash; the number of polymorphic bands, PPB (%) - the percentage\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003eof polymorphism; PIC \u0026ndash; polymorphism information content for each starter\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. SCoT-PCR data analysis\u003c/h2\u003e\u003cp\u003eThe SCoT banding patterns were scored using a binary system, assigning a value of 1 for the presence and 0 for the absence of bands. Only distinct, bright, and well-resolved bands were considered, while faint bands were excluded to minimize scoring artefacts. For each primer, the numbers of monomorphic and polymorphic amplification products were recorded. Polymorphism Information Content (PIC) was calculated following Ghislain et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) using the formula: PIC\u0026thinsp;=\u0026thinsp;1 \u0026ndash; p\u003csup\u003e2\u003c/sup\u003e \u0026ndash; q\u003csup\u003e2\u003c/sup\u003e, where p is the band frequency and q is band absence. Genetic distances among all accessions were estimated according to Nei and Li (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1979\u003c/span\u003e). A dendrogram was constructed using the unweighted pair group method with arithmetic mean (UPGMA) via Treecon version 3.1 software (Van de Peer and Wachter, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Branch support was evaluated by bootstrap analysis with 2000 replicates.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Genome size\u003c/h2\u003e\u003cp\u003eSignificant statistical differences in genome size were observed among the studied \u003cem\u003ePhacelia\u003c/em\u003e accessions. The 2C nuclear DNA content ranged from 1.00 pg in \u003cem\u003eP. ciliata\u003c/em\u003e to 3.61 pg in \u003cem\u003eP. egena\u003c/em\u003e, corresponding to 978 Mbp and 3531 Mbp, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Notably, intraspecific genome size variation was detected, as exemplified by three accessions of \u003cem\u003eP. crenulata\u003c/em\u003e: the 2C DNA content was 1.36 pg in \u003cem\u003eP. crenulata\u003c/em\u003e, 1.70 pg in \u003cem\u003eP. crenulata\u003c/em\u003e var. \u003cem\u003eambigua\u003c/em\u003e, and 1.96 pg in \u003cem\u003eP. crenulata\u003c/em\u003e var. \u003cem\u003ecorrugata\u003c/em\u003e. Similarly, genome size differences between \u003cem\u003eP. campanularia\u003c/em\u003e (1.25 pg/2C) and \u003cem\u003eP. campanularia\u003c/em\u003e subsp. \u003cem\u003evasiformis\u003c/em\u003e (1.50 pg/2C) enabled clear discrimination of these genotypes. The majority of accessions (78%) exhibited genome sizes within the range of 1.02 to 1.99 pg/2C, reflecting significant variation both within and between species. According to Soltis et al. (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) classification, 35 accessions possessed a very small genome, whereas five displayed a small genome. The coefficient of variation (CV) was low (\u0026lt;\u0026thinsp;5), indicating high reliability and reproducibility of the flow cytometric measurements.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.2. SCoT markers\u003c/h2\u003e\u003cp\u003eTen SCoT primers were screened to study the genetic relationships among \u003cem\u003ePhacelia\u003c/em\u003e species. All primers produced reproducible polymorphic bands across all 40 \u003cem\u003ePhacelia\u003c/em\u003e accessions. An image of the SCoT amplification generated by one primer of all studied accessions is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. A total of 212 polymorphic bands were amplified across the studied species (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The sizes of the amplified fragments ranged from approximetely100 to 3300 bp. All tested primers revealed 100% polymorphism. The lowest and highest numbers of polymorphic bands were 20 for SCoT-35 and 28 for SCoT-36, respectively, with an average of 24 polymorphic bands per primer. The polymorphism information content (PIC) values for the 10 SCoT primers ranged from 0.289 (SCoT-11) to 0.381 (SCoT-36), with an average PIC of 0.338 per primer. Primers SCoT-5, SCoT-12, SCoT-32, and SCoT-55 proved to be particularly effective, allowing for the discrimination of even closely related genotypes.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe genetic distance analysis among the studied accessions revealed distinguished variation in genetic similarity across the examined \u003cem\u003ePhacelia\u003c/em\u003e species. The lowest genetic distance (0.28) was observed between \u003cem\u003eP. capitata\u003c/em\u003e and \u003cem\u003eP. brachyloba\u003c/em\u003e. In contrast, the highest genetic distance values, reaching 0.85, were recorded between \u003cem\u003eP. rupestris\u003c/em\u003e and \u003cem\u003eP. coerulea\u003c/em\u003e, as well as between \u003cem\u003eP. rupestris\u003c/em\u003e and \u003cem\u003eP. pachyphylla\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Using the binary matrix derived from SCoT marker data, a dendrogram was constructed to visualize the genetic relationships among the accessions. The dendrogram effectively grouped the studied species into distinct clusters, each containing two, three or four accessions. Notably, four species - \u003cem\u003eP. grisea\u003c/em\u003e, \u003cem\u003eP. integrifolia\u003c/em\u003e, \u003cem\u003eP. pachyphylla\u003c/em\u003e, and \u003cem\u003eP. rupestris \u0026ndash;\u003c/em\u003e were not grouped into any of the clades (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Furthermore, the analysis demonstrated strong clustering of species represented by two or three accessions, reflecting their genetic correlation. For instance, \u003cem\u003eP. ramosissima\u003c/em\u003e clustered tightly with its variety \u003cem\u003eP. ramosissima\u003c/em\u003e var. \u003cem\u003elatifolia\u003c/em\u003e, underscoring their close genetic affinity. Similarly, \u003cem\u003eP. cicutaria\u003c/em\u003e grouped closely with its variety \u003cem\u003eP. cicutaria\u003c/em\u003e var. \u003cem\u003ehispida\u003c/em\u003e. The species \u003cem\u003eP. campanularia\u003c/em\u003e and its subspecies \u003cem\u003eP. campanularia\u003c/em\u003e ssp. \u003cem\u003evasiformis\u003c/em\u003e also clustered together, highlighting their taxonomic similarity. Additionally, the three accessions of \u003cem\u003eP. crenulata\u003c/em\u003e were grouped within a single cluster, further supporting the genetic consistency within this species (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe genus \u003cem\u003ePhacelia\u003c/em\u003e exhibits considerable morphological variability and ecological adaptability, which complicates accurate taxonomic classification. Understanding the genetic diversity within and among \u003cem\u003ePhacelia\u003c/em\u003e species is essential for effective species preservation and conservation strategies. Flow cytometry, as a rapid and reliable method for estimating genome size, serves as an essential preliminary step in species identification, providing valuable cytogenetic data that can distinguish closely related taxa. Using flow cytometry, genome size screening was performed on 40 \u003cem\u003ePhacelia\u003c/em\u003e accessions. To the best of our knowledge, this study represents the first report of 2C DNA content determination for 39 of these accessions. The measured 2C DNA content across all accessions ranged from 1.00 to 3.61 pg/2C, with approximately 88% of accessions exhibiting very small genome sizes (\u0026lt;\u0026thinsp;2.25 pg/2C). Until now, the 2C DNA content had only been reported for \u003cem\u003eP. tanacetifolia\u003c/em\u003e by Zonneveld (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), with a value of 0.22 pg higher than that obtained in the present study (1.26 pg/2C). Variations in genome size enabled the discrimination of nearly 18% of the examined \u003cem\u003ePhacelia\u003c/em\u003e accessions. Notably, intraspecific differences in 2C DNA content allowed clear differentiation between \u003cem\u003eP. campanularia\u003c/em\u003e (1.25 pg) and \u003cem\u003eP. campanularia\u003c/em\u003e ssp. \u003cem\u003ecampanularia\u003c/em\u003e (1.50 pg), as well as among three accessions of \u003cem\u003eP. crenulata\u003c/em\u003e (1.36 pg), \u003cem\u003eP. crenulata\u003c/em\u003e var. \u003cem\u003eambigua\u003c/em\u003e (1.70 pg), and \u003cem\u003eP. crenulata\u003c/em\u003e var. \u003cem\u003ecorrugata\u003c/em\u003e (1.96 pg). Conversely, nuclear DNA content did not permit distinction between \u003cem\u003eP. cicuriata\u003c/em\u003e and \u003cem\u003eP. cicuriata\u003c/em\u003e var. \u003cem\u003ehispida\u003c/em\u003e, nor between \u003cem\u003eP. ramosissima\u003c/em\u003e and \u003cem\u003eP. ramosissima\u003c/em\u003e var. \u003cem\u003elatifolia\u003c/em\u003e, where genome size differences were minimal (0.03 pg). The literature contains numerous reports demonstrating the successful application of flow cytometry as a reliable and efficient method for species identification, particularly in herbaceous plants where morphological differentiation is minimal or ambiguous (e.g. Jedrzejczyk, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Jedrzejczyk, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Jedrzejczyk and Rewers, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn cases where flow cytometry is insufficient to discriminate closely related species or varieties due to overlapping 2C DNA content, the incorporation of molecular markers serves as a complementary tool that provides a more precise and reliable means of genotype differentiation. The application of molecular markers such as SCoT markers offers a powerful approach for resolving taxonomic ambiguities, particularly among morphologically similar accessions (Jedrzejczyk, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Rewers et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In the present study, 10 SCoT primers were successfully employed, which amplify conserved genomic regions flanking the ATG start codon, thereby generating gene-targeted polymorphisms with potential functional relevance (Collard and Mackill, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Tabasi et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This marker system facilitates the detection of genetic variation at coding sequences, providing higher discriminatory power for intraspecific differentiation compared to genome size estimation applied alone. To the best of our knowledge, this is the first report of the application of SCoT molecular markers for genetic diversity assessment within the genus \u003cem\u003ePhacelia\u003c/em\u003e or even in the Boraginaceae family. In our study, all primers used in SCoT-PCRs revealed 100% polymorphism between all accessions, highlighting the high efficiency and discriminatory power of SCoT markers in capturing genetic variation within the \u003cem\u003ePhacelia\u003c/em\u003e genus. Using these primers, it was possible to identify all tested species and varieties.\u003c/p\u003e\u003cp\u003eThe highest genetic similarity observed between \u003cem\u003eP. capitata\u003c/em\u003e and \u003cem\u003eP. brachyloba\u003c/em\u003e indicated a close genetic relationship between these accessions, which was also confirmed by similar genome size, 1.87 pg/2C and 1.86 pg/2C, respectively. This genetic similarity could reflect retained ancestral states, relatively recent divergence, or gene flow within the genus, despite these ecological distinctions. Conversely, the lowest similarity was detected between \u003cem\u003eP. rupestris\u003c/em\u003e and both \u003cem\u003eP. coerulea\u003c/em\u003e and \u003cem\u003eP. pachyphylla\u003c/em\u003e, reflecting significant genetic divergence within these accessions, probably due to geographic isolation or ecological specialization. These findings correspond with prior observations of genetic subdivision and limited gene flow within \u003cem\u003ePhacelia\u003c/em\u003e populations (Castillo, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Glass, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The mapping of genome size onto the molecular tree highlights both parallel and divergent evolutionary trends within \u003cem\u003ePhacelia\u003c/em\u003e. In the majority of clades, relative genome size shifts were observed, suggesting the occurrence of polyploidy or chromosomal rearrangements. Even closely related accessions, in some clades (e.g. \u003cem\u003eP. campanularia, P. crenulata\u003c/em\u003e), differ substantially in genome size, indicating independent events of genome expansion or contraction. Although genome size generally aligns with SCoT-based clustering, additional evolutionary and ecological factors probably influenced genome size variation within the genus.\u003c/p\u003e\u003cp\u003eThe phylogenetic tree constructed using SCoT molecular markers provides clear insights into the genetic relationships among the examined \u003cem\u003ePhacelia\u003c/em\u003e species. The topology reveals several well-supported clades, grouping taxa that are congruent with previously proposed sectional or infrageneric classifications based on morphological or molecular evidence (Ferguson, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Luebert et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The clustering of species such as \u003cem\u003eP. crenulata\u003c/em\u003e, \u003cem\u003eP. crenulata var. corrugata\u003c/em\u003e, and \u003cem\u003eP. crenulata var. ambigua\u003c/em\u003e further aligns with their recognized taxonomic treatment as closely related taxa (Wilken, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), supporting the resolution power of SCoT markers for both species-level and infra-species differentiation. Furthermore, the grouping of \u003cem\u003eP. campanularia\u003c/em\u003e with its subspecies \u003cem\u003eP. campanularia ssp. vasiformis\u003c/em\u003e, and the close placement of species pairs like \u003cem\u003eP. fremontii\u003c/em\u003e and \u003cem\u003eP. douglasii\u003c/em\u003e, suggested a robust relationship between morphologically similar or geographically proximate taxa.\u003c/p\u003e\u003cp\u003eNotably, some clusters contain species that are morphologically diverse or distributed across different sections in traditional taxonomy, which supports previous molecular studies indicating that morphological convergence or plasticity is relatively common in \u003cem\u003ePhacelia\u003c/em\u003e (Hileman et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). For instance, taxa such as \u003cem\u003eP. californica\u003c/em\u003e, \u003cem\u003eP. cicutaria\u003c/em\u003e and its variety \u003cem\u003eP. cicutaria var. hispida\u003c/em\u003e appear in a distinct and well-separated branch, reflecting their unique evolutionary lineage, as noted in earlier phylogenetic reconstructions based on ITS and chloroplast sequences (Ferguson, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). The observed arrangement of \u003cem\u003eP. integrifolia\u003c/em\u003e and \u003cem\u003eP. glandulosa var. glandulosa\u003c/em\u003e in separate branches despite some morphological similarities echoes earlier findings pointing to complex evolutionary patterns within the genus (Ferguson, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Luebert et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOverall, the SCoT-based phylogeny corroborates and refines previous hypotheses regarding the genetic relatedness and taxonomic boundaries within \u003cem\u003ePhacelia\u003c/em\u003e. These findings are consistent with the conclusions drawn from earlier analyses employing nuclear and chloroplast markers (Ferguson, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Luebert et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), underscoring the value of SCoT markers for resolving fine-scale phylogenetic relationships within the genus. Furthermore, the results emphasize the importance of integrating multiple molecular marker systems to fully capture the evolutionary complexity of \u003cem\u003ePhacelia\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eSeveral well-supported clades in the SCoT-based phylogenetic tree correspond closely to sections and subsections recognized in classical taxonomy. However, certain taxa traditionally assigned to the same sectional or subsectoral groups are dispersed across different branches of the tree. This incongruence indicates that the relationships inferred from SCoT markers do not always fully align with morphological classification schemes, pointing to a more complex evolutionary history for the genus. Possible explanations for such discrepancies include convergent evolution of morphological traits, incomplete lineage sorting, or differences in the resolution power among molecular markers. Similar patterns of discordance have been reported in other molecular phylogenetic studies of \u003cem\u003ePhacelia\u003c/em\u003e (Ferguson, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Luebert et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), highlighting the necessity of integrative approaches that combine diverse molecular datasets with comprehensive morphological analyses to achieve a more robust and accurate understanding of phylogenetic relationships within the genus.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eThis study provides a comprehensive assessment of genetic diversity and genome size variation within the genus \u003cem\u003ePhacelia\u003c/em\u003e, combining flow cytometry and SCoT molecular markers for the first time in this taxon. The integration of flow cytometry and molecular marker analyses enables precise assessment of genetic relationships and species delimitation, complementing traditional morphological methods. This combined strategy enhances the accuracy of phylogenetic studies, germplasm characterization, and breeding programs, ultimately facilitating the optimized utilization of \u003cem\u003ePhacelia\u003c/em\u003e species in ecological restoration, sustainable agriculture, and pharmaceutical research.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIJ:\u003c/strong\u003e Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing \u0026ndash; Original Draft, Writing \u0026ndash; Review \u0026amp; Editing, Visualization, Resources, Supervision, Project administration. \u003cstrong\u003eAL\u003c/strong\u003e: Investigation. \u003cstrong\u003eMR:\u0026nbsp;\u003c/strong\u003eConceptualization, Methodology, Validation, Formal analysis, Investigation, Writing \u0026ndash; Original Draft, Writing \u0026ndash; Review \u0026amp; Editing, Visualization.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by the Minister of Science under the program \u0026ldquo;Regional Initiative of Excellence\u0026rdquo; (RID/SP/0017/2024/01). The authors are gratefully thankful to the GRIN-ARS-USDA gene bank for supplying \u003cem\u003ePhacelia\u003c/em\u003e seeds.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAppendix A. Supporting information\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary data associated with this article can be found in the online version at \u0026hellip;.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBaldwin, B.G., Goldman, D.H., Keil, D.J., Patterson, R., Rosatti, T.J., Wilken, D.H., 2011. The Jepson Manual: Vascular Plants of California, 2nd Edition. University of California Press, Berkeley, CA.\u003c/li\u003e\n\u003cli\u003eCalifornia Native Plant Society, 2024. Inventory of Rare and Endangered Plants of California [online]. https://www.cnps.org/rare-plants (accessed July 2024)\u003c/li\u003e\n\u003cli\u003eCastillo, R., 1994. 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Geobot. 8, 24\u0026minus;78. https://doi.org/10.3264/FG.2019.1022.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-applied-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"joag","sideBox":"Learn more about [Journal of Applied Genetics](https://www.springer.com/journal/13353)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/joag/default.aspx","title":"Journal of Applied Genetics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Boraginaceae, DNA content, flow cytometry, genetic diversity, molecular identification","lastPublishedDoi":"10.21203/rs.3.rs-7542683/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7542683/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe genus \u003cem\u003ePhacelia\u003c/em\u003e comprises numerous species with significant ecological, agricultural, and ornamental value. Despite its diversity, the genetic relationships and genome size variation within \u003cem\u003ePhacelia\u003c/em\u003e have remained poorly characterized. In this study, the nuclear DNA content (genome size) of 40 accessions representing 35 \u003cem\u003ePhacelia\u003c/em\u003e species was estimated using flow cytometry (FCM), and genetic diversity was assessed using Start Codon Targeted (SCoT) molecular markers. The 2C nuclear DNA content ranged from 1.00 pg (\u003cem\u003eP. ciliata\u003c/em\u003e) to 3.61 pg (\u003cem\u003eP. egena\u003c/em\u003e), with notable inter- and intra-specific variation observed. SCoT marker analysis generated clear and reproducible banding patterns, enabling the construction of a UPGMA dendrogram that resolved the accessions into distinct groups, largely consistent with morphological and taxonomic relationships. Some closely related taxa, such as \u003cem\u003eP. capitata\u003c/em\u003e and \u003cem\u003eP. brachyloba\u003c/em\u003e, exhibited both high genetic similarity and comparable genome sizes, while significant divergence was observed among others. Although several well-supported clades identified in the SCoT-based phylogeny corresponded closely to established sections and subsections, certain taxa traditionally classified together were found in separate branches, suggesting complex evolutionary histories and potential morphological convergence. The results demonstrate that combining SCoT markers and genome size estimation provides valuable insights into the genetic diversity and evolutionary relationships within \u003cem\u003ePhacelia\u003c/em\u003e, underscoring the importance of integrative molecular approaches for taxonomic and conservation studies in this genus.\u003c/p\u003e","manuscriptTitle":"Molecular profiling of 35 Phacelia species using SCoT molecular markers and genome size estimation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-24 06:01:44","doi":"10.21203/rs.3.rs-7542683/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revisions Needed","date":"2026-01-26T08:48:02+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-09-29T22:46:17+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-15T17:57:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-10T08:47:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Applied Genetics","date":"2025-09-05T05:08:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-applied-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"joag","sideBox":"Learn more about [Journal of Applied Genetics](https://www.springer.com/journal/13353)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/joag/default.aspx","title":"Journal of Applied Genetics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b79a035e-79ac-4e1d-b42c-b5c277c625da","owner":[],"postedDate":"September 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-07T16:09:11+00:00","versionOfRecord":{"articleIdentity":"rs-7542683","link":"https://doi.org/10.1007/s13353-026-01055-w","journal":{"identity":"journal-of-applied-genetics","isVorOnly":false,"title":"Journal of Applied Genetics"},"publishedOn":"2026-03-31 15:58:27","publishedOnDateReadable":"March 31st, 2026"},"versionCreatedAt":"2025-09-24 06:01:44","video":"","vorDoi":"10.1007/s13353-026-01055-w","vorDoiUrl":"https://doi.org/10.1007/s13353-026-01055-w","workflowStages":[]},"version":"v1","identity":"rs-7542683","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7542683","identity":"rs-7542683","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}